Encyclopedia of Nanoscience and Nanotechnology 9781588830654, 1588830659

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Encyclopedia of Nanoscience and Nanotechnology Volume 9 Number 1 2004 Polymeric Nanoparticles for Drug and Gene Delivery

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M. N. V. Ravi Kumar; M. Sameti; C. Kneuer; A. Lamprecht; C.-M. Lehr Porphyrin-Based Chemical Sensors

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Roberto Paolesse; Federica Mandoj; Alessia Marini; Corrado Di Natale Preparation of Vesicles (Liposomes)

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Peter Walde Protein-Doped Nanoporous Silica Gels

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Stefano Bettati; Barbara Pioselli; Barbara Campanini; Cristiano Viappiani; Andrea Mozzarelli Quantum Dot Atoms, Molecules, and Superlattices

105

Hiroyuki Tamura; Hideaki Takayanagi; Kenji Shiraishi Quantum Dot Infrared Photodetector

131

Jamie Phillips; Adrienne Stiff-Roberts; Pallab Bhattacharya Quantum Dots: Artificial Atoms and Molecules

143

Philippe Matagne; Jean-Pierre Leburton Quantum Well Infrared Detectors

179

W. Lu; Y. Fu Quantum Well Infrared Photodetectors: Theoretical Aspects

199

A. F. M. Anwar; Kevin R. Lefebvre Raman Scattering in Nanostructures

225

C. E. Bottani; C. Castiglioni; G. Zerbi Raman Spectroscopy of Quantum Wires and Quantum Dots

273

V. Wagner; J. Geurts; W. Kiefer Raman Spectroscopy in Carbon Nanotubes

307

M. S. Dresselhaus; A. M. Rao; G. Dresselhaus Resists for Nanolithography

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P. Argitis Resonant Tunneling Devices

357

A. F. M. Anwar; Mirza M. Jahan Room-Temperature Ballistic Nanodevices

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Aimin M. Song

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Scanning Probe Techniques for Semiconductor Nanostructures

391

Thomas Ihn Scanning Tunneling Microscopy of Carbon Nanotubes

415

László P. Biró; Philippe Lambin Self-Assembled Monolayers on Semiconductor Surfaces

427

D. Zerulla Self-Assembled Nanobiomaterials

459

Steve S. Santoso; Shuguang Zhang Self-Assembled Nanofibers

473

Hirotaka Ihara; Makoto Takafuji; Toshihiko Sakurai Self-Assembled Nanostructures at Silicon Surfaces

497

D. Y. Petrovykh; F. J. Himpsel Self-Assembled Organic/Inorganic Nanocomposites

529

Byron McCaughey; J. Eric Hampsey; Donghai Wang; Yunfeng Lu Self-Assembled Porphyrin Arrays

561

Kazuya Ogawa; Yoshiaki Kobuke Self-Assembled Porphyrinic Nanoarchitectures

593

Xin Chen; Charles Michael Drain Self-Organization of Colloidal Nanoparticles

617

Joydeep Dutta; Heinrich Hofmann Self-Organized Nanostructure Formation on Surfaces

641

Andrew T. S. Wee Semiconductor Nanodevice Modeling

653

Eric A. B. Cole Semiconductor Nanoparticles for Photocatalysis

669

W. Li; S. Ismat Shah Semiconductor Nanotransistors

697

Y. Fu Semiconductor Quantum Dots

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Lucjan Jacak; Arkadiusz Wójs; Pawe Machnikowski III/V Semiconductor Quantum Dots

735

M. Guzzi; S. Sanguinetti; M. Gurioli Semiconductor Quantum Wires

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Xing-Quan Liu; Xue-Lun Wang; C. Jagadish; M. Ogura

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SiGe/Si Heterostructures

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C. W. Liu; L. J. Chen Silicon Nanocrystals in SiO2 Thin Layers

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A. G. Nassiopoulou Silicon Nanowires

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Klaus Sattler Silicon Quantum Dots

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Anri Nakajima Silicon Surface Nanooxidation

859

D. Stiévenard; B. Legrand Single-Electron Devices

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Konstantin Likharev Single-Electron Dynamics

885

Toshimasa Fujisawa Single-Electron Transistors

903

Jia Grace Lu Single Wall Carbon Nanotubes

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Ákos Kukovecz; Zoltán Kónya; Imre Kiricsi Sliding, Rotation, and Rolling of Nanoparticles

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K. Miura Copyright © 2004 American Scientific Publishers

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Encyclopedia of Nanoscience and Nanotechnology

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Polymeric Nanoparticles for Drug and Gene Delivery M. N. V. Ravi Kumar, M. Sameti, C. Kneuer, A. Lamprecht, C.-M. Lehr Saarland University, Saarbarucken, Germany

CONTENTS 1. Introduction 2. Features of Polymeric Particles 3. Preparation and Characterization of Nanoparticles 4. Recent Developments in Nanoparticle Technology 5. Nanoparticles for Some Specific Applications Glossary References

1. INTRODUCTION There is a plethora of reasons why the simple capsule containing pure active drug is not an acceptable delivery system for a drug at any stage of its life (Fig. 1). Conventional formulations are usually thought of as oral (tablets and capsules), topical (ointments, creams, etc.), and injections. It is often desirable for a drug to be delivered at a steady, uniform rate; zero order is ideal. This implies that the concentration of the drug in blood or tissue is independent of the concentration of the drug at the site of absorption. None of the conventional formulations meet these demands. Controlled drug delivery occurs when a polymer, whether natural or synthetic, is judiciously combined with a drug or other active agent in such a way that the active agent is released from the material in a predesigned manner. The release of the active agent may be constant over a long period, it might be cyclic over a long period, or it may also be triggered by the environment or other external events. Over the past few decades, there has been considerable interest in developing biodegradable nanoparticles as potential candidates for controlled drug delivery. Various polymers [1] have been used in drug delivery research as they are expected to be capable of delivering drugs to target site and thus increase the therapeutic benefit, while minimizing side effects [2]. ISBN: 1-58883-065-9/$35.00 Copyright © 2004 by American Scientific Publishers All rights of reproduction in any form reserved.

