Nanoparticles in medicine and environment: Inhalation and health effects [1 ed.] 9048126312, 9789048126316, 9789048126323

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Nanoparticles in Medicine and Environment

Jan C.M. Marijnissen

l

Leon Gradon´

Editors

Nanoparticles in Medicine and Environment Inhalation and Health Effects

Editors Jan C.M. Marijnissen Delft University of Technology Dept. Chemical Engineering Julianalaan 136 2628 BL Delft Netherlands

Leon Gradoń Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warynskiego 1 00-645 Warsaw Poland

ISBN 978-90-481-2631-6 e-ISBN 978-90-481-2632-3 DOI 10.1007/978-90-481-2632-3 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009941459 # Springer Science+Business Media B.V. 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Cover design: Boekhorst Design Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Introduction

Currently a huge effort is put into nanoparticle research and the production of nanoparticles. In many cases it is unavoidable that during the production processes or during the use of the particles or a product made from these particles, nanoparticles are released into the environment. It is also realized that combustion processes, including traffic and power plants release nanoparticles into the atmosphere. However it is not known how nanoparticles interact with the human body, especially upon inhalation. At the same time research activities are devoted to understand how nano-sized medicine particles can be used to administer medicines via inhalation. In any case it is absolutely necessary to know how the nanoparticles interfere with the inhalation system, how they deposit in and affect the human system. During May 30 and 31, 2008, a group of scientists met in the Jablonna Palace near Warsaw in Poland to discuss all aspects from the origin/production of the nanoparticles till the interaction with the lungs and the toxic/therapeutic effects. This workshop, the fourth in a series of very specialized workshops on aerosol particles and the human body, brought together top-experts from different disciplines but all in the field related to aerosol nanoparticle release/production and nanoparticles inhalation and the effects. The subjects were assembled in three main themes: Sources and Production, Inhalation and Deposition, Toxicological and Medical Consequences. The workshop was concluded with an overview and a roundtable discussion. The chapters in this book, including the last one, which reports the overview and discussions, make clear how complex the subject is and that it only can be attacked by an interdisciplinary approach. It also made evident that much is still unknown. Yet we are confident that this book presents the state of the art in the field and that it sets directions on how to proceed in each different part of this important health issue. It also made clear how crucial it is to work together with all the different subdisciplines. As mentioned, the Jablonna 4 workshop was held in the beautiful Renaissance Jablonna Palace, surrounded by a big park, which contributed greatly in making this very fruitful scientific meeting again a very pleasant get-together. The Polish v

vi

Introduction

hospitality was highly appreciated. Finally the fantastic music, from the Dutch composer/graphic artist Juriaan Andriessen, realized by the Dutch pianist Jetje van Wijk with contributions of the Polish puppeteer Andrzej Bocian and adorned with visionary paintings of the composer as made into a slide show by Allert Schallenberg, completed the wonderful workshop. The workshop was sponsored by the Warsaw University of Technology, Delft University of Technology, Alistore-ERI, the German Gesellschaft fu¨r Aerosol Forschung and some industries. Leon Gradon´ Jan C.M. Marijnissen

Warsaw University of Technology Delft University of Technology

Contents

1

The Origin and Production of Nanoparticles in Environment and Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Heinz Burtscher

2

Characterization of Combustion and Engine Exhaust Particles . . . . . . . . 19 M. Matti Maricq

3

Medicine Nanoparticle Production by EHDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Jan C.M. Marijnissen, Caner U. Yurteri, Jan van Erven, and Tomasz Ciach

4

Electrospray and Its Medical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Da-Ren Chen and David Y.H. PUI

5

Generation of Nanoparticles from Vapours in Case of Exhaust Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Markku Kulmala and Mikko Sipila¨

6

Measurement and Characterization of Aerosol Nanoparticles . . . . . . . . . . 91 Wladyslaw W. Szymanski and Gu¨nter Allmaier

7

Inhalation and Deposition of Nanoparticles. Fundamentals, Phenomenology and Practical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Arkadiusz Moskal, Tomasz R. Sosnowski, and Leon Gradon´

8

Dosimetry of Inhaled Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Wolfgang G. Kreyling and Marianne Geiser

9

Particles of Biomedical Relevance and Their Interactions: A Classical and Quantum Mechanistic Approach to a Theoretical Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Ewa Broclawik and Liudmila Uvarova vii

viii

Contents

10

Health Effects of Nanoparticles (Inhalation) from Medical Point of View/Type of Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Robert Baughman and Michal Pirozynski

11

Effects of Cigarette Smoke and Diesel Exhaust on the Innate Immune Function of the Airway Epithelium . . . . . . . . . . . . . . . . . . 203 P.S. Hiemstra

12

The Potential Harmful and Beneficial Effects of Nanoparticles in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Karen G. Schu¨epp

13

Targeting Drugs to the Lungs – The Example of Insulin . . . . . . . . . . . . . 227 S. Ha¨ussermann, G. Scheuch, and R. Siekmeier

14

Protection of the Respiratory System Against Nanoparticles Inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Albert Podg´orski

15

Overview and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Jan C.M. Marijnissen, Leon Gradon´, and Bob W.N.J. Ursem

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

Contributors

Gu¨nter Allmaier Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164-IAC, A-1060, Vienna, Austria Robert Baughman University of Cincinnati Medical Center, 1001 HH Eden Avenue and Albert Sabin Way, P.O. Box 670565, Cincinnati, OH 45267-0001, USA Ewa Broclawik Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Krakow, Poland, [email protected] Heinz Burtscher Institute for Aerosol and Sensor Technology, University of Applied Sciences, Northwestern Switzerland, CH-5210 Windisch, Switzerland Tomasz Ciach Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warsaw, Poland Da-Ren Chen Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO USA Jan van Erven Nano Structured Materials, TU Delft, Juliananlaan 136, 2628 BL Delft, the Netherlands Marianne Geiser Institute of Anatomy, University of Bern, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland ix

x

Contributors

Leon Gradon´ Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warynskiego 1 00 – 645 Warsaw, Poland S. Ha¨ussermann Air Liquide Research Center CRCD, Paris, France P. S. Hiemstra Department of Pulmonology, Leiden University Medical Center, Leiden, the Netherlands, [email protected] Wolfgang G. Kreyling Institute for Inhalation Biology and Focus-Network Nanoparticles and Health, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, D-85764 Neuherberg/Munich, Germany Markku Kulmala Department of Physics, University of Helsinki, P.O. Box 64 (Gustaf Ha¨llstro¨minkatu 2), FI-00014, University of Helsinki, Finland Helsinki Institute of Physics, P.O. Box 64, FI-00014, University of Helsinki, Finland M. Matti Maricq Research and Advanced Engineering, Ford Motor Company, MD 3179, P.O. Box 2053, Dearborn, MI 48121, USA, [email protected] Jan C.M. Marijnissen Faculty of Applied Sciences, Delft University of Technology, Delft, the Netherlands Arkadiusz Moskal Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warynskiego 1 00-645 Warsaw, Poland Michal Pirozynski Department of Anesthesiology and Intensive Therapy, CMKP, 241 Czerniakowska Street, 00-416 Warsaw, Poland Albert Podgo´rski Faculty of Chemical and Process Engineering, Warsaw University of Technology Waryn´skiego 1, 00-645 Warsaw, Poland David Y. H. PUI Director of Particle Technology Laboratory, Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN USA

Contributors

xi

G. Scheuch Activaero GmbH, Gemu¨nden, Germany Karen G. Schu¨epp Department of Paediatric Respiratory Medicine, University Children’s Hospital, Bern, Switzerland and Swiss Paediatric Respiratory Research Group, Switzerland R. Siekmeier Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany Mikko Sipila¨ Department of Physics, University of Helsinki, Finland, P.O. Box 64 (Gustaf Ha¨llstro¨minkatu 2), University of Helsinki, Finland and Helsinki Institute of Physics, P.O. Box 64, FI-00014 University of Helsinki, Finland. Tomasz R. Sosnowski Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warynskiego 1 00 – 645 Warsaw, Poland Wladyslaw W. Szymanski Faculty of Physics, University of Vienna, Aerosol Physics, Biophysics and Environmental Physics Research Group, Boltzmanngasse 5, A-1090 Vienna, Austria Bob W.N.J. Ursem Faculty of Applied Sciences, Delft University of Technology Delft, Netherlands L. Uvarova Department of Applied Mathematics, Moscow State University of Technology “STANKIN”, Moscow, Russia Caner U. Yurteri Faculty of Applied Sciences, Delft University of Technology, Delft, the Netherlands

Chapter 1

The Origin and Production of Nanoparticles in Environment and Industry Heinz Burtscher

1.1

Introduction

Together with nitrogen oxides (NOX) particulate matter (PM) is considered one of the most important pollutants in ambient air. Many toxicological and epidemiological studies established adverse health effects by particulate matter. In most of these studies the particle mass in terms of PM10 or PM2.5 is used. There is increasing evidence that several health effects are associated with the ultra fine particles with diameters below 100 nm (Brown et al. 2001). Recent research shows that they can penetrate the cell membranes, enter into the blood and even reach the brain (Oberdo¨rster et al. 2004). Some investigations indicate that particles can induce heritable mutations (Somers et al. 2004). Usually approximately 90% of PM consists of fine and ultrafine particles (UBA 2005). Particulate matter in the ambient air is a mixture of directly emitted primary aerosol particles and secondary aerosol particles formed in the atmosphere. Coarse particles from primary aerosols originate mainly from mechanical processes (construction activities, road dust, re-suspension, wind, etc.) whereas fine particles are particularly produced through combustion (WHO 2006). So far the discussion is focused on vehicle emissions from diesel engines. Due to their adverse health effects and their abundance in the vicinity of roads, in particular in urban areas, they have become of great concern in the past years (Lighty et al. 2000, Wichmann and Peters 2000). However, other combustion systems as for example such for biomass combustion also have a significant contribution. Secondary aerosols are formed in the atmosphere through conversion of gaseous precursors such as sulphur oxides (SO2, SO3), nitrogen oxides (NO, NO2), ammonia

H. Burtscher 1 Institute for Aerosol and Sensor Technology, University of Applied Sciences, Northwestern Switzerland, CH-5210, Windisch, Switzerland e-mail: [email protected]

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_1, # Springer ScienceþBusiness Media B.V. 2010

1

2

H. Burtscher

(NH3) and Non-Methane Volatile Organic Compounds (NMVOC). Reaction products among many others are ammonium sulphates and ammonium nitrate, which often dominate PM mass in ambient air. According to (UBA 2005) important sources of precursors for secondary aerosols are agriculture (NH3), Diesel engines (NOX), other combustion processes (SO2, SO3 and NO, NO2) and the use of solvents, chemical industry and petro chemistry (NMVOC). In the next section some data on particulate matter in terms of mass concentrations will be given, and then emissions from some important contributors and measures taken to reduce these emissions will be discussed. This will by far not be complete, but I hope that the most important contributors are mentioned. The focus will lie on the submicron fraction.

1.2

Particulate Matter (PM10), Sources and Composition

Most available data for ambient air particulate matter concerns total mass concentrations (PM10, PM2.5). Only these are routinely measured and limited. Number concentrations, size distributions, and detailed chemical composition are determined only in research work. Main components of the ambient aerosol are l

l

l

l

l

l

Salts (most abundant ammonium sulphate and nitrate): This is secondary aerosol formed from gaseous precursors, mainly NH3, SO2 and NOx. The salts are found in the accumulation mode. Elemental carbon (EC): EC mainly arises from incomplete combustion, important sources being diesel engines. In ambient air EC usually is found as agglomerates in the accumulation mode with a number of volatile species adsorbed on the surface. Coarse mode EC particles are due to tire wear. Organic carbon (OC): In incomplete combustion (combustion engines, biomass combustion) a variety of organic species are produced, which are emitted in the gas phase and condense on solid cores or nucleate when the exhaust cools. More volatile material may form secondary aerosol upon oxidation in the atmosphere. Most OC is found in the fine fraction. Anthropogenic coarse OC may for example be due to tire wear or wood processing. Minerals (aluminium silicate, calcium carbonate, gypsum): From construction, road dust, agriculture, coarse particles. Sodium chloride: Near coasts from sea salt, during winter time road salt for de-icing. Metal particles: From traffic, engine wear, break wear and industrial processes.

Figure 1.1 shows examples for the composition of urban (Fig. 1.1a) and rural (Fig. 1.1b) PM10, measured at two locations in Switzerland. The urban site is in the city of Berne, the rural in Chaumont. Composition data from many other locations are similar.

1 The Origin and Production of Nanoparticles in Environment and Industry

a

17%

7%

b

10%

13%

ammonium 8%

4%

nitrate

12%

2% 5%

11%

3

sulfate EC

9%

OC 29%

17%

30%

21%

mineral dust trace elements unidentified

5%

Fig. 1.1 Typical (a) urban (city of Bern) and (b) rural (Chaumont) PM10 composition (data from EKL 2007)

domestic 11%

industry agriculture and forrestry traffic

29%

30%

30%

Fig. 1.2 Contribution of different sources to PM10 (from EKL 2007)

Important producers of anthropogenic particulate matter are l

l l

l

Road traffic (soot emissions, mainly from diesel engines, tire-, brake- and clutch wear, road dust). In urban areas traffic is the main source. Mobile off road diesel engines (forest work, agriculture, construction). Stationary sources: Combustion of wood, coal and other fuels, industrial emissions. In addition to primary aerosol these sources also emit gaseous precursors leading to the formation of secondary aerosol in the atmosphere (NH3, NOX, sulphur compounds, VOC).

The quantitative contribution of important sources to the primary aerosol mass is shown in Figs. 1.2 and 1.3. In a recent paper presenting data from India (Delhi) the contribution of traffic is much higher (Srivastava et al. 2008). Figure 1.4 shows emissions of the major precursor gases. NOX is mainly due to traffic, SOX and NMVOC are dominated by industrial emissions and NH3 stems almost completely from agriculture.

4

H. Burtscher wood combustion diesel

8%

gasoline wood combustion in forrest other combustion

17% 1% 57%

non combustion

7%

10%

Fig. 1.3 Contribution of combustion and non combustion to PM10 (from EKL 2007)

NMVOC

NOx

6%

13%

6%

22%

13% 22%

23%

domestic

23%

industry

6% 6%

57% 57% 14%

agriculture and forrestry

59%

14%

traffic

59% NH3

0% 2% 2%

SOx

1%

96%

9%

23%

67%

Fig. 1.4 Contribution of different emitters to the major precursors of secondary aerosol (NOX, non methane VOC (NMVOC), NH3 and SOX) (data from EKL 2007)

1 The Origin and Production of Nanoparticles in Environment and Industry

1.3

5

Particles from Diesel Engines

Diesel particles as well as particles from other combustion sources are a complex mixture of elemental carbon, a variety of hydrocarbons, sulphur compounds and other species. Particles differ in size, composition, solubility and therefore also in their toxic properties. Figure 1.5 shows the typical composition of particles from diesel engines (Kittelson 1998). The data shown in Fig. 1.5 are average values over a number of heavy-duty engines measured during a transient cycle. Operating conditions and engine type strongly influence the composition. For example the EC fraction may be greater than 80%, in particular in high load conditions. The exhaust contains a significant volatile fraction. Depending on temperature and other conditions the volatile fraction may l l l

Remain in the gas phase Condense on existing solid particles Nucleate and form new particles

Different amounts of the species mentioned above will consequently be measured as ‘particulate’ emissions, depending on how samples are taken (location of sampling, temperature, dilution, etc.). The sample contains not only particles, formed in the combustion process, but also secondary particles, formed during cooling in the exhaust and sampling lines. In addition to these ‘directly condensing’ volatile material secondary aerosol is formed later in the atmosphere. According to work by Robinson et al. (2007) the importance of aerosol is formed later in the atmosphere has been strongly underestimated up to now. On the other hand, much of the previously condensed material re-evaporates in the atmosphere according to the results of these authors. Diesel particles are agglomerates consisting of mainly spherical primary particles of about 15–40 nm in diameter. A study by Su et al. (2004) indicates that the primary particles emitted from modern engines fulfilling the EURO IV limits are smaller than those from older engines. These authors also find differences in

Fig. 1.5 Composition of diesel particles, average values for heavy duty engines during transient test cycle (from Kittelson 1998)

6

H. Burtscher 1.5E+13 2000RPM/100% 1400RPM/100% 2000RPM/50%

1E+13

part/kwh

1400RPM/50%

5E+12

0 10

100

d [nm]

1000

Fig. 1.6 Size distribution of diesel particles at different operating conditions (different speed and 50% or 100% load)

the particle microstructure. Whereas for conventional engines amorphous and graphitic structures are dominant, they observed a higher fraction of fullerenelike soot primary particles for the modern engine they tested. They also found that these particles can be oxidized more easily. The number size distribution of the agglomerated particles (accumulation mode) peaks almost always in the range of 60 to 120 nm. An example for size distributions of particles in the exhaust of a diesel engine used in machines for building or street construction, e.g. diggers, in different operating conditions is given in Fig. 1.6. As shown by this example the size is relatively insensitive to the operating conditions of the engine. Only few extreme conditions lead to significantly different size distributions. There is also no strong dependence of the particle size on the type of engine. The size distributions are lognormal with an almost constant geometric standard deviation of 1.8–1.9 (Harris and Maricq 2002). As will be shown later the accumulation mode can be accompanied by a ‘nucleation mode’, consisting of much smaller particles. Modern trap technology allows a very efficient removal of solid particles, also of the nanometer-sized fraction. Volatile material passes the trap in the gas phase. As the solid surface to condense on has been removed by the trap, nucleation becomes much more probable. The number concentration of particles downstream a particle trap is often dominated by volatile particles in the nucleation mode (see Fig. 1.7). Nucleation is further enhanced by oxidation due to catalytic active devices (catalytic converters or catalysts used for trap regeneration). For example the oxidation of SO2 to SO3, in combination with water may lead to the formation of sulphuric acid droplets. Whereas a strong correlation of the occurrence of nucleation and the fuel sulphur content is observed, chemical analysis of nucleation mode particles by thermal desorption particle beam mass spectrometry shows that the sulphur content of these particles is only a few percent or even less (Sakurai et al. 2003).

1 The Origin and Production of Nanoparticles in Environment and Industry

7

1000000

without trap

concentration [#/cm3]

100000

10000 with trap

1000

100 10

100 diameter [nm]

1000

Fig. 1.7 Size distribution of diesel particles with and without particle trap

They mainly consist of organic material. The finger print of the measured mass spectra indicates that this material stems mainly from lubricant oil and only to a small fraction from the fuel (Sakurai et al. 2003). This indicates that the first step in nucleation particle formation is the nucleation of sulphuric acid and water, followed by particle growth by condensation of organic species. A detailed theoretical investigation of this process has been done by Vouitsis et al. (2008). Downstream a trap the particle composition is dominated by volatile material (Burtscher 2005). Engine optimization led to a significant reduction in emitted mass; however, the number concentration is not significantly reduced. Comparing the size distribution of EURO3 to EURO5 engines shows that mainly the tail on the large particle side is reduced for modern engines (Mayer et al. 2007). The same study also shows that by open traps, introduced recently, only a small reduction in particle emissions is achieved. A significant reduction is only obtained by closed traps. They remove more than 99.9 %. Catalytic aftertreatment devices (oxidation catalysts to reduce hydrocarbon emissions, catalytically active filters to assist filter regeneration) also oxidize NO, which means that direct NO2 emissions are significantly increased (Du¨nnebeil et al. 2007), if such devices are applied. This leads to an increase in NO2 concentrations measured near highways. As NO is rapidly oxidized in the atmosphere anyway this effect is only observed in the direct vicinity of frequented roads. Emissions from very large diesel engines for marine applications or electrical power production differ significantly from those of smaller engines. This is on the one hand due to the different fuel (much higher sulphur and ash content) but also to

8

H. Burtscher

different operation conditions as for example the much lower speed. The fraction of volatile organic material is significantly higher, whereas the EC fraction is small. The average particle diameter is only about 50 nm (Kasper et al. 2007).

1.4 1.4.1

Partilces from Other Combustion Sources Spark Ignition Engines

Particles from gasoline engines are smaller than those from diesel engines (typically 40 nm average diameter) and they contain a large fraction of volatile material. Measurements from a small engine for electrical power production show a mean diameter of 40 nm and a fractal dimension df of three, when measured at ambient temperature. This indicates compact particles. If these particles are heated to a temperature of about 250 C, they become significantly smaller (mean diameter 20 nm) and df decreases from 3 to about 2.2. An explanation for this observation may be that at ambient temperature the solid, fractal part of the particles is encapsulated in volatile material. If this volatile material is removed by heating, the fractal core becomes ‘visible’ (Burtscher 2000). Emissions from well maintained port injection spark ignition engines are relatively low during stationary operation of the warm engine. However, they increase by orders of magnitude during acceleration (Kasper et al. 2005). Emissions from the cold engine are also much higher. The situation is very different for direct injection spark ignition engines (lean engines). These engines have been introduced because their fuel consumption in partial load conditions is lower. However, their particle emissions are somewhere in between those from conventional port injection gasoline engines and diesel engines (Aufdenblatten et al. 2002; Andersson et al. 2007). Two Stroke engines, for example in scooters, emit very high concentrations of particles, consisting mainly of organic carbon (Czerwinski et al. 2006).

1.4.2

Wood Combustion

Wood combustion in small domestic furnaces or stoves contributes significantly to ultrafine particle concentrations in ambient air. If the conditions for the combustion are good, most particles are in the submicron range. Emissions are high in the start up phase. Later the number concentration decreases and the particles become smaller. Typical size distributions in different phases of the burning cycle are shown in Fig. 1.8. The situation completely changes, if the furnace is not operated properly, in particular if the air supply is too low. In this case particle emissions may be dramatically higher and the size distribution is shifted to much larger particles (see for example Nussbaumer et al. 2008).

1 The Origin and Production of Nanoparticles in Environment and Industry

9

9.0E+07

start up

dn/dlog(d)(cm–3)

intermediate

6.0E+07

burn out

3.0E+07

0.0E+00 10

100 d [nm]

1000

Fig. 1.8 Size distribution of particles from wood combustion (log wood stove) at different phases of the combustion

Particles from wood combustion mainly consist of three fractions: (1) an inorganic fraction (minerals and salts, dominated by potassium and calcium compounds), (2) elemental carbon (EC) and (3) organic carbon (OC). For bad operating conditions in particular the organic fraction may become very high. The OC/EC ratio is much higher than for diesel engines. Pellet and chip furnaces allow a very good combustion. In this case the inorganic fraction is dominant. Typical mass emission factors are 30–60 mg/MJ. Emission factors for wood log stoves range from 10 mg/MJ for optimal operation up to several 1000 mg/MJ. In Fig. 1.9 total mass (TM) is plotted versus total organic carbon mass (OC) for different furnaces and operating conditions. The plot shows that TM and OC decrease both, when optimizing the combustion. After a base level is reached a further reduction of organic material by better combustion has no more significant influence of the total mass, which is now dominated by inorganic non-combustible material (Johansson et al. 2004). Toxicology tests (Klippel and Nussbaumer 2007) indicate that particles emitted from well operated furnaces (mainly minerals and salts) are less toxic than diesel particles, but those from high emitters (dominated by organic material) are much worse. Recently small electrostatic precipitators became available, which allow a reduction of emitted particles up to 90% (Schmatloch and Rauch 2005). The efficiency depends mainly on the flow rate of the exhaust gas.

10

H. Burtscher 10000 Wood pellets Wood logs

TM (mg/MJ)

1000

inorganic fraction organic fraction

100

10

1 1

10

100 OC (mg/MJ)

1000

10000

Fig. 1.9 Total mass (TM) emission factor versus emission factor for organic carbon (OC) (data from Johansson et al. 2004)

1.4.3

Waste Incineration

Waste incineration used to be a relevant source for emission of dust, heavy metals, acids, and many other species. Meanwhile the plants have been equipped with efficient flue gas cleaning devices, which led to a significant reduction of the above mentioned pollutants. Flue gas cleaning is done by the following devices: l

l l

Electrostatic precipitators (usually dry filters, sometimes wet filters are applied) or fabric filters for removal of particles. Wet scrubbers (removal of SO2, HCl, HF, Heavy metals, Aerosol particles). DeNOX system. Selective non catalytic reduction (SNCR) of NOX by NH3 injection (NOx is reduced at about 900 C) or more frequently Selective Catalytic Reduction (SCR) of NOx also by NH3 injection. For SCR significantly lower temperatures are sufficient. The catalyst usually is the last stage of the cleaning system.

The typical capacity of one combustion line of a waste incineration plant is in the order of 100000 Nm3/h. The plants have 2–3 lines. Figure 1.10 shows an example of size distributions, measured in the raw exhaust, after the electrostatic precipitator (ESP) and in the stack (after wet scrubber and SCR, Burtscher et al. 2002). The raw emissions are very high, but already the ESP removes about 99.9 % of the particles. The increase at the small particle side most probably has to be ascribed to nucleation of volatile material. The wet scrubber reduces the particle concentration by another order of magnitude. The resulting

1 The Origin and Production of Nanoparticles in Environment and Industry

11

1.E+09 solid: SMPS, dots: OPC

1.E+08

raw gas

dN/d log(d) [cm–3]

1.E+07 1.E+06

after ESP

1.E+05 1.E+04 1.E+03

stack

1.E+02 1.E+01 10

100

1000

10000

d [nm]

Fig. 1.10 Particles in the exhaust of a waste incineration plant. Concentrations in the raw gas, after the electrostatic precipitator, and in the stack are plotted

stack concentration is in the order of ambient air concentrations. Measurements at a number of plants corroborated these results. This demonstrates that the flue gas cleaning system of modern waste incineration plants is very efficient concerning fine particle removal. Properly maintained state of the art waste incineration plants are no relevant source for particulate matter.

1.4.4

Boilers and Furnaces

Particle emissions from domestic heating other than wood combustion are very small. Well maintained gas- and oil heatings allow an almost complete combustion. The same can be said about gas turbines. A comparison of the particle size distributions in the exhaust of a number of combustion systems can be found in Nussbaumer (2004). Table 1.1 shows a comparison of emissions from gas, oil and wood.

1.5

Noncombustion Particles

Traffic contributes to particulate pollution not only via tail pipe emissions, but also by road dust, tire wear and so on. In terms of PM10 emissions the contribution of this ‘non-combustion’ fraction is significant. Figure 1.11 shows the relative

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H. Burtscher

Table 1.1 Emission factors for particle mass and number concentration for heating systems with different fuels

Mass (mg/MJ) Natural Gas 90%) of the soot (Dec and Kelly-Zion 2000). The above scenario applies to the majority of carbon based fuels ranging from methane to coal. The main difference between pure molecular fuels and practical fuels, such as diesel fuel and coal, is the presence of impurities including sulfur and metals. The latter produce metal oxides, which when their concentrations are low condense onto soot surfaces (Miller et al. 2007). But under high metal to carbon ratios, as might occur with fuel-born additives, nucleation of metal nanoparticles can occur during combustion (Miller et al 2007; Kasper et al. 1999). As a result of extensive agglomeration and oxidation, soot/ash particles emerge from many combustion processes with a ubiquitous lognormal size distribution (Harris and Maricq 2001). Due to their high temperature origin these particles are nonvolatile and, therefore, relatively robust to sampling methodology, with the main concerns being losses and coagulation. Sampling plays a much more important role with respect to the semivolatile components of combustion aerosols. The precursors of this PM, including unburned fuel, lube oil, and partial combustion products, exit the combustion source as gases and in some cases are further transformed, for example the conversion of SO2 to SO3 over a diesel oxidation catalyst (DOC). As the exhaust cools some substances approach their saturation vapor pressures, whereupon two possibilities ensue: they condense onto existing soot particles or nucleate to form new particles. This can occur already in the exhaust system, or as the exhaust exits into the atmosphere (or dilution air of a sampling system). The degree to which each path is followed depends sensitively on a host of conditions such as temperature, dilution rate, humidity, soot concentration, fuel composition, and the presence of aftertreatment devices (Abdul-Khalek et al. 1998, 1999; Shi and Harrision 1999; Vaaraslahti et al. 2004). Condensation is the principal pathway in situations where soot dominates the PM. This is the case in conventional diesel engine exhaust, where condensed material often only modestly affects soot characteristics. As the ratio of condensable material to soot increases, nucleation predominates. At moderate levels this produces a bimodal particle size distribution, but at high levels it can alter the nature of the PM to one dominated by liquid heavy hydrocarbon/sulfuric acid droplets. Sampling conditions, too, can affect the propensity for nucleation and thereby alter the appearance of PM. In the following sections we will explore more deeply sampling methods and their influence on PM measurements, the methods used to characterize combustion particles, and the picture this provides of the soot and nuclei modes of particles from flames and internal combustion engines.

2.2

How Sampling Impacts PM Measurement

Ordinarily to study aerosol processes, for example how soot evolves in a flame, the goal is to probe non-invasively, or to use sampling methods that preserve the nascent aerosol characteristics. But how is this possible in the case of combustion

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emissions, when some particles are produced in the flame or engine, whereas others may only form later? Approaches such as light scattering and laser induced incandescence (LII) (Dec et al. 1991; Snelling et al. 1999) probe particles as they exist in a flame, but they miss semivolatile PM that subsequently condenses or nucleates as the exhaust cools. Combustion PM characterization and sampling methods are, therefore, inexorably linked. But this is only one sampling issue. Here, at least, the emissions are combustion produced; the complexity lies in interpreting the gas–particle partitioning. A second issue concerns the possibility, depending on design, that the sampling system itself acts as a particle source, generating artifacts that cannot always be readily distinguished from combustion source PM.

