Chitosan Nanoparticles for Non-Viral Gene Therapy - ACS Publications

Chapter 9

Chitosan Nanoparticles for Non-Viral Gene Therapy 1

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Downloaded by STANFORD UNIV GREEN LIBR on July 27, 2012 | http://pubs.acs.org Publication Date: June 22, 2006 | doi: 10.1021/bk-2006-0934.ch009

Julio C. Fernandes , Marcio José Tiera , and Françoise M . Winnik

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Orthopedic Research Laboratory, Hôpital du Sacré-Cœur de Montréal, Université de Montréal, Montréal, Québec H4J 1C5, Canada Departamento de Química e Ciências Ambientais, UNESP-Universidade Estadual, Paulista, Brazil Faculty of Pharmacy and Department of Chemistry, Université de Montréal, Montréal, Québec, Canada

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The cationic polysaccharide chitosan has been widely used for non-viral transfection in vitro and in vivo and has many advantages over other polycations. Chitosan is biocompatible and biodegradable and protects D N A against DNase degradation. However following administration the ChitosanD N A polyplexes must overcome a series of barriers before D N A is delivered to the cell nucleus. This paper describes the most important parameters involved in the chitosan-DNA interaction and their effects of on the condensation, shape, size and protection of D N A . Strategies developed for chitosanD N A polyplexes to avoid non-specific interaction with blood components and to overcome intracellular obstacles as the crossing of the cell membrane, endosomal escape and nuclear import are presented.

© 2006 American Chemical Society

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In Polysaccharides for Drug Delivery and Pharmaceutical Applications; Marchessault, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

178 Introduction


Basic Concepts Gene therapy involves the introduction of exogenous genes into target cells where production of the encoded protein will occur. In the case of acquired or inherited genetic disorders, this enables the replacement of a missing or defective gene, leading to normal cell function. Moreover, the introduction of D N A encoding antigenic proteins into cells has been studied for immunotherapy of cancer and viral infections (1,2). It is also considered a promising approach for tissue engineering, especially in the field of orthopedics, where the delivered gene could be used as a short-term local enhancer for repair of injured musculoskeletal tissue (3). D N A is delivered to cells either by the viral or nonviral systems. Viral vectors include retroviruses, adenoviruses, adenoassociated viruses, herpes simplex virus and lentivirus (4). Naked D N A , cationic liposomes, cationic lipids and polymers, as well as DNA/cationic liposome/polycation complexes are utilized in the nonviral approach (5-7). Although viral systems have demonstrated higher transfection efficiency compared to non-viral vectors, they suffer from a number of drawbacks that severely hinder their use in vivo (8). Such limitations include their rapid clearance from the circulation, the reduced capacity to carry a large amount of genetic information and the associated risks of toxicity and immunogenicity, which limits the possibility of subsequent administrations. The main advantages of the non-viral method resides in its being a safer alternative, demonstrating no immunogenicity, negligible toxicity, having the ability to carry large therapeutic genes and a reduced production cost (9,10). For these reasons, there is an increased interest in the development of a safe and efficient non-viral gene delivery system that can circumvent the limitations seen with the viral approach. However to effectively produce a therapeutic effect a non viral vector must overcome multiple biological barriers, ie, i) avoid degradation by Dnase in the extracellular (as well as intracellular) environment, ii) interact effectively with the cell surface in order to be internalized by endocytosis or pinocytosis , iii) the endocytosed vector has to protect the D N A promoting the escape from endosome/ lysosomes, iv) D N A has to reach the nucleus by diffusion within the cytosol v) Entry of D N A in the nucleus and gene expression followed by m R N A being transported out the nucleus vi) Protein translation from mRNA. (Figure 1). In the following sections we will discuss all these steps presenting alternatives to overcome these barriers by employing chitosan as a non-viral D N A carrier.

Chitosan : Structure and General properties Chitosan ( a (1 -> 4) 2-amino-2-deoxy-p-D-gIucan is obtained by alkaline deacetylation of chitin. Chitin, a polysaccharide found in the exoskeleton of


179 Polymeric


Plasmid DNA

nanovector

χ

Figure 1: Schematic gene therapy mechanism. (A) Bioavailability and extracellular trafficking (1) gene-vector complexation; (B) internalization and endocytosis by the cell membrane; (C) intracellular trafficking (2) uptake of the vector complex into intracellular endolysosome; (3) DNA-chitosan release from the endosome into the cytoplasm; (4) Internalization of the complex into the nucleus; (D) gene expression; (5) DNA dissociation from the vector; (6) ARNm transcription from the gene; (7) protein translation from ARNm. The protein can be secreted out of the cell, be released into the cytoplasm, orfixedonto the membrane. (Adaptedfrom Mansouri et al, (10))

crustaceans and insects (77) (Figure 2). It is a copolymer of N-acetyl-Dglucosamine and D-glucosamine, which behaves as a weak base. The pKa value of the D-glucosamine residue of about 6.2-7.0. Chitosan is insoluble in water at neutral and alkaline pH values. In acidic medium, the amine groups will be positively charged, conferring to the polysaccharide a high charge density (72). Chitosan excels in enhancing the transport of drugs across the cell membrane. Its cationic polyelectrolyte nature provides a strong electrostatic interaction with mucus, negatively charged mucosal surfaces and other macromolecules such as

D N A (12,13).