Nanoparticles were first developed by Speiser and co-workers [3] around 1970 and are defined as solid colloidal particles, less then 1
m in size, that consist of macromolecular compounds. Since then, a considerable amount of work on nanoparticles is being carried out around the world in the field of drug/gene delivery (Fig. 2). They were initially devised as carriers for vaccines and anticancer drugs [4]. The use of nanoparticles for ophthalmic and oral delivery was also investigated [5]. Drugs or other biologically active molecules are dissolved, entrapped, or encapsulated in the nanoparticles or are chemically attached to the polymers or adsorbed to their surface. The selection of the appropriate method for preparing drug-loaded nanoparticles depends on the physicochemical properties of the polymer and the drug. On the other hand, the procedure and the formulation conditions will determine the inner structure of these polymeric colloidal systems. Two types of systems with different inner structures are possible: (i) a matrix-type system composed of an entanglement of oligomer or polymer units, defined here as a nanoparticle or nanosphere; (ii) a reservoir-type system, consisting of an oily core surrounded by a polymer wall, defined here as a nanocapsule. Various colloidal nanoparticulate systems in use for drug/gene delivery are as shown in Figure 3. The term nanoparticle shall be used to refer to both systems including nanoparticles as well as nanocapsules. In this chapter we discuss preparation techniques, characterization, and some reported nanoparticulate delivery systems and their applications.

2. FEATURES OF POLYMERIC PARTICLES 2.1. Small Size and Volume It is practically impossible to fabricate a polymeric particle less than 5 nm in size, considering the fact that polymer molecules usually have a molecular weight higher than 10,000 Da. Particles composed of 1000 units of polymers with molecular weight 10,000 Da would be 30 nm in size.

Encyclopedia of Nanoscience and Nanotechnology Edited by H. S. Nalwa Volume 9: Pages (1–19)

Polymeric Nanoparticles for Drug and Gene Delivery

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Plasma Drug Concentration

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Time Figure 1. Comparison of typical pharmacokinetic profiles seen for conventional vs controlled release formulations. (Source: Science Finder, reports included until July 2002)

Their small volume enables the whole body of particles to respond quickly to stimuli. The relaxation time of volume change of a gel was indicated to be proportional to the square of its radius [6]. Therefore, fine particles can be a microreactor with a high reaction rate.

2.2. Large Specific Surface Area The large surface area of the particles would be available for sites of absorption and desorption, chemical reactions, light scattering, etc. The total surface area of 1 g of particles having a diameter of 0.1
m would be about 60 m2 . The total surface area is inversely proportional to the diameter.

2.3. High Diffusibility and Mobility Polymer colloids have a low viscosity and high fluidity compared to solutions containing the same amount of solid. The viscosity of polymer colloids is a universal function of apparent volume fraction of the particles. The apparent volume fraction of particles can be changed by environmental conditions such as pH and temperature for some polymer colloids [7]. In dispersion the particles can move macroscopically through the medium by gravity, electric field, etc. and microscopically via Brownian motion. These movements keep a 1978

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Gene Delivery Systems

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Figure 2. Updates on nanoparticle research (Source: Science Finder).

Figure 3. Various colloidal particles in use for numerous pharmaceutical applications.

fresh interface between the particles and the medium. Particles having soft layers on the surface allow water to penetrate the layer and meet less resistance when they move through water. Such particles occasionally exhibit a fairly high electrophoretic mobility even if they have little charge [8]. This explains the extraordinarily high electrophoretic mobility of cells and other biocolloids.

2.4. Stable Dispersions The potential energy and stability of the polymer particles in the dispersion are decided collectively by the three factors, viz., electrostatic repulsive forces, van der Waals attractive forces, and steric repulsive forces among the particles. If the particle has a potential energy larger than 15 kT, it has a high stability ratio W and is stable enough to be stored for a relatively long period, say, more than one month [9]. The stability ratio [W ] is expressed as W = Kq /Ks , where Kq and Ks are the rate constants for rapid and slow flocculation, respectively. The surface potential energy is affected significantly by environmental factors such as ionic strength and pH. The minimum concentration of a salt to flocculate the particles is termed flocculation concentration [10]. Steric repulsive interactions are a crucial force for microspheres covered with polymer layers protruding in solution. The steric stabilization effect consists of both an enthalpy and an entropy effect. The overlap of the surface layers of two vicinal particles results in a shift from the equilibrium state of the layer (i.e., enthalpy gain) and a decrease in the conformational freedom of solvated polymer chains (i.e., entropy loss) [11]. If the polymer layers on the particles are sensitive to temperature, the stability of the dispersion due to the steric stabilization effect is influenced by temperature. The critical temperature for flocculation, in such systems, is called the critical flocculation temperature. Dissolved, but nanoadsorbable, polymer molecules cause the coagulation of particles. This phenomenon is referred to as the depression effect [12, 13]. This effect may work in many practical uses of polymer colloids, although it has not been considered extensively. The high stabilities of polymer colloids may bring about some difficulties in the separation of particles from the dispersion medium. Centrifugation sometimes fails

Polymeric Nanoparticles for Drug and Gene Delivery

for dispersions of very fine particles because the sedimentation rate is proportional to the square of the diameter. Coagulation with salt is one of the most conventional methods to separate particles from water. Magnetic or electric fields are sometimes applied for collecting particles from dispersion.