2.2.1

Conventional Approach – Dilution Tunnels

The standard approach to motor vehicle PM emissions measurement collects test vehicle exhaust into a dilution tunnel (Hildemann et al. 1989; Code of Federal Regulations 2008). Figure 2.2 illustrates two such tunnels in a chassis dynamometer facility. Historically one is reserved for diesel and the other for gasoline vehicles based on their disparate emissions; however, the advent of diesel particulate filters (DPF) has blurred this distinction. Vehicle exhaust is combined in the tunnel with filtered, temperature and humidity controlled, dilution air (38 C and 9 C dew point) at a constant total volume. This implies a dilution ratio that varies during transient emissions tests, such as the US Federal Test Procedure (FTP) or New European Drive Cycle (NEDC). But it confers the advantage that emission rates per distance traveled are directly computed from the tunnel flow rate and PM concentration. The weakness of this approach lays not so much with the dilution tunnel as with the need to convey the exhaust from tailpipe to tunnel. This typically occurs

Fig. 2.2 Dilution tunnels used for PM measurement in a chassis dynamometer test cell

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Fig. 2.3 Comparison of port fuel injection gasoline vehicle PM size distributions measured at the tailpipe versus from the dilution tunnel. The large nanoparticle peak originates from the sampling system transfer hose

through a heated corrugated stainless steel hose that can be meters in length and, therefore, introduces delays of a second or longer prior to dilution. Engine PM does not remain unchanged during this transit; particles are lost by diffusion and thermophoresis, and coagulation reduces their numbers while increasing their sizes. Moreover, hot vehicle exhaust can desorb materials deposited on the transfer hose walls, which subsequently nucleate in the dilution tunnel to generate an aerosol artifact difficult to distinguish from engine PM (Maricq et al 1999). This is illustrated in Fig. 2.3 by gasoline vehicle test data recorded after sustained driving at 100 km/h. The dilution tunnel measurement exhibits an intense nanoparticle peak, which is absent in the sample drawn directly via ejector diluter (at 200 C) from the tailpipe. One can argue, as discussed below, that the nanoparticles are real; ejector sampling simply suppresses their nucleation. However, this peak appears only after many minutes at the 100 km/h speed, and then dissipates over many minutes after the vehicle is returned to low speed. This lag correlates with transfer hose temperature and suggests that these nanoparticles arise from heat release of stored precursors, most likely heavy hydrocarbons.

2.2.2

Direct Sampling Methods

Redesigning the dilution tunnel to enable dilution at, or near, the tailpipe can alleviate artifact particle formation (Maricq et al. 2003). Alternatively, a number of options exist to sample combustion aerosols directly at the source including the rotating disc diluter (Matter Engineering) and ejector pump diluters (Dekati Ltd.). These methods permit control of sampling conditions to help avoid biases and artifacts, for example by the use of heated lines and heated first stage dilution to reduce thermophoretic losses and suppress storage–release mechanisms. Sampling is especially critical directly from a flame or engine cylinder where temperatures of ~2000 K and particle concentrations on the order of 1010 cm3 require immediate

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dilution to quench combustion chemistry and particle agglomeration (Zhao et al. 2003). But sampling conditions can impact also the combustion aerosol itself, hence the argument with respect to Fig. 2.3 that one cannot distinguish without additional data whether direct sampling via the heated ejector pump prevents an artifact or suppresses nucleation of material from the engine exhaust. While it may complicate a precise definition of combustion PM, the ability to control sampling conditions can aid in aerosol characterization. The principal control parameters are temperature, residence time, and dilution factor (Abdul-Khalek et al. 1999). The EU Particulates Project, for example, analyzed motor vehicle exhaust via so called “wet” and “dry” branches to distinguish semivolatile and nonvolatile PM components (Ntziachristos et al. 2004). Sampling is done through a “perforated tube” diluter, which permits the use of cold dilution air while avoiding thermophoretic deposition. Cold dilution air enhances nucleation and condensation, hence the label “wet” branch. Passing this aerosol through a thermo denuder (Burtscher et al 2001), a heater followed by activated charcoal adsorbent, creates the “dry” branch, which yields the nonvolatile component of the combustion PM. Comparison to the “wet” branch then reveals the semivolatile component. In essence this accomplishes via sampling what thermal elemental carbon/organic carbon (EC/OC) analysis provides for filter collected PM (Chow et al. 2001). Furthermore, the “dry” branch forms the basis for the proposed European Particle Measurement Program (PMP) number based particle emissions standard (Kasper 2004; Giechaskiel et al. 2008). The sampling system design employs a heated residence chamber to evaporate semivolatile PM, and relies on dilution to prevent re-nucleation.

2.3

Particles Formed by Combustion

Bearing in mind sampling’s potential influences, we now turn to examine the nature of combustion PM. Recent reviews of physical and chemical characterization of diesel PM provide a detailed picture of current progress in this field (Burtscher 2005; Maricq 2007). The present discussion focuses on four qualities: morphology, density, volatility, and electrical charge, which are useful to distinguish the two modes prevalent in both flame and engine generated particles. These modes are illustrated in Fig. 2.4 in the case of a light duty diesel vehicle run over the transient FTP drive cycle. The peaks in time arise primarily from the increased exhaust flow during vehicle acceleration. As demonstrated below, the 20–200 nm mode can be associated with soot, that is, particles primarily formed in the engine cylinder or flame. The 2–20 nm mode remains more inscrutable; in some cases it is born from combustion chemistry and in others subsequently produced by nucleation. The present section examines the morphology and effective density of the so-called soot mode, whereas the next section investigates the nuclei mode via its volatility and electrical charge.

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Fig. 2.4 Time resolved bimodal particle size distributions recorded from the exhaust of a light duty diesel vehicle (DOC and low sulfur fuel) via a dilution tunnel. Mobility equivalent diameter measured by Engine Exhaust Particle Sizer (TSI Inc.)

2.3.1

Size and Morphology

Transmission electron microscopy (TEM) has been much applied to the study of soot particle physical characteristics, usually via thermophoretic sampling directly from the flame (see for example (Dobbins 2007) and references therein). Here we examine soot that is first size selected with a differential mobility analyzer (DMA). Figure 2.5 displays TEM images of rich premixed ethylene flame soot particles at four distinct sizes ranging from 25 to 200 nm in mobility diameter, and produced with equivalence ratios from F ¼ 2.05–2.45. The latter two of this series represent particles that are larger than the vast majority actually present in these premixed flames, even at heights well past the flame front (see Fig. 2.9a for the flame soot size distribution at 20 mm above the burner). The 100 and 200 nm particles are grown by allowing soot sampled from the flame, quenched but not highly diluted (~5  109 particles/cm3), to coagulate for 3 s in a residence chamber at near room temperature. One consequence of electrical mobility sizing is that multiply charge particles appear smaller to the DMA than they actually are by a factor of approximately n½, where n represents the number of charges. Figure 2.5b displays one such outsized particle, presumably transmitted by the DMA due to multiple charges. Aside from this charge dependence, the chosen mobility diameter in each case matches closely, within ~10%, the projected area equivalent diameter of the corresponding TEM images. The major feature of Fig. 2.5 is the striking change in morphology that accompanies increasing soot size. At 25 nm the particles are at best disfigured spheres, with primary particle structure barely discernable due to filling in by surface growth. By 50 nm primary particle structure is clearly visible, but the aggregates

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Fig. 2.5 TEM images of mobility size selected soot particles formed by a rich premixed ethylene flame. Soot from the flame was allowed to coagulate for up to 3 s in a residence chamber to grow the aggregates. The odd oversize particle in panel B is a doubly charged particle with the same electrical mobility as the 50 nm particles

remain compact, with growth dominated by aggregate–primary particle collisions. The 100 and 200 nm particles increasingly display the fractal-like structure commonly associated with soot, and arising from aggregate–aggregate collisions. Figure 2.6 displays nominally 60 nm mobility selected particles collected from the exhaust of an idling diesel vehicle; but clearly larger, multiply charged, particles are also evident. Compared to their flame counterparts these soot particles appear less fractal-like. They possibly contain condensed hydrocarbons, but morphology changes that might indicate evaporation were not observed under the TEM. Condensed material is not unexpected here as idle and low load are engine operating conditions often associated with a higher semivolatile to soot emissions ratio owing to lube oil or fugitive heavy end fuel components.

2.3.2

Effective Density

Another manifestation of particle morphology is revealed through measurement of its density. When particles assume complex shape, density takes on two interpretations: one is the material density and the other is the concept of effective density

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Fig. 2.6 TEM images of 60 nm mobility size selected soot particles from a light duty diesel vehicle run at idle. Larger particles transmitted by the DMA on account of their multiple charge are also displayed

(Kelly and McMurry 1992). In the first case soot particle density remains constant with size and shape except if changes in composition take place, for example hydrocarbons condensed onto diesel soot particles were found to lower their density from 1.8 to 1.3 g/cm3 (Park et al. 2004). In contrast, effective density, defined here as particle mass divided by its mobility equivalent volume, decreases with increasing particle size owing to the rising fraction of voids caused by aggregation. As per definition, effective density can be determined by simultaneous measurement of a particle’s mobility diameter and mass. One procedure (Park et al. 2003) accomplishes this directly via a tandem DMA – aerosol particle mass analyzer (APM). Another approach is to select particles of known mobility diameter and sequentially, or in parallel, measure their aerodynamic diameter (Schleicher et al. 1995; Maricq and Xu 2004; Virtanen et al. 2004). The latter quantity is connected with the particle’s settling time and is therefore dependent on its mass. Effective density is related to the two particle equivalent diameters via re ðdm ÞCc ðdm Þdm2 ¼ ra Cc ðda Þda2

(2.1)

where ra is assigned unit density and Cc is the Cunningham slip correction (Hinds 1999). Figure 2.7a compares aerodynamic and mobility diameter measurements of mobility selected particles at dm ¼ 141 nm. The line marked “DMA” depicts the mobility diameter recorded using a second DMA. In decreasing order, the three peaks correspond to 141 nm particles having n ¼ 1, 2, and 3 charges as a result of passing through a 210Po neutralizer prior to the second DMA. In contrast, the aerodynamic diameter (histogram), as measured by electrical low pressure impactor (ELPI), is substantially smaller than the mobility diameter. By Eq. 2.1 this implies an “effective” density below 1 g/cm3, which is considerably lower than the commonly accepted 1.8 g/cm3 material density of soot (Park et al. 2004).

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Fig. 2.7 Effective density measurement of flame and diesel soot particles. Panel A: Aerodynamic (ELPI) versus mobility (DMA) diameter of 141 nm particles. Panel B: Flame soot density from low and moderately sooting flames. Panel C: Diesel soot density at various vehicle speeds

Figures 2.7b and c display the mobility diameter dependence of soot effective density for flame and diesel soot, respectively. These data follow closely the power law expression re ¼ r0 ðdm =d0 ÞðDf 3Þ

(2.2)

expected for fractal-like particles, except at small diameter where the aggregate primary particle number approaches unity. Here d0 and r0 are the primary particle diameter and density, and Df is the fractal dimension. Particles from a moderately sooting flame, F ¼ 2.4, sampled from 20 mm above the burner exhibit a fractal dimension of Df ¼ 1.9, within the range found by light scattering and TEM (Dobbins 2007). Lowering the height to 10 mm and the equivalence ratio to F ¼ 2.0 produces a younger soot. The fractal dimension increases to Df ¼ 2.3, reflecting the reduced time for aggregation and consequently more spherically shaped particles. That these particles also exhibit a lower overall effective density suggests a decrease in their C/H ratio. Light duty diesel vehicle soot effective density also follows a Df ¼ 2.3 dependence, with only little variation between idle and 112 km/h operation, similar to what is found for heavy duty diesels by Park et al. 2003. Its higher fractal dimension relative to the more aged flame soot may originate from semivolatile hydrocarbon condensation filling voids in the fractal-like structure of dry soot (Ristima¨ki and Keskinen 2006), or from the partial soot oxidation that occurs in the latter stages of diesel combustion (Dec and Kelly-Zion 2000) leaving more compact structures. Condensation has little effect on the small particles, which already have a re ffi 1 g/cm3 close to that of hydrocarbons, but it would tend to increase the effective density of larger aggregates, where re falls well below 1 g/cm3. One practical application of effective density is that it enables calculation of PM mass from particle size distribution data, N(dm), via 1 ð p ddm re ðdm Þdm3 Nðdm Þ (2.3) M¼ 6 0

The soot (solid particle) mode of diesel PM exhibits a characteristic lognormal soot mode, with a width (sg ¼ 1.75) nearly independent of engine operation and fuel

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Fig. 2.8 Particle number versus PM mass emission rates from various light duty engine technologies

choice (Harris and Maricq 2001) (in some cases high hydrocarbon/sulfate emissions can significantly broaden this or yield bimodal distributions). This leaves two free parameters to describe diesel soot, namely particle number and geometric mean diameter. Mean size varies with engine speed/load, level of exhaust gas recirculation, and fuel composition, but typically remains within the 50–100 nm range. Soot emissions from spark ignition engines, port as well as direct fuel injection, deviate somewhat from this characteristic size distribution, but not extensively. As a result soot mode mass and number emissions are correlated, as demonstrated in Fig. 2.8 (light duty vehicles). Here PM mass is recorded gravimetrically, so gaseous adsorption artifacts on the filter media are responsible for some of the data scatter at low emissions levels. Likewise, not applying strict PMP protocols to nuclei particle removal may contribute to some of the scatter in particle number. Nevertheless, a quite good correlation between PM mass and number emissions encompasses a variety of engine/aftertreatment technologies, the emission levels of which stretch over three orders of magnitude. One implication of this correlation is that the European EU5a number limit of 6x1011 particles/km for diesel vehicles is substantially more stringent than the 4.5 mg/km mass emissions standard.

2.4

Nuclei Mode Characterization

Although different in detail, soot modes from most combustion processes exhibit a qualitative similarity that derives from their high temperature origin. Differences originate principally from variations in ash content and from the extent of semivolatile material that condenses onto the soot. The situation with nucleation is different. As demonstrated below, a mode of 2–20 nm particles can appear either during combustion or as the exhaust subsequently cools. Size alone is insufficient to distinguish these two situations, but the volatility and electrical characteristics of the nuclei mode can help.

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2.4.1

M. Matti Maricq

Particle Volatility

A variety of methods exists to examine the semivolatile versus solid components of aerosol particles, the common element of which involves heating the PM. The most widespread practice, EC/OC analysis, heats particulate matter collected onto quartz filters first in an inert atmosphere, and subsequently in an oxidizing atmosphere (Cadle et al. 1980; Huntzicker et al. 1982). The first stage evaporates semivolatile material, the carbon in which is then quantified by conversion to CO2 (or CH4). The second stage directly oxidizes the remaining nonvolatile carbon to CO2. Considerable efforts have been paid to ascertain the extent of pyrolysis during the first stage and to correct the resulting overestimate of EC (Chow et al. 2001). Unless separate provisions are made, non-carbonaceous constituents, such as sulfate and ash are not detected. Standard EC/OC analysis does not distinguish between different PM modes. A straightforward way to study the size dependence of particle volatility is via parallel “wet” and “dry” branch measurements (Ntziachristos et al. 2004). One limitation of this procedure is that it does not examine the EC/OC composition of individual particles; it reveals only the net contribution of semivolatile components to the overall size distribution. Another limitation is the small effect that the evaporation of condensed material has on a fractal-like particle’s mobility diameter, unless this represents a significant fraction of the particle (Ristima¨ki and Keskinen 2006). Alternatively, detailed examination of particle volatility is possible by a tandem DMA – evaporation tube – DMA approach (Sakurai et al. 2003; Kwon et al. 2003; Wehner et al. 2004). As in the measurement of effective density, the first DMA selects a narrow range of particle size for study. The selected particles are heated to a set temperature, and what remains of them after evaporation and desorption of the semivolatile components is recorded by a second DMA. Figure 2.9 illustrates the application of each method to the case of a sooting premixed flame. The parallel approach in Fig. 2.9a reveals that both the nucleation and accumulation modes appear to be nonvolatile, at least up to 330 C. The small reduction in particle concentration at 330 C, the “dry” branch, occurs because of thermophoretic and diffusion losses in the evaporation tube, which increase as temperature increases and particle size decreases. Figure 2.9b presents data taken with the tandem method that provide a closer look at the effect of heat on particles in the nuclei mode. As the temperature in the heat pipe increases from 25 C to 620 C, the mobility diameter decreases from 5.8 to 3.5 nm, and the originally monodisperse peak broadens. At this small size accounting for losses makes it difficult to determine the exact fraction of particles that survive to 620 C, but clearly a significant fraction does. This nuclei mode appears in relatively cooler flames as a result of new particle formation, which can continue well beyond the flame front (Maricq 2006a). These incipient soot particles are reported to have a C/H ratio lower than that of the soot aggregates that comprise the accumulation mode of larger particles (Sgro et al. 2007), an interpretation consistent with the decreased effective density observed in Fig. 2.7 for particles in the more lightly sooting flame. Thus, a

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Fig. 2.9 Ethylene flame soot volatility. Panel A: Soot size distributions in “wet” branch (40 C) versus “dry” branch (330 C). Panel B: Tandem DMA volatility data for 5.8 nm particles from the nuclei mode

Fig. 2.10 Diesel exhaust PM volatility. Panel A: Semivolatile nuclei mode. Panel B: Nonvolatile nuclei mode. Measurements are made by passing non-size selected exhaust particles through heat pipe and then measuring their size distribution

plausible explanation of why the nuclei mode particles examined in Fig. 2.9b shrink, is that heating the particles increases their carbonization and leads towards a more graphitic structure (Dobbins 2002). Interestingly, nuclei mode particles in engine exhaust exhibit two volatility behaviors depending on engine operation, and perhaps other factors. The predominant case is the one of semivolatile particles illustrated in Fig. 2.10a. Here, heating the aerosol to above 200 C removes the nuclei mode particles, leaving behind the lognormal soot mode. The interpretation is that the former mode arises from semivolatile exhaust components as the exhaust cools, either from dilution into

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a sampling system or emission into the atmosphere. This conclusion is supported by particle mass spectrometry, which reveals a composition primarily (>95%) of lube oil derived hydrocarbons in heavy duty diesel exhaust (Tobias et al. 2001), and of sulfate and heavy hydrocarbons for light duty diesel vehicles (Schneider et al. 2005). Under some conditions, for example at idle, diesel vehicles can also produce nonvolatile nuclei mode particles (De Filippo and Maricq 2008), as illustrated in Fig. 2.10b by their persistence to 450 C. This light duty vehicle exhaust is sampled upstream of any exhaust aftertreatment devices, and is diluted with hot (200 C) air, which tends to suppress condensation. Thus, here the mode appears almost entirely nonvolatile. Thermophoretic losses in the heat pipe are likely primarily responsible for the decrease the mode’s intensity with increasing temperature. The reduction in mean diameter from 9.5 to 7.5 nm could be due to removal of some condensed material or to restructuring from carbonization. Nonvolatile nuclei mode particles have also been observed as cores of much larger particles in the exhaust of a heavy duty diesel engine designed to meet EURO IV emissions standards without use of a diesel particulate filter. In that case condensed material consisted of semivolatile hydrocarbon emissions (Ro¨nkko¨ et al. 2007).

2.4.2

Particle Electrical Charge

Electrical charge represents another characteristic that is useful in tracing particle origins. Combustion chemistry includes chemiionization reactions, whereby at high temperatures two neutral molecules combine to produce positive and negative ions (Calcotte 1981). The ions rapidly collide with any particles that may be present, at rates enhanced by attractive image charge forces, and deposit their charge. Subsequent aggregation of the soot particles brings their charge into a size dependent Boltzmann distribution (Hinds 1999; Maricq 2006b) fdm ðzÞ ¼



KE e2 pdm kT

1=2

  KE z2 e2 exp dm kT

(2.4)

where KE ¼ 9.0  109 Nm2/C2 in SI units, e is the charge of an electron, k is Boltzmann’s constant, and T represents temperature. As seen in Fig. 2.11, the soot mode (~20–200 nm) consistently exhibits a substantial fraction (40–60%) of charged particles, essentially evenly balanced between positive and negative charges. In the flame case, a temperature of ~1700 K describes the distribution of charges, which matches the flame temperature (Maricq 2006a). However, the characteristic temperature of the diesel soot electric charge is 800–1200 K, considerably lower than expected from diesel combustion temperatures (~2200 K) (Maricq 2006b). This is explained by the redistribution of charge to a lower temperature by the initially rapid particle aggregation that occurs

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Fig. 2.11 Electrical charge of combustion particles. Panel A: rich premixed flame. Panel B: Light duty diesel vehicle, case 1 – semivolatile nuclei mode. Panel C: Light duty diesel vehicle, case 2 – nonvolatile nuclei mode. Panel D: DPF equipped diesel

as the soot exits the engine cylinder and cools in the exhaust system, but which cannot keep up with the exhaust cooling rate because aggregation drives down the particle collision rate. The nuclei mode again exhibits two contrasting behaviors. In Fig. 2.11a, the nuclei mode of incipient flame particles appears electrically neutral, even though it is formed within the flame. The hypothesis is that by 20 mm above the burner all the ions have already attached to the existing soot particles; hence, none are left to charge these newly born nuclei particles. The same does not appear to be the case for the nonvolatile diesel exhaust nuclei particles in Fig. 2.11c. Here, the fraction of charged particles ranges from ~1% for each polarity at 3 nm to ~10% each at 10 nm, which is consistent with a temperature of ~850 K. This value is similar to what is observed for the diesel soot mode, suggesting that these nuclei particles arise during combustion, and not subsequently in the exhaust system. Only the beginning of the accumulation mode is evident at ~50 nm in Fig. 2.11c, and it is likewise charged. The diesel exhaust nuclei particles in Fig. 2.11b and d are electrically neutral. In the former case, the volatility of this mode is the same as displayed in Fig. 2.10a; namely the particles are removed at temperatures above about 200 C. The conclusion is that this nucleation mode forms from organic compounds and sulfate that surpass their saturation vapor pressures as the exhaust temperature drops. This occurs at exhaust temperatures below ~200 C, at which point there is no mechanism to charge the resulting aerosol; hence, it remains electrically neutral. The situation shown in

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Fig. 2.11d is identical, except that these particles are sampled downstream of a diesel particulate filter. The filter reduces the soot mode concentration, as evident from its much lower intensity. Without soot particles as a sink, semivolatile hydrocarbons and sulfate nucleate. At some point condensation onto the newly formed nuclei exceeds new particle nucleation and leads to particle growth, which explains why the mean nucleation mode diameter in panel D is the largest in Fig. 2.11.

2.5

Discussion and Conclusion

The size categories PM10, PM2.5, and ultrafine particles play a central role in the areas of ambient PM measurement, regulations, and health effects. However, with few exceptions, such as fly ash, combustion particles all fall well below the PM2.5 cutoff, and they are ineffectively distinguished by the conventional 100 nm ultrafine demarcation. Instead, they more naturally divide into nucleation and accumulation (soot) modes, the former generally extending from 2 to 20 nm and the latter from 10 to 300 nm. Because these two modes arise from distinct chemical and physical processes, they are not solely differentiated by size, but also by morphology, density, volatility, electrical charge, and chemical composition, although not always in a mutually exclusive manner. Posing questions within this more natural framework of particle modes may improve insight into issues of PM measurement, regulation, and health effects. The current method for engine PM emissions measurement is by dilution tunnel sampling onto filter substrates held at 47  5 C, followed by gravimetric analysis. Present day stack sampling from stationary sources follows EPA Method 5 to collect PM isokinetically onto glass fiber filters held at 120  14 C. As this discriminates against semivolatile particles, Method 202 is applied to collect condensable PM via impingers. Besides these mass based metrics, the European Union is introducing a particle number based emission standard for motor vehicles (GRPE 2007). It specifies hot dilution (150 C) followed by an evaporation tube held at 300 C to accelerate semivolatile particle removal, and stipulates a 23 nm lower cutpoint to communize an otherwise ill-defined limit for what is counted as a particle. Because the mass based methods are operationally defined, they do not provide consistent measures of PM. When soot dominates, this issue may be minor. These particles are solid, and so not much affected by sampling conditions. But these methods will register semivolatile components differently. The PMP number based approach is distinct, not only by the choice of metric, but also in the decision to specifically target solid exhaust particles, and thereby move away from an operational definition. In the absence of significant semivolatile PM mass, the solid particle number count is related to traditional mass measurement via soot size and effective density, a relationship supported by the correlation in Fig. 2.8 for light duty vehicle PM. As emissions regulations tighten, aftertreatment devices and ultralow sulfur fuel will change the exhaust PM character, primarily by a relative increase in semivolatile content. Currently, more thought is needed to identify

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quantification methods specific to the characteristics of semivolatile combustion PM, and less dependent on operationally based definitions. Particulate matter remains too varied and complex to allow ready classification. But as illustrated here via morphology, effective density, volatility, and electrical charge, investigating their characteristics provides numerous benefits. It can help elucidate their origins, whether in the engine, as nucleation/condensation in the exhaust, or as a sampling artifact. It aids health effects studies, where the fate of respired particles depends on size, whether they are liquid or solid, and presumably on composition. And it provides the basis for designing new PM measurement instrumentation. These new methods may not always conform to the historical definition of PM, but they may suggest new ways to think about combustion aerosols. Acknowledgments The author would like to thank Mike Loos, Adolfo Mauti, Sandip Shah, and Joseph Szente (Ford Motor Co.) for their generous help with the motor vehicle emissions measurements, and Yi Liu (Wayne State University) for his gracious help producing the TEM images.

