Preparation, characterization and biodistribution of ultrafine chitosan nanoparticles

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Abstract

Chitosan nanoparticles cross-linked with glutaraldehyde have been prepared in AOT/n hexane reverse micellar system. The cross-linking in the polymeric network has been confirmed from FTIR data. Because of the adhesive nature of these particles, their sizes, as measured by QELS, have been found dependent on the particle density in aqueous buffer. The particle size has also been found to vary with the amount of cross-linking. The actual particle size of these chitosan nanoparticles with a particular degree of cross-linking has been determined at infinite dilution of particles in water. The particle size at infinite dilution is ≈30 nm diameter, when 10% of the amine groups in the polymeric chains have been cross-linked and it shoots up to 110 nm diameter when all the amine groups are cross-linked (100% cross-linked). TEM pictures show that these particles are spherical in shape and remain in the form of aggregation. The biodistribution of these particles after intravenous injections in mice showed that these particles readily evade the RES system and remain in the blood for a considerable amount of time. The γ image of the rabbit after administration of 99mTechnetium (99mTc) tagged chitosan nanoparticles also confirms the above observation, as the blood pool is readily visible even after 2 h. The γ picture shows distribution of particles in the heart, liver, kidneys, bladder and the vertebral column. Interestingly, the biodistribution studies of the chitosan nanoparticles have indicated that these particles are distributed in the bone marrow also, implying the possibility of using these nanoparticles for bone imaging and targeting purpose.

Introduction

Chitosan is an interesting natural material occurring in abundance in the environment. Its excellent biocompatibility and several advantages due to its unique polymer cationic character renders it highly useful for pharmaceutical application (Thanoo et al., 1992, Illum, 1998). A polysaccharide comparable to cellulose, comprising copolymers of glucosamine and N-acetyl glucosamine linked by β-(1–4) linkages, chitosan can be derived by partial deacetylation of chitin from crustacean shells. The primary amino groups lead to special properties that render chitosan very interesting for pharmaceutical applications (Kristl et al., 1993). In contrast to most other natural polymers, it has a positive charge and is mucoadhesive (Berscht et al., 1994).

Besides other applications (Felt et al., 1998), chitosan has been extensively examined for its potential in the development of controlled release drug delivery systems (Kawashima et al., 1985, Thanoo et al., 1992, Akbuga, 1993) and these controlled release formulations have been made in the form of chitosan gels (Kristl et al., 1993), tablets (Nigalaye et al., 1990) and capsules (Tozaki et al., 1997), in addition to microspheres and microcapsules (Nishioka et al., 1990, Aiedeh et al., 1997). Nanoparticles have a special role in targeted drug delivery in the sense that they have all the advantages of liposomes including the particle size, but unlike liposomes, nanoparticles have a long shelf life and can usually entrap more drugs than liposomes. Although nanoparticles made of hydrophobic polymers encapsulating hydrophobic drugs have been reported in the literature, very few studies have been made on the preparation of drug-loaded hydrogel nanoparticles (Mitra et al., 2002). Nanoparticles made of hydrophobic polymers are usually taken up by RES and have short residence time in blood (Douglas et al., 1986). The surfaces of these hydrophobic nanoparticles are made hydrophilic by conjugating with the polyethylene glycol (PEG) type of molecules to make these particles long circulating in blood. We have recently optimized a method of preparation of ultra low size nanoparticles of hydrogel polymers and have been able to load water-soluble drugs into them (Maitra et al., 1999). These hydrophilic nanoparticles evade RES and remain in circulation for a couple of hours without PEG conjugation on the particle surface. These hydrogel polymers can have reactive groups on the surface which enable the nanoparticles to be converted to stimuli responsive particles and they can also be made targetable by attaching receptor specific ligands. The preparation of nanoparticles from chitosan, a hydrogel polymer, for drug delivery can have several advantages over the use of chitosan microspheres and microcapsules. Nanometer range particles have easy accessibility in the body, being transported via the circulation to different body sites. Extremely small nanoparticles, of <100 nm diameter with hydrophilic surface, have been found to have longer circulation in blood (Allemann et al., 1993). Such systems should allow the control of the rate of drug administration that prolongs the duration of the therapeutic effect, as well as the targeting of the drug to specific sites.

Preparation of nanoparticles with this versatile hydrogel material has been attempted by several workers with the purpose of utilizing its mucoadhesive properties to transport drugs and DNA across mucosal surfaces (Alonso et al., 1998, Roy et al., 1999). The polycationic nature of deacetylated chitosan results in polycondensation in the presence of anionic macromolecules. Thus, chitosan–DNA nanoparticles can be prepared by the method of coacervation (Roy et al., 1999). Besides, the reactive free amino group on the particle surface makes it possible to chemically conjugate various other reactive groups, such as polyethylene glycol (PEG) derivatives, different ligands, antibodies and other pH and temperature sensitive moieties (MacLaughlin et al., 1998). Several methodologies have also incorporated other polymers in the preparative procedure, with the intent of preparing smart hydrophilic nanoparticles under extremely mild conditions (Calvo et al., 1997).

Considering the advantages of using ultra low sized hydrophilic nanoparticles for drug delivery, we have focussed on the preparation of chitosan nanoparticles of <100 nm diameter. Preparation of nanoparticles using reverse micelles as a medium makes it possible to produce ultrafine particles with narrow size distribution (Leong and Candau, 1982, Munshi et al., 1995). The aqueous core of the reverse micellar droplets can be used as nanoreactor to prepare these particles, since the size of the reverse micellar droplets is in the nanometer range and these droplets are highly monodispersed (Maitra, 1984). The present paper describes the preparation of cross-linked chitosan nanoparticles of size <100 nm diameter using reverse micelles as the media and their physicochemical characterization as well as biodistribution.

Section snippets

Materials

Chitosan (M.W. 400 kDa), glutaraldehyde, surfactant i.e. sodium bis(ethylhexyl) sulfosuccinate (AOT), were purchased from Sigma (St. Louis, MO). All other reagents used were of analytical grade. Sodium pertechnetate separated from Molybdenum -99 by solvent extraction method was procured from the Regional Center for Radiopharmaceutical (Northern Region), Board of Radiation and Isotope Technology, Delhi, India.

Preparation of cross-linked chitosan nanoparticles

Chitosan nanoparticles were prepared by modifying the reverse micelle medium using a

Size of the nanoparticles

We have determined the particle size by quasi-elastic light scattering measurements. A representative spectrum is shown in Fig. 2. Chitosan is an adhesive hydrogel polymer (Robert et al., 1988). Therefore, in aqueous solution, these nanoparticles interact among themselves through their Brownian motion to form clusters. For interacting particles, the average particle size is always found higher than the actual size of the particles. The diffusion coefficient, D, of these interacting particles

Conclusions

Ultra-low size (<100 nm diameter) nanoparticles of water-soluble hydrogel polymer, such as chitosan, can be prepared in the aqueous core of reverse micellar droplets and can be cross-linked through glutaraldehyde. The size of the particles depends on the particle density as well as on the degree of cross-linking. For a particular degree of cross-linking, the particle size has been determined at infinite dilution of the particle solution. The particles have high level of radio labeling

Acknowledgements

Dr T. Lazar Mathew, ex-Director, INMAS and Gen. T. Ravindranath, Director, INMAS are gratefully acknowledged for extending help and for their continuous encouragement and assistance throughout this study.

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