2.5. Uniformity Techniques for the preparation of monodisperse polymer colloids received greater attention during 1980s [14, 15]. The use of monodisperse particles makes it possible to give sharp, reliable, and reproducible results for their respective applications. Emphasis should be on their uniformity and size; the chemistry and morphology of the particles also deserve attention. Technologies to satisfy these conditions have been developed with an understanding of the principles for particle nucleation and growth in particle-forming polymerizations.

2.6. Variety Polymer particles can be prepared by physical methods, such as emulsification, coacervation, and spray-drying, and by chemical methods like heterogeneous polymerization. These preparative methods give a variety of particles in terms of size, surface chemistry, composition, surface texture, and morphology.

3. PREPARATION AND CHARACTERIZATION OF NANOPARTICLES 3.1. Cross-Linking of Amphiphilic Macromolecules Nanoparticles have been prepared from polysaccharides, proteins, and amphiphilic macromolecules by inducing their aggregation followed by stabilization by either heat denaturation or chemical cross-linking. It can be done by a water-in-oil emulsion system or in aqueous environment. The cross-linking technique was first used by Kramer et al. in 1974 [16]. In this technique, an aqueous solution of protein was emulsified in oil using a high-speed homogenizer/sonicator and the water-in-oil emulsion was then poured in a hot oil having a temperature greater than 100  C and held for a specific time (to denature the protein), thereby leading to the formation of submicroscopic particles. These particles were finally washed with organic solvents and subsequently collected by centrifugation. The crucial factors in nanoparticle production were emulsification energy and stabilization temperature; however, the limitation of high stabilization temperature was overcome by adding a chemical cross-linking agent (e.g., glutaraldehyde) to the system. To achieve the variable size of the nanosphere, several formulations were adopted and optimized. Protein and polysaccharide nanoparticles can be obtained by a phase separation process in aqueous medium. This can be induced by desolvation of the macromolecule, by change in pH or temperature, or by adding counterions in acid medium [17].

3 3.2. Polymerization of Acrylic Monomers Couvreur et al. [18, 19] carried out extensive investigations on nanoparticles and nanospheres composed of polyalkylcyanoacrylate (PACA) polymers, which are bioresorbable and were used as surgical glues for several years. PACA nanoparticles have been prepared by an emulsion polymerization method in which droplets of water-insoluble monomers are emulsified in aqueous/acidic phase containing a stabilizer. The monomers polymerize relatively faster by an anionic mechanism, the rate of polymerization being pH dependent. The system is maintained under magnetic agitation while the polymerization reaction takes place. The duration of polymerization reaction is determined by the length of the alkyl chain varying from 2 to 12 h for ethyl and hexylcyanoacrylate, respectively. Finally, the colloidal suspension is neutralized and lyophilized following incorporation of glucose as a cryoprotectant. Water-soluble drug may be associated with PACA nanosphere either by dissolving the drug in the aqueous polymerization medium or by incubating blank nanospheres in an aqueous solution of the drug. The drug loading efficiency is dependent on various factors, including the pKa and polarity of the drug, size and surface charge of the nanospheres, and the drug concentration in aqueous medium [18]. In another method of encapsulation of lipophilic drugs into PACA polymers, the monomers and the drug have been dissolved in a mixture of a polar solvent (acetone or methanol), an oil (coconut oil or benzyl benzoate), and a lipophilic surfactant, such as lecithin. The organic phase is added into aqueous phase having a hydrophobic surfactant (e.g., Poloxamer 188) under magnetic agitation. Thus, diffusion of the polar solvent into aqueous phase and the polymerization of the monomer at the oil–water interface take place simultaneously. Polymerization is initiated by the hydroxyl anions and leads to the formation of nanocapsules having an oily core surrounding by a polymer coat. The organic solvent is eliminated completely from the colloidal suspension. The selection of the oil has a great role to play, as it influences the size of the nanocapsules, the molecular weight of the polymer coat, and the stability of the suspension after storage [19].

3.3. Polymer Precipitation Solvent precipitation techniques have been generally applied for hydrophobic polymers, except for dextran nanospheres. Several techniques described in the literature are based on the mechanism of polymer precipitation.

3.3.1. Solvent Extraction–Evaporation In this technique, hydrophobic polymer is dissolved in an organic solvent, such as chloroform, ethyl acetate, or methylene chloride, and is emulsified in an aqueous phase having a stabilizer [e.g., polyvinyl alcohol (PVA)]. Just after the formation of nanoemulsion, solvent diffuses to the external phase until saturation. The solvent molecules that reach on the water–air interphase evaporate, which leads to continuous diffusion of the solvent molecules from the inner

4 droplets of the emulsion to the external phase. Simultaneously, the precipitation of the polymer leads to the formation of nanospheres. The extraction of solvent from the nanodroplets to the external aqueous medium can be induced by adding an alcohol (e.g., isopropanol), thereby increasing the solubility of the organic solvent in the external phase. A purification step is required to assure the elimination of the surfactant in the preparation. This technique is most suitable for the encapsulation of lipophilic drugs, which can be dissolved in the polymer solution.

3.3.2. Solvent Displacement or Nanoprecipitation In this method, the organic solvent selected is completely dissolved in the external aqueous phase; thus there is no need for evaporation or extraction for polymer precipitation. Polymer and drug are dissolved in acetone, ethanol, or methanol and incorporated under magnetic stirring into an aqueous solution of surfactant. The organic solvent diffuses instantaneously to the external aqueous phase followed by precipitation of the polymer and drug. After the formation of nanoparticles, solvent is eliminated and the suspension concentrated under reduced pressure. This method is surfactant-free. However, this method is limited only to the drugs that are highly soluble in a polar solvent.