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Dec JE, zur Loye AO, Siebers DL (1991), Soot Distribution in a D. I. Diesel Engine Using 2-D Laser-Induced Incandescence Imaging, SAE Technical Paper 910224 De Filippo A, Maricq MM (2008) Diesel nucleation mode particles: Semivolatile or solid? Environ Sci Technol 42:7957–7962 Dobbins RA (2002) Soot inception temperature and the carbonization rate of precursor particles. Combust Flame 130:204–214 Dobbins RA (2007) Hydrocarbon nanoparticles formed in flames and diesel engines. Aerosol Sci Technol 41:485–496 Frenklach M, Wang H (1991) Detailed modeling of soot particle nucleation and growth. Symp (Int) Combust 23:1559–1566 Giechaskiel B, Dilara P, Andersson J (2008) Particle measurement programme (PMP) light-duty inter-laboratory exercise: Repeatability and reproducibility of the particle number method. Aerosol Sci Technol 42:528–543 GRPE (2007) ECE/TRANS/WP.29/GRPE/2007/8 – (United Kingdom) Proposal for draft Supplement 7 to the 05 series of amendments to Regulations No. 83 (Emissions of M1 and N1 categories of vehicles). http://www.unece.org/trans/doc/2007/wp29grpe/ECE-TRANS-WP29GRPE-2007-08e.pdf Harris SJ, Maricq MM (2001) Signature size distributions for diesel and gasoline engine exhaust particulate matter. J Aerosol Sci 32:749–764 Health Effects Institute (2003) Revised analyses of time-series studies of air pollution and health. Special Report, Health Effects Institute, Boston, MA Hildemann LM, Cass GR, Markowski GR (1989) A dilution stack sampler for collection of organic aerosol emissions: design, characterization and field tests. Aerosol Sci Technol 10:193–204 Hinds WC (1999) Aerosol Technology. Wiley, New York Huntzicker JJ, Johnson RL, Shah JJ, Cary RA (1982) Analysis of organic and elemental carbon in ambient aerosol by a thermal-optical method. In Wolff GT, Klimisch RL (eds) Particulate carbon: atmospheric life cycle. Plenum, New York IPCC (2007) Climate change 2007: synthesis report. contribution of Working Groups I, II and III to the fourth assessment report of the intergovernmental panel on climate change [Core Writing Team, Pachauri, R.K and Reisinger, A. (eds.)]. IPCC, Geneva, Switzerland, 104 pp, http://www.ipcc.ch Kasper M (2004) The number concentration of non-volatile particles – Design study for an instrument according to the PMP recommendations, SAE Technical Paper 2004-01-0960 Kasper M, Sattler K, Siegmann K, Matter U, Siegmann HC (1999) The influence of fuel additives on the formation of carbon during combustion. J Aerosol Sci 30:217–225 Kelly WP, McMurry PH (1992) Measurement of particle density by inertial classification of differential mobility analyzer-generated monodisperse aerosol. Aerosol Sci Technol 17:199–212 Kittelson DB, Watts WF, Johnson JP (2006) On-road and laboratory evaluation of combustion aerosols Part 1: Summary of diesel engine results. J Aerosol Sci 37:913–930 Kwon S-B, Lee KW, Saito K, Shinozaki O, Seto T (2003) Size-dependent volatility of diesel nanoparticles: chassis dyamometer experiments. Environ Sci Technol 37:1794–1802 Maricq MM (2006a) A comparison of soot size and charge distributions for ethane, ethylene, acetylene, and benzene/ethylene premixed flames. Combust Flame 144:730–743 Maricq MM (2006b) On the electrical charge of motor vehicle exhaust particles. J Aerosol Sci 37:858–874 Maricq MM (2007) Chemical characterization of particulate emissions from diesel engines: a review. J Aerosol Sci 38:1079–1118 Maricq MM, Xu N (2004) The effective density and fractal dimension of soot particles from premixed flames and motor vehicle exhaust. J Aerosol Sci 35:1251–1274 Maricq MM, Chase RE, Podsiadlik DH, Vogt R (1999) Vehicle exhaust particle size distributions: a comparison of tailpipe and dilution tunnel measurements, SAE Technical Paper 1999-01-1461 Maricq MM, Chase RE, Xu N, Podsiadlik DH (2003) A constant volume rapid exhaust dilution system for motor vehicle PM number and mass measurements. J Air Waste Manage Assoc 53:1196–1203

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Miller A, Ahlstrand G, Kittelson D, Zachariah M (2007) The fate of metal (Fe) during diesel combustion: morphology, chemistry, and formation pathways of nanoparticles. Combust Flame 149:129–143 Ntziachristos L, Giechaskiel B, Pistikopoulos P, Samaras Z, Mathis U, Mohr M, Ristima¨ki J, KeskinenJ, Mikkanen P, Casati R, Scheer V (2004) Overview of the European “Particulates” Project on the characterisation of exhaust particulate emissions from road vehicles: results for light-duty vehicles, Vogt R, SAE Technical Paper 2004-01-1439 Park K, Cao F, Kittelson DB, McMurry PH (2003) Relationship between particle mass and mobility for diesel exhaust particles. Environ Sci Technol 37:577–583 Park K, Kittelson DB, Zachariah MR, McMurry PH (2004) Measurement of inherent material density of nanoparticle agglomerates. J Nanoparticle Res 6:267–272 Ristima¨ki J, Keskinen J (2006) Mass measurement of non-spherical particles: TDMA-ELPI setup and performance tests. Aerosol Sci Technol 40:997–1001 Robinson AL, Donahue NM, Shrivastava MK, Weitkamp EA, Sage AM, Grieshop AP, Lane TE, Pierce JR, Pandis SN (2007) Rethinking organic aerosols: Semivolatile emissions and photochemical aging. Science 315:1259–1262 Ro¨nkko¨ T, Virtanen A, Kannosto J, Keskinen J, Lappi M, Pirjola L (2007) Nucleation mode particles with a nonvolatile core in the exhaust of a heavy duty diesel vehicle. Environ Sci Technol 41:6384–6389 Sakurai H, Park K, McMurry PH, Zarling DD, Kittelson DB (2003) Ziemann, PJ, Size-dependent mixing characteristics of volatile and nonvolatile components in diesel exhaust aerosols. Environ Sci Technol 37:5487–5495 Schleicher B, Ku¨nzel S, Burtscher H (1995) In situ measurement of size and density of submicron aerosol particles. J Appl Phys 78:4416–4422 Schneider J, Hock N, Weimer S, Borrmann S, Kirchner U, Vogt R, Scheer V (2005) Nucleation particles in diesel exhaust: composition inferred from in situ mass spectrometric analysis. Environ Sci Tech 39:6153–6161 Sgro LA, De Filippo A, Lanzuolo G, D’Alessio A (2007) Characterization of nanoparticles of organic carbon (NOC) produced in rich premixed flames by differential mobility analysis. Proc Combust Inst 31:631–638 Shi JP, Harrision RM (1999) Investigation of ultrafine particle formation during diesel exhaust dilution. Environ Sci Technol 33:3730–3736 Snelling DR, Smallwood GJ, Sawchuk RA, Neill WS, Gareau D, Clavel D, Chippior WL, Liu F, ¨ L Bachalo WD (1999) Particulate Matter Measurements in a Diesel Engine Exhaust Gu¨lder O by Laser-Induced Incandescence and the Standard Gravimetric Procedure, SAE Technical Paper 1999-01-3653 Tobias HJ, Beving DE, Ziemann PJ, Sakuri H, Zuk M, McMurry PH, Zarling D, Waytulonis R, Kittelson DB (2001) Chemical analysis of diesel engine nanoparticles using a nano-DMA/ thermal desorption particle beam mass spectrometer. Environ Sci Technol 35:2233–2243 United States Environmental Protection Agency (2007) The original list of hazardous air pollutants. http://www.epa.gov/ttn/atw/188polls.html Vaaraslahti K, Virtanen A, Ristima¨ki J, Keskinen J (2004) Nucleation mode formation in heavyduty diesel exhaust with and without a particulate filter. Environ Sci Technol 38:4884–4890 Virtanen A, Ristima¨ki J, Keskinen J (2004) Method for measuring effective density and fractal dimension of aerosol agglomerates. Aerosol Sci Technol 38:437–446 Wehner B, Philippin S, Wiedensohler A, Scheer V, Vogt R (2004) Variability of non-volatile fractions of atmospheric aerosol particles with traffic influence. Atmos Environ 38:6081–6090 Zhao B, Yang Z, Wang J, Johnston MV, Wang H (2003) Analysis of soot nanoparticles in a laminar premixed ethylene flame by scanning mobility particle sizer. Aerosol Sci Technol 37:611–620

Chapter 3

Medicine Nanoparticle Production by EHDA Jan C.M. Marijnissen, Caner U. Yurteri, Jan van Erven, and Tomasz Ciach

3.1

Introduction

Chemical products, in general, can be produced in the liquid or gas phase. However for most medicines, with their complex molecules, the liquid route will be the appropriate one. To make these medicines into nanoparticles can also be done via the wet route (colloids) or a dry route. To separate the nanoparticles from the liquid phase will be almost impossible without some contamination, so to avoid contamination a dry method might be favourable. So to be considered is the disintegration of bigger structures into (nano) fractions. Depending on the phase of the structures, liquid or solid, different disintegration techniques exist, such as grinding, liquid atomization, lithography and etching, and evaporation/condensation. Only attention will be given here to liquid atomization with the consequent droplet to particle conversion. From the several atomization methods we are only interested in methods which break up into rather uniform droplets, so we limit ourselves to jet breakup in the laminar flow region (Lefebvre 1989). Another limitation is the size of the initially generated droplets. To produce nanoparticles the initial droplet size should be already fairly small, because otherwise the begin concentration has to be unacceptably low. One should realize that the diameter of the final particle after drying equals the diameter of the initial droplet times the cube root of the volumetric concentration of the non-volatile material (van Erven et al. 2005). So in case of very low concentrations of the product material, the role of impurities might become very important. For methods, where atomization is brought about by J.C.M. Marijnissen and C.U. Yurteri Faculty of Applied Sciences, Delft University of Technology, Delft, the Netherlands e-mail: J.C.M. [email protected] J. van Erven Nano Structured Materials, TU Delft, Juliananlaan 136, 2628 BL Delft, the Netherlands e-mail: [email protected] T. Ciach Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warsaw, Poland

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_3, # Springer ScienceþBusiness Media B.V. 2010

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forcing liquid through a thin nozzle or orifice, such as for the Vibrating Orifice Aerosol Generator (TSI Model 3450) the generated droplet size will be about two times the orifice diameter, while the orifice size is restricted by clogging risk. So the best option is a method, which produces mono sized droplets with a diameter smaller than the inside nozzle diameter. Such a method is found in: ElectroHydrodynamic Atomization (EHDA) or Electrospraying. EHDA is a method to produce very fine droplets from a liquid (atomization) by using an electric field. By applying the right conditions, monodisperse droplets from nanometers to several micrometers can be produced. By means of an example, i.e., the production of nano platinum particles, a generic way to produce nanoparticles from a multitude of different precursors is given. After that, several examples of medicine particles made by EHDA will be given, in the nano- and micro-range, with different properties, such as controlled release, high porosity and elongated shape. Also a method, bipolar coagulation, where two sprays of opposite electrical potential are used will be discussed. In this method, each combination of a positive and a negative charged droplet can be seen as a nano- or micro-reactor, so being able to produce new chemical compounds in a liquid, aerosolized condition. Bipolar coagulation can also be used to apply nanoparticles on a carrier. Finally some attention will be given on EHDA instrumentation and out-scaling methods.

3.2

Electro Hydrodynamic Atomization and the Production of Nanoparticles

EHDA refers to a process where a liquid jet breaks up into droplets under influence of electrical forces. Depending on the strength of the electric stresses in the liquid surface relative to the surface tension stress, and on the kinetic energy of the liquid leaving the nozzle, different spraying modes will be obtained (Cloupeau and Prunet-Foch 1994; Grace and Marijnissen 1994). For the production of nanoparticles in our case the so called Cone-jet mode is the relevant one. In this mode a liquid is pumped through a nozzle at low flow rate (mL/h to mL/h). An electric field is applied between the nozzle and some counter electrode. This electric field induces a surface charge in the growing droplet at the nozzle. Due to this surface charge, and due to the electric field, an electric stress is created in the liquid surface. If the electric field and the liquid flow rate are in the appropriate range, then this electric stress will overcome the surface tension stress and transform the droplet into a conical shape, the Taylor cone (Taylor 1964). The tangential component of the electric field accelerates the charge carriers (mainly ions) at the liquid surface toward the cone apex. These ions collide with liquid molecules, so accelerating the surrounding liquid. As a result, a thin liquid jet emerges at the cone apex. Depending on the ratio of the normal electric stress over the surface tension stress in the jet surface, the jet will break up due to axisymmetric instabilities, also called varicose instabilities or due to varicose instabilities and also lateral instabilities, called kink instabilities (Hartman et al. 2000). At a low stress ratio in the varicose break-up mode the desired monodisperse droplets are produced. The droplets produced by EHDA carry a high electric charge close to the Rayleigh charge limit (Hartman et al. 2000). To avoid Rayleigh disintegration of

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41

the droplets (Davis and Bridges 1994; Smith et al. 2002), the droplets have to be completely or partially neutralized. Rayleigh disintegration happens when the mutual repulsion of electric charges exceeds the confining force of surface tension, a result here of the evaporation of the droplets. Neutralization is also desirable to make the droplets manageable, A possible method of discharging is with ions of opposite charge created by corona discharge. To estimate the right conditions and operational parameters to produce nanodroplets of a certain size, scaling laws can be used. de la Mora and Loscertales (1994) and Gan˜a´n-Calvo et al. (1997) developed scaling laws which estimate the produced droplet size (or jet diameter) and the electric current required for a liquid sprayed in the Cone-Jet mode as function of liquid flow rate and liquid properties. Hartman refined the scaling laws for EHDA in the Cone-Jet mode using his theoretically derived models for the cone, jet, and droplet size (Hartman et al. 1999, 2000). For the current scaling for liquids with a flat radial velocity profile in the jet, which is appropriate here because of the high conductivity of the solution, he derived the following relation 1

I ¼ bðgKQÞ2 ;

(3.1)

where Q is the flow rate (m3/s), I is the current through the liquid cone (A), g is the surface tension (N/m), K is conductivity (S/m), and b is a constant, which is approximately 2. The droplet diameter for the varicose break-up mode is given by Eq. 3.2:

dd;v

re0 Q4 ¼c I2


1 6

;

(3.2)

where dd,v is the droplet diameter for varicose break-up and c is a constant, which is approximately 2. Substituting Eq. 3.1 into Eq.3.2 yields:

dd;v ¼




16re0 Q3 gK

1 6

(3.3)

For a spherical particle, the diameter of the (final) particle (dp) is related to the droplet diameter (Eq. 3.3) by Eq. 3.4: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi rdroplet 3 d dp ¼ 3 f rparticle droplet

(3.4)

where f is the mass fraction of the material in the solution ( ), rdroplet is the density of the solution and rparticle is the density of the final (product) particle (kg/m3).

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This chapter describes the production of (medical) nanoparticles by EHDA. Other authors report already on the production of nanoparticles by EHDA (Rulison and Flagan 1994; Hull et al. 1997; Ciach et al. 2002; Lenggoro et al. 2000) but besides presenting two methods to produce specific nanoparticles by EHDA, our methods can according to us be seen as generic ways to produce well-defined nanoparticles of many different compositions on demand. Before we describe the production of medical nanoparticles, we start with an example, i.e., the production of Pt nanoparticles, to explain each step involved in detail, including chemical reactions. The two different EHDA configurations which have been used for the production of Pt nanoparticles, relate to the two different routes of the decomposition step of the platinum precursor into platinum. In the first one the precursor droplets are collected on a support and heat treated afterward. In the second route the produced precursor droplets are kept in airborne state, neutralized and heat treated before collection.

3.2.1

Experimental

Two production routes of platinum nanoparticles are used as described by van Erven et al. (2005). In both routes the droplets are produced from a solution of chloroplatinic acid (H2PtCl6.6H2O Alfa-Aesar 99.9%) in ethanol. When heated above 500 C the platinum precursor will decompose into platinum, gaseous hydrochloric acid, and chlorine (Hernandez and Choren 1983). In the first route the by EHDA produced chloroplatinic acid particles are deposited on a carrier support. After deposition the support is placed in a tubular furnace and the particles are decomposed forming platinum nanoparticles. In the second route the produced droplets are neutralized and ducted in an airborne state through a tubular furnace where they decompose. After the furnace the particles are deposited on a substrate, such as a TEM grid. The two different routes have different set ups. The first one, is referred to as the Capillary-plate set-up and the second one, as the Aerosol reactor set up.

3.2.2

Capillary-Plate Set-Up

The capillary-plate set-up is shown in Fig. 3.1. Droplets are produced by pumping (Harvard PHD2000) a 1 wt% solution of chloroplatinic acid in ethanol (K ¼ 0.04 S/ m, g ¼ 0.022 N/m) through a capillary (B). The flow rate of the solution was 13 mL/h. The required electrical field is created by applying a voltage between the capillary (B) (inner diameter 60 m, outer diameter 160 mm) and a grounded counter electrode (D) using a high voltage power supply (C) (FUG HCL 14-12500). For the experiments the potential difference between B and D was 1.26 kV and the distance between the tip of the capillary (B) and the carrier support (E) was 1 mm.

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Fig. 3.1 (a) Capillary-plate set-up. A – syringe; B – metal capillary; C – high voltage power supply; D – grounded plate; E – Si/SiO2 support. (b and c) SEM images of particles produced by capillary-plate set-up before and after 10 min decomposition at 700 C

The droplets are deposited on the carrier support (E) which in principle can be any material which is heat resistant at the decomposition temperature of chloroplatinic acid and is conductive to discharge the droplets. In this study thin plates of silicon, with an 0.4 mm oxidized top layer, of about 20 by 20 mm were used as carrier support. The set up was operated at room temperature. After evaporation of the solvent the support with the chloroplatinic acid nanoparticles was placed in a tubular furnace for 10 min at T ¼ 700 C to decompose the deposited chloroplatinic acid particles into platinum particles. The particles were examined before and after decomposition by a SEM (Hitachi Model S-4700).

3.2.3

Aerosol Reactor Set Up

The aerosol reactor set up is shown in Fig. 3.2a. The set up can be divided in two sections, A and B. Section A is the production part which is based on the Delft Aerosol Generator (Meesters et al. 1992). In section B the chloroplatinic acid particles are decomposed, in the airborne state, during their transport through the tubular furnace. A blow up of the production area, section A, is shown in the upper part of Fig. 3.2a. A 0.2 wt% solution of chloroplatinic acid in ethanol (K ¼ 0.01 S/ m, g ¼ 0.022 N/m) was pumped (Harvard PHD2000) through a metal capillary (I.D. 60 mm, O.D. 160 mm) with a flowrate of 8 mL/h. In this set up a ring is used as counter electrode. The ring is connected to a high voltage power supply (FUG HCL 14 12500), but at a lower voltage than the capillary, respectively 5.57 and 8.8 kV. The distance between the ring and capillary is approximately 15 mm. The potential difference between the nozzle and the ring creates the field to produce the droplets, which will pass through the ring. In this way the droplets are not deposited as in the capillary-plate set-up, but are kept airborne.

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Fig. 3.2 (a) Aerosol reactor set up. In section A the particles are generated and dried. In section B the dried chloroplatinic particles are decomposed to form platinum particles, (b) single Pt particle of 7 nm, (c) Cluster of 3 Pt particles, (d) TEM-EDX spectrum of a platinum particle (Cu and Ni peaks are from TEM grid)

To discharge the highly charged droplets a grounded needle is used in this set up. The needle has a sharp tip and the high electric field strength there, creates a corona discharge, so supplying ions of opposite charge for the neutralization. The distance between the tip of the needle and the ring is 60 mm. The chloroplatinic acid particles are then ducted into a tubular furnace (T = 700 C) with filtered air (fv ¼ 1.5 L/min). The residence time is estimated to be 2 min. After the furnace the platinum nanoparticles are deposited on a TEM grid. The deposition takes place by two phenomena; thermophoresis and diffusion. In the beginning thermophoresis is important because the TEM grid is cold compared to the gas. When the grid has been heated up, diffusion will be the dominant process of deposition. After deposition the nanoparticles are examined by a HR-TEM (Philips CM30UT).

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3.2.4

45

Results of Pt Particles Production

A small area of a Si/SiO2 substrate with chloroplatinic acid particles, produced by the capillary-plate set-up, is shown in Fig. 3.1b. The surface concentration was obtained by spraying for 5 s. The spot sizes as seen in Fig. 3.1b vary between 80 and 120 nm. Substituting the values of the different variables as described in the experimental section in the scaling laws (Eq. 3.3) and using Eq. 3.4, yields a particle size of 63 nm (here in Eq. 3.4 f is the mass fraction of the chloroplatinic acid in ethanol, rdroplet is the density of ethanol and rparticle is the density of chloroplatinic acid). Realizing that some deformation might occur during deposition of still wet particles, the measured and calculated values are in reasonable agreement. Figure 3.1c shows the particles after the decomposition of the chloroplatinic acid in a tubular furnace for 10 min at 700 C. It can be seen that the original chloroplatinic acid particles are formed into clusters of supposedly platinum particles of 5–15 nm. This is caused by the fact that platinum does not evaporate at 700 C, while the other decomposition products are gaseous. Platinum particles produced by the aerosol reactor set up with the settings mentioned in Section 3.2.3 are shown in Fig. 3.2. In Fig. 3.2b, a TEM micrograph of a single particle of approximately 7 nm is shown. The produced particles are not charged and can therefore form agglomerates. An example of such an agglomerate is shown in Fig. 3.2c. Elemental analysis using EDX (Fig. 3.2d) showed that the particles only contain platinum. The TEM pictures also prove that the platinum particles are crystalline. Using the values of the variables as described in the experimental section the scaling laws (Eqs. 3.3 and 3.4) predict a particle size of 13 nm. By observing different areas of the TEM grid, we noticed that the particle size of non-agglomerated particles was very similar. To get an estimation of the size a limited number of particles was measured giving an average size in the order of 10 nm.

3.3

Medicine Nano- and Micro-Particles Produced with EHDA

In the preceding part a general introduction was given to produce nanoparticles with EHDA. From now we will focus on medicine particles. Drug particles with a narrow size distribution have the unique advantage of providing more regular and predictable drug release profiles from batch to batch compared to particles with the same mean size but wider distribution. Electrospraying is the ideal route for the production of such drug particles either in pure or polymer blended form. In this case a drug or a polymer/drug combination dissolved in a suitable solvent is electrosprayed. The aerosol reactor setup shown in Fig. 3.1 is used to demonstrate that nanosized medical particles or polymer blended combinations can be generated. Paclitaxel

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Fig. 3.3 (a) Taxol; 1.0% in EtoH at 22 mL/h (21oC/38% RH), 60 s (spray time), 3 cm (spray to substrate distance), 2.1 kV (high voltage), (b) PVP; 0.3% in EtoH at 10 mL/h (22oC/47% RH), 60 s, 3 cm, 2.4 kV, (c) Taxol + PVP; 0.1% in EtoH at 20 mL/h (21oC/64% RH), 120 s, 3 cm, 2.0 kV

(Taxol) is selected to illustrate an example. Taxol is used to treat various forms of cancer such as breast, lung, ovarian, and it is also used as a way of prevention against restenosis. Taxol can be dispensed alone or blended with a biodegradable polymer such as PVP, PLA, or PLGA. Taxol (Sigma-Aldrich) is dissolved in Ethanol in a 1% mass ratio. With a flow rate of 22 mL/h and a potential difference of 2.1 kV, droplets are produced and targeted to an SEM stub placed 3 cm downstream of the nozzle. Based on the initial droplet diameter of 1.5 mm, a droplet evaporates to deposit as a dry Taxol particle in the size of 300 nm on the surface of the SEM stub, Fig. 3.3a. When the flow rate is lowered to 10 mL/h, particle sizes were in the order of 200 nm or less as confirmed by SEM (Philips XL20). In order to show the fabrication of polymer and mixed polymer/drug nanoparticles a PVP solution and a PVP/Taxol solution is utilized. In order to fabricate polymer sub¯ S-M.W. 1300000, K85-95) is utilized. Experiments are micron spheres PVP (ACRO carried out in ambient conditions. Figure 3.3b and c is an example of pure and Taxol blended polymer nanoparticles. Spray conditions for fabricating these particles are listed in the caption of Fig. 3.3. The size of the particles in Fig. 3.3 is between 200 and 300 nm.

3.3.1

Slow Release and Low Density Particles

Besides the production of medical nanoparticles as such, EHDA also offers the possibility to produce more complex particles such as slow release and low density particles, which e.g. can be used in inhalation treatment. The two following examples consider micrometer sized particles, but the methods can be equally used for the production of nanoparticles (Ciach et al. 2002) First we discuss the use of biodegradable polymer solutions for the production of slow release medicine particles. We selected poly-(lactic-co-glycolic acid) (PLGA) (50:50, Aldrich) as the primary polymer and Polyethylene glycol (PEG) as an additive to modify the decomposition rate. As an example of drug paclitaxel (taxol) was used again. As solvent a dichloromethane acetone mixture (4:1 weight) was employed. The solution was atomized with a set-up as in Fig. 3.2, but instead of a tube furnace a heating element around the outlet tube of the production part was used.

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Relative cumulative release [-]

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3

PLGA PLGA + PEG 4.6k

0.2 0.1 0.0 0

10

20

30

40

50

60

Time [days]

Fig. 3.4 (a) PLGA particles containing paclitaxel produced by EHDA. (b) Relative cumulative release of paclitaxel from polymer microparticles made of PLGA (poly(lactic-co-glycolic acid)) and PEG (polyethylene glycol).Conditions: room temperature, pH ¼ 7

Figure 3.4a shows PLGA particles containing paclitaxel, produced in the described way. As can be seen the size distribution is narrow but additional small particles are present. The contribution of these particles in the total mass of the system is negligible but some effort will be made in the future to avoid formation of these small particles. To investigate the drug release characteristics, particles together with the filter on which they were collected were immersed in a 200 mL buffer solution of pH ¼ 7 at room temperature with a small addition of sodium azide to prevent bacteria growth. To measure the paclitaxel release into the liquid as a function of time, samples of 1 mL of the solution were taken at certain time intervals and after passing them through a membrane filter analysed on the paclitaxel content with liquid chromatography. In the buffer solution slow release of the medicine takes place. The involved mechanisms are supposed to be hydrolytic decomposition of the polymer matrix followed by dissolution of medicine entrapped in the polymer. In addition, diffusion of active compound to the surface and dissolution probably also takes place. The results of the paclitaxel release as a cumulative release with time is presented in Fig. 3.4b. It is clear that the cumulative release of the medicine is rather linear with time, with some faster release in the first few days and a slowing down after about 35 days. This initial burst of the active substance could originate from decomposition of small particles and/or from the drug available on the particle surface. For a higher time span (some 30 days) the release rate is more or less constant. As can be seen choosing a proper polymer mixture (here PLGA + PEG) can serve as a tuning method for particle decomposition time. To produce low density particles with EHDA, we have used two different ways. Hollow or balloon like particles can be obtained with the right evaporation conditions and concentration. In reality also other factors play an important role, such as mechanical properties and porosity of the formed solid shell as well as the surface tension of the solution and the presence of surface-active compounds. If we do not

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Fig. 3.5 (a) Budesonide1 particles produced by EHDA. The bar length in the picture is 5 mm. (b) Inflated PEG particle attached to a wire. (c) Surface of the inflated PEG particle

choose the composition of the droplets or the conditions of solvent evaporation properly, we can get the wrong particle structure such as small solid particles or remains of collapsed shells. There is still no scientific way to accurately predict if the solution we have will produce hollow spheres. The only way is still trial and error. An example of particles obtained from a 1% (wt) solution of Budesonide1 in a water-ethanol (1:10, wt) mixture, is shown in Fig. 3.5a. On the picture we can see shell-like particles. The calculated volumetric fraction of the walls in relation to the whole particle is about 1%. Some of the particles have broken walls. Among big shells we see small particles, which may have been formed from satellite droplets. Another way to obtain low-density particles is to inflate them by releasing a gas inside the polymer structure after particle formation. As a substance that can release gas we use NaHCO3 or (NH4)2CO3. These inflating agents decompose at elevated temperature (about 60) releasing carbon dioxide. At this temperature the polymer is already soft. The gases are formed inside the polymeric structure of the particle and the whole process can be compared with baking a cake where the biodegradable polymer is the dough. To verify this idea we used a solution of PEG (10 kDa M.W.) containing 0.5% (wt) of NaHCO3 and 0.1%(wt) of surfactant (related to the weight of the polymer). In a first test we created droplets on a 50 mm wire by immersing the wire in the solution. This resulted in tiny droplets hanging at the end of the wire. After evaporation of the solvent we put the wire with the particles for five minutes in an oven at 60. At this temperature the polymer became soft and the inflating agent decomposed, releasing CO2. The gas expanded the particle. An example particle is shown in Fig. 3.5b. We can see in Fig. 3.5c that the particle has a spongy structure with pores. The measured porosity of this particle is about 80%. The particle is slightly collapsed. We also try to accomplish the same process in the aerosol state by heating airborne particles. Particles are produced by EHDA and after solvent evaporation they pass a heated chamber where, we expect that gas is released inside the polymeric particles.

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Fig. 3.6 (a) Elongated polymer particles, (b) smooth PVP nanofibers, (c) TiO2 nanofibers, (d and e) electrospun nanotubes obtained by co-spinning olive oil/PVP-TiO2 precursor

3.3.2

Different Shapes

For certain medical applications it might be advantageous to use particle shapes different from spheres. EHDA is able to produce elongated shapes and fibres. Depending on the concentration, type of polymer and solvent and drying conditions it is possible to produce elongated particles see e.g. Fig. 3.6a. It is even possible to make fibres (electro spinning), see Fig. 3.6b and c. If fibres or particles are formed depends probably especially on the degree of chain entanglement of the polymer. By using a coaxial spinning system it is also possible to produce (nano) tubes of a certain material (polymer or ceramic) filled or not with another material (Fig. 3.6d and e).

3.4

Bipolar Coagulation and Carrier Particles

It is also possible to use two sprays of oppositely charged droplets. If they are directed towards each other coagulation between the droplets takes place through the electrical attraction between them. The two sprays are created using EHDA in the cone-jet mode. The coagulation can be used just to neutralize the droplets, but also a chemical reaction can take place in the newly formed droplet obtaining the desired product, see Fig. 3.7. If the right conditions are chosen, it is also possible to coat one material with another.

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Fig. 3.7 Bipolar coagulation

More or less the same method can be used to load carrier particles with nano- or microsized medicine particles. This can be of high interest for the pharmaceutical industry. The electrospraying of nanoparticle laden liquids resolves an apparent problem of effectively dispersing nanoparticles. Electrospraying such suspension generates a spray of charged droplets that are seeded with nanoparticles. Thus electrospraying offers a solution for dispersing and depositing nanoparticles on a substrate. In order to enhance the efficiency of deposition, the charged nature of the nanoparticles can be exploited to coat host particles or to coat them with other droplets. EHDA leads to the formation of unipolarly charged suspensions of nanoparticles, while host particles can be charged with opposite polarity by means of tribocharging, corona or inductive charging, or get their charge due to the use of EHDA. When these particles are brought into contact in an appropriate way, the mutual electrostatic attraction force between the negative and positive charge will cause a coating to be deposited on the surface. Interaction can be realized in three ways: nanoparticles can be embedded in host particles, host particles can be encapsulated with a polymer and nanoparticles, and nanoparticles can be discretely deposited on the surface of host particle. We have studied several possibilities for mutually interacting oppositely charged particles in order to deposit nanoparticles on micro ones as an example of the latter case as depicted in Figs. 3.8a and b and 3.9a. These processes can be named as the grounded moving target (GMT) method (Dabkowski 2006; Dabkowski et al. 2007), falling curtain method (Coppens 2007; van Ommen et al. 2008), and vibrating dish method respectively. In the GMT method, in which 165 mm alumina host particles were coated with 65 nm PS nanoparticles, the host particles were charged by tribocharging on a particle feeder, while the suspension of nanoparticles were charged with opposite polarity by means of electrospraying. In the first experiment the charged host particles were fed onto the conveyor, which in this case was stationary, giving a deposition pattern as shown in Fig. 3.8c. We see good targeting of PS particles on the alumina due to the mutual attraction between the oppositely charged particles. Although in this stationary case a very high degree of deposition can be achieved, we prefer a continuous deposition method. The coating level then can be controlled by changing the residence time of the host particles in the

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Fig. 3.8 (a) A schematic representation of the Grounded Moving Target (GMT) set-up, (b) schematic representation of falling curtain setup, (c) Stationary coated 165 mm alumina with 65 nm PS, (d) GMT coated alumina; Three types of depositions are distinguished: single, in groups and agglomerates, (e) 200 mm glass beads coated with 500 nm PS in a falling curtain setup (conditions; mean counter air velocity of 1.26 m/s and two EHDA nozzles)

spraying zone via conveyor speed and changing the concentration of the suspension. When host particles are in motion, three types of nanoparticle deposits were identified: single, in groups and in agglomerates, Fig. 3.8d. The latter type is presumable explained by the deposition of droplets with a high concentration of nanoparticles. Tribo charging of host particles can be improved by constructing the particle feeder/charger out of a material, which is far away from the host particle in the tribo series, e.g. for glass host particle, the particle feeder is made out of Teflon. However, charging the host particles too high causes particles to stick on the feeder making it difficult to supply them to the conveyor for coating them on the conveyor. Too high charge also causes sticking of the particles to the conveyor. The falling curtain set up (Fig. 3.8b) omits the contact of particles with the conveyor surface. However the particle residence time is also reduced. Applying multiple electrosprays as well as applying a counter air flow increases the residence time of the particles (glass beads in this case) in the spray zone and thus enhances targeting of the nanoparticles on the glass beads. Figure 3.8e is an example of coating with counter air flow and two EHDA sprays. Besides these continuous processes, batch type processes are investigated to get more insight in the process involved using a vibrating dish setup as shown in Fig. 3.9a. One gram of 45 mm glass beads are tribo charged using a PTFE vibrating dish. These particles form an almost single layer of vibrating particles. Due to their confinement in the dish the electrostatic interaction with the spray is enhanced. Two cases are considered, in the first case the particles are charged by vibration and then the vibration is stopped. A small amount of these

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Fig. 3.9 (a) Schematic of the vibrating dish coating unit, (b) 45 mm glass beads stationary coated with 100 nm PS spheres (c) 45 mm glass beads dynamically coated in a vibrating dish with 100 nm PS

particles are mounted on a SEM stub and exposed to electrosprayed nanoparticles, Fig. 3.9b. In the second case the vibration is continued and nanoparticles are directly sprayed on the vibrating host particles, Fig. 3.9c. In this setup a needle – ring configuration is used to avoid the influence of charged particles on the spray formation process.