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Chitosan is a non-toxic biodegradable polycationic polymer with low immunogenicity (14). It is a good candidate for gene delivery system because when positively charged, it can be complexed with negatively charged D N A (15,16). Chitosan can effectively bind D N A and protect it from nuclease degradation (17,18). Moreover chitosan/DNA complexation takes place without sonication and does not require the use of organic solvents, therefore minimizing possible damage to D N A during complexation. DNA-loaded chitosan microparticles were found to be stable during storage (19). The application of DNA-chitosan nanospheres has advanced in vitro D N A transfection research and data have been accumulating that shows their usefulness for gene delivery

(20,21).

Condensation, Shape, Size and Protection of DNA-Chitosan Polyplexes The Chitosan-DNA interaction is driven mainly by the electrostatic interaction between the amino groups of chitosan and the charged phosphate groups of D N A (22, 23) and, although different methodologies have been published on the preparation of chitosan nanoparticles, for gene therapy, the complex coacervation has been the most employed procedure. In general the encapsulation process is entirely performed in aqueous solution at low temperatures preserving the bioactivity of the plasmid D N A . Stable complexes are formed only when chitosan is added in molar excess relative to D N A , with zeta potential values between 10 and 20 mV, depending upon the degree of excess. However it is well documented that molecular weight, charge ratio (+/-) and pH are important parameters in providing the needed protection, as well as in determining the nanoparticles shape. Transmission electronic microscopy measurements have provided evidence that chitosan of 8 kDa condenses plasmid into toroids and rod shaped particles whose sizes were estimated about 66 nm by light scattering measurements (24). Although condensation of the D N A by the chitosan is of great importance, the plasmid D N A must remain intact to assure its functionality once inside the cell. A widely used method to monitor the D N A condensation and the effect of such conditions on the integrity of the plasmid D N A is that of gel electrophoresis. Chitosan at a concentration of 0.02%, used for synthesis of the complexes, is viewed in Figure 3 (20). The intact D N A , before complexation, is seen in lane 2, while complexes of chitosan-DNA of molecular weights 150, 400 and 600 kDa were loaded in lanes 3, 5 and 7, respectively. The D N A in these three lanes is unable to migrate and remains in the gel loading wells, indicating a strong attachment of the D N A to the chitosan. No unbound D N A is seen in these lanes. Following digestion with chitosanase and lysozyme, the integrity of plasmid D N A can be evaluated (viewed in lanes 4, 6, and 8). The plasmid is intact and shows no signs of degradation. This suggests that the coacervation process does not affect the integrity of the condensed D N A . The size and morphology of the chitosan-DNA nanoparticles


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χ > y

c=>

CHITOSAN


* < y « = > ί CHITIN Figure 2: Chemical structure of chitin and chitosan and the deacetylation process to produce the highly deacetylatedproduct. In the 2-amino-2-deoxy-d~ glucopyranose ring is shown the commonly used numbering for the carbon atoms.

Figure 3. Electrophoresis of chitosan-DNA nanoparticles to determine plasmid integrity following synthesis. Nanoparticles were digested with chitosanase and lysozyme after synthesis and the released DNA was visualized with ethidium bromide. Lane 1: Molecular weight marker; lane 2: VR1412 plasmid DNA; lane 3: nanoparticles composed of 150 kDa chitosan; lane 4: lane 3 + digestion; lane 5: nanoparticles composed of400 kDa chitosan; lane 6: lane 5 + digestion; lane 7: nanoparticles composed of600 kDa chitosan; lane 8: lane 7 + digestion. (Reproduced with permission from ref 20)



182 can be vizualised by electron microscopy. The nanoparticles are embedded in Epon, sectioned and the D N A stained with uranyl acetate and lead citrate, giving it a darker appearance (Figure 4). The micrographs demonstrate a homogenous distribution of D N A within the particles. On the other hand, atomic force microscopy ( A F M ) revealed the size and morphology of the synthesized particles. The complexes made employing a chitosan 150 kDa appear spherical with a mean size that is inferior to 100 nm ( Figure 5). Recently Danielsen et al. (25-26) have confirmed by A F M that polyplexes made by mixing plasmid D N A with chitosan from 10 to 200 kDa yielded a blend of toroids and rods. The ratios between the fractions of toroids and rods were observed to decrease with increasing acetylation degree (FA) of the chitosan indicating that the charge density of chitosan, proportional to (1 - F A ) , is important in determining the shape of the compacted D N A . The amount of chitosan required to fully compact D N A into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low degree of polymerization (DP) chitosan could compensate for the reduced interaction strength of the individual ligands with D N A . The stability of these D N A chitosan complexes was studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy ( A F M ) and ethidium bromide (EtBr) fluorescence assay. Studies of the polyplex stability when challenged by H A showed that whereas H A was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation(2

Chitosan Nanoparticles for Non-Viral Gene Therapy - ACS Publications

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