3.3.3. Salting Out A technique based on the precipitation of a hydrophobic polymer is useful for the encapsulation of either hydrophilic or hydrophobic drugs because of a variety of solvents, including polar (e.g., acetone or methanol) and nonpolar (methylene chloride or chloroform) solvents, can be chosen for dissolving the drug. This procedure is just like nanoprecipitation; however, the miscibility of both phases is prevented by the saturation of the external aqueous phase with electrolytes. Precipitation occurs when a sufficient amount of water is added to allow complete diffusion of the acetone in the aqueous phase.

3.4. Nanocapsule Preparation We considered PACA while discussing the preparation and characterization techniques of nanoparticles for the sake of convenience. Nanocapsules of PACA are obtained via an interfacial polymerization process in emulsion. Nanocapsules are formed by mixing an organic phase with an aqueous phase. The organic phase is generally an ethanolic solution of the monomer mixed together with the oily core material and the lipophilic drug to be encapsulated and occasionally, soya bean lecithin is added as an additional surfactant. Oils, viz., Miglyol, benzylbenzoate, ethyl oleate and lipiodoal, have been frequently used in preparation of nanocapsules. The encapsulation efficiency of a lipophilic drug is dependent on its partition coefficient between the oil and the aqueous phase, so the oil must be chosen accordingly. The aqueous phase is a solution of a nonionic surfactant, usually Synperonic PE-F68 at 0.5% at a pH between 4 and 10. Nanocapsules are formed by adding the organic phase dropwise into the aqueous phase under stirring, through a wide-bore syringe needle or a

Polymeric Nanoparticles for Drug and Gene Delivery

micropipette tip. The mixture immediately becomes opalescent. After stirring for 15–30 minutes the ethanol is removed by evaporation under reduced pressure. If required, the nanocapsules can be further concentrated by evaporation; this allows them to be rediluted in a physiological buffer for injection. Nanocapsules formed by this way have a mean diameter between 200 and 300 nm with a narrow polydispersity [20]. The speed of the magnetic stirring has no influence on the particle size, which depends solely on the nature and the volume of the oil and on the volume of the diffusing organic phase. The proportion of the monomer must be correctly chosen to avoid simultaneous formation of either flakes of polymer or of a single oily emulsion. The presence of surfactant in the aqueous phase is, in fact, not necessary for the successful formation of nanocapsules but does prevent them from aggregating on storage to a cake, which is difficult to disperse. Nanocapsules formed in this way are physically stable for several years at ambient temperature and may be sterilized by autoclaving at 120  C for 20 min. However, as a result of their vesicular character, nanocapsules are not easily lyophilized, since they tend to collapse releasing the oily core [20]. The colloids formed by the interfacial polymerization of PACA could be influenced by many factors, viz. the nature of the aqueous phase, the pH, the composition of the organic phase (monomer, oil, ethanol), the ratio of monomer to the aqueous phase, and the emulsification conditions. The degree of polymerization, and therefore the molecular weight, depends on a balance between initiation, propagation, and termination [21]. The number of growing chains depends on the concentration of initiators. For the same quantity of monomer, when the number of live chains is high, the degree of polymerization (DP) is low. However, DP is reduced when the concentration of the terminating agents is high [22, 23]. The concentration of the initiating and the terminating agents available for the monomer depends on the physicochemical nature of the system in which the components are dispersed at the moment of the formation of the colloid. In the case of a system composed of two immiscible phases, such as an emulsion, the presence and the concentration of the solutes in the different phases is a function of the polarity of the solute molecules and of the dielectric constant of the medium. In contrast, at an interface between an aqueous and an organic medium, there can be large local variations in properties, which can themselves cause changes in the interfacial properties in a dynamic system. Nevertheless, the degree of polymerization depends on the propagation reaction; other characteristics of the colloids, viz. particle size and morphology, depend on interfacial phenomena inducted by the dynamic mixing of an organic phase with an aqueous phase. Since the preparation of nanocapsules of PACA is rather similar to the preparation of PACA nanospheres, it is necessary to verify that the colloidal suspension does not consist of a simple mixture of an oil-in-water emulsion containing polymeric nanospheres of similar size. Rollot et al. by their various experiments confirmed that the preparation indeed was composed of oil-filled nanocapsules [24].

Polymeric Nanoparticles for Drug and Gene Delivery

3.5. Nanosuspensions The nanosuspension technique is an alternative and promising approach for the production of drug nanoparticles, where the drugs are poorly soluble in both aqueous as well organic medium. The major advantages of this technology are its general applicability to most of the drugs and its easy fabrication/handling. The disintegration principle for obtaining nanosuspensions is the cavitation forces in high pressure homogenizers (e.g. piston-gap homogenizers like APV gaulin types). The preparation method involves the dispersion of the drug powder in an aqueous surfactant solution by high speed stirring. The obtained macrosuspension is then passed through a high pressure homogenizer applying typically 1500 bar and 3 to 10 up to a maximum of 20 passes (=homogenization cycles). The suspensions pass a very small homogenization gap in the homogenizer, typically having a width of 25
m at 1500 bar. Due to the narrowness of the gap the streaming velocity of the suspension increases tremendously with an increase in fluid pressure. Simultaneously, the static pressure on the fluid decreases below the boiling point of water at room temperature [25, 26]. As a consequence, water starts boiling at room temperature due to the high pressure; gas bubbles are formed which implode cavitation when the fluid leaves the homogenization gap. These cavitation forces are strong enough to break the drug microparticles to drug nanoparticles [25]. The mean particle size in the nanometer range obtained by this procedure depends on the pressure and number of cycles applied; in addition it is affected by the hardness of the drug itself. Mean diameters of 330 and 600 nm are reported for paclitaxel nanosuspensions [26] and clofazemine [27] respectively. Müller and co-workers pioneered the field of nanosuspensions [28–32]. Müller [33] et al. in their recent article reviewed the preparation of nanosuspensions on a laboratory scale, and large scale, physical and chemical properties of nanosuspensions, surface modification of nanosuspensions, biological properties, and perspectives were also discussed.