3.5

Production Equipment

A typical EHDA setup for powder production is presented in Fig. 3.10. The set-up consists of a cylindrical glass tube of 10–20 cm diameter. One end acts as an inlet for filtered air and the other end directs the produced particles to a filter for collection. Sometimes a heating step before collection is necessary to evaporate the solvent. The EHDA spraying nozzle is positioned in a glass side tube in which also the counter electrode ring is placed close to the main glass cylinder. A corona discharge needle is inserted in the glass cylinder opposite to the spraying nozzle to neutralize the droplets generated. A more detailed description is already given in Section 3.2.3. Another set-up is proposed by Ciach (2007), which was designed to have better long term production stability, see Fig. 3.11. The reactor consists of a glass cylinder, 20 cm in diameter and 50 cm long. The EHDA nozzle is placed on top of the cylinder and is surrounded by a counter electrode in the shape of a tube with rounded edges. This tube also acts as an inlet for the air stream, which carries the particles away. Four or six corona discharge electrodes of opposite polarity as the nozzle and the ring, are placed symmetrically some distance from the bottom of the cylinder to neutralize the droplets. By carefully selecting the corona current, the particles will not be completely neutralized. They follow the air stream, which enters through the tube electrode and small holes in the upper cover near the cylinder wall (not shown). Due to their charge the particles are efficiently collected on a grounded collecting plate downstream of the reactor. The collecting plate is a disc of 18 cm diameter placed 2 cm below the cylinder outlet rim which rotates slowly. While the disc rotates slowly a Teflon

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Fig. 3.10 Setup for powder production by EHDA

Fig. 3.11 EHDA particle production setup by Ciach (not on scale)

scraper directs the particles to the powder container. The particles should be preferably dry before collection. The reactor produces half a gram of powder per hour and operates stably for at least 24 h. To avoid problems related to particle discharging, drying, and accumulation on the setup walls, particles can be collected in a liquid, see Fig. 3.12 (Ciach 2007). In this setup particles are atomized by EHDA 10–20 cm above the collecting solution surface. The grounded collecting liquids act as the counter electrode, in which the particles are immersed. Obviously particles should not be soluble in the collecting liquid. This method can also be used to produce porous particles and for encapsulation of poorly water soluble drugs like taxol. It is also possible to place the spraying nozzle in a liquid, with the nozzle on a high potential and a submerged

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Fig. 3.12 Collection of EHDA particles in a liquid

Liquid feed HV

Collecting solution

grounded counter electrode. In this case emulsions, can be made. It is clear that the liquid in which is sprayed (the continuous phase of the emulsion) must have a low conductivity.

3.6

Future of EHDA – Out Scaling

As shown, Electrospraying enables controlled atomization. Therapeutic aerosols with a narrow size distribution can be generated of a desired size, chemical composition, charge and morphology, hence providing a safe and controlled way of respiratory drug delivery. Besides for the production of inhalation particles, EHDA can be used to coat particles or surfaces with medical nanoparticles in a very efficient way. This leads to cost savings in expensive pharmaceutical materials. However, industrial implementation still suffers from low production rates although much effort is put in up-scaling the production process. In order to generate small sized particles, low flow rates are required. For example, a flow rate of less than 0.1 mL/h for a single nozzle is needed to obtain droplets in the micrometer diameter range. To obtain a desired size is mainly determined by flow rate and conductivity of the liquid as dictated by the scaling laws (Eq. 3.3). For the same droplet size it is impossible to increase the production rate by increasing the flow rate. Thus an out-scaling rather than up-scaling is needed by means of using multiple sprays. There are many efforts reported on out-scaling methods including the use of an array of capillaries, an array of holes in combination of non-wetting material, serrations, grooves, multi jet mode operation as summarized by Deng and Gomez (2007). Increasing the number of capillary nozzles seems to be a simple and effective way of increasing the number of droplets. However, out-scaling mainly suffers from flow rate and field intensity variations and thus droplet size changes from nozzle to nozzle. The design may also be dependent on the nature of the liquid.

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There is therefore a need for systematic design tools. The challenge is a uniform delivery of the liquid and having an equal field intensity in each spraying point. Studies of Snarski and Dunn (1991) and Rulison and Flagan (1994) show that the voltage required for the steady cone-jet mode increases with a decrease in distance between the capillary nozzles. As the distance decreases in order to increase the nozzle packing density further issues will arise. Space charge, a dense charged droplet cloud, decreases the field strength at the nozzles, and so may cease the cone jet spraying of one or more nozzles. So for steady spraying a higher voltage setting is needed. So the electric field at the tip of a capillary nozzle is more often influenced by the nearby nozzles’ electric field. If the influence between the nozzles is large, also the radial component of the electric force acting on a cone is not negligible and the electric force deforms the cone at the tip of the capillary nozzle leading to no or interrupted droplet break up. As already discussed the droplets are highly charged and to avoid Rayleigh disintegration, they have to be discharged. The more jets result in the higher space charge in the gap between the cones and the counter electrodes. The higher the space charge is, the higher the required potential difference necessary for the formation of the cones. The space charge in the setup could also lead to differences in the electric field at the nozzles. The problem of the electric field can be solved by introducing a ring electrode close to the nozzle just as for a single nozzle. In that case, the electric field is determined by the field between the nozzle and the ring. Neighbouring nozzles have no longer an influence on the field at the nozzle. The problem of space charge can be solved in two ways; collecting the particles

Fig. 3.13 Schematic of multi nozzle system after Hartman

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immediately after their generation on a conducting surface (counter electrode), or discharging and transporting the particles with a carrier gas flow. Figure 3.13 shows a schematic representation of a multiple nozzle system as suggested and realized by Hartman (1998), in where all the requirements have been fulfilled.

References Ciach T (2007) Application of Electrohydrodynamic atomization in drug delivery: a review. J Drug Deliv Sci Technol 17(6):367–375 Ciach T, Geerse KB, Marijnissen JCM (2002) EHDA in particle production. In: Kanuth P, Schoonman J (eds) Nanostructured materials. Kluwer Academic, Boston Cloupeau M, Prunet-Foch B (1994) Electrohydrodynamic spraying functioning modes: a critical review. J Aerosol Sci 25:1021–1036 Coppens PF (2007) Coating of tribocharged model particles with nanoparticles using EHDA, MS Thesis, Delft University of Technology, Faculty of Applied Sciences, Nanostructured Materials Research Group, Process and Product Engineering Dabkowski MF (2006) Coating of particles with nanoparticles by means of electrostatic forces, MS Thesis, Delft University of Technology, Faculty of Applied Sciences, Nanostructured Materials Research Group, Process and Product Engineering Research Group Dabkowski MF, van Ommen JR, Yurteri CU, Hochhaus G, Marijnissen JCM (2007) The coating of particles with nanoparticles by means of electrostatic forces. In: Schreglmann C, Peukert W (eds) Partec 2007 – CD proceedings, Nuernberg, Germany, paper S37_2 Davis EJ, Bridges MA (1994) The Rayleigh limit of charge revisited – light-scattering from exploding droplets. J Aerosol Sci 25(6):1179–1199 De la Mora JF, Loscertales IG (1994) The current emitted by highly conducting taylor cones. J Fluid Mech 260:155–184 Deng W, Gomez A (2007) Influence of space charge on the scale up of multiplexed electrosprays. J Aerosol Sci 38:1062–1078 Gan˜a´n-Calvo AM, Davila J, Barrero A (1997) Current and droplet size in the electrospraying of liquids. Scaling laws. J Aerosol Sci 28:249–275 Grace JM, Marijnissen JCM (1994) A review of liquid atomization by electrical means. J Aerosol Sci 25(6):1005–1019 Hartman RPA (1998) Electrohydrodynamic atomization in the cone-jet mode. From physical modeling to powder production. PhD thesis, Delft University of Technology Hartman RPA, Brunner DJ, Camelot DMA, Marijnissen JCM, Scarlett B (1999) Electrohydrodynamic atomization in the cone-jet mode physical modeling of the liquid cone and jet. J Aerosol Sci 30(7):823–849 Hartman RPA, Brunner DJ, Camelot DMA, Marijnissen JCM, Scarlett B (2000) Jet break-up in electrohydrodynamic atomization in the cone-jet mode. J Aerosol Sci 31(1):65–95 Hernandez JO, Choren EA (1983) Thermal stability of some platinum complexes. Thermochimica Acta 71(3):265–272 Hull P, Hutchison J, Salata O, Dobson P (1997) Synthesis of nanometerscale silver crystallites via a room-temperature electrostatic spraying process. Adv Mater 9(5):413–417 van Erven J, Moerman R, Marijnissen Jan CM (2005) Platinum nanoparticle production by EHDA. Aerosol Sci Technol 39(10):929–934 Lefebvre AH (1989) Atomization and sprays. Hemisphere Publishing, WA Lenggoro I, Okuyama K, de la Mora J, Tohge N (2000) Preparation of ZnS nanoparticles by electrospray pyrolysis. J Aerosol Sci 31(1):121–136 Meesters G, Vercoulen PHW, Marijnissen JCM, Scarlett B (1992) Generation of micron-sized droplets from the Taylor cone. J Aerosol Sci 23(1):37–49

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Rulison AJ, Flagan RC (1994) Synthesis of Yttria powders by electrospray pyrolysis. J Am Ceramic Soc 77:3244–3250 Smith JN, Flagan RC, Beauchamp JL (2002) Droplet evaporation and discharge dynamics in electrospray ionization. J Phys Chem A 106(42):9957–9967 Snarski SR, Dunn PF (1991) Experiments characterizing the interaction between two sprays of electrically charged liquid droplets. Exp Fluids 11(4):268–278 Taylor GI (1964) Disintegration of water drops in an electric field. Proc R Soc A280:383–397 van Ommen JR, Beetstra R, Nijenhuis J, Yurteri CU, Marijnissen JCM (2008) Coating of tribocharged host particles with nanoparticles using electrospraying, Particulate processes in the pharmaceutical industry II, San Juan, Puerto Rico, 3–7 February

Chapter 4

Electrospray and Its Medical Applications Da-Ren Chen and David Y. H. PUI

4.1

Introduction

Electrohydrodynamic atomization, commonly called “electrospray (ES)”, has recently attracted a great deal of interests in research communities. It is because of its enormous potential in practical applications. In traditional applications, the process has been applied to the surface coating (Hines 1966; Paul 1985; van Zomeren et al. 1994), agricultural treatments (Coffee 1964), emulsion (Nawab and Mason 1958) or supermicron aerosol production, fuel spraying (Jones and Thong 1971), micro-encapsulation (Langer and Yamate 1969), ink-jet printers (Tomita et al. 1986), and colloid micro-thrusters (Huberman et al. 1968). More recently new applications have been explored. Examples include (1) using the electrospray as ion sources for mass spectrometry (ES MS) for the macromolecular detection (Yamashita and Fenn 1984; Fenn et al. 1989; Thompson et al. 1985; Cole 1997; Smith et al. 1997; Dulcks and Juraschek 1999), (2) monodisperse nanoparticle generation (Chen et al. 1995), (3) biomolecule detection using gas-phase electrophoretic mobility molecular analyzer (GEMMA) (Kaufman et al. 1996; Kaufman 1998, 1999; Koropchak et al. 1999; Scalf et al. 1999; Bacher et al. 2001), (4) enhancement of droplet mixing by inter-electrospray (Dunn and Snarski 1991; Snarski and Dunn 1991; Dunn et al. 1994), (5) targeted drug delivery by inhalation (Tang and Gomez 1994), (6) micro-mixing for drug powder production (Borra et al. 1999), (7) inorganic nanoparticle preparation by electrospray pyrolysis (Lenggoro et al. 2000), (8) preparation of non-structured ceramic thin films (Chen et al. 1999), (9) electrospray gene transfection (Chen et al. 2000), (10) compound-jet D.-R. Chen (*) Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO USA e-mail: [email protected] D.Y.H. PUI Director of Particle Technology Laboratory, Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN USA

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_4, # Springer ScienceþBusiness Media B.V. 2010

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electrospray for the nano-encapsulation and enhancement of targeted lung delivery of nano-medicines (Chen and Pui 2000). The general reviews of the electrostatic atomization and applications are covered in the books written by D. Michelson (1990) and by A. G. Bailey (1988). More advanced discussion on the functional modes in the process are presented in the works of Cloupeau and Prunet-Foch (1989, 1990, 1994), and Jaworek and Krupa (1999). Among all the electrospray operating modes, the most commonly used and studied one is the so-called “cone-jet mode”. It is because the cone-jet mode operation has the capability of producing monodisperse particles with the same electrical polarity. It means particles generated by the conejet electrospray are monodisperse and non-agglomerated. Many applications can be made possible or benefit from particles with such properties, especially for those making use of particles in the nanometer size range. The most common configuration of ES system is the point-to-plate (or orifice plate) arrangement. A single capillary with one end serving as the “point” is used to deliver the spray liquid into the spray chamber. The spray liquid is often delivered into the capillary by either a syringe pump or gravity force. High voltage is applied either at the capillary with the grounded plate, or on the plate with the grounded capillary. At a proper voltage the shape of liquid meniscus at the capillary end will form the conical shape with a tiny jet issued from the cone tip (so-called cone-jet mode). For some of ES systems an orifice plate is used in place of the solid plate, allowing generated particles to exit the spray chamber for further particle conditioning. The systems consisting of a single capillary is named as the single-capillary ES system in this article. Many applications have been explored with single-capillary ES systems. Several limitations on the ES operation exist for the single-capillary ES systems. First, the electrical conductivity of spray liquid or solution needs to be conditioned within a proper range in order to electrospray them in a stable operation. For some liquids, especially for non-polar solvents, conductivity conditioners (or ion additives) are difficult to be identified. For some cases only limited range of electrical conductivity can be varied even if the proper conditioners were found. Secondly, the particle size and the associated electrical charges cannot be controlled independently. Additional charge conditioner will then be needed if the charge level on ES-generated particles were critical for applications. Lastly, the coating of colloidal particles with a different material (e.g., polymers) by single capillary ES systems is limited for the cases that particles and coating material can stably co-exist in the same solvent. To further extend the applications for ES technique dual-capillary ES systems are thus proposed. In such a system a dual-capillary assembly (one served as the outer cylinder and the other as inner tube) is used. The outer and inner tubes are often coaxially aligned. The coaxial tubing arrangement creates two flow channels for introducing two liquids. One flow channel is in the annual spacing between the outer and inner tubes, and the other in the inner tube. The use of the dual-capillary assembly in ES systems expands its potential to overcome the limit of singlecapillary ES systems and opens for more applications based on the ES technique. The dual-capillary configuration of ES systems was first proposed to introduce biomaterial into cells for gene transfection (Chen and Pui 2000). It makes use of the

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space charge effect to propel biomaterial particles at high speed prior to reaching target cells. Here, the dual-capillary ES system was proposed to introduce more electrical charges on the particles by sheathing the biomaterial suspension (in the inner capillary) with highly conductive and volatile liquid (in the outer capillary) during the spray process. From the calculation it is found that the particle velocity in such an application is primarily attributed by the space charge effect, resulting from the production of highly charged particles at high concentration, upon the particle impact on the cell membrane. Using the coaxial electrospray, Loscertales et al. (2002) further demonstrated the production and control of monodisperse capsules in submicron sizes, varying from 0.15 to 10 mm. They also found that the diameter of capsules produced by coaxial electrospray is influenced not only by the operational parameters, such as liquid feed flowrates, but also by the physical properties of spray liquids as well as the interaction between inner and outer liquids during the spray process. Lopez-Herrera et al. (2003) investigated electrified coaxial jets of two immiscible liquids issuing from a structured Taylor cone. To interpret the experimental observation, they introduced the concept of a driving liquid and presented the linear scaling law for the compound jet diameters. Chen et al. (2005) studied compound jet electrospray modes using an ethanol–glycerol– tween80 (polysorbate detergents) mixture and cooking oil, two immiscible liquids. They found that the spray phenomena were mainly controlled by the property of outer liquid, which was very viscous and electrically conductive. When compared with other liquid atomization techniques the uniqueness of ES technique is its ability to produce highly-charged, non-agglomerated and monodisperse particles in a wide size range from the nanometer to supermicron range. The unique properties of ES-generated particles offer great control on the dispersion and deposition of particles. Such features on the technology opens up many modern medical and biological applications where the precise control of particle size, morphology, and electrical charges associated with, as well as the control on the particle dispersion and deposition are needed. In this chapter we will briefly introduce the electrospray technology and its variation. Examples of medical and biological applications using the electrospray technique will then be discussed. Note that the intention of this chapter is to make readers aware of the electrospray technology and its application in medical/ biological areas. A comprehensive review of the subject is not the intention of the authors for this chapter.

4.2 4.2.1

Basics of Single- and Dual-Capillary Electrospray Basics for Single-Capillary ES

All recent applications described herein are operating the electrospray in this mode, so-called “cone-jet electrospray”. Consequently the following review is primarily focused on the cone-jet electrospray. The fundamental characteristics of cone-jet

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electrospray are based on the spray electrical current and droplet size produced. With the experimental observations and dimensional analysis of the parameters in a cone-jet electrospray, de la Mora and Loscertales (1994), and Rosell-Llompart and de la Mora (1994) proposed that the spray current, I, can be formulated as f(k) (GKQ/k)1/2 and the main droplet size, Dd, can be scaled with r* (the charge relaxation length) (where K is electrical conductivity of liquids, G liquid surface tension, m liquid absolute viscosity, and k relative dielectric constant of liquids, Q the feeding flow rate). The charge relaxation length, r* is defined as the characteristic traveling distance for electrical charges to make up its loss due to the jet breakup; it can be estimated as (Qt)1/3 where t is the electric charge relaxation time and is inversely proportional to the electrical conductivity of spray solutions). The function of f(k) and the proportionality for Dd were later experimentally determined by Chen and Pui (1997). Taking a different approach, Gan˜a´n-Calvo (1994) published a set of new scaling laws based on the asymptotic analysis of the 1-D governing equations for electrical and flow fields in the near region where the jet is emitted from a liquid cone. They are summarized as I ¼ CI*k1/4(gKQ/k)1/2 and Dd ¼ Cd*k1/6(Qt)1/3 for polar liquids; I ¼ KI*(QKg3/r)1/4 and Dd ¼ Kd*Q1/2(r/ gK)1/6 for non-polar liquids. However the proposed scaling laws were revised in later works of the same author (Gan˜a´n-Calvo 1997, 1999). On the other hand, the recent experimental and numerical work by Hartman et al. (2000) suggested that Dd ~ Q1/2 if varicose breakup involved and Dd ~ Q1/3 if whipping break-up process occurred. Unfortunately, the transition between two jet breakups cannot be confirmed or predicted by the proposed model. The experimental determination of this transition also presents a difficulty because of the poor quality of collected data. Table 4.1 summarizes the scaling laws on the droplet size and emitted current proposed in various publications. The other important spray characteristic is the mean charges of individual droplets produced by a cone-jet electrospray. The knowledge is of importance in many practical applications such as precision deposition of particles, droplet micromixing, detection of macromolecules, gene transfection, drug delivery and so on. However, disagreements exist among the literatures. According to the models Table 4.1 Summary of scaling laws for droplet size and emitted current in the electrospray operated at the cone-jet mode Emitted current, I References Droplet diameter, Dd f(k) (gKQ/k)1/2 Rosell-Llompart et al. (1994); Chen and g(k)(Qt)1/3 Pui (1997a) CI k1/4(gKQ/k)1/2 Gan˜a´n-Calvo (1994) Cd k1/6 (Qt)1/3 polar liquids KI (QKg3/r)1/4 Kd Q1/2(r/gK)1/6 non-polar liquids 3.78p 2/30.6Q1/2(re0/ 4.25(gQK/ln(Q/Q0)1/2)1/2 Gan˜a´n-Calvo (1997) Kg)1/6 2.6 (QK/g)1/2 Gan˜a´n-Calvo (1999) 2.9 e0p 2/3(rgQ3/K)1/6 1/2 I ~ (QK)1/2 Hartman et al. (2000) Dd ~ Q , varicose breakup Dd ~ Q1/3, whipping break-up

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proposed by Vonnegut and Neubauer (1952), Ryce and Patriarche (1965), Pfeifer and Hendricks (1967), the mean droplet charge was about 50% of the Rayleigh limit. Experimental data were provided for the confirmation. Although the principle applied in these models was questioned by Krohn (1973) the result was often cited in the literature. The analysis given by Jones and Thong (1971) predicted that the charge-to-mass ratio for electrosprayed droplets was a linear function of Dd 1 in the small particle size range. No experimental data was given to verify the result until the works published by Cloupeau and Prunet-Foch (1989), Gomez and Tang (1993) for heptane droplets in 5–10 mm, Tang and Gomez (1994) for water droplets in 2–10 mm size range, and Chen et al. (1995) for water droplets of the sizes ranging from 0.1 to 1 mm. Gomez and Tang (1993) also found that for heptane droplets of the sizes larger than 10 mm, the charge-to-mass ratio could be characterized by the power function of Dd 3/2. Meanwhile, based on the revised scaling laws, Gan˜a´nCalvo (1999) suggested that the maximal surface electric charge on electrosprayed droplets had a universal value independent of the jet size and the liquid flow rate, and could be given as 0.53*21/2p1/3(e0g2rK2)1/6. It should be noted that in all these previous studies the mean charge on individual droplets was derived from the droplet size and spray current given by scaling laws or obtained by measurements. The assumption underlying the derivation was that the charge distribution on electrosprayed particles was relatively narrow if relatively monodisperse droplets were produced. Thus the droplet production rate was estimated from the liquid mass flowrate with the droplet size given by the scaling law or measurements. The mean electrical charges were then calculated from the spray current and droplet production rate. However, the assumption was questioned in the experimental work of de la Mora (1997). The assumption was further challenged with the observation that the gas surrounding the cone-jet had an important effect on the spray current (Aguirre-de-Carcer and de la Mora 1995). Moreover, the charge-to-mass characterization is complicated by the presence of satellite droplets. They are often observed in the production of primary droplets due to the nonlinear dynamics and breakup of liquid jets. For neutral jets they came about through the mechanisms of pinching singularity (Eggers 1997a). Prior to the breakup a tiny neck is usually formed between the jet and drop. The singularity is initially localized and producing pinchoff at the location where the neck is attached to the drop. Since only a small amount of fluid is involved, it acts on time scales much shorter than the growth of disturbances on the jet, once it sets in. Due to the asymmetrical singularity the only way the liquid neck can be matched onto an outer solution is by pinching off again at the point where the neck is linked with the jet. Hence the liquid neck is pinched off from both sides and eventually contracts into a satellite droplet. The detail process was photographed and further simulated using the 1-D models (Eggers 1997b). For electrically charged jets similar process was numerically observed in the work of Setiawan and Heister (1997). However, electric charges carried by these satellites remain largely uncharacterized. Meanwhile, satellite droplets could also be produced by the droplet fission. Recent works on electrosprayed droplets of supermicron sizes have shown that under the influence of aerodynamic forces droplet fission occurred already at charge states of

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about 70–80% of Rayleigh limit (Taflin et al. 1989; Gomez and Tang 1993; Shrimpton 2005). All experimental investigations have shown that there was no “explosion” of the droplet but the droplet surface formed a cone-like shape from the tip of which a number of smaller droplets was ejected. Measurements showed that this “uneven fission” the primary droplet lost about 15% of the charge and about 2% of its mass. However, whether this scenario can be taken for droplets in submicron and nanometer size range remains unknown. The mean charge characteristics are even more complex if evaporating droplets are produced. As the droplet size is continuously reduced through either solvent evaporation or droplet fission electrical charges may release from droplets (as ion emitters). It is because of the buildup of the electric field strength at the droplet surface (Iribarne and Thomson 1976). The scenario is often called “ion evaporation”.

4.2.2

Dual-Capillary ES

The operation of dual-capillary ES systems is more sophisticated than that of single-capillary ES systems. The main reason is due to the involvement of two liquids coaxially introduced into a highly non-uniform electrical field. The scientific knowledge on the liquid behavior under such a condition is generally insufficient. From the operational point of view the first question to be addressed for using the dual-capillary ES systems is under which condition a stable compound cone-jet mode can be established similar to that established in the single-capillary ES system to generate monodisperse particles. This question has been partially answered by Mei and Chen (2008). They found that a stable compound cone-jet mode can be easily established for miscible and partially miscible liquid pairs. For an immiscible liquid pair, the liquids should satisfy two sufficient conditions to form a stable compound cone-jet mode: (1) a liquid of high dielectric constant should be used as the inner one; and (2) the surface tensions of liquid pair should satisfy the spreading-coefficient criterion for the engulfing and partial engulfing cases in three-phase interaction. For the first condition, the density of electric flux emitted from the outer liquid cone base would be more than that from the inner liquid cone base, if the dielectric constant of the outer liquid was higher than that of the inner liquid. As a result, the normal electric force along the interface would be greatly reduced or negligible. The formation of the inner cone, based on the balance of normal electric stress and interfacial tension, thus became impossible. For the second condition, the dynamic condition of the liquid pair used in a dual-capillary ES system should not depart much from the static condition of three-phase interaction, because of the slow fluid motion in the liquid cones in the stable compound cone-jet mode. The 2nd question to be addressed is under which condition the encapsulation of droplets will occur in dual-capillary ES system. Based on the study of Mei and Chen (2007) it was found that two different types of droplet size distributions, e.g., uni-modal and bi-modal types were produced by dual-capillary ES systems. For the examples of ethanol–olive oil, TBP–olive oil, ethanol–mineral oil pairs (i.e., inner

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liquid–outer liquid), unimodal size distributions were generally observed. In these cases, the size of the produced capsules linearly increased with an increase of the inner liquid flowrate in the low flowrate regime. It implied that varicose or kink instability was the dominant mechanism in the low flowrate regime. After exceeding a certain value of inner liquid flowrate, the particle size remained constant, implying that the jet breakup was significantly influenced by the inertia effect of inner liquid at high flowrate. For other test cases in the study, bi-modal size distributions or polydisperse but uni-modal size distributions of produced particles were observed, which suggested uncontrollable or failed encapsulation for such liquid pairs. For the cases with bi-modal size distributions an increase of the inner liquid flowrate decreased only the concentration of particles in the large size peak but their sizes remained constant. Through the data analysis they identified the criteria to predict the formation of capsules by the dual-capillary ES process. Two domains (i.e., controllable and uncontrollable encapsulation) were indicated in the R*O/R*I vs r*O/r*I plot, where R* was the inertia length of liquid, r* the charge relaxation length of liquid, and the subscript o and I indicating outer and inner liquids used in the ES systems. The finding of two domains on the plot implied that the encapsulation using the dual-capillary ES mainly accounted for combined effects of the relative importance of the inertial and electrical force for the inner and outer liquids. Further work and study need to be performed on the investigation of spray current and droplet size produced by the dual-capillary ES systems. The authors are only aware of two studies on these subjects. Interested readers can refer to the publications to find out the details of both studies. In summary limited liquid pairs were tested in the study performed by Lopez-Herrera et al. (2003). Empirical models on spray current and droplet size generated from the dual-capillary ES system had been proposed to fit the data collected in the study. Unfortunately the proposed models cannot be applied to all the studied liquid pairs at the same category. A broader range of liquid pairs were studied in the dissertation performed by Mei (2008). This study also advances our understanding on the subjects but it is far from what we know for single-capillary systems.

4.3

4.3.1

Examples of Medical/Biological Applications Using Electrspray Drug Reformulation

Most newly synthesized medicines are poorly soluble in water. Because of their poor solubility, some have been abandoned for further development and others require the patients to take high dose of the medicines with potential negative side effects. One way to increase the solubility is to reduce the size of medicine particles and consequently increase the surface area of particles. The single-capillary

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4.0

Impactor D50

3.5 3.28e+005 dN/dlogDp (#/cm³)[e5]

3.0 2.5 2.0 1.5 1.0 0.5 0.0

10

Diameter (nm)

100

1000

Fig. 4.1 Particle size distribution produced by single-capillary electrospray technique. TSI scanning mobility particle sizer (Model # 3936NL25) was used to measure the size distribution of particles

electrospray technique has been proposed to produce nanometer-sized pharmaceutical particles. An example of such application is shown in Fig. 4.1. The size distribution of steroid particles produced by single capillary ES technique was measured by the scanning mobility particle sizer (SMPS). It is evidenced that steroid particles of 13 nm diameter can be produced by the ES technique. In this case the solvent used was water and nitric acid was used to adjust the electrical conductivity of spray solutions to 520 mO/cm. The liquid feed flow rate was 0.1 mL/min. The issue of using the electrospray technique for drug reformulation is on the mass throughput of the ES systems. More development work needs to be done in this area to address the issue. One idea to increase the mass throughput of the singlecapillary ES system is to use multiple capillaries in parallel. However the tight packing of multiple capillaries presents a challenge on the successful implementation of the multi-capillary systems. It is primarily because of the space charge effect resulted from highly charged particles produced. At present all the successful works were accomplished for solutions with low surface tension and low electrical conductivity. For such solutions the electrical charges associated with ES-produced particles are much less than those produced by electrospraying highly conductive solutions of high surface tension.

4.3.2

Nanoparticle Dispersion for Toxicity Study

Nanoparticles are encountered in many industrial systems utilizing aerosol reactors. Such reactors are used in industries to make a wide variety of particulate

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commodities, such as carbon black, pigments, and materials for high technology applications such as optical waveguides and powders for advanced ceramics (Stamatakis and Natalie 1991). Kruis et al. (1998) have described newly proposed applications in nanotechnology. To realize these new applications, nanoparticles of different physical and chemical properties are synthesized through different routes with unknown toxicity. A similar scenario is encountered in many other systems, where a large quantity of the so-called “undesirable” aerosols is produced. Biswas and Wu (1998) cited municipal waste incinerators, hazardous waste incinerators, welding systems, exhausts, coke ovens, smelters, nuclear reactor accidents, utility boilers, and the exhausts from automobile, diesel engine, and jet aircraft as examples. In manufacturing, nanoparticles need to be collected to fabricate parts for applications. In the case of waste particle generation, the particles could be potentially very toxic, and their emission to the ambient atmosphere needs to be prevented. With recent increasing findings that nanoparticles may be associated with deleterious health effects (Wolfgang et al. 2006), it will be necessary to study the toxicity of nanoparticles. Most engineered nanoparticles are in the powder form or colloidal suspension in liquids. The dispersion of nanoparticles in gaseous phases is needed to investigate the toxicity of nanoparticles through the in-vivo and in vitro routes. Unfortunately the dispersion of nanoparticles from suspensions cannot be accomplished by conventional pressure atomization. As an example the suspension of PSL particles of 28 nm diameter was atomized using the Collison atomizer. Shown in Fig. 4.2a is the particle size distribution measured by SMPS when the diluted PSL suspension was dispersed. It is obvious that PSL nanoparticles cannot be isolated from the measured particle size spectrum. One of the reasons resulted in such observation is the impurity from the water and the original PSL suspension (e.g., the surfactant used to keep PSL nanoparticles apart). The other reason is due to the nature of pressure atomization. The pressure atomization technique often produced droplets with a broad size range. With the impurity presence in the colloidal suspension, atomizer-produced droplets in which only impurity is present will form residue particles in nanometer size range. Shown in Fig. 4.2b is the size distribution measured by electrospraying diluted PSL suspension. By tuning the droplet size slightly larger than 28 nm in diameter it is possible to isolate the PSL nanoparticles from the SMPS-measured size distribution. A further example to demonstrate the capability of ES technique to disperse nanoparticles suspension is given in Fig. 4.2c. In this case the slurry used in CMP (Chemo-mechanical polishing) process was used. The sprayed slurry was diluted from the original suspension by a factor of ten in concentration. A programmable syringe pump was used to linearly vary the liquid feed flow rate into the ES capillary. The size distribution shown in the figure was accomplished by continuously varying the feed flowrate (consequently varying the droplet size produced) to synchronize with SMPS scanning process. Nanoparticles of three different sizes were detected. Nanoparticles of three different sizes in the diluted slurry were also confirmed by the SEM imaging of the sprayed sample.

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Fig. 4.2 (a) SMPS-measured particle size distribution by dispersing 28 nm PSL suspension with collison atomizer. (b) SMPS-measured Particle size distribution by dispersing 28 nm PSL suspension with single-capillary electrospray (c) SMPS-measured size distribution of particles airborne by electrospraying the diluted CMP slurry

4.3.3

Electrospray Inhaler for Asthma Patient

Inhalers are critical devices to deliver medicine into the target location of asthma patient’s lung for effective reduction of the asthma symptom. Research in developing a better inhaler has been progressing for a couple of decades. Different atomization techniques were proposed to accomplish the delivery task. Most of these techniques produce polydisperse particles. However, the particles size and

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electrical charges associated with them are two important factors to deposit medicine particles in the target lung area. With polydisperse particles generated by many atomization techniques some (or majority for some cases) of the particles would not be deposited on the target lung area. Because of the monodispersity of ES produced particles it has been proposed for inhaler applications. Battelle Memorial Institute has been a key player for the commercialization of MysticTM inhaler which is based on the electrospray technology. A spin-off company, Ventaira Pharmaceuticals Inc. was established to further develop and market the inhaler. The design of the MysticTM inhaler is based on the patents filed by Coffee et al. (2004). In addition to the electrospray component for atomizing medicines a corona discharge compartment is included in the inhaler to reduce the electrical charges on ES-produced droplets for the particle delivery. The product was initially scheduled to be introduced commercially in 2008 but was delayed due to the issues encountered in the Phase I clinical trial. The Ventaira Company is now ceased to exist and was purchased back by Battelle Memorial Institute.

4.3.4

Polymer Coating for the Control of Drug Release Rate

Coating of medicine particles for the controlled drug release is one of modern medical applications. By controlling the medicine release rate the patients can take the medicine on the daily, weekly or even monthly basis. The drug release control also allows the application of medicine when needed. The encapsulation of pharmaceutical particles can be accomplished by the dual-capillary ES technique (Mei 2008, 1991). The bio-degradable polymers, e.g., PEG and PLGA are often used as the coating material. For other medical applications proteins can be used as the coating material.

Fig. 4.3 (a) A typical stent for the human use. (b) Coated stent prepared by electrospray technique

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Fig. 4.4 (a) The porous film morphology of the coated layer, prepared by electrospray technique, on the stents. (b) The continuous film morphology of the coated layer, prepared by electrospray technique, on the stents

4.3.5

Coating of Medical Devices

The coating of stents with medicines and/or polymers used the dual-capillary electrospray system is one of the examples. The bypass surgery was often performed to treat clogged blood vessel in the human hearts in the past. The surgery is not performed regularly with the invention of stents. Stents are often made by laser carving stainless steel tubes of tiny diameters to form a wire structure. An SEM image of a stent used in human heart is shown in Fig. 4.3a. The wire structure of stents allows it to be significantly expanded in volume. The heart surgeon can thus insert the un-expanded stent into the blood vessel through a tiny cut on the patient body and expand it after proper positioning it in the clogged vessel. In stent restenosis is an issue, expected in 6 months following its installation, leading to the clogging of the blood vessel again. One way to resolve this issue is to coat stents with the medicine to prevent the vessel tissue from growing around the opened stent wall. In the current practice the stent coating is done by the ultrasonic atomization of the medicine solution and depositing particles on the stent by inertial impact. Because of the size and porosity of stents majority of sprayed particles are not deposited on the stent and are wasted. Due to the nature of the electrospray technology it offers great improvement on the waste reduction and yield increase over the current practice. With the presence of electrical field in the ES system charged particles in sub-micrometer sized range will follow the existing electric field and deposited on the stent wall. Because of the electric field around the stent wire wall particles will be deposited around the wall wires, instead of merely on the outer side of the stent wall. The coating around the stent wall wires is possible when coating particles in the submicron sizes. Shown in Fig. 4.3b is the coated stent done by the electrospray process. The uniformity of coating layer around the stent wall wires is evidenced. Further, as demonstrated in Fig. 4.4a and b, the morphology of coated film on the stent can be controlled by using the dual-capillary ES technique.

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Fig. 4.5 (a) Nanogradient thin film of neuron growth factor, prepared by electrospray technique. (b) Guided neuron growth using the nanogradient thin film of neuron growth factor

4.3.6

Formation of Nano-Gradient Growth Factor for Guided Neuron Growth

Nano-scaled variation of the concentration of particles deposited on the substrate can be accomplished using the single-capillary ES technique. The task of creating the pattern with the deposited material in nano-gradient concentration on a substrate can be done by either direct electrospray writing or the use of patterned mask to filter electrosprayed particles. The ability of making the pattern with nano-scaled gradient of concentration opens its potential medical application. An example given herein is for the guided growth of neuron cells. Shown in Fig. 4.5a is the gradient line of the neuron growth factor prepared by electrospraying and masking growth factor with the careful control of the substrate moving. Fig. 4.5b evidences the guided growth of frog neuron cells after placing them on the prepared growth factor nano-gradient lines.

4.3.7

Gene Transfection Using the Electrospray Technique

Gene transfection at the cellular level offers much application and potential in plant improvement, cancer therapy and other applications in biology. Many techniques have been proposed to introduce genes into the cells. The electrospray technique offers an alternative way to accomplish the same task. An exploratory study was published in Chen et al. (2000). It has been demonstrated that the particles, produced by the dual-capillary electrospray with the introduction of additional electrical charges by outer ionized liquids, have sufficient velocities to enable them penetrating through the membrane of animal cells.

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Summary

The uniqueness of the electrospray techniques relies on its ability to produce highly charged, monodisperse particles in the diameter range from nanometer to supermicrometer. Due to the electrical charges of the same polarity electrosprayed particles are non-agglomerated. The technology allows users to gain greater control on the generation, dispersion and deposition of particles over other atomization techniques. It allows users to tailor particles to the desired size, morphology and construction for improved particle function and transport properties, making the technique suitable for many particle applications, especially for medical and biological applications. In this chapter we have briefly reviewed the history and the evolution of the electrospray technique. Single- and dual capillary electrospray techniques were introduced. The up-to-date basics on the operation of single- and dual- capillary ES techniques were also summarized. The last part of this chapter has been devoted to provide example applications of the electrospray technique in medical and biological areas. Many studies have been reported on using the electrospray technique for medical applications. More work is needed to take this wonderful technology to the next level for practical applications. One issue is related to the mass throughput of the electrospray technique. Limited success has been reported using the multiple capillary systems. All the reported works with multiple capillary systems involved spraying solutions of low surface tension and electrical conductivity, resulting in less electrical charges on particles and thus lower space effect attributed by the charged particles. The space charge effect resulted from the charged particles in high concentration will eventually limit the number of capillaries that can be deployed in the multiple capillary ES systems. The mass throughput of multiple capillary systems cannot be scaled up indefinitely. A breakthrough design for implementing the multiple capillary systems will be needed in the future. Another concern of using electrospray for medical application is related to the viability of biomaterials after spraying even though the viability of some biomolecules has been established (Kwok et al. 2008; Clarke and Jayasinghe 2008). Potential damage to the bio-molecular structure exists, especially for fragile bio-molecules. To evaluate the bio-viability of sprayed biomaterial is always a necessary step in the technology development. The situation also calls for the development of a soft electrospray technique to ensure no damage to the sprayed bio-materials. Lastly, the control of electrical charges on the electrosprayed particles is often accomplished through the use of corona discharge devices, either DC or AC. It is because of the gradually tightened safety regulation for the use of radioactive materials for neutralizing the charged particles. Unfortunately, ozone is also produced in the corona discharge process. The strong oxidation ability of ozone presents the threat of bio-material damage; it also has adverse health effect on the patient if ES was used to deliver the medicine into the human lung. An alternative approach to controlling the charges on the electrosprayed particles will be much needed for the medical application of the electrospray technique.

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Acknowledgement This work was partially supported by a grant from the U.S. Department of Defense (AFOSR) MURI Grant (FA9550-04-1-0430) “Relationship between Physicochemical Characteristics and Toxicological Properties of Nanomaterials”.

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Chapter 5

Generation of Nanoparticles from Vapours in Case of Exhaust Filtration Markku Kulmala and Mikko Sipila¨

5.1

Introduction

Generation, investigation and manipulation of nanostructured materials are of fundamental and practical importance for several disciplines including materials sciences and medicine. Recently, atmospheric new particle formation in the nanometer size range has been found to be a global phenomenon (Kulmala et al. 2004). The processes related to nanomaterials and atmospheric nanoparticles are at least similar and in some cases even identical. However, the detailed mechanisms for nucleation and nanoparticle formation are mostly unknown, largely depending on the incapability to generate and measure nanoparticles in a controlled way. In recent experiments an organic vapour (n-propanol) condenses on molecular ions as well as charged and uncharged inorganic nanoparticles via initial activation by heterogeneous nucleation (Winkler et al. 2008). In these experiments a smooth transition in activation behaviour as a function of size has been found, and activation did occur well before the onset of homogeneous nucleation. Furthermore, nucleation enhancement for charged particles and a significant negative sign preference were quantitatively detected. While fresh aerosol particle formation has been observed to take place almost everywhere in the atmosphere (Kulmala et al. 2004), several gaps in our knowledge regarding this phenomenon still exist. These gaps range from the basic processlevel understanding of atmospheric aerosol formation to its various impacts on atmospheric chemistry, climate, human health and environment. Until recent years nucleation pathways have been poorly understood even though several different mechanisms have been suggested (Kulmala 2003; Kulmala et al. 2006). Main

M. Kulmala (*) and M. Sipila¨ Department of Physics, University of Helsinki, P.O. Box 64 (Gustaf Ha¨llstro¨minkatu 2), FI-00014, University of Helsinki, Finland Helsinki Institute of Physics, P.O. Box 64, FI-00014, University of Helsinki, Finland e-mail: [email protected]

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_5, # Springer ScienceþBusiness Media B.V. 2010

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reason for that has been the instrumental inability to detect particles below 3 nm in diameter. Direct measurements of both the nucleation process itself and the initial growth of the clusters are crucial in order to resolve the detailed pathways of the particle formation. Only very recently observations of atmospheric neutral particles and clusters below 3 nm have shed light on the first steps of particle formation (Kulmala et al. 2005, 2007a). Those observations were made using newly developed instruments designed for maximal detection efficiency of small clusters, like UF02proto swirling flow condensation particle counter pair (Mordas et al. 2005, 2008) and an Air Ion Spectrometer (Mirme et al. 2007) equipped with an aerosol charger i.e. Neutral Cluster and Air Ion Spectrometer (NAIS) (Kulmala et al. 2007a). Here we first describe the recent instrumentation for physical detection of nanoparticles and nanoclusters (Section 5.2). In Section 5.3 we describe aerosol generation, and in Section 5.4 we present how filtration will affect on generation of nanoparticles both theoretically and experimentally. The concluding remarks are given in Section 5.5.

5.2

Detection of Nanoparticles

Studying nanoparticles e.g. during atmospheric aerosol formation or particle generation in laboratories requires the measurements of both physical and chemical properties of nucleation mode particles (3–20 nm) and clusters (90% of the MP are associated with lung-surface macrophages at any long-term retention time after inhalation. Unfortunately, there are no human long-term lymphatic clearance data. However, according to the striking similarity of the predominant mechanisms of human and canine particle clearance kinetics, namely the progression of lung retention and lung-surface macrophage-mediated long-term particle clearance towards the mucociliary escalator and larynx, a similar interstitial retention of MP in human lungs as that shown in dogs is expected. Still unclear is the role of bronchus associated lymphatic tissue (BALT) by which particles would find their way back to the airway epithelium through the lymphatic drainage, as it was hypothesized very early on (Macklin 1955; Adamson and Bowden 1981, Bowden and Adamson, 1984). In summary, the major differences in the clearance kinetics of MP between rodents and man may relate to morphological (length of respiratory bronchioles) as well as to functional differences (on top or below the epithelium).

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Since the effective transport rates of lung-surface macrophage-mediated clearance is the same for MP and NP in rodent lungs (Semmler-Behnke et al. 2007a), one could extrapolate from both lines of evidence that NP also penetrate the human epithelium for long term retention in the interstitial spaces and that the clearance kinetics of NP in humans is not faster but as slow as that of MP. However, the underlying mechanisms are not yet fully understood and presumably more complex. Note, however, that due to the very slow particle clearance kinetics in humans, with declining particle clearance rates over increasing retention time, there is an estimated fraction of 10–20% of insoluble particles which will never be cleared out of the human lungs under physiological conditions (Kreyling and Scheuch 2000). In cases of very high particle exposure, like smoking or in some occupational settings (mining, milling, etc.), the fraction of never-cleared particles may be substantially enhanced and associated with fibrotic pathogenesis. We have recently assessed the clearance of inhaled 20-nm TiO2 NP by lungsurface macrophages at the individual NP level by EFTEM (Geiser et al. 2008). The data from this study in rats showed that lung-surface macrophages do not efficiently phagocytose these NP but take them up rather sporadically and unspecifically within the first 24 h after NP inhalation: 1. There was only 0.06–0.12% of the TiO2 NP taken up by lung-surface macrophages within 24 h after aerosol inhalation, compared to >10% of MP that were phagocytosed already within the first hour (Geiser 2002) and >80% within 24 h (Geiser et al. 1990, 2000a, b; Lehnert and Morrow 1985; Sorokin and Brain 1975). 2. As little as 0.2% and 1.7% of the BAL macrophage populations, respectively, contained NP at 1 h and at 24 h after the aerosol inhalation, which are about two orders of magnitudes less than what was shown for 3 to 6-mm particles of different materials (Geiser 2002; Geiser et al. 1994). 3. The TiO2 NP in BAL macrophages were not tightly enclosed by the vesicular membrane, as it is known from phagocytic uptake of MP. Instead, they were located in large vesicles compared to NP size and the vesicles contained other material (Fig. 8.8). This also points to a rather sporadic uptake of TiO2 NP by lung-surface macrophages, maybe during the process of phagocytic uptake of other material. Hence, there is evidence that, at least within the first 24 h after aerosol inhalation, NP bypass the most important clearance mechanisms for particles deposited in the alveoli, namely phagocytic uptake by lung-surface macrophages. Consequently, the probability of NP uptake by lung epithelial cells and/or the translocation of NP through the thin epithelial barrier increases. Particle Transport to Regional Lymph Nodes. In a previous review (Kreyling and Scheuch 2000) we pointed out species differences between rodents versus humans, dogs and monkeys, i.e. there is less MP accumulation found in lymphatic nodules adjacent to airways in rodents than in large animals and humans. This may be physiologically plausible in the case where most long-term retained MP are

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Fig. 8.8 TEM micrograph of BAL macrophage with TiO2 NP located in a large phagolysosome containing other phagocytosed material. Bar ¼ 500 nm

phagocytosed by macrophages residing on the (rodent) lung epithelium. But it is not conceivable for NP being long-term retained in interstitial spaces with minimal lymphatic clearance. In large species, there are only MP clearance data for dogs available. These studies demonstrated that under physiological conditions, only a small, inter-subject variable fraction of 1–5% of deposited MP was drained to the regional lymph nodes (Kreyling et al. 1986; Kreyling and Scheuch 2000), although these MP were retained in interstitial spaces right next to the lymphatic drainage system. There are no newer particle kinetic data available for larger species and particularly not for human lungs. Particle Transport from the Interstitium. There is evidence for re-appearance of MP on the epithelium of man and dog, because the most prominent clearance mechanism was shown to be lung-surface macrophage-mediated and directed towards the larynx (Kreyling et al. 2001). Note that there is negligible interstitial retention of MP in rodents (Ellender et al. 1992; Lehnert et al. 1990). While there are no human or large species data available on the long-term retention and clearance of insoluble NP from the alveolar region, in rodents NP retention in the interstitium is prominent and NP re-appear on the lung epithelium for lung-surface macrophage-mediated clearance towards the larynx (Semmler-Behnke et al. 2007a). It remains to be resolved whether NP re-appear on the alveolar epithelium at large from the interstitial connective tissue or from BALT, as discussed much earlier on (Macklin 1955; Adamson and Bowden 1981; Bowden and Adamson 1984). The re-appearance of NP on the alveolar epithelium from the interstitium is likely to be macrophage-mediated as discussed above.

8.3.4

Translocation of Nanoparticles to the Blood and Their Subsequent Accumulation in Secondary Target Organs

Since about a decade, epidemiological studies continue to indicate associations between exposure to increased concentrations of ambient fine and ultrafine particles

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and adverse health effects in susceptible individuals (Ibald-Mulli et al. 2002; Peters and Pope 2002; Pope 2004; Schulz et al. 2005). Cardio-vascular effects observed in these studies triggered the discussion on enhanced translocation of ultrafine particles from the respiratory epithelium towards the blood circulation and subsequently to target organs, like the heart, liver and brain, eventually causing adverse effects on cardiac function and blood coagulation, as well as on functions of the central nervous system (Oberdo¨rster et al. 2005). From the latter review it appears that NP are subjected to transport across membrane boundaries, which has not been reported for MP. A possible mechanism provides the formation of complexes of NP with proteins of the lung-lining layer such that this complex acts like a ferry boat for NP. In contrast, MP would not be able to make use of those ferry boats because of their much larger size (Kreyling et al. 2007; Cedervall et al. 2007; Lynch and Dawson 2008). In addition, since MP are rapidly phagocytosed by lung-surface macrophages, they are only shortly available for protein-mediated transport. In fact, modifications of the surface of NP are currently intensively investigated in the discipline of Nanomedicine aiming to design, test, and optimize specific biokinetic behaviors of medicinal NP as diagnostic and therapeutic tools to reach high target organ specificity (ESF 2005); for example, drug delivery to the central nervous system via circulating NP requires surface modifications facilitating receptor-mediated translocation across the tight blood–brain barrier (e.g., apolipoprotein-E coating for LDL-receptor–mediated endocytosis in brain capillaries) (Kreuter 2001, 2004; Kreuter et al. 2002). Such highly desirable properties of NP must be carefully weighed against potential adverse cellular responses to targeted NP drug delivery; a rigorous risk assessment is mandatory. Besides the large surface area that can be modified, the large number of NP allows to disperse them into many more cells and intracellular compartments than MP of the same mass. For instance, a particle mass of 100 ng corresponds to only 2.4  104 particles (spheres of unit density) of 2 m in diameter, but to 2.4  108 particles of 20 nm in diameter or 2.4  1011 particles of 2 nm in diameter. Note that 20-nm particles comprise a major fraction of the number concentration of ambient aerosol particles (Kreyling et al. 2003), and 2 to10-nm particles are the primary particles originating from many combustion processes of which aggregated ambient ultrafine particles are made of. When we assume a NP mass of 100 ng to have accumulated in a secondary target organ like the heart or the brain, this mass would usually not be considered to be of any toxicological relevance for low toxicity particle; particularly, if one considers their low number in the entire organ, when they are 2-m in diameter. However, if they are 2-nm NP, they are to exceed the number of cells in the organ easily by a factor of ten (cell estimate is based on a 100 g organ and an average cellular volume of 6.5  10 11 cm corresponding to a cell of 5 m in diameter), and the triggering of adverse effects is more likely. Of course, the induction of such effects depends on other factors as well, including NP localization within subcellular structures, NP chemistry and surface characteristics. Although there is a huge set of literature on in-vitro NP-cell interaction usually the number of NP per cell were not estimated but only the total NP mass added to the cellular system is provided. Rough

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estimates indicate that most of these in vitro studies used NP-to-cell ratios far beyond 1000:1. It would be most relevant indeed to provide such an estimate of these ratios to better understand the relevance of those in vitro studies to the NP doses per cell under real exposure conditions.

8.3.4.1

Experimental Translocation Studies

Human Studies. The comprehensive analysis of NP accumulation and retention in organs and tissues is practically impossible in humans; because of ethical reasons, but also because of the limiting resolution of existing detection systems. Currently, there are no reliable human experimental data reporting a translocated NP mass fraction of more than 1% of the dose delivered to the lungs (Brown et al. 2002; Wiebert et al. 2006a, b; Mills et al. 2006; Mo¨ller et al. 2008a). However, there is indirect evidence of NP translocation in humans from recent exposure studies in healthy subjects using diluted Diesel exhaust (Mills et al. 2005). Inhalation of dilute diesel exhaust was found to impair two major functions of the vasculature, i.e. the regulation of the vascular tone and fibrinolysis. In addition, in a similar study in healthy volunteers, spontaneous alterations of EEG signals in the frontal cortex were observed during and until 1 h after exposure to Diesel exhaust (Cruts et al. 2008). From these studies it is not clear, whether the observed effects were initiated by the translocated NP or by mediators released from the lungs upon their interaction with deposited NP. However, neither of the initiation pathways can be excluded. Until now, classical pathology has reported substantial MP loads in secondary target organs only for long term and massive particle exposure conditions; e.g. the accumulation of tar in the lungs of smokers leading to increasing blackening of the lungs, or the accumulation of particles or fibres in the liver and other organs of the reticuloendothelial system in coal miners and asbestos workers (Auerbach et al. 1980; LeFevre et al. 1982). Thereby, particle translocation via the lymphatic system into the blood circulation was assumed. Similarly, in overload conditions particle translocation and accumulation particularly in the reticulo-endothelial organs were observed in experimental animals (dogs: Bianco et al. 1974; rodents: reviewed by Miller 2000). Animal Studies. There is evidence for the translocation of NP such as gold, silver, TiO2, polystyrene and carbon, in the size range of 5–100 nm, across the air– blood barrier from animal experiments. These studies demonstrated NP either in the blood circulation (Berry et al. 1977; Kapp et al. 2004; Geiser et al. 2005) and in secondary target organs (Oberdo¨rster et al. 2002; Kreyling et al. 2002, Takenaka et al. 2001, 2006; Semmler et al. 2004, 2007a, b), or they revealed thrombogenic effects (Nemmar et al. 2002; Silva et al. 2005; Khandoga et al. 2004). However, it still remains unclear, whether the translocated NP fractions exceeded 5% of the delivered lung dose (see also Kreyling et al. 2004, 2006b). Recently, Chen et al. (2006) reported an estimated translocated fraction of 1–2% of 50 and 200 nm polystyrene particles.