3.6. Characterization Nanoparticles as a colloidal carriers mainly depend on the particle size distribution, surface charge, and hydrophilicity. These physicochemical properties affect not only drug loading and release, but also the interaction of these particulate carriers with biological membranes.

3.6.1. Particle Size Analysis Two main techniques have been used to determine the particle size distribution of colloidal systems: photon correlation spectroscopy (PCS) and electron microscopy including both scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The quasi electron light scattering technique for Brownian moment measurement offers an accurate procedure for measuring the size distribution of nanoparticles. The PCS technique does not require any particular preparation for analysis and is excellent due to its efficiency and accuracy. However, its dependency on Brownian movement of particles in suspended medium may affect the particle size determination.

5 Electron microscopy provides an image of the particles to be measured. In particular, SEM is used for vacuum dried nanoparticles that are coated with a conductive carbon–gold layer for analysis and TEM is used to determine the size, shape, and inner core structure of the particles. TEM in combination with freeze–fracture procedures differentiates among nanocapsules, nanospheres, and emulsion droplets. Atomic force microscopy (AFM) is an advanced microscopic technique and its images can be obtained in aqueous medium. AFM images nowadays are powerful support for the investigation of nanoparticles in biological media.

3.6.2. Surface Charge and Hydrophobicity The interaction of nanosphere with biological environment and electrostatic interaction with biological compounds occur due to the charge on the surface (e.g., negative charge promotes the adsorption of positively charged drug molecules such as aminoglucosides as well as enzymes and proteins). The surface charge of colloidal particles can be determined by measuring the particle velocity in an electric field. Nowadays laser light scattering techniques, in particular laser Doppler anemometry, are fast enough to measure the surface charge with high resolution. Hydrophobicity of the nanoparticles can be determined by the methods including adsorption of hydrophobic fluorescent or radiolabeled probes, two phase partitions, hydrophobic interaction chromatography, and contact angle measurements. Recently, X-ray photoelectron spectroscopy has been developed which offers the identification of chemical groups in the 5-Å-thick coat on the external surface of nanospheres. Gref et al. [34] have characterized the poly(ethylene glycol) (PEG)-coated poly(lactide-co-glycolide) (PLGA) nanosphere and identified the PEG chemical elements that were concentrated on the nanosphere’s surface.

3.6.3. Methods of Changing Particle Size and Surface Characteristics The fate of colloidal particles inside the body depends on three factors: particle size, particle charge, and surface hydrophobicity. Particles with a very small size (less than 100 nm), low charge, and a hydrophilic surface are not recognized by the mononuclear phagocytic system and, therefore, have a long half-life in the blood circulation. In general, nature and concentration of the surfactant play an important role in determining the particle size, as well as the surface charge (e.g., nanospheres with mean size of less than 50 nm were prepared by increasing concentration of Poloxamer 188 [35]). The approaches for modifying surface charge and hydrophilicity were initially based on the adsorption of hydrophilic surfactants, such as block copolymers of the poloxamer and poloxamine series. The in vivo studies of hydrophilic nanospheres limit their usefulness due to their toxicity in intravenous injection. Lately, the idea of using diblock copolymers made of poly(lactic acid) (PLA) and poly(ethylene oxide) (PEO) is widely accepted due to the safety and stability of the hydrophilic coat. For this purpose, the copolymer is dissolved in an organic solvent and then emulsified in an external aqueous phase, thereby orienting the PEO toward the aquous surrounding medium, while in

6 another method PLA–PEO copolymer is adsorbed onto preformed PLGA nanoparticles. It is found to be efficient in prolonging the nanosphere circulation time following intravenous administration.

3.7. Drug Incorporation and Adsorption For a nanoparticulate system to be highly successful it should have a high drug loading capacity, which in turn reduces the quantity of carrier required for administration. Two theoretical curves can be proposed to describe the adsorption of drugs onto nanoparticles: Langmuiriantype and constant partitioning-type isotherms. In fact, it was found that nanoparticles can entrap a drug according to a Langmurian adsorption mechanism, because of their large specific area [36]. The drug can either be incorporated into nanospheres during the polymerization process or adsorbed onto the surface of preformed particles. There are reports on vidarabine, an antiviral agent, whose nucleophile N in positions 3 and 7 may play a role of initiator for the anionic polymeric mechanism of the cyanoacrylic monomer [37], which suggests drug–polymer interaction as a covalent linkage. A similar study in this direction was described with peptide compounds such as growth hormone-releasing factor (GRF) [38]. In these studies it was demonstrated that if the drug was added within 5 min of the polymerization process, 50% of the peptide was found to be covalently linked to the polymer. In contrast to these findings, the drug loading capacity was found to be very low when the peptide was added after 60 min during polymerization, but no chemical modification was observed. These findings suggest that there is a narrow window of time for the addition of GRF to the polymerization medium resulting satisfactory drug-loading capacity as well as preservation of chemical structure of the peptides [38]. Generally, the longer the alkyl chain length, the higher the affinity of the drug. Moreover, the Langmurian isotherm states that the percentage of drug adsorption generally decreases with the quantity of drug dissolved in the polymerization medium [39]. Reports were also available on the attempts made for the association of synthetic fragments of deoxy ribonucleic acid (DNA) to PACA nanospheres [39]. The association of antisense oligonucleotides with nanoparticles was achieved only in the presence of a hydrophobic cation, such as triphenylphosphonium or quaternary ammonium salts [40]. The poor yield of oligonucleotide association without cations could be explained by the hydrophilic character of nucleic acid chains that are known to be soluble in water. The adsorption efficiency of oligonucleotide–cation complexes on nanospheres was found to be highly dependent on several parameters: oligonucleotide chain length, nature of the cyanoacrylic polymer, hydrophobicity of the cations used as ion-pairing agents, and ionic concentration of the medium [39].