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Quantitative Assessment of NP Translocation. Quantitative NP biokinetics as described in the Methods section of this chapter allows for a rather precise determination of total and organ-specific translocated NP fractions. Such data currently exist for the following NP: – Inhaled iridium NP, 20 and 80 nm, in rats and mice (Kreyling et al. 2002; Semmler et al. 2004; Semmler-Behnke et al. 2007a) – Inhaled carbon NP, 25 nm, spiked with radio-labeled primary iridium NP in rats (Kreyling et al. 2008 submitted) – Instilled gold NP, 1.4 and 18 nm, in rats (Semmler-Behnke et al. 2007b, 2008) From the inhalation studies with iridium NP it became clear that NP accumulate not only in secondary target organs but also in soft (connective) tissue and skeletal bone including bone marrow. In these studies, 20-nm NP accumulation in all secondary target organs (liver, spleen, kidneys, heart, brain, reproductive organs) was in the range of 1–2% of the deposited dose at 24 h after administration. A similar fraction was found in the skeleton and up to 5% in the soft tissue. Hence, the total translocated NP fraction reached just about 10% (Kreyling et al. 2008 submitted). Furthermore, 20-nm iridium NP were poorly cleared from secondary target organs such that six months after a single one-hour NP inhalation exposure, the total NP fraction in the secondary target organs was still close to 1% of the initial NP deposit in the lungs, and all organs studied still contained NP (Semmler et al. 2004; Semmler-Behnke et al. 2007a). Unfortunately, there are no further data on longterm translocation of NP. There were no significant differences in NP translocation and accumulation in secondary target organs at 24 h after inhalation observed between rats and mice (Semmler-Behnke and Kreyling, personal communication 2008). Even in the fetuses of pregnant rats in their third trimester, small but detectable translocated NP fractions were registered (Semmler-Behnke et al. 2007b). NP Size Dependence. There is evidence from experimental studies that the translocation and accumulation of NP in secondary target organs depend on their size; i.e. inhaled 80-nm iridium NP were shown to translocate about one order of magnitude less than the 20-nm iridium NP, including accumulation in the skeleton and soft tissue (Kreyling et al. 2002; Kreyling et al. 2008 submitted). Additionally, significant differences in the translocation and accumulation between 1.4-nm and 18-nm gold NP have been observed, with total translocated fractions of 8% and 0.2%, respectively, at 24 h after intratracheal NP instillation (Semmler-Behnke et al. 2008). Both NP sizes were found in all secondary target organs investigated. NP Material Dependence. Consequences of different materials on NP translocation and 24-h accumulation in secondary organs can be derived from inhalation studies with 20-nm iridium and 25-nm carbon NP. There were significant, 5–10 times lower carbon than iridium NP fractions found in any of the secondary target organs (except in the liver) studied, as well as in the skeleton or soft tissue, clearly indicating a material dependence. Note that both NP types are chain agglomerates made up of either 2 to 5-nm iridium or 5 to 10-nm carbon primary particles. Caution is required when these data are compared with those of 18-nm gold NP of similar

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size: there is not only the material difference, but gold NP are spherical and have a smooth surface. In addition, the methods of NP administration to the lungs was different (inhalation versus instillation). Yet, the total difference of almost 10% of translocated iridium and about 2% carbon chain agglomerates compared to 0.2% of the spherical gold NP within 24 h is striking, when considering NP material dependence only. There are more quantitative data required to better understand biokinetics associated with different NP materials. Summary Remarks on NP Translocation Across the Air–Blood-Barrier. Besides the discussed importance of size and material, other NP characteristics such as the surface charge (zeta potential) and surface structures are very likely to influence the NP biokinetics. They determine the interactions of NP with proteins and cellular components and thereby the transport mechanisms responsible for NP translocation and accumulation in extra-pulmonary organs. However, it needs to be emphasized that according to the current knowledge, NP translocation and accumulation in extra-pulmonary organs is a minor clearance pathway for NP from the lungs compared to lung-surface macrophages mediated NP clearance towards the larynx. Yet, while the latter pathway leads to NP excretion via the gastro-intestinal tract, NP translocation into the blood circulation distributes NP in the body and permits access to e.g. the cardio-vascular system, the central-nervous system and the reticulo-endothelial, i.e. the immune system. Despite the potential toxicological consequences for the organism when NP interact with these organ systems, it is still unknown whether it is the translocated NP that cause the epidemiologically established adverse effects. Particularly, it remains to be shown whether chronic exposure leads to sufficiently high NP doses to trigger or mediate responses leading to initiation and/or progression of disease. In addition, the release of mediators into the blood circulation needs thorough investigations: these mediators may be triggered or modulated by the well-known oxidative stress and pro-inflammatory responses to NP. Yet, even the importance of the dose metric in the lungs or in extra-pulmonary organs is still debated: if NP mass is the effect-determining metric, it appears very unlikely to reach sufficiently high doses in extra-pulmonary organs by inhalation. However, if NP number and (reactive) surface are the cause-effect-determining metrics, then chronic NP exposure may well be a health hazard; particularly, in susceptible individuals such as infants, the elderly and individuals with pre-existing cardiovascular and lung diseases. Furthermore, the interaction of NP with the organism has to be studied at cellular and molecular levels, in lungs as well as in those secondary target organs which receive a sufficiently high NP dose. Microscopic analyses of organs from animal inhalation experiments may give more detailed information about possible pathways for (adverse) effects by inhaled NP. It will be important to know what cell and tissue types or what intracellular compartments NP interact with and what NP properties are responsible for these interactions. Unrestricted crossing of the cellular membranes by NP facilitates not only NP translocation into basically any organ but also provides access to any subcellular compartment. While unexpected NP access to secondary target organs at non negligible doses on a macroscopic scale as well as unexpected NP access to parenchymal and

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immune cells and to their subcellular structures like mitochondria and nuclei may result in adverse health effects, these interactions and pathways provide unforeseeable opportunities in the design of NP for diagnostic or therapeutic medical use in the new field of Nanomedicine. Acknowledgements This work was supported in part by EU FP6 PARTICLE_RISK 012912 (NEST), U.S. National Institutes of Health grant HL070542, the German Research Foundation FOR 627, the Swiss National Science Foundation grant 3200B0-105419 and the Swiss Society for Cystic Fibrosis. We acknowledge B. Kupferschmid, M. Casaulta and C. Wigge and B. Krieger for their microscopic and technical contributions to the graphics.

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Laden F, Neas LM, Dockery DW, Schwartz J (2000) Association of fine particulate matter from different sources with daily mortality in six U.S. cities. Environ Health Perspect 108:941–947 Laden F, Schwartz J, Speizer FE, Dockery DW (2006) Reduction in fine particulate air pollution and mortality: extended follow-up of the Harvard Six Cities study. Am J Respir Crit Care Med 173:667–672 LeFevre ME, Green FH, Joel DD, Laqueur W (1982) Frequency of black pigment in livers and spleens of coal workers: correlation with pulmonary pathology and occupational information. Hum Pathol 13:1121–1126 Lehnert BE, Morrow PE (1985) Association of 59Iron oxide with alveolar macrophages during alveolar clearance. Exp Lung Res 9:1–16 Lehnert BE, Valdez YE, Tietjen GL (1989) Alveolar macrophage-particle relationships during lung clearance. Am J Respir Cell Mol Biol 1:145–154 Lehnert BE, Ortiz JB, London JE, Valdez YE, Cline AF, Sebring RJ, Tietjen GL (1990) Migratory behaviors of alveolar macrophages during the alveolar clearance of light to heavy burdens of particles. Exp Lung Res 16:451–479 Lynch I, Dawson KA (2008) Protein-nanoparticle interactions. Nano Today 3:40–47 Macklin CC (1955) Pulmonary sumps, dust accumulations, alveolar fluid and lymph vessels. Acta Anat 23:1–33 Miller FJ (2000) Dosimetry of particles in laboratory animals and humans in relationship to issues surrounding lung overload and human health risk assessment: a critical review. Inhal Toxicol 12:19–57 Mills NL, Tornqvist H, Robinson SD, Gonzalez M, Darnley K, MacNee W, Boon NA, Donaldson K, Blomberg A, Sandstrom T, Newby DE (2005) Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis. Circulation 112:3930–3936 Mills NL, Amin N, Robinson SD, Anand A, Davies J, Patel D, de la Fuente JM, Cassee FR, Boon NA, MacNee W, Millar AM, Donaldson K, Newby DE (2006) Do inhaled carbon nanoparticles translocate directly into the circulation in humans? Am J Respir Crit Care Med 173:426–431 Mo¨ller W, Felten K, Sommerer K, Scheuch G, Meyer G, Meyer P, Haussinger K, Kreyling WG (2008a) Deposition, retention, and translocation of ultrafine particles from the central airways and lung periphery. Am J Resp Crit Care Med 177:426–432 Mo¨ller W, KreylingWG, Schmid O, Semmler-Behnke M, Schulz H (2008b) Deposition, retention and clearance, and translocation of inhaled fine and nano-particles in the respiratory tract. In Gehr P, Blank F, Mu¨hlfeld C, Rothen-Rutishauser B (eds) Particle-Lung Interactions Second Edition, Series: Lung Biology in Health and Disease Volume 241 Nemmar A, Hoylaerts MF, Hoet PH, Dinsdale D, Smith T, Xu, H, Vermylen J, Nemery B (2002) Ultrafine particles affect experimental thrombosis in an in vivo hamster model. Am J Respir Crit Care Med 166:998–1004 Oberdo¨rster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, Kreyling WG, Cox C (2002) Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats. J Toxicol Environ Health 65:1531–1543 Oberdo¨rster G, Oberdo¨rster E, Oberdo¨rster J (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113:823–839 Peters A, Pope CA III (2002) Cardiopulmonary mortality and air pollution. Lancet 360:1184–1185 Pope CA III (2004) Air pollution and health – good news and bad. N Engl J Med 351:1132–1134 Rytting E, Nguyen J, Wang X, Kissel T (2008) Biodegradable polymeric nanocarriers for pulmonary drug delivery. Expert Opin Drug Deliv 5:629–639 Scha¨ffler A, Menche N (1999) Mensch Ko¨rper Krankheit. Urban and Fischer, Munich, Germany Schulz H, Harder V, Ibald-Mulli A, Khandoga A, Koenig W, Krombach F, Radykewicz R, Stampfl A, Thorand B, Peters A (2005) Cardiovascular effects of fine and ultrafine particles. J Aerosol Med 18:1–24 Schu¨rch S, Gehr P, Im Hof V, Geiser M, Green F (1990) Surfactant displaces particles toward the epithelium in airways and alveoli. Respir Physiol 80:17–32

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Semmler M, Seitz J, Erbe F, Mayer P, Heyder J, Oberdo¨rster G, Kreyling WG (2004) Long-term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organs. Inhal Toxicol Jun 16:453–459 Semmler-Behnke M, Takenaka S, Fertsch S, Wenk A, Seitz J, Mayer P, Oberdo¨rster G, Kreyling WG (2007a) Efficient elimination of inhaled nanoparticles from the alveolar region: Evidence for interstitial uptake and subsequent reentrainment onto airways epithelia. Environ Health Perspect 115:728–733 Semmler-Behnke M, Fertsch S, Schmid O, Wenk A, Kreyling WG (2007) Uptake of 1.4 mm versus 18mm Gold particles by secondary target organs is size dependent in control and pregnants rats after intertracheal or intravenoiz application. In: Euro nanoforum: nanotechnology in industrial applications, pp 102–104, http://www.euronanoforum2007.de/download/Proceedings%20ENF2007.pdf. Semmler-Behnke M, Bolle I, Moeller W,.Schulz H, Takenaka S, Tsuda A, Kreyling WG (2007) Ultrafine particle deposition differs consistently between the developing and adult rat lung. European Aerosol Conference 2007, Salzburg, Abstract T08A013 Semmler-Behnke M, Kreyling WG, Lipka J, Fertsch S, Wenk A, Takenaka S, Schmid G, Brandau W (2008) Biodistribution of 1.4 nm and 18 nm Gold particles in rats. Small 4(12):2108–2111 Silva VM, Corson N, Elder A, Oberdo¨rster G (2005) The rat ear vein model for investigating in vivo thrombogenicity of ultrafine articles (UFP). Toxicol Sci 85:983–989 Sims DE, Westfall JA, Kiorpes AL, Horne MM (1991) Preservation of tracheal mucus by nonaqueous fixative. Biotech Histochem 66:173–180 Sorokin SP, Brain JD (1975) Pathways of clearance in mouse lungs exposed to iron oxide aerosols. Anat Rec 181:581–626 Sterio DC (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc 134:127–136 Takenaka S, Karg E, Roth C, Schulz H, Ziesenis A, Heinzmann U, Schramel P, Heyder J (2001) Pulmonary and systemic distribution of inhaled ultrafine silver particles in rats. Environ Health Perspect 109 (Suppl 4):547–551 Takenaka S, Karg E, Kreyling WG, Lentner B, Moller W, Behnke-Semmler M, Jennen L, Walch A, Michalke B, Schramel P, Heyder J, Schulz H (2006) Distribution pattern of inhaled ultrafine gold particles in the rat lung. Inhal Toxicol 18:733–740 Thurston RJ, Hess RA, Kilburn KH, McKenzie WN (1976) Ultrastructure of lungs fixed in inflation using a new osmium-fluorocarbon technique. J Ultrastruct Res 56:39–47 Weibel ER (1984) Morphometric and stereological methods in respiratory physiology including fixation techniques. In: Otis AB (ed) Techniques in the life sciences. Elsevier Scientific, Ireland, pp 1–35 West MJ, Østergaard K, Andreassen OA, Finsen B (1996) Estimation of the number of somatostatin neurons in the striatum: an in situ hybridization study using the optical fractionator method. J Comp Neurol 370:11–22 Wiebert P, Sanchez-Crespo A, Falk R, Philipson K, Lundin A, Larsson S, Mo¨ller W, Kreyling WG, Svartengren M (2006a) No significant translocation of inhaled 35-nm carbon particles to the circulation in humans. Inhal Toxicol 18:741–747 Wiebert P, Sanchez-Crespo A, Seitz J, Falk R, Philipson K, Kreyling WG, Mo¨ller W, Sommerer K, Larsson S, Svartengren M (2006b) Negligible clearance of ultrafine particles retained in healthy and affected human lungs. Eur Respir J 28:286–290 Yoneda K (1976) Mucous blanket of rat bronchus. Am Rev Respir Dis 114:837–842

Chapter 9

Particles of Biomedical Relevance and Their Interactions: A Classical and Quantum Mechanistic Approach to a Theoretical Description Ewa Broclawik and Liudmila Uvarova

9.1

Introduction

The need for theoretical modeling in biological and medicinal chemistry stems from many reasons. Experimental results are often difficult to interpret and the assignment of the observed signals to certain species is arbitrary in many cases. Theoretical models give direct insight into the electronic, energetic and geometric properties of the chemical systems (molecules and their aggregates), and processes they undergo, These then allow relating the obtained quantities with the measured ones. Direct investigation of materials of biological importance by modern experimental techniques is usually hardly possible, at most time- and funds consuming processes. Computational methods allow investigating a variety of hypothetical structures, where the most promising ones may be selected for further study. In addition, unstable or extremely short-living species, crucial for overall processes but being hardly proven by experiments, may be still investigated by theoretical tools. However, it should be noted that theoretical studies are performed not on actual biological systems, but rather on relevant model systems. Thus the reliability of theoretical results may strongly depend on the quality of the model, that mimics the biological system. When it comes to implementations of the theoretical and computational models, one must, for practical reasons, employ certain level of uncertainty. Therefore, modeling results must always be verified by experiments. As a rule of thumb, computational methods can be done either very accurate but feasible only for small molecules, or fast but much less accurate. The latter one is often the only possibility for the computational treatment of large systems, e.g. for tissues, enzymes or heterogeneous catalysts. It should be noted, however, that in E. Broclawik (*) Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Krakow, Poland e-mail: [email protected] L. Uvarova Department of Applied Mathematics, Moscow State University of Technology “STANKIN”, Moscow, Russia

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_9, # Springer ScienceþBusiness Media B.V. 2010

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many cases the current theoretical methods are able to predict chemical and/or biological properties with experimental accuracy, even for quite large and complex systems. In conclusion, experimental and theoretical methods should be treated as complementary approaches to gain a feasible overall view of the studied problems. Proper links between the two may be provided by mathematical models of mesoscopic phenomena, based either on atomistic scale modeling or phenomenological approaches for these physical processes. Interesting perspectives of this emerging field of quantum medicines is given in a monograph edited by Carloni and Alber 2003.

9.2 9.2.1

Theoretical Approaches Basics of Quantum Chemistry

Experiments from the beginning of the twentieth century showed that atoms and subatomic particles cannot be described by classical physics, but should obey the laws of quantum mechanics (QM) (Atkins and Friedman 2005; Szabo and Ostlund 1996). Quantum chemistry (QC) in that regard, evolved as the application of the quantum mechanics for chemical systems, namely atoms, molecules or their aggregates. The basic concept of the quantum theory is a wavefunction (WF), being in general the complex function of the coordinates, spin and time. For systems without the time dependent interactions WFs may be defined in a 4N-dimensional spinconfiguration space (with accuracy to the time dependent phase shift) C ¼ Cðq1 ; q2 ; . . . qN Þ;

q ¼ x; y; z; s

(9.1)

A WF itself has no physical meaning, but the square of the WF determines linearly the probability density of finding the species under study, i. e. giving the probability of finding the system with coordinates in the range between rN and rNþdrN and with spin value sN. It is further stressed that a WF must obey the Schro¨dinger equation (SE), which for the time independent and nonrelativistic case has the form: H^ C ¼ EC

(9.2)

where H^ is the total energy operator (Hamiltonian) and E is total energy of the system. Despite the apparent simplicity, SE can only be solved exactly for simple model problems. One of the approximations commonly used in QC to make solving SE possible, comes from the fact that nuclei are much heavier than electrons, thus move much slower and can be separated from electrons. In this case, the Hamiltonian as defined in Eq. 9.2 can be simplified to the electronic Hamiltonian H^e , which depends only on the electronic coordinates, with the nuclear coordinates becoming fixed parameters. In the BO approximation, the set of electron energies for the various nuclei positions constitutes the potential energy surface (PES), hence, providing the relevant data for molecular geometry, vibrational properties and reaction pathways.

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The strict solutions of SE are known for hydrogen atoms and hydrogen-like ions, containing one nucleus and one electron. One-electron functions, being the solutions, are called spinorbitals. The simplest approximation of the WF for the N-electron systems could be assumed as the product of the N one-electron WF (spinorbitals) keeping in mind that electrons are the fermions (they have spin equal to one half). The Pauli principle for fermions says that WFs of N fermions must be antisymmetrical with respect to the odd permutation of fermions. The simplest function fulfilling this requirement is the determinant function (determinant is the ‘antisymmetrised product’) The variational method of searching for the best spinorbitals to construct such a wave function, i.e. by minimizing the energy, is known as the Hartree–Fock (HF) method (Szabo and Ostlund 1996). HF is the mean field approximation, because within this approach electrons do not interact among each other directly, instead, each electron interacts with the averaged electric field originating from the remaining (N 1) electrons and from the Coulomb field of the nuclei. The HF procedure of energy minimization leads to the set of one-electron equations: ^ i ðri Þ ¼ Ei ’i ðri Þ h’

(9.3)

where h^ is one-electron energy operator and ’i(ri) are the orbitals, i.e. spatial parts of the spinorbitals. Equation 9.3 is still too complex to be solved even for systems of very small molecules and certainly for systems containing species such as particles. For more complex molecular systems, molecular orbitals are expanded as the linear combinations of atomic orbitals (Linear Combination of Atomic Orbitals – Molecular Orbitals): ’i ðrÞ ¼ Sk Cik wk ðrÞ

(9.4)

and the whole HF procedure is then simplified so as to find an optimal set of coefficients of the expansion. Here, HF is used as a mean field approach, which means that electrons do not interact instantaneously: they experience the averaged electric field of the other electrons. As a result, HF neglects the correlation, coming from additional repulsion between the charges of equal sign and various spins. The correlation energy is defined as the difference between the exact nonrelativistic energy and the limit of the HF energy. Although being the small fraction of the total energy, the correlation energy may bring large contribution to the relative energies, which are the object of interest in chemistry. The idea to represent the total energy as the functional relation of the particle density (observable defined in physical space), instead of abstract WF, was proposed to overcome the typical drawbacks of traditional QC and so as to avoid problems with electron correlation (Parr 1994). Such an approach has appeared to be quite successful in solid state physics, enabling a qualitatively acceptable description of many properties. However, the errors in the predicted relative energies were far from sufficient in comparison to the accuracy required in chemistry. Moreover, this density based approach had no formal justification for a long time.

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In 1964 Hohenberg and Kohn (1964), and later, in more general form, Levy (Levy 1982), showed that energy (or any other observable parameter) can be equivalently described in the terms of WF formalism and as the functional relations of the particle density (the electron density for chemical systems in the BO approximation). Hohenberg–Kohn theorems form the background of the density functional theory (DFT). The next important step in the development of DFT was the Kohn–Sham (KS) method (Kohn and Sham 1965), being now the most commonly applied variant of DFT in computational chemistry. A detailed description of the KS method may be found in many handbooks (Parr 1994; Koch and Holthausen 2001) and review articles (Baerends and Gritsenko 1997; Jones and Gunarsson 1987). In formulae, the result is a set of KS one-electron equations: KS KS KS h^ ’KS i ðri Þ ¼ ei ’i ðri Þ

where

(9.5)

rðrÞ ¼ Sk nk ’KS kðrÞ =2

Kohn–Sham equations (Eq. 9.5) have similar form as the Hartree–Fock equations, nevertheless, differences between the HF and KS approaches are essential. It must be noted that, despite the use of orbitals in Eq. 9.5, KS is the density functional method where the electron density is the direct outcome. The total energy is calculated without any assumption about the form of the exact WF of a real system (while in HF it is approximated by the one-electron approximation). The great popularity of DFT in KS formulation comes from the fact that this is the only correlated computational method with low computational cost (time of calculations). As for the accuracy, the DFT results are typically much better than HF, comparable to the very advanced time-consuming methods. DFT is usually able to give at least qualitatively correct results for strongly correlated systems, for which HF completely fails (for example in bulk metals). In the field of modeling of large systems, like in periodic solids or biological macromolecules, DFT is the only way to obtain reasonable results.

9.2.2

Molecular Mechanics and Molecular Dynamics

Facing the fact, that Schrodinger equation is solvable only for the simple model systems and finding even approximate solutions for the complex ones is difficult, the simplified theoretical method for macromolecules and extended systems has been proposed and developed parallel to QM, namely molecular mechanics (MM) (Kettering et al. 1930; Jensen 2006). Within this approach an electronic structure does not appear explicitly. Instead, the total energy of the investigated system is presented as dependent on a certain force field, being the function of nuclear positions (treated as the mass centre of the atoms). The detailed form of this scalar field is expressed by assumed classical formulas and includes a number of arbitrary

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parameters, characterizing the interatomic interactions. These parameters are fitted either to the experimental data or to the theoretical results, obtained by more accurate computational methods for model systems. The functions appearing in the described above formula for energy are called force fields (FF). FF with properly fitted parameters is supposed to predict selected chemical properties, such as the geometries of local minima on PES for defined set of investigated systems. The expectation of limited transferability of parameters is justified by certain experimental results. For example, it is known from infrared spectroscopy that vibrational modes of some group of atoms are very similar for different molecules containing given groups. For example the stretching mode of C¼C bond is very similar for different hydrocarbons. From this observation one can expect that properties of this bond can be approximately described by a single empirically fitted parameterization for a wide range of molecules containing C¼C bonds. The main advantage of MM is a very short time of calculations in comparison to the QM methods. Therefore MM may be easily applied for large systems, even those not tractable at QM level. MM formalism is not only applicable for the simple search of the local minima of energy, but it can be easily extended to the investigation of time evolution. Such an approach, called the classical molecular dynamics (MD), considers solving classical equations of motion (Newton or Lagrange) for the system of species interacting via FF. Classical MD calculations may be performed on large systems and relatively long time scales. MM calculations are often used for initial optimizations of the structures, which are to be calculated then at QM level. Very attractive are combined QM/MM methods. The limitations of classical MM/MD are, however, numerous. First of all, the transferability of MM parameters is limited, thus caution must be paid to the choice of a proper set of parameters. Due to the simplified nature of MM approach, often only qualitative agreement with experiment can be obtained. There is no simple prescription which FF should be applied for the studied system to give reasonable values. The quality of parameters strongly depends on the fitting procedure and the database of quantities used for fitting. For example FF parameters fitted for some set of experimental structures may produce accurate geometrical properties of studied systems, but give poor relative energies or vibrational frequencies. Traditional MM/ MD is not able to describe bond breaking or formation; therefore it cannot be applied for the modeling of chemical reactions, like search for the transition states. The direct combination of DFT with classical molecular dynamics, called Car– Parinello molecular dynamics (CP-MD, Car and Parrinello 1985), widely extended the scope of applications towards medicinal chemistry (Carloni et al. 2002).

9.2.3

Phenomenological Approaches

In spite of the rapid development of methods focused on investigating the system of interest on an atomistic scale, vast variety aspects of reality, especially in life and environmental sciences, remain out of reach at that level and require phenomenological

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treatment. The rapid development of the modern applied science and the technical and technological components of the civilization lead in particular to the increase of atmosphere pollution. Therefore a lot of scientific studies were devoted to the problems of an interaction of nanoparticles, particles with magnetic properties, and drug drops with a human organism, or aerosol particles with the lung (Korn and Krause 2007; Pauluhn 2005; Frampton et al. 2006). Nanoparticles effectively spread in all parts of the lung tract at an inhalation that can lead to complex consequences. For the description of e.g. the transport of the clusters in the lung systems it is necessary to investigate the interaction of the clusters with the lung wall. This may be accomplished by the use of phenomenological models in addition to atomistic investigation by QM or MD methods. One example is the Hydrodynamics Transport Model (Kleinstreuer and Zhang 2007; Shi and Kleinstreuer 2007). The hydrodynamics transport model relates derivatives of the velocity components, ui on the density r, the pressure p, the coefficient of viscosity n, and the roughness-viscosity nR in a manner similar to the Eddy-viscosity concept in turbulent flow models. By introducing nR, the effect of the wall roughness on the laminar flow profile was included in the airflow simulations. For the correct calculations it is also necessary to add the heat transport equation which must be considered for agglomerates. One of possible methods is based on the set theory (Uvarova 2007). It is interesting to find the particle dimensions for which the macroscopic models may be used. As an example, the calculation experiments carried out for nicotine drops may be invoked. The internal energy was determined for different number of molecules constituting a drop, beginning with two molecules. The potential energy calculation was carried out using binary interactions of a molecule with any other molecule. The potential of the interaction was assumed in the form of the Lenard–Jones modification taking chemical interactions into account. For the most effective calculations, the Metropolis’s scheme was used. It was found that by adding subsequently molecules, the energy in one drop stabilizes at approximately 110 molecules. Similar calculations for a particle of other substances (water, metals and other materials) were carried out as well (Vnukova 2008). The experiments here showed that the energy stabilizes at 40–130 molecules by adding one molecule to a drop. It allowed determining the particle dimensions for which using of the continuum models then should be acceptable. The transport processes of small particles with sizes close to the molecule dimensions in alveolus, may be studied in this way, also by the molecular dynamics (MD) method. The force was determined with the help of the surface potential given in the form of a modified Lennard–Jones potential. The characteristics for membranes were taken from the lipids parameters (Zieder 2008). The calculation experiments for water clusters were carried out by Vnukova 2008 (Vnukova, 2008; Babarin and Uvarova 1997). The water clusters moved and precipitated on the surface of the alveolus. The geometry for the alveolus was simulated by the cone with two different diameters, i.e. 12 and a 10 nm, respectively (Patton 1996). The initial quantities for the co-ordinates and particle velocity came from a random numbers generator. In a given case (for water clusters) the density of the clusters at n ¼ 3, 4, . . . , 10 exceeded the density of water in its normal state. As a result,

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clusters with increased density precipitated on the surface more quickly then clusters of the appropriate diameter with the water density. The cluster structure and orientation differed too. Calculation experiment results agreed with the experimental data on the precipitating particles (Nel et al. 1996) where a significant percentage of the precipitation of the nanoparticles was shown.

9.3

Selected Theoretical Case-Studies for Systems of Biological Importance

Theoretical enzyme chemistry took a major step forward about 10 years ago when it became possible to attack mechanistic problems involving enzyme active sites containing transition metal centers (Siegbahn and Borowski 2006). This is a very important part of biochemistry containing enzymes such as photosystem II of photosynthesis, cytochrome c oxidase in the respiratory chain, ribonucleotide reductase in DNA synthesis, and methane monooxygenase for converting alkanes to alcohols. Quantum chemical methods have now been used to examine these enzymes and many others, and appropriate methods and models have eventually emerged. In the present section, examples will be given where theory has provided significant new contributions, with the emphasis on problems where theory has been shown to be particularly useful. In this context, comparison of different systems yielding trends is a major point. Another advantage with theory is that the proposed mechanisms sometimes can be shown relatively easy to be unlikely and that new suggestions need to be made. To prove that a certain mechanism is the preferred one is quite a task, both experimentally and theoretically, and will usually require a long combined effort. In this context, theory has risen in importance during the past decade to become a part that needs to be considered before a mechanism can be fully accepted.

9.3.1

Iron-Containing Enzymes

Since the advent of a robust computational techniques based on density functional theory (DFT) quantum mechanical modeling has provided extensive information on catalytic cycle of cytochromes. This included transformation of the initial, inactive form of the enzyme into the active oxyferryl form, model ligand binding and subsequent metabolism. Recently a few comprehensive reviews have been published on the subject (Sono et al. 2006, Denisov et al. 2005; Shaik et al. 2005). The cytochrome P450 (CYP) enzymes are membrane bound proteins that catalyze primary oxidations of endobiotics and xenobiotics. CYP3A4 is a major CYP450 isoform and contributes extensively to human drug metabolism due to its high level of expression in the liver and broad capacity to oxidize structurally diverse

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substrates. It metabolizes 50% of the drugs used in human being. Hydroxylation of the C–H bond of the drug is one of the most important metabolism steps that can influence their bioavailability by transforming them either to active form or to toxic compounds. A detailed understanding of this metabolism step and prediction of metabolites is thus a major challenge being crucial to screen drugs in an early stage of a leading development. Since experimental investigation of the catalytically active species in the metabolism requires already the presence of a substrate to initiate the reaction cycle, computational methods are very important to accomplish this task. Such techniques involve docking in the active site, pharmacophore modeling, quantitative structure activity relationship (QSAR) and/or quantum chemical/molecular mechanical (QM/MM) studies. Apart from practical significance, the recognition of the mechanism making enzymatic oxidation a highly potential tool capable of activating the inert C–H bond in hydrocarbons, and is in itself of high scientific interest, and, thus, deserves strong attention. One example discussed here (Shaikh et al. 2006, 2007) is focused on the compound (S)-N-[1-(3-morpholin-4-yl phenyl)ethyl]-3-phenylacrylamide, the novel KCNQ2 potassium channel opener that was found to have significant oral activity in a cortical spreading depression model of migraine. The substrate has excellent oral bioavailability in dogs and rats; however, CYP3A4 MDI studies indicated that it forms reactive intermediate after metabolism. On the other hand, its difluoro analogue, (S)-N-[1-(4-fluoro-3-morpholin-4-yl phenyl)ethyl]3-(4-fluorophenyl)acrylamide was found to be orally bioavailable KCNQ2 opener free of CYP3A4 MDI. The existence of a pair of closely related compounds with different MDI properties provides promising material and good guidance for examining details of selected steps in their metabolism; it also confirms the position of primary oxidation of the phenyl ring. Another example is clavaminic acid synthase (CAS) (Borowski et al. 2007), a remarkable nonheme iron dioxygenase that catalyzes three separate oxidative reactions in the biosynthesis of clavulanic acid, a clinically used inhibitor of serine b-lactamases. Notably, all three oxidative reactions, i.e. hydroxylation, cyclization, and desaturation, take place at the single active site of CAS, which in the native state hosts a single high-spin ferrous ion coordinated by the 2-histidine-1-carboxylate binding motif and three water molecules. CAS belongs to a large superfamily of 2-oxoglutarate (2-OG) dependent oxygenases, a group of mononuclear non-heme iron enzymes that couples the oxidative decarboxylation of the 2-oxoglutarate cosubstrate to the 2-electron oxidation of a primary organic substrate. The results of the classical MD simulations suggest that the active site region of the CAS-Fe (IV)¼O-succinate-PCA has a well-defined structure consistent with the concept of “negative catalysis” which proposes that enzymes with highly reactive intermediates achieve product selectivity via ablation of unproductive/undesirable pathways. Interestingly, in this structure the hydroxyl group of PCA lies substantially closer to the oxoferryl group than the C40 -bound hydrogen of PCA. Thus, this structure, together with a repulsive PMF calculated for an approach of 40 (S) hydrogen of PCA toward the oxo group, suggests that the alcohol group of PCA is oxidized first by the

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reactive oxoferryl species. The DFT investigations indeed show that oxidation of an alcohol group is markedly easier than activation of the C40 –H bond. Moreover, based on the DFT results, a novel mechanism is proposed for the cyclization reaction by CAS. This new mechanistic hypothesis involves O-radical fragmentation, ylide formation, and 1,3-dipolar cycloaddition with aldehyde as dipolarophile. Importantly, this new mechanism is consistent with the isotope kinetics data and predicts formation of a relatively long-lived intermediate, whose accumulation might be experimentally verifiable.

9.3.2

Quantitative Structure–Activity Relationship

In the next example, Quantitative Structure–Activity Relationship (QSAR) of a series of novel phenanthrene-based tylophorine derivatives with anticancer activity has been studied (Liao et al. 2008) by using the Density Functional Theory (DFT), Molecular Mechanics (MM), and statistical methods. The established model shows not only significant statistical quality, but also predictive ability. It was found that the anticancer activity expressed as pIC50, which is defined as the negative value of the logarithm of necessary molar concentration of this series of compounds to cause 50% growth inhibition against the human A549 lung cancer cell line, closely relates with the energy of the Highest Occupied Molecular Orbital, the net charge of the terminal H atom of substituent R2 (QHR2), the hydrophobic coefficient of substituent R2 (log PR2 ), and the net charges of the first atom of substituent R1 (QFR1 ). The same model was further applied to predict the pIC50 for six recently reported congeneric compounds as external test set, and the predicted pIC50 values are close to the experimental ones, and thus it further confirms that this QSAR model has high predictive ability. The theoretical results can offer some useful references for understanding the action mechanism and designing new compounds with anticancer activity. Based on this QSAR equation, ten new compounds with higher anticancer activity have been theoretically designed and are expecting experimental confirmation, hopefully soon. Antagonists of the 5-HT2A receptor studied by Borowski et al. (2000), are used to treat many psychiatric disorders. The work focuses on a group of 27 antagonists possessing varying affinities toward the receptor. These are 26 title compounds and clozapine as a reference antagonist. The active conformers of the conformationally flexible ligands were proposed by using the active rigid analogue approach and performing similarity calculations. The calculations involved genetic neural network (GNN) computations deriving QSAR from similarity matrices with crossvalidated correlation coefficients exceeding 0.92. The performance of neural networks with variety of architectures was studied. As the computations were performed for cations and neutral molecules separately, the relevance of the ligand charging is discussed.

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Miscellaneous Examples of Atomistic Simulation for Biosystems

The problem of designing new potent and selective muscarinic agonists, which could be utilized in treatment of Alzheimer disease, is among the top problems in medicinal chemistry nowadays. The most promising way towards potential and selective drugs is driven by understanding the receptor–ligand interactions. The work by Broclawik and Borowski (2000), focuses on electronic and conformational structure of the bicyclic analogues of arecoline and sulfoarecoline – muscarinic receptor agonists structurally related to 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) and its S-methylsulfonium derivative (DHTO). Conformational freedom of six-member rings containing sulphur and nitrogen has been investigated by means of the semiempirical AM1 method. Interaction between ‘cationic heads’ of two representative compounds and a carboxyl group of Asp in the muscarinic receptor has been modeled using the DFT method. The electrostatic potential (ESP) around the studied complexes and ligands with an extra electron (simulation of complex formation) was analyzed. The position and depth of the ESP minima in a series of studied ligands correlated well with their activity as muscarinic agonists. On the basis of these results the mechanism of the ligand–binding site interaction was hypothesized. The calculations allowed also for the comparison of bicyclic analogues of arecoline with an already existing model for muscarinic pharmacophore and to rationalize the model parameters. In the next example (Watanabe et al. 2007), the conformational changes of p47phox–p22phox complexes of wild-type and three mutants, which have been detected in CGD patients, have been analyzed using molecular dynamics (MD) simulations. The phagocyte NADPH oxidase complex plays a crucial role in the host defense against microbial infection through the production of superoxides. The chronic granulomatous disease (CGD) is an inherited immune deficiency caused by the absence of certain components of the NADPH oxidase. Key to the activation of the NADPH oxidase is the cytoplasmic subunit p47phox, which includes the tandem SH3 domains (N-SH3 and C-SH3). In active phagocytes, p47phox forms a stable complex with the cytoplasmic region of the membrane subunit p22phox that forms a left-handed polyproline type-II (PPII) helix conformation. It was found that in the wild-type, two basal planes of PPII prism in cytoplasmic region of p22phox interacted with N-SH3 and C-SH3. In contrast, in the modeled mutants, the residue at the ape of PPII helix, which interacts simultaneously with both of the tandem SH3 domains in the wild-type, moved toward C-SH3. Furthermore, interaction energies of the cytoplasmic region of p22phox with C-SH3 tended to decrease in these mutants. All these findings allowed concluding that interactions between N-SH3 of p47phox and PPII helix, which is formed by cytoplasmic region of p22phox, may play a significant role in the activation of the NADPH oxidase. The last example (Snyder and Madura 2008) deals with silica dust particles known to promote pulmonary diseases. The workers with exposures to certain respirable dusts are at high risk of developing diseases such as silicosis or coal

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workers pneumoconiosis. Fine respirable-sized crystalline silicon dioxide mineral dusts (quartz or other polymorphs) are well documented etiological agents for pulmonary fibrosis by epidemiological studies of human occupational exposures and by animal model inhalation or installation studies. Moreover, there is sufficient evidence that crystalline silica exposures can increase the risk of lung cancer. The precise mechanism of silica cytotoxicity at the molecular level is not completely known. Upon deposition in the lung, the respirable dust surfaces may be conditioned by interaction with biological fluids and materials such as surfactant components of the pulmonary bronchiole-alveolar surface. Hence, adsorption processes on silica dusts play an important role in understanding the pathogenic origin of pneumoconiosis. Crystalline silica exposures cause silicosis, while silica in the form of aluminum silicates (clays, kaolin) does not. It was proposed that quartz and kaolin have a comparable membranolytic potential on a specific surface area basis and a comparable cytotoxic potential for lavaged pulmonary macrophages. To develop some insight into this phenomenon, the interaction between a phospholipid and silica particles was examined by performing ab initio DFT calculations on clusters constructed with representative parts of the silicate surface and the phospholipid head group. Fully optimized geometries of the complexes were used to determine binding energies, –OH vibrational frequency shifts, and NMR chemical shieldings. Results indicated that the interaction of an unprotonated aluminol group (Al–OH) with the phospholipid head group is stronger compared to that with a silanol group (Si–OH). The presence of the choline moiety increased the magnitude of the –OH vibrational frequency shifts, and the shifts were significantly larger in complexes with protonated aluminol groups relative to silanol complexes. Calculated 31 P NMR chemical shieldings were increased slightly by the presence of the choline unit and were also larger in aluminol complexes relative to those in silanol complexes. These results shed some light on differences in toxicity observed for silica versus aluminosilicate surfaces.

9.4

Conclusions

Quantum chemical calculations emerged nowadays as a key element in biological research and computational medicinal chemistry. They can aid the formulation of hypotheses that provide connecting links between experimentally determined structures and biological functions. The calculations can be used to understand e.g. enzyme mechanisms, hydrogen bonding, ligand binding and other fundamental processes both in normal and aberrant biological contexts. The fundamental assumption of the rational developing new therapeutics is that beneficial effects of drugs come from molecular recognition and binding of ligands to the active sites of specific targets, such as enzymes, receptors, and nucleic acids. The effect of binding can be either promotion or inhibition of signal transduction, enzymatic activity, or

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molecular transport. The design of small molecules able to affect the biological function of the latter is one of the major aims in the future of medicinal chemistry. Obviously, the choice of the computational strategy depends on the ability of the method (i.e. the types of atoms and/or molecules, and the type of property that can be treated satisfactorily) and the size of the system to be investigated. In biochemical applications the method of choice – if we are interested in the dynamics and effects of temperature on an entire protein with, say, 10,000 atoms – is classical molecular dynamics (MD) simulation. The key problem then is to choose a relevant force field. On the other hand, if we are interested in electronic and/or spectroscopic properties or explicit reaction with bond breaking and formation in an enzymatic active site, we must introduce quantum chemical methods where electrons are treated explicitly. Among the various quantum chemical approaches available, the density functional theory (DFT) has become a key method over the past decade, with applications ranging from interstellar space, to the atmosphere, the biosphere and the solid state. In this chapter we presented an introduction to the theory and exemplified the wide range of problems that can be addressed, with some illustrative results taken from our work and other recent works in the field of drug design (quantum chemistry) and particle transport in physiological tracts.

References Atkins PW, Friedman RS (2005) Molecular Quantum Mechanics, 4th edn. Oxford University Press, Oxford Babarin SS, Uvarova LA (2007) Mathematical modeling of non-equilibrium processes in nano volume the happening as the Casimir’s force acting. Dynamical heterogeneous sys, Moscow 29 (1), 11:60–69 (in Russian). Borowski T, de Marothy S, Broclawik E, Schofield C, Siegbahn PEM (2007) Mechanism for cyclization reaction by clavaminic acid synthase. Insights from modeling studies. Biochem-US 46:3682–3691 Borowski T, Krol M, Brocławik E, Baranowski TC, Strekowski L, Mokrosz MJ (2000) Application of similarity matrices and genetic neural networks in quantitative structure–activity relationships of 2- or 4-(4-Methylpiperazino)pyrimidines: 5-HT2A receptor antagonists. J Med Chem 43:1901–1909 Baerends EJ, Gritsenko OV (1997) A quantum chemical view of density functional theory. J Phys Chem A 101:5383–5403 Broclawik E, Borowski T (2000) Characteristics of the ligand–binding site interaction for a series of arecoline-derived muscarinic agonists: a quantum chemical study. Comp Chem 24:411–420 Car R, Parrinello M (1985) Unified approach for molecular dynamics and density-functional theory. Phys Rev Lett 55:2471–2474 Carloni P, Rothlisberger U, Parrinello M (2002) The role and perspective of ab initio molecular dynamics in the study of biological systems. Acc Chem Res 35:455–464 Carloni P, Alber F (eds) (2003) Quantum medicinal chemistry: methods and principles in medicinal chemistry, vol 17. Wiley-VCH, Weinheim

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Denisov IG, Makris TM, Sligar SG, Schlichting I (2005) Structure and chemistry of cytochrome P450. Chem Rev 105:2253–2278 Frampton MW, Stewart JC, Oberdo¨rster G, Morrow PG, Chalupa D, Pietropaoli AP, Frasier LM, Speers DM, Cox C, Huang L, Utell MJ (2006) Inhalation of ultrafine particles alters blood leukocyte expression of adhesion molecules in humans. Environ Health Perspect 114(1):51–58 Hohenberg P, Kohn W (1964) Inhomogeneous electron gas. Phys Rev 136:B864 Jensen F (2006) Introduction to computational chemistry. Wiley-VCH, Weinheim Jones RO, Gunarsson O (1989) The density functional formalism, its applications and prospects. Rev Modern Phys 61:681–746 Kettering CF, Shutts LW, Andrews DH (1930) A representation of the dynamic properties of molecules by mechanical models. Phys Rev 36:531–543 Koch W, Holthausen MC (2001) A chemist guide to density functional theory. Wiley-VCH, Weinheim Kohn W, Sham LJ (1965) Self-consistent equations including exchange and correlation effects. Phys Rev 140:A1133 Korn K, Krause E (2007) Cell-based high content screening of small-molecule libraries. Curr Opin Chem Biol 11:503–511 Levy M (1982) Electron densities in search of Hamiltonians. Phys Rev A 26:1200–1208 Li Z, Kleinstreuer C, Zhang Z (2007) Particle deposition in the human tracheobronchial airways due to transient inspiratory flow patterns. J Aerosol Sci 38:625–644 Si-Yan L, Jin-Can C, Qian L, Shen Y, Kang-Cheng Z (2008) QSAR studies and molecular design of phenanthrene-based tylophorine derivatives with anticancer activity. QSAR Comb Sci 27:280–288 Nel A, Xia T, Madler L, Li N (2006) Toxic potential of materials at the nanolevel. Science 311:622–627 Parr RG, Yang W (1994) Density-functional theory of atoms and molecules. Oxford University Press, Oxford Patton JS (1996) Mechanisms of macromolecule absorption by the lungs. Adv Drug Deliv Rev 19:3–36 Pauluhn J (2005) Overview of inhalation exposure techniques: strengths and weaknesses. Exp Toxicol Pathol 57:111–128 Shi H, Kleinstreuer C, Zhang Z (2007) Modeling of inertial particle transport and deposition in human nasal cavities with wall roughness. J Aerosol Sci 38:398–419 Siegbahn Per EM, Borowski T (2006) Modeling enzymatic reactions involving transition metals. Acc Chem Res 39:729–738 Shaik S, Kumar D, de Visser SP, Altun A, Thiel W (2005) Theoretical perspective on the structure and mechanism of cytochrome P450 enzymes. Chem Rev 105:2279–2328 Shaikh AR, Broclawik E, Tsuboi H, Koyama M, Endou A, Takaba H, Kubo M, Del Carpio CA, Miyamoto A (2007) Oxidation mechanism for the metabolism of (S)-N-[1-(3-morpholin-4ylphenyl)ethyl]-3-phenylacrylamide on oxyferryl active site in CYP3A4 enzyme: DFT modeling. J Mol Model 13:851–860 Shaikh AR, Broclawik E, Ismael M, Tsuboi H, Koyama M, Endou A, Takaba H, Kubo M, Del Carpio CA, Miyamoto A (2006) Hyperconjugation with lone pair of morpholine nitrogen stabilizes transition state for phenyl hydroxylation in CYP3A4 metabolism of (S)-N-[1-(3morpholin-4-yl phenyl) ethyl]-3-phenylacrylamide. Chem Phys Lett 419:523–527 Snyder JA, Madura JD (2008) Interaction of the phospholipid head group with representative quartz and aluminosilicate structures: an ab initio study. J Phys Chem B 112:7095–7103 Sono M, Roach MP, Coulter ED, Dawson JH (1996) Heme-containing oxygenases. Chem Rev 96:2841–2888 Szabo A, Ostlund NS (1996) Modern quantum chemistry: introduction to advanced electronic structure theory, Dover Publications Uvarova l A (2007) Mathematical modeling of heat transport in aerosol systems with particles of complex geometry. In: European Aerosol Conference. Salzburg, Abstract T12A015

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Vnukova KV (2008) Mathematical model of physical clusters with using density functional theory. In: Fundamental physical and mathematical problems and modeling for technical and technological systems. Moscow, Yanus –K 11, 51–54 (in Russian) Watanabe Y, Tsuboi H, Koyama M, Kubo M, Del Carpio CA, Broclawik E, Ichiishi E, Kohno M, Miyamoto A (2006) Molecular dynamics study on the ligand recognition by tandem SH3 domains of p47phox, regulating NADPH oxidase activity. Comput Biol Chem 30:303–312 Zieder A (2008) Thermodynamic studies and binding mechanisms of cell-penetrating peptides with lipids and glycosaminoglycans. Adv Drug Deliv Rev 60:580–597

Chapter 10

Health Effects of Nanoparticles (Inhalation) from Medical Point of View/Type of Diseases Robert Baughman and Michal Pirozynski

10.1

Introduction

Our understanding of the functioning of the human body at the molecular and nanometer scale has improved tremendously, our diagnostic and therapeutic options for the effective treatment of severe and chronic diseases have increased only slowly over the past. Diseases like cancer, interstitial lung diseases, airway disorders, diabetes, lung and cardiovascular problems, inflammatory and infectious diseases, and neurological disorders are serious challenges to be dealt with. Applied nanotechnology to medical problems – nanomedicine – can offer new concepts. Understanding the role of nanoparticles in pathogenesis of diseases, but also their use in therapy may be the future of medicine. The respiratory system, skin and intestinal tract are always in direct contact with the environment. These organs are the first entry ports for all particles. Inhalation is the most significant route of exposure for airborne particles including the smallest – the nanoparticles. The lung consists of two functional compartments – the conducting zone made up of airways (trachea, bronchi, and bronchioles) and respiratory zone – alveoli (gas exchange units). The conducting zone consists of the first 16 generations of airways (trachea, main, lobar, segmental, subsegmental bronchi, subdividing into smaller bronchi and finally bronchioles). The respiratory zone consists of all structures that participate in gas exchange beginning with respiratory bronchioles and ending with the alveoli. The human lung is made of approximately 2300 km of airways and 500 million alveoli (Stone et al. 1992). R. Baughman (*) University of Cincinnati Medical Center, 1001 HH Eden Avenue and Albert Sabin Way, P.O. Box 670565, Cincinnati, OH 45267-0001, USA e-mail: [email protected] M. Pirozynski Department of Anesthesiology and Intensive Therapy, CMKP, 241 Czerniakowska Street, 00-416, Warsaw, Poland

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_10, # Springer ScienceþBusiness Media B.V. 2010

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The effect of small particles can be beneficial to humans but more often it is harmful. Air pollution is a cocktail of different components, gaseous and particulate. The particulate component of air pollutant is measured as particle mass (PM) including PM10 and PM2,5. Particles deposit in different regions of the respiratory system depending on their aerodynamic diameter (Fig. 10.1). Levels of PM in any area (urban, rural) vary temporally (a fluctuating level around a mean) and spatially (depending on level of traffic or local industrial sources). Most health effects of PM occur at the level seen in modern cities. The most common adverse events are shown in (Table 10.1). Toxicological studies have helped to understand the mechanism of the adverse effect on the respiratory and cardiovascular systems. The complexicity and variability of ultrafine particles (UFPs) present in the air have a heterogeneous effect on the respiratory system. Particles such as sea salt, ammonium nitrates and sulfates, road dust, and crustal dust exhibit low potency in causing inflammation in the lungs. The most harmful are primary combustionderived nanoparticles (PCDNs), derived predominantly from automobiles, especially diesel, known to cause pulmonary inflammation in humans and animals.

Fig. 10.1 Deposition curves. The relationship between particle size and deposition in different anatomical structures of the respiratory system Table 10.1 Human adverse health effects of increased ultrafine particle levels

Increased mortality from cardiovascular and respiratory causes Increased admission to hospitals due to respiratory and cardiovascular causes Exacerbation of asthma and COPD Increased asthma symptoms Increased asthma medication usage Decrease of lung function Increase incidence of lung cancer

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Nanoparticles are extremely small particles with a diameter less than 100 nm, and those found in the general environment are principally derived from traffic. They have enhanced ability to generate highly reactive “free radical” molecules that damage and activate lung cells to produce proinflammatory mediators. In long-term, high-dose inhalation studies, UFPs of various types have been shown to cause chronic pulmonary inflammation, increased chemokine expression, epithelial cell hyperplasia, pulmonary fibrosis, and lung tumor (Dasenbrock et al. 1996; Driscoll et al. 1996; Nikula et al. 1995; Oberdorster et al. 1994). Short-term, low-dose exposure studies on ultrafine carbon black (CB) led to more inflammation than the larger fine CB. This was associated with increased oxidative stress, and modulation of one of the coagulation systems in normal rats (Ferin and Oberdorster 1992; Peters et al. 1997a, b). Particle size and surface area of the nanomaterials are important characteristics from a toxicological viewpoint. Carbon black nanoparticles of similar mass and composition but with different specific surface areas (300 versus 37 m2/g) were compared. It is recognized that the biologically available area was a critical parameter of the effects of nanomaterials. Also particle surface chemistry, biodegradability, number, shape, and solubility were all found to be significant factors in determining harmful biological effects (King et al. 2001). Biological effects such as inflammation, genotoxicity, and histology were related to surface area and not particle mass. Similar findings have been reported regarding tumorogenic effects of inhaled particles (Driscoll et al. 1997). Nanoparticle deposition in the respiratory system is determined by diffusional displacement due to thermal motion of air molecules interacting with the inhaled and exhaled air stream. Deposition occurs in all regions of the respiratory system and depends on particle size, shape and ventilation parameters. With decreasing particle diameter below 500 nm the deposition increases in all regions of the lung. Nanofibers with a small diameter will penetrate deeper into the lung while long fibers (>20 mm) deposit mainly in the upper airways. The fate of inhaled nanoparticles depends on regional distribution in the lung. Depending on where they deposit, the nanoparticles interact with the mucous lining fluid of the airways or the surfactant layer within the alveoli. Airway mucous (5 mm in depth) is a complex aqueous layer comprised of cell debris, electrolytes, proteins, and glycoproteins. The mucous varies depending on environmental and disease states. The surfactant layer (10–20 nm in thickness) covers the alveolar surface. Both airway and alveolar surface liquids are coated with a monolayer of highly surface active lung surfactant comprised of water insoluble long chain phospholipids. After deposition the nanoparticles are submerged in the lining fluid regardless of their nature. The smaller the particles in size, the more they can be incorporated into the surfactant layer. This does not affect the surface pressure of the surfactant, thus nanoparticles do not destabilize the lung surfactant film. Once deposited on to the lining fluid the particles act differently depending on their solubility in the fluid. Particles that are either lipid soluble or soluble in intracellular or extracellular fluids undergo chemical dissolution in situ. The kinetics of the diffusion in the alveoli is much faster than in the small airways. Only a small fraction of the nanoparticles are

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absorbed from the tracheobronchial tree. Inhaled nanoparticles that are insoluble are not able to be rapidly absorbed and undergo physical translocation depending on the lung region in which they are deposited (Oberdorster et al. 2005). Immersion of the slowly dissolving or insoluble nanomaterials in the airway lining fluid leads to close association with the epithelial cells and cells of the host defense system. This phenomenon may induce the inflammatory cascade in the lungs (Geiser et al. 2003) Inflammation plays a dominant role of all small particles’ effect on the respiratory system. Small particles exert their proinflammatory effects in the airways but also in the interstitium of the lung. PM has shown to activate nucleus factor kappa B and other proinflammatory signaling pathways in the lungs to increase the levels of proinflammatory mediators. These effects help to explain the increased exacerbation of asthma and COPD seen during increased levels of pollution(Arenz et al. 2006; Nel et al. 1998; Riedl and Nel 2008; Takano et al. 1997). Inflammation and systemic effects of ultrafine particles are the driving force of cardiovascular effects. There is evidence of systemic inflammation following

Fig. 10.2 Pathways from inflammation to pulmonary and cardiovascular injury

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increases in fine particle levels as shown by elevated levels of C-reactive protein, blood leukocytes, platelets, fibrinogen and increased plasma viscosity (Fig. 10.2).

10.2

Nanoparticle Toxicity

Nanoparticles may also cause pulmonary toxicity. As noted, particles such as silica are known to cause pulmonary fibrosis. Interstitial lung disease and emphysema have been described in animals exposed to nanoparticles (Donaldson et al. 2006; Sayes et al. 2007; Warheit et al. 2007). To date, neither has been described in humans. Most of the information regarding pulmonary toxicity from nanoparticles is based on animal testing. In vitro testing allows for screening for potential toxicity of a wide range of materials against various cell lines and primary cells from the airways and the alveoli (Sayes et al. 2007). These studies allow one to test for cell cytokine release and cytotoxicity. However, these studies must be verified by whole host response. The most common testing for pulmonary toxicity with nanoparticles is by intratracheal administration, although there are inhaled studies as well. Assessment of response is usually performed by bronchoalveolar lavage (BAL) at fixed time points. Figure 10.3 summarizes the percentage of neutrophils of the nucleated cells of rats exposed to either phosphate buffered saline (saline) or carbonyl iron (CI), two relatively bland substances. These are

Fig. 10.3 The percentage of neutrophils in the nucleated cells retrieved by BAL in rats exposed to either saline, carbonyl iron, nano zinc oxide (5 mg/kg), mined silica (5 mg/kg) or nano quartz (5 mg/kg dose). Lavages were performed at the specified time points. Adapted from studies by Sayes et al. (2007) and Warheit et al. (2007)

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compared to other particles, including mined silica, nano sized zinc oxide and nano quartz, all at 5mg/kg dose (Sayes et al. 2007; Warheit et al. 2007). One day after instillation, all groups have increased neutrophils in the BAL. By day 7, animals exposed to saline or carbonyl iron no longer have increased percentage of neutrophils in the BAL. For those exposed to nano zinc oxide, the BAL by day 30 is now normal. For those animals exposed to silica particles, increased neutrophils were found in the BAL through day 90. Those animals exposed to mined silica have a higher percentage of neutrophils than those exposed to the manufactured nano quartz. This figure demonstrates that larger particles (mines silica) may lead to larger reactions than nano particles. However, one can still demonstrate inflammatory reaction if a large enough dose is instilled. A variable reaction has been reported with other nanoparticles (Warheit et al. 2006; Zhu et al. 2008). Pathologic studies have confirmed that the inflammatory reaction found in the BAL is associated with inflammatory changes in the lung parenchyma (Warheit et al. 2007). Foamy macrophages are also seen in the alveolar space. These suggest that alveolar macrophage activation may be occurring. This has been associated with increased production of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor-alpha (Sayes et al. 2007). This inflammatory reaction can be quite intense. Chen et al. found that titanium oxide nanoparticles led to not only alveolar inflammation, but also emphysematous changes (Chen et al. 2006). The authors demonstrated that the emphysematous changes were associated with apoptosis of the epithelial cells. In humans, progressive massive silicosis can be associated with emphysematous changes (Gamble et al. 2004). To date, there have been no reports of bronchiolitis from nanoparticles. This represents an area of the lung which leads to fairly nonspecific symptoms such as cough and dyspnea. Routine chest roentgenogram and pulmonary function are of limited value for mild to moderate disease. Since nanoparticles will deposit in the bronchioles, the potential for bronchiolitis does exist. The most extensively studied disorders resulting from nanoparticles interaction with the respiratory system are of allergic diseases. The specific effects of diesel exhaust particles (DEPs) and its nanoparticles on allergic respiratory disease have been explored in a number of animal, in vitro, and human clinical studies. The most striking finding is the profound adjuvant effects of DEPs on the development and intensity of allergic inflammation. Animal studies (instilment of diesel particles in the upper airways) have demonstrated an increase in total and antigen-specific IgE levels, as well as increases in IL-4, IL-5, and GM-CSF levels in response to DEP exposure. In addition, DEPs reproducibly induce increased airway eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity (AHR) in murine models of asthma (Nel et al. 1998; Takano et al. 1997). All these pathophysiological findings are consistent with clinical signs of asthma and chronic bronchitis. Asthma is a disease characterized by periodic airflow limitation, airway inflammation, and airway hyperresponsiveness (AHR) (Bochner and Busse 2005).

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Evidence on the effects of particulate air pollution on asthma exacerbation and hospital admissions is increasing. A panel study on subjects with asthma found that UFP number concentration correlated closely with alterations in lung function; furthermore, variations in the concentration of fine particles (PM 2.5) and UFP correlated with use of asthma medications (Peters et al. 1997b).