3.7.1. Biodegradation and Drug Release The rate of cyano copolymer degradation is dependent on their alkyl chain length. The dominating mechanism of particle degradation was found to be surface erosion [40], resulting via the hydrolysis by the enzymes of the ester side chain of the polymer [41]. This process keeps the polymer

Polymeric Nanoparticles for Drug and Gene Delivery

chain intact, but it becomes more and more hydrophilic until it is water soluble. The degradation pathway is consistent with the production of alcohol during the bioerosion of PACA nanospheres in vitro in the presence of esterases [41]. Indeed, the action of rat liver microsomes and tritosomes on the ester hydrolysis of polyisobutylcyanoacrylate nanospheres was clearly demonstrated [41]. Since the biodegradability of polyalkycyanoacrylate depends on the nature of the alkyl chain, it is possible to choose a monomer whose polymerized form has a biodegradability corresponding to the established program for the drug release [41]. Lenaerts et al. [42] demonstrated that the drug release from nanospheres was a direct consequence of the polymers bioerosion, by using a double radiolabeled technique. Later this was confirmed using GRF, another model drug [42]. No drug release was reported in the absence of esterase with this peptide compound, whereas in the nanosphere suspension turbidity remained unchanged, indicating no polymer bioerosion. Drug release appeared to be esterase dependent. In other studies, dextran sulfate was employed as a stabilizer in the preparation of PACA nanospheres, which slowed down the release rates of drug rose Bengal [42].

4. RECENT DEVELOPMENTS IN NANOPARTICLE TECHNOLOGY Other than the commonly used synthetic hydrophobic polymers, various other polymers like chitosan, sodium alginate, gelatin, etc. are also being explored in drug delivery [43]. A drug delivery system is most often associated with fine particulate carriers, such as emulsion, liposomes, and nanoparticles, which are designed to localize drugs in the target site. From the clinical point of view, they might have to be biodegradable and/or highly biocompatible. In addition, high drug content is desirable, because in many cases actual drug loading efficiency is often too low to secure an effective dose at the target site. Biodegradable nanoparticles have received considerable attention as potent vehicles for targeting a site and controlled release of drugs/bioactive components [43–47]. Various nanoparticlulate systems composed of different materials are constantly being explored in the areas of drug and gene delivery as shown in Table 1. We hereby discuss some nanoparticulate systems developed by various researchers.

4.1. Doxorubicin Nanoparticle Conjugates Natural and synthetic polymers have widely been used for conjugating the drugs for improved circulating times in the bloodstream, targeting a specific organ, and sustained release at the injection site [48–50]. The polymer conjugation approach, along with formulations with polymeric micelles and liposomes, is an attractive means to selectively suppress tumor growth [51, 52]. Some of the earlier studies suggested the use of high molecular weight polymers would eventually cause problems [53, 54]. Yoo et al. [55] reported doxorubicin-poly(d, l-lactic-co-glycolic acid) (PLGA) conjugates (Fig. 4). Doxorubicin was chemically conjugated to a terminal end group of PLGA by an ester linkage and the doxorubicin–PLGA conjugate was later formulated into nanoparticles. They have observed a 1-month release of

Polymeric Nanoparticles for Drug and Gene Delivery

7

Table 1. Nanoparticle types and their applications. Nanoparticles

Application

Poly(lactide-co-glycolide) Chitosan Solid lipids Liposomes Block copolymers Poly(ethylene glycol) Polycaprolactone Polycyanoacrylate Dextran Poly-l-lysine Silica Gelatin Poly(aspartic anhydride-co-ethylene glycol) Atelocollagen Alginate

drug/gene delivery drug/gene delivery drug/gene delivery drug/gene delivery drug/gene delivery drug/gene delivery drug delivery drug/gene delivery drug/gene delivery drug/gene delivery drug/gene delivery drug/gene delivery gene delivery drug/gene delivery drug/gene delivery

conjugated doxorubicin from the nanoparticles vs a 5 day release of unconjugated doxorubicin. In vivo antitumor activity assay also showed that a single injection of the nanoparticles had comparable activity to that of free doxorubicin administered by daily injection [55]. Recently, Mitra et al. [56] and Janes et al. [57] reported dextran–doxorubicin conjugates using chitosan as the nanoparticle carrier.

4.2. Lipid Coated Cisplatin Nanocapsules Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The poor solubility of the cisplatin results in low O

OH

O

OH

O

O

OH

OH

OH OMeO

OH Fmoc-OSu, DMF

OH

OH

OMeO

3 h, 88%

O

O

H 3C

H 3C NH 2

NHFmoc

OH

OH

PLGA500 5 PyBroP

O

OH

DMAP, TEA CH 2 Cl2 , 15 h O

O O O

OMeO

O

O

OH

H n

CH 3

OH O Piperidine 5 min DMF

H 3C NHFmoc OH O

OH

O

O O

O

OH O OMeO

O

H n

encapsulation efficiency [58, 59]. Researchers have tried to use lipophilic derivatives of cisplatin to improve the encapsulation efficiency [60]. Recently, Burger et al. [61] reported a novel procedure to efficiently encapsulate native, nonderivatized cisplatin in lipid formulations. The methodology is based on simple freeze thawing of concentrated solution of cisplatin in the presence of negatively charged phospholipids [61]. The authors claim this procedure as novel with maximum encapsulation efficiency with cisplatin aggregates being covered with a single lipid bilayer. It was found that the in vitro cytotoxicity was raised 1000-fold with this new technology [61].

4.3. Poly(dl-lactide-co-glycolide) Nanoparticles The most widely used emulsion solvent evaporation method for preparation of nanoparticles using PLGA requires surfactants to stabilize the dispersed particle [62]. This method often has a problem in that the surfactant remains at the surface of the particles and hence is difficult to remove especially where PVA is used as surfactant. Other surfactants such as span series or tween series, PEO, etc. are also used to stabilize with some disadvantages like removal of solvents, toxicity, low particle yield, consumption of more surfactant, and multisteps. The most important factor that needs to be considered while using surfactants, is that they are nonbiodegradable and nondigestible and tend to affect humans with allergic reactions. Recently, Jeon et al. [63] proposed a surfactantfree method for preparation of PLGA nanoparticles as an alternative. The surfactant-free PLGA nanoparticles were prepared by dialysis method using various solvents and their physiochemical properties were investigated against used solvent. Release kinetics of norfloxacin showed that a higher drug content leads to larger particle size and slow release [63]. Kwon et al., [64] reported estrogen loaded PLGA nanoparticles employing emulsification-diffusion method using PVA or didodecyl dimethylammonium bromide (DMAB) as stabilizers. They have studied the influence of process variables on the mean particle size of the nanoparticles. The particle size was less than 100 nm when DMAB was used a stabilizer [64]. Stabilizers like D--tocopheryl polyethylene glycol 1000 succinate (vitamin E-TPGS) were also tried by other groups [65]. Santos-Magalhaes et al. [66] reported PLGA nanocapsules/nanoemulsions for benzathine pencillin G. Nanoemulsions were produced by spontaneous emulsification and nanocapsules by interfacial deposition of preformed polymer. They have observed similar release kinetics from both formulations [66].

CH 3

OH O

H 3C NH 2 OH

Figure 4. Synthetic route of DOX–PLGA conjugate. Reprinted with permission from [55], H.-S. Yoo et al., J. Control. Release 68, 419 (2000), © 2000, Elsevier Science.

4.4. Poly(ethylene oxide)-poly(l-lactic acid)/poly(
-benzyl-l-aspartate) Polymeric micelles are expected to self-assemble, when block copolymers are used for their preparation [67]. Micelles of biocompatible copolymer, viz., PEO with PLA or with poly(-benzyl-l-aspartate) (PBLA), have been reported in the literature [68, 69]. The synthetic process of

Polymeric Nanoparticles for Drug and Gene Delivery

8

polypropylene (PPO)-PEO block copolymer or with tetrafunctional (PEO-PPO)2 –N -CH2 -CH2 -N –(PPO-PEO)2 [72]. Such coats are bound to the core of the nanosphere by the hydrophobic interactions of the PPO chains, while PEO chains protrude into the surrounding medium and form a steric barrier, which hinders the adsorption of certain plasma proteins onto the surface of such particles. On the other hand, the PEO coat enhances adsorption of certain other plasma compounds. As a consequence, the PEO-coated nanospheres are not recognized by macrophages as foreign bodies and are not attacked by them [73].

O CH2COCH2

O

HN O β-benzyl-L-aspartate-N-carboxyanhydride (BLA-NCA) CHCl3 / DMF R ~ H or CH3 350

RO-(-CH2CH2O-)m-(CH2)3-S-(CH2)2NH2 α-hydroxy-ω-amino PEO and α-methoxy-ω-amino PEO

(a)

O

C2H5-O

C2H5-O CHCH2CH2OH

C2H5-O H 2C

C2H5-O C2H5-O

C2H5-O

CHCH2CH2OK

C2H5-O

C2H5-O

CHCH2CH2O[CH2CH2O]m-K

C2H5-O C2H5-O

CHCH2CH2O[CH2CH2O]m-[COCH(CH3)O-]n-K

sonification in water C2H5-O C2H5-O

Allock and co-workers developed derivatives of the phosphazene polymers suitable for biomedical applications [74, 75]. Long-circulating in the blood, 100– 120 nm in diameter, PEO-coated nanoparticles of the poly(organophospazenes) containing amino acid have been prepared. PEO–polyphosphazene copolymer, or poloxamine 908 (a tetrafunctional PEO copolymer), has been deposited on their surface [76]. Chemical formulae of such polyphosphazene derivatives are shown in Figure 6.