10.3

Screening for Toxicity

Since nanoparticles may cause interstitial and airway disease, screening for toxicity seems reasonable. The highest risk population would be workers involved in manufacturing the particles. These workers may be exposed during routine operation as well as exposures after accidents such as spills, which could lead to high levels of exposure. A lower risk would be individuals exposed to single doses for medical or other uses. However, patients treated with nanoparticles for underlying lung disease may be at risk because of underlying damage to the lung. An example of this would be acute exacerbation in the IPF patient. Table 10.2 lists the potential methods one could use to detect toxicity. Various factors need to be considered when evaluating these tests. For example, a respiratory questionnaire has low cost and low risk. However, the questionnaires will only be positive when a patient becomes symptomatic. They are of limited value for trying to detect subclinical disease. Pulmonary function studies are perhaps the most useful screening method for detecting early airway and lung disease. Serial testing allows for detecting trends, such as a drop of FEV-1. Decreasing FEV-1 may be occurring in response to an exposure, even when the individual test results remain within the predicted normal range (Kreiss et al. 2002). For interstitial lung disease, the earliest changes have

Table 10.2 Potential tests for detecting interstitial and airway lung disease Test Interstitial Bronchiolar Respiratory questionnaire Good sensitivity Fair sensitivity Not specific Not specific Serial Good sensitivity Fair sensitivity Spirometry Fair specifity Fair specifity Serial DLCO Good sensitivity Fair sensitivity Good specifity Fair specifity HRCT Very good sensitivity Very good sensitivity Good specifity Good specifity Bronchoscopy with BAL Fair sensitivity Fair sensitivity Fair specifity Not specific Open lung biopsy Excellent sensitivity Excellent sensitivity Excellent specifity Excellent specifity Exhaled gas condensate measurement Good sensitivity Good sensitivity Fair specifity Fair specifity

Cost Very low Low Moderate High High Very high Moderate

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been noted with the DLCO (Murphy et al. 1971). This test is more expensive and less reproducible than spirometric measurements of the FEV-1 and FVC. The DLCO is also not useful in detecting bronchiolitis. Radiologic evaluation is another method for screening for lung disease. Chest roentgenographic reading has proved useful in detecting various dust related lung diseases, especially coal workers pneumoconiosis. With specific training, the films can be interpreted with good reproducibility (Lawson et al. 2001). However, the chest roentgenogram has less sensitivity and specificity than HRCT. This technique has proved useful in detecting interstitial (American Thoracic Society 2000; Baughman et al. 1991) and bronchiolar (de Jong et al. 2006) disease. In addition, the findings of HRCT are relatively specific for individual lung diseases (Wells and Hansell 2004). The major differences between chest roentgenogram and HRCT are cost and radiation risk (Chodick et al. 2007). However, newer protocols for HRCT have limited the amount of radiation exposure (Sone et al. 2007). As noted, bronchoalveolar lavage (BAL) has been useful in detecting disease in animal models. The technique has been widely used for diagnosis of infectious and noninfectious interstitial lung diseases (Meyer 2007). However, for occupational lung diseases, BAL lacks specificity. For example, in farmer’s lung, a form of hypersensitivity pneumonitis, increased lymphocytes in the BAL are routinely found (Leatherman et al. 1984). However, increased lymphocytes in the BAL are also found in asymptomatic farmers. Pulmonary evaluation of these asymptomatic farmers more than 5 years after the initial evaluation found no evidence for interstitial lung disease despite abnormal BAL findings (Lalancette et al. 1993). Surgical lung biopsy provides the most sensitive and specific information regarding interstitial and bronchiolar lung disease. However, the technique is associated with toxicity, including death (Utz et al. 2001). Therefore, the technique is usually reserved for those in whom a specific diagnosis would have impact on treatment. Bronchiolitis can have a subtle presentation. The diagnosis of occupational bronchiolitis in workers at microwave popcorn and other flavor manufacturers required a fair amount of detective work. The studies in this area demonstrated that there was at least two fold increase in respiratory symptoms for those workers exposed to diacetyl(Kreiss et al. 2002). These symptoms include cough, shortness of breath, wheezing, and asthma symptoms. These are fairly nonspecific complaints. The authors did find that the highest level of exposure to diacetyl was associated with the lowest FEV1 by quartile. However, the FEV1 for those with the highest exposure were still over 80% of predicted, therefore fitting into the normal range. Thus screening by symptoms and pulmonary function studies alone may fail to detect early disease. On the other hand, HRCT may be useful in providing a more specific diagnosis in these patients. Figure 10.4a demonstrates the mosaic pattern suggesting bronchiolitis in a worker at a flavor manufacturer. She had developed increasing dyspnea. As the Fig. 10.4b shows, she has gone on to develop fibrosis as well. An open lung biopsy demonstrated both fibrosis and bronchiolitis. The fibrosis was as an end-stage product of the bronchiolitis.

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Fig. 10.4 HRCT inspiratory and expiratory views of worker in flavour manufacturing plant who developed increasing dyspnea and cough. Patient was a non-smoker. Her inspiratory film demonstrates areas of ground glass density (circled) surrounded by areas of decreased density (mosaic pattern). This was accentuated on the expiratory view (circled)

10.4

Nanoparticles for Therapy

The use of nanoparticles to treat interstitial and bronchial lung disease seems quite reasonable (Bergamaschi et al. 2006). One of the advantages of nanoparticles is their area of deposition. There are several factors that affect the area of deposition. One is the size of the particles; another is the charge of the particles, and finally the properties of particles. For example, hydroscopic particles, which absorb water rapidly, will deposit a larger part of the airways. The size of the nanoparticle should deposit for the most part in the bronchioles and in the alveoli with minimal deposition in the nose and larynx. This, therefore, would make them an ideal treatment for interstitial and bronchial lung disease. There are several diseases of the small airways and interstitium which could be potential targets for therapy with nanoparticles. At the same time, the nanoparticles could cause disease in these same areas. We will discuss diseases of the small airways and interstitium and point out potential toxicity and therapy for these areas. Bronchiolitis is a reflection of the small airways of the lung. This was often referred to as the silent part of the lung. It can lead to both obstruction and restriction of disease. Unfortunately, bronchiolitis can be very difficult to diagnose and treat. There are several conditions, which have led to bronchiolitis that have been relatively well studied: chronic lung rejection to transplant and the bronchiolitis after exposure to diacetyl by workers in flavor manufacturing plants (de Jong et al. 2006; Kreiss et al. 2002) (Fig. 10.4). Lung transplant patients who develop chronic rejection of the new lung have bronchiolitis obliterans. Advanced bronchiolitis obliterans is a significant cause of death in the lung transplant population and several methods have been proposed to detect early disease. Monitoring with serial pulmonary function tests has been

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useful in indicating possible disease. A drop in the forced expiratory volume in one second (FEV-1) of less than 80% of post transplant values usually leads to more evaluation (de Jong et al. 2006). The use of high resolution CT scan (HRCT) has been useful in detecting bronchiolitis obliterans. The usual findings for bronchiolitis obliterans are areas of ground glass mixed with areas of increased attenuation (mosaic pattern) (de Jong et al. 2006). The localized air trapping is often more obvious on an expiratory HRCT (Fig. 10.4b). A lung transplant patient with dropping FEV-1 or increasing dyspnea will often undergo bronchoscopy with transbronchial biopsies and bronchoalveolar lavage. Unfortunately, the bronchoscopy techniques have not been reliable for detecting bronchiolitis. However, they are quite useful in detecting opportunistic infections. A recent report has suggested analysis of exhaled gases for markers of increased inflammation may be useful for detection of early bronchiolitis (Van et al. 2007). Since this technique is noninvasive, it may prove a useful screening method in the appropriate population. Interstitial lung disease can be caused by several processes. One is an acute inflammation, which is a pneumonia-like pattern with increased neutrophils. This is usually self-limited. However, it can occur on top of other interstitial diseases and cause much shortness of breath. An example is an acute decompensation of idiopathic pulmonary fibrosis (Collard et al. 2007). Patients with acute hypersensitivity pneumonitis will have cough, wheezing, and squeaks. Symptoms can be more chronic, with just unexplained cough and gradually worsening of dyspnea. For both acute and chronic pulmonary function studies demonstrate both obstruction and restriction, with a reduced DLCO being an early feature. HRCT findings include nodularity and areas of ground glass. Hypersensitivity pneumonitis tends to be an upper lobe disease. Fibrosis is more prominent in chronic disease (Adler et al. 1992). One can also have chronic inflammation such as granulomatous lung disease, including sarcoidosis and hypersensitivity pneumonitis (Baughman et al. 2003). Fire fighters at “ground zero” after the 9/11 disaster in United States were noted to have a marked increase in the rate of a sarcoidosis-like disease (Izbicki et al. 2007). Inhalation of particle dust appears to be related to granulomatous disease. Animal studies have found that carbon nanoparticles can cause granulomatous reactions (Donaldson et al. 2006). Sarcoidosis is a granulomatous disease of unknown etiology affecting multiple organs. Lung involvement is identified in more than 90% of patients at time of diagnosis (Baughman et al. 2001). While the HRCT of sarcoidosis patients can be similar to hypersensitivity pneumonitis (Fig. 10.5). While many patients with sarcoidosis will have remission within 2 years of presentation, a quarter of patients require long term treatment (Baughman et al. 2006b). Novel treatments for chronic sarcoidosis include cytotoxic agents such as methotrexate and monoclonal antibodies which block tumor necrosis factor (Baughman et al. 2006a). Nanoparticles represent a potential approach to deliver immune modifiers to the lung of patients with chronic refractory sarcoidosis. Interstitial lung disease can be predominantly fibrotic. Among the chronic fibrotic lung diseases are silicosis and asbestosis. Inhalation of silica and other dusts are well

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known to cause lung disease in humans (Chong et al. 2006). The profile for clinically significant silicosis is in a young individual with heavy exposure (Castranova and Vallyathan 2000). Changes in lung function include both restrictive and obstructive disease (Gamble et al. 2004). Cigarette smoking appears to be an important cofactor leading to more extension disease (Gamble et al. 2004). Over the past few years, a group of diseases causing progressive pulmonary fibrosis have been studied. Among the idiopathic interstitial lung diseases is idiopathic pulmonary fibrosis (IPF) (2000). This disease is associated with digital clubbing and crackles on auscultation of the chest. Pulmonary function studies tend to show just restriction, with DLCO decrease out of proportion to the loss in lung volume. High-resolution CT (HRCT) shows diffuse interstitial disease changes, with a basilar prominence for many of the conditions. The presence of subpleural honeycombing is a characteristic feature seen in patients with idiopathic pulmonary fibrosis (Fig. 10.6) (2000). IPF is a progressive disease. There is no effective treatment for all patients. Some patients appear to have a response to antiinflammatory agents such as corticosteroids and/or cytotoxic drugs (Demedts et al.

Fig. 10.5 Areas of patchy infiltrate in patient with biopsy confirmed sarcoidosis

Fig. 10.6 Patient with idiopathic pulmonary fibrosis. Subpleural honeycombing is identified. Disease is more extensive at the base of the lung (b) than in mid lung (a)

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2005; Raghu et al. 1991). However, it is not clear that any of the currently available agents change the natural course of the disease for most patients (Walter et al. 2006). Progressive pulmonary fibrosis can lead to respiratory failure in patients with scleroderma and other collagen vascular diseases. These patients have a difference in their clinical presentation with more ground glass and traction bronchiectasis, and less subpleural honeycombing than seen with IPF (Chan et al. 1997). It is a progressive disease and if untreated, the patients go on to die, although not as rapidly as in IPF. Recent studies have demonstrated a modest benefit from the use of the cyclophosphamide, a cytotoxic agent associated with significant morbidity (Tashkin et al. 2006). Pulmonary fibrosis is an increasing problem for the last 20 years (Gribbin et al. 2006). Part of this has been increased recognition on the basis of high-resolution CT scan. However, a true rise in cases appears due to our aging population. Idiopathic pulmonary fibrosis is the disease of the elderly with the average age greater than 65. The disease is associated with significant morbidity and a mortality of greater than 50% within 5 years of diagnosis (Walter et al. 2006). Agents that have been used as anti-inflammatory drugs have limited effectiveness for IPF. Nanotechnology has the advantage of being able to provide drugs that will be delivered directly to the lung. This would minimize the toxicity of other drugs given by intravenous dose. One of the limitations of nanotechnology for pulmonary fibrosis is aerosol deposition. The traction bronchiectasis in honeycomb leads to areas in which there is limited ventilation. While drug delivery may be problematic if immediate treatment by the aerosol was planned. However, the potential for prolonged deposition in the

Fig. 10.7 Patient with idiopathic pulmonary fibrosis who was clinically stable over a 6 month period (Baseline). She developed acute decompensation and respiratory failure (Acute Decompensation). Her repeat HRCT now shows diffuse ground glass opacification. She underwent multiple studies, including bronchoscopy, but no infectious agent could be identified. She subsequently died of respiratory failure

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lung and movement into areas not well ventilated may enhance effectiveness of agents delivered by nanoparticles. Patients with pulmonary fibrosis can also develop an acute exacerbation (Collard et al. 2007). Figure 10.7 demonstrates a patient with idiopathic pulmonary fibrosis with mild subpleural honeycombing and some mild ground glass on the left. She subsequently developed respiratory failure. Evaluation for infection, including bronchoscopy, was negative. HRCT demonstrates new diffuse ground glass with air bronchograms. This patient went on to die of respiratory failure. The cause of acute exacerbation is often unknown. Any aerosol treatment would have to allow for the possibility of enhancing pulmonary toxicity. While there are several treatment options for bronchiolitis, granulomatous and other interstitial lung disease available, all of these have their limitations. Nanoparticles have the potential of offering new treatment for all of these diseases. However, nanoparticles themselves have the potential of causing toxicity in these areas of the lung. The respiratory system is an attractive route for non-invasive drug delivery with advantages for both systemic and local applications . Incorporating therapeutics with polymeric nanoparticles offers additional degrees of manipulation for delivery systems, providing sustained release and the ability to target specific cells and organs. However, nanoparticle delivery to the lungs has many challenges including formulation instability due to particle–particle interactions and poor delivery efficiency due to exhalation of low-inertia nanoparticles. Also their potential toxic effects should be recognized. Further exploration into the effect of particle physicochemical properties (e.g. nanoparticle size and material) on extending particle persistence in the lungs and their influence on particle fate is necessary to aid the design of improved systems. The development of inhalable nanoparticle-based delivery systems should draw from the extensive nanoparticle research for injectable applications, including surface modification to target-specific sites. Further coordination with environmental health research is the key to understanding the implications that particle fate and toxicology might have on drug delivery.

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Driscoll KE, Deyo LC, Carter JM, Howard BW, Bertram TA (1997) Effects of particle exposure and particle elicited inflammatory cells on mutation in rat epithelial cells. Carcinogenesis 18:423–430 Ferin J, Oberdorster G (1992) Polymer degradation and ultrafine particles: potential inhalation hazards for astronauts. Acta Astronaut 27:257–259 Gamble JF, Hessel PA, Nicolich M (2004) Relationship between silicosis and lung function. Scand J Work Environ Health 30(1):5–20 Geiser M, Schurch S, Gehr P (2003) Influence of surface chemistry and topography of particles on their immersion into the lung’s surface-lining layer. J Appl Physiol 94(5):1793–1801 Gribbin J, Hubbard RB, Le JI, Smith CJ, West J, Tata LJ (2006) Incidence and mortality of idiopathic pulmonary fibrosis and sarcoidosis in the UK. Thorax 61(11):980–985 Izbicki G, Chavko R, Banauch GI, Weiden MD, Berger KI, Aldrich TK, Hall C, Kelly KJ, Prezant DJ (2007) World Trade Center “sarcoid-like” granulomatous pulmonary disease in New York City Fire Department rescue workers. Chest 131(5):1414–1423 King TE Jr, Schwarz MI, Brown K, Tooze JA, Colby TV, Waldron JA Jr, Flint A, Thurlbeck W, Cherniack RM (2001) Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am J Respir Crit Care Med 164(6):1025–1032 Kreiss K, Gomaa A, Kullman G, Fedan K, Simoes EJ, Enright PL (2002) Clinical bronchiolitis obliterans in workers at a microwave-popcorn plant. N Engl J Med 347(5):330–338 Lalancette M, Carrier G, Laviolette M, Ferland S, Rodrique J, Begin R, Cantin A, Cormier Y (1993) Farmer’s lung Long-term outcome and lack of predictive value of bronchoalveolar lavage fibrosing factors. Am Rev Respir Dis 148(1):216–221 Lawson CC, LeMasters MK, Kawas LG, Simpson RS, Rice CH, Lockey JE (2001) Reliability and validity of chest radiograph surveillance programs. Chest 120(1):64–68 Leatherman JW, Michael AF, Schwartz BA, Hoidal JR (1984) Lung T cells in hypersensitivity pneumonitis. Ann Intern Med 100(3):390–392 Meyer KC (2007) Bronchoalveolar lavage as a diagnostic tool. Semin Respir Crit Care Med 28 (5):546–560 Murphy RL, Jr, Ferris BG, Jr, Burgess WA, Worcester J, Gaensler EA (1971) Effects of low concentrations of asbestos. Clinical, environmental, radiologic and epidemiologic observations in shipyard pipe coverers and controls. N Engl J Med 285(23):1271–1278 Nel AE, az-Sanchez D, Ng D, Hiura T, Saxon A (1998) Enhancement of allergic inflammation by the interaction between diesel exhaust particles and the immune system. J Allergy Clin Immunol 102(4 Pt 1) 539–554 Nikula KJ, Snipes MB, Barr EB, Griffith WC, Henderson RF, Mauderly JL (1995) Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats. Fundam Appl Toxicol 25(1):80–94 Oberdorster G, Cherian MG, Baggs RB (1994) Correlation between cadmium-induced pulmonary carcinogenicity, metallothionein expression, and inflammatory processes: a species comparison. Environ Health Perspect 102(Suppl 3):257–263 Oberdorster G, Oberdorster E, Oberdorster J (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113(7):823–839 Peters A, Dockery DW, Heinrich J, Wichmann HE (1997a) Short-term effects of particulate air pollution on respiratory morbidity in asthmatic children. Eur Respir J 10(4):872–879 Peters A, Wichmann HE, Tuch T, Heinrich J, Heyder J (1997b) Respiratory effects are associated with the number of ultrafine particles. Am J Respir Crit Care Med 155(4):1376–1383 Raghu G, Depaso WJ, Cain K, Hammar SP, Wetzel CE, Dreis DF, Hutchinson J, Pardee NE, Winterbauer RH (1991) Azathioprine combined with prednisone in the treatment of idiopathic pulmonary fibrosis: a prospective double-blind, randomized, placebo-controlled clinical trial. Am Rev Respir Dis 144(2):291–296 Riedl MA, Nel AE (2008) Importance of oxidative stress in the pathogenesis and treatment of asthma. Curr Opin Allergy Clin Immunol 8(1):49–56

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Chapter 11

Effects of Cigarette Smoke and Diesel Exhaust on the Innate Immune Function of the Airway Epithelium P. S. Hiemstra

11.1

Introduction

The lung is exposed to a large volume of inhaled air that contains numerous inhaled particles and gases. This way potential pathogenic micro-organisms reach the epithelial surface and need to be dealt with by host defense mechanisms to prevent severe lung infections (Bals and Hiemstra 2004; Diamond et al. 2000). The same inspired air that contains these respiratory pathogens may also contain cigarette smoke and air pollutants. These toxic compounds have been shown to cause lung inflammation and affect the ability of the lung to mount an efficient host defense response against these pathogens. In addition to impairing host defense, lung injury caused by inhalation of toxic substances may also pave the way for respiratory infections, because of the increased ability of pathogens to adhere to the injured lung mucosa. This way, smoke and air pollutants contribute to the development of airways inflammation and respiratory infections. These respiratory infections are a major cause of morbidity and mortality worldwide. In the past decades intensive research has led to a major increase in our understanding of the mechanisms that are involved in host defense against respiratory infections. An increasing number of clinical and basic science studies are addressing the way that inhaled toxic substances present in cigarette smoke and air pollutants affect this defense system and contribute to both respiratory infections and chronic lung disease. This review provides a selected overview of this area with a focus on the airway epithelium and addresses some of the remaining gaps in our knowledge.

P.S. Hiemstra Department of Pulmonology, Leiden University Medical Center, Leiden, the Netherlands e-mail: [email protected]

J.C.M. Marijnissen and L. Gradon´ (eds.), Nanoparticles in Medicine and Environment, DOI 10.1007/978-90-481-2632-3_11, # Springer ScienceþBusiness Media B.V. 2010

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Host Defense Against Infection in the Lung

Despite the fact that we daily inhale large numbers of pathogenic micro-organisms, respiratory infections are relatively rare. Host defense mechanisms that prevent respiratory infections are operative in our lungs, and the observation that respiratory infections are rare indicates that these mechanisms are very effective. Various cell types and mechanisms are involved in protecting the lung from respiratory pathogens. These include not only phagocytic macrophages and neutrophils, but also a range of other inflammatory and immune cells. Although the focus of this paragraph and chapter is on the airway epithelium, it should be noted that cigarette smoke and diesel exhaust may also directly or indirectly affect the host defense function of these other cell types in the lung. The airway epithelium is crucial to an effective host defense system because of the large epithelial surface of the lung, and airway epithelial cells are the first cell type to interact with airborne pathogens. It provides both a passive barrier against entry of inhaled pathogens, and is increasingly recognized as an essential element of immunity in the lung (Diamond et al. 2000; Strieter et al. 2003; Bals and Hiemstra 2004) (Table 11.1). The various types of epithelial cells that constitute the pseudostratified epithelium that lines the conducting airways provide mucociliary clearance. Mucus produced by specialized airway epithelial cells and submucosal glands forms a layer on top of the airway epithelium that traps inhaled particles including micro-organisms (Bals and Hiemstra 2004; Thornton et al. 2008). This layer is positioned on top of a thin fluid layer that covers the airway epithelium, and that allows the ciliated cells of the airway epithelium to move the mucus layer that contains the trapped substances. This clearance mechanism is further supported by coughing. Airway epithelial cells also produce a range of molecules that kill micro-organisms that may have passed the mucus layer or reach the epithelial surface where little or no mucus is present (e.g. in the alveoli) (Bals and Hiemstra 2004; Rogan

Table 11.1 Mechanisms that contribute to host defense against infection by airway epithelium and submucosal glands Mechanism Function Barrier formation Prevention of penetration of micro-organisms Mucociliary clearance Trapping and removal Production of antimicrobial substances Killing and growth inhibition (such as AMPs, ROI and RNI) Cytokine and chemokine production Recruitment and activation of inflammatory and immune cells; antiviral activity of interferons Transport of antibodies (submucosal Prevention of adherence, complement-mediated glands) killing and opsonization AMPs, antimicrobial peptides and proteins; ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates.

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et al. 2006). These molecules include peptides and proteins such as lysozyme and lactoferrin, and are collectively referred to as antimicrobial peptides and proteins (AMPs). Whereas most of these AMPs were originally discovered based on their direct antimicrobial activity, it is now well established that these compounds also display a range of other activities and are involved in inflammation, immunity and repair processes. The major classes of antimicrobial peptides present in human airway secretions are the defensins and the cathelicidins. Based on their structure, the human defensin family can be divided into the a- and the b-defensin subfamilies. Human airway secretions contain a-defensins that are produced and secreted by neutrophils, whereas b-defensins present in these secretions are mainly derived from epithelial cells. In contrast to other species that contain a range of cathelicidin antimicrobial peptides, in humans only one member of the cathelicidin family is expressed: hCAP18/LL-37. Airway secretions contain this hCAP18/ LL-37 that is derived from neutrophils, but also e.g. epithelial cells and mast cells may contribute to its production. Antimicrobial proteins detected in airway secretions include lysozyme, lactoferrin, cationic serine proteinase inhibitors (secretory leukocyte proteinase inhibitor (SLPI) and elafin), and surfactant proteins SP-A and SP-D. In addition to these AMPs, reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) that are formed by specialized enzyme systems present in the epithelium and in recruited inflammatory cells contribute to killing of micro-organisms. The expression of some of the components of this defense system show constitutive expression, thus providing a constant level of protection. The expression of other components is inducible following microbial exposure, inflammation and/or tissue repair processes. Airway epithelial cells may signal the presence of microbial exposure using so-called pattern recognition receptors that detect conserved molecular patterns present on pathogens (Akira et al. 2006; Bals and Hiemstra 2004). The cells respond to this exposure by e.g. increasing the production of certain AMPs, and by producing cytokines and chemokines. Cytokines signal to a vast range of cell types, including endothelial cells that line the vessels. This signaling results in increased expression of adhesion molecules on these endothelial cells that facilitate the recruitment of inflammatory and immune cells, such as the phagocytic neutrophils and monocytes, antigen-presenting dendritic cells and a range of other cell types. Chemokines produced by the airway epithelium form a chemotactic gradient along which the inflammatory and immune cells can migrate to the site of infection. In addition, viral exposure leads to the epithelial production of interferons with direct and indirect antiviral activities. Another mechanism that contributes to host defense against infection is the transport of antibodies that are produced by the adaptive immune system to mucosal secretions, which primarily occurs in the submucosal glands. These secreted antibodies (mainly of the IgA and IgM classes) prevent adherence of micro-organisms, activate the complement system and cause opsonization that facilitates recognition and removal by phagocytic cells.

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Chronic Obstructive Pulmonary Disease – Role of Cigarette Smoking and Air Pollution in Chronic Inflammatory Lung Disease

Chronic obstructive pulmonary disease (COPD) is a major health problem on a global scale (Pauwels and Rabe 2004; Mannino and Buist 2007; Barnes 2008). COPD is a very common disease characterized by progressive and irreversible airways obstruction, and its incidence is increasing world-wide. The major cause is cigarette smoking, but also indoor air pollution that is caused by cooking and heating in poorly ventilated houses may contribute to the development. COPD is characterized by chronic airway inflammation caused by inhalation of the toxic compounds present in cigarette smoke and air pollution, leading to the development of lung injury and irreversible airflow obstruction. Airway inflammation is markedly increased during acute episodes; these exacerbations are frequently accompanied by respiratory infections. Bacterial and viral infections are associated with COPD both in clinically stable patients as well as during exacerbations, and may contribute to the decline in lung function. At present no effective pharmaceutical intervention is available that slows the progressive loss of lung function, and smoking cessation is by far the most effective treatment. A large number of epidemiological studies have shown an association between levels of various air pollutants and hospital admissions for a variety of reasons, including pulmonary disorders (Sydbom et al. 2001; Riedl and az-Sanchez 2005). Traffic emissions that result from the combustion of fossil fuels constitute a major source of this air pollution. Despite advances in technology aimed to reduce the emission of hazardous compounds, diesel engines contribute more to traffic-related air pollution than gasoline engines. Combustion of diesel fuel results in the generation of diesel exhaust particles (DEPs), as well as various gaseous compounds that have adverse health effects. A large number of animal and human studies have shown that diesel causes pulmonary inflammation (Sydbom et al. 2001).

11.4

Modulation of Host Defense

Cigarette smoke has marked effects on epithelial gene expression, and these effects are thought to be involved in the development of lung cancer as well as COPD. The effect of cigarette smoke on airway epithelial gene expression has been explored in animal models and using in vitro cultures of human airway epithelial cells. These studies have shown that induction of goblet cell hyperplasia is an important consequence of smoke exposure that leads to increased mucus production in the airways. This effect has been shown to be mediated by smoke-induced activation of the epidermal growth factor receptor (EGFR) (Takeyama et al. 2001). Other studies have used epithelial cells derived from never smokers, current and ex-smokers to identify genes that are associated with the acute effects of cigarette smoke and those

11 Effects of Cigarette Smoke and Diesel Exhaust on the Innate Immune Function Table 11.2 Mechanisms involved in impaired host defense resulting from exposure to cigarette smoke or diesel exhaust

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Direct Decreased mucociliary clearance l Increased microbial adherence to epithelial surfaces l Decreased production of antimicrobial molecules l Decreased activity of phagocytes Indirect l Increased inflammation l

associated with long-term effects of smoke exposure, and studied whether or not changes in gene expression are reversible after smoking cessation (Spira et al. 2004; Chari et al. 2007; Beane et al. 2007). These smoke-induced changes in gene expression were identified using gene expression profiling techniques such as microarray and serial analyis of gene expression (SAGE). The studies showed that in vivo smoke exposure not only affects mucin expression, but also changes expression of a range of genes involved in xenobiotic metabolism and antioxidant responses, as well as several oncogenes. Cigarette smoke not only causes airways inflammation, but also markedly affects host defense against respiratory infections. The increased risk of smokers and those exposed to environmental tobacco smoke (ETS; also referred to as “passive smoke”) to respiratory infections is well-documented (Arcavi and Benowitz 2004; Sopori 2002). A variety of respiratory infections are associated with smoking, including community-acquired pneumonia (CAP) which is an important cause of morbidity and mortality (Almirall et al. 2008; Almirall et al. 1999; Baik et al. 2000). Animal models of respiratory infections have confirmed the effect of smoke exposure on sensitivity to infection. Cigarette smoke has a suppressive effect on many elements of the innate and adaptive immune system (Sopori 2002) (Table 11.2). Smoke-induced inflammation itself may impair an efficient defense against respiratory pathogens by causing tissue injury and impairing effective clearance by phagocytes. Cigarette smoke decreases mucociliary clearance by inhibiting ciliary activity and increasing mucus production. It also increases bacterial adherence to epithelial surfaces (Ozlu et al. 2008) and thus promotes infection. The remodeling process of the epithelium that occurs during smoke- and/or inflammation-induced injury and subsequent repair has been shown to favor bacterial infections (Puchelle et al. 2006). Finally, cigarette smoking may decrease local defense mechanisms as shown by studies on alveolar macrophage function in smokers (Hodge et al. 2007). This study demonstrated a decreased phagocytic ability of macrophages from smokers, that was partially reversed by smoking cessation in COPD patients. In another study in COPD patients, a decreased ability to ingest non-typeable Haemophilus influenzae was reported. Both active smoking and COPD itself (by investigating ex-smokers with COPD) were identified as determinants in the decreased ability of macrophages from smokers with and without COPD to ingest Haemophilus influenzae (Berenson et al. 2006). Diesel exhaust particles (DEPs) constitute another important environmental risk factor for respiratory infections. The majority of DEPs are found in the size range classified as fine (0.1–2.5 mm) and ultrafine (