C+ O O

CHCH2CH2O[CH2CH2O]m-K

CHCH2CH2OK

CH 2 O

CHCH3

C2H5-O

4.6. Polyphosphazene Derivatives

, K+

+

C2H5-O

CH2C-O

RO-(CH2CH2O)n-(CH2)3-S-(CH2)2NH-(COCHNH)m-H CH2

CHCH2CH2O[CH2CH2O]m-[COCH(CH3)O-]n-H Micelles with acetal groups on their surface H+ freeze dried micelles dissolved in H2O and acidified with HCl

4.7. Poly(ethylene glycol) Coated Nanospheres Nanospheres of PLA, PLG, or poly( -caprolactone) coated with PEG may be used for intravenous drug delivery. PEG and PEO denote essentially identical polymers. The only difference between the respective notations is that methoxy groups in PEO may replace the terminal hydroxyls of PEG. PEG coating of nanospheres provides protection against interaction with the blood components, which induce removal of the foreign particles from the blood. PEG

O=CHCH2CH2O-[-CH2CH2O-]m-[-COCH(CH3)O-]n-H

(b) Figure 5. (a) Poly(ethylene oxide)-co-b-benzyl-l-aspartate and (b) poly(ethylene oxide)-co-l-lactide micelles with aldehyde groups on their surface. Reprinted with permission from [67], J. Jagur-Grodzinski, React. Funct. Polym. 39, 99 (1999). © 1999, Elsevier Science.

such nanospheres with functional groups on their surface is shown in Figure 5. Aldehyde groups on the surface of the PEO–PLA micelles might react with the lysine residues of a cell’s proteins and may facilitate attachment of the amino-containing ligands. These hydroxyl groups on the surface of the PEO– PBLA micelles can be further derivatized and conjugated with molecules capable of targeting the modified micelles to specific sites of a living organism. Such nanospheres have been tested as vehicles for delivery of anti-inflammatory and antitumor drugs [70, 71].

4.5. Poly(lactide-co-glycolide)–[(propylene oxide)-poly(ethylene oxide)] Biocompatible and biodegradable poly(lactide-co-glycolide) (PLG) nanoparticles (80–150 nm) have been prepared by the following nanoprecipitation technique [72]. The nanoparticles were coated with a 5–10 nm thick layer of

NH(R')OC2H5 N

P

NH(R')OC2H5 R = 40% C H - C + 60% C H2C

n

O

O

(a)

NH(R") 95% CH2COC2H5 P

N

NH(R") n

O R" = + 5% (CH2CH2O)m

n= 7000 m= 60

(b) Figure 6. Structures of polyphosphazenes for medical applications. (a) Poly([(phenylethylalanine ethylester)40%(glycine ethyl ester)60%] phosphazene) PF(GL-PhAL). Reprinted with permission from [75], H. R. Allock et al., Macromolecules 10, 824 (1997). © 1997, American Chemical Society.

Polymeric Nanoparticles for Drug and Gene Delivery

9

coated nanospheres may function as circulation depots of the administered drugs [34, 77]. Slow release of the drugs into plasma alters the concentration profiles leading to therapeutical benefits. PEG-coated nanospheres (200 nm), in which PEG is chemically bound to the core, have been prepared, in the presence of monomethoxy PEG, by ring opening polymerization (with stannous octoate as a catalyst) of such monomers as -caprolactone, lactide, and/or glycolide [77]. Ring opening polymerization of these monomers in the presence of such multifunctional hydroxy acids as citric or tartaric acid, to which several molecules of the monomethoxy monoamine of PEG (MPEG-NH2  have been attached, yields multiblock (PEG)n –(X)m copolymers. PEG– PLA copolymer in which NH2 terminated methoxy PEG molecules have been attached to tartaric acid is shown in Figure 7. It has been demonstrated that morphology, degradation, and drug encapsulation behavior of copolymers containing PEG blocks strongly depends on their chemical composition and structure. Studies of nanoparticles composed of the diblocks of the PLG with the methoxy terminated PEG (PLG–PEG) or of the branched multiblocks PLA–(PEG)3 , in which three methoxy terminated PEG chains are attached through a citric acid residue, suggested that they have a corecorona structure in an aqueous medium. The polyester blocks form the solid inner core. The nanoparticles, prepared using equimolar amounts of the PLLA–PEG and PDLA–PEG stereoisomers, are shaped as discs with PEG chains sticking out from their surface. Their hydrophobic/hydrophilic content seems to be just right for applications in cancer and gene therapies. Such nanospheres are prepared by dispersing the methylene chloride solution of the copolymer in water and allowing the solvent to evaporate [77]. By attaching biotin to its free hydroxyl groups and complexing it with avidin, cell specific CH3(OC2H4) NHCO

COOH

n

CHOH

2 CH3(OC2H4) NH2 + CHOH n

CHOH

CHOH

CH3(OC2H4) NHCO n

COOH

H3CHC

CO

O

CH3(OC2H4) NHCO n

CH3

CH3

CHO(COCHO)

COCHOH

CHO(COCHO)

COCHOH

m-1

m-1

CH3(OC2H4) NHCO n

CH3

CH3

Figure 7. Multiblock (PEG)n –(X)m copolymers. Amino terminated methoxy poly(ethylene glycol) molecules attached to tartaric acid with PLA side chains. Reprinted with permission from [77], R. Gref and A. J. Domb, Adv. Drug Del. Rev. 16, 215 (1995). © 1995, Elsevier Science.

delivery may be attained. Nuclear magnetic resonance studies of such systems [78] revealed that the flexibility and mobility of the thus attached PEG chains are similar to those of the unattached PEG molecules dissolved in water. Recently, PLG microspheres, with the PEG–dextran conjugates attached to their surface, have been investigated as another variant of the above-described approach. Microspheres with a diameter of 400–600 nm have been prepared [79].

4.8. Poly(isobutylcynoacrylate) Nanocapsules Intragastric administration of insulin-loaded poly(isobutylcyanoacrylate) nanocapsules induced a reduction of the glycemia to normal level in streptozotocin diabetic rats [80] and alloxan induced diabetic dogs [81]. The hypolglycemic effect was characterized by surprising events incl