Development and characterization of a new plasmid delivery system based on chitosan–sodium deoxycholate nanoparticles

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Abstract

Chitosan is one of the most promising polymers for drug delivery through the mucosal routes because of its polycationic, biocompatible, and biodegradable nature, and particularly due to its mucoadhesive and permeation-enhancing properties. Bile salts are known to interact with lipid membranes, increasing their permeability. The addition of bile salts to chitosan matrices may improve the delivery characteristics of the system, making it suitable for mucosal administration of bioactive substances. In the present study we have developed chitosan nanoparticles using sodium deoxycholate as a counter ion and evaluated their potential as gene delivery carriers. Chitosan–sodium deoxycholate nanoparticles (CS/DS) obtained via a mild ionic gelation procedure using different weight ratios were used to encapsulate plasmid DNA (pDNA) expressing a “humanized” secreted Gaussia Luciferase as reporter gene (pGLuc, 5.7 kDa). Mean particle size, polydispersity index and zeta potential were evaluated in order to select the best formulation for further in vitro studies. The nanoparticles presented an average size of 153–403 nm and a positive zeta potential ranging from +33.0 to +56.9 mV, for nanoparticles produced with CS/DS ratios from 1:4 to 1:0.6 (w:w), respectively. The pDNA was efficiently encapsulated and AFM studies showed that pDNA-loaded nanoparticles presented a more irregular surface due to the interaction between cationic chitosan and negatively charged pDNA which results in a more compact structure when compared to empty nanoparticles. Transfection efficiency of CS/DS–pDNA nanoparticles into moderately (AGS) and well differentiated (N87) gastric adenocarcinoma cell lines was determined by measuring the expression of luciferase, while cell viability was assessed using the MTT reduction. The CS/DS nanoparticles containing encapsulated pDNA were able to transfect both AGS and N87 cell lines, being more effective with AGS cells, the less differentiated cell line. The highest enzymatic activity was achieved with 20% pDNA encapsulated and after 24 h of transfection time. Low cytotoxicity was observed for the CS/DS nanoparticles either with or without pDNA, suggesting this could be a new potential vehicle for mucosal delivery of pDNA.

Graphical abstract

AFM morphology and cross section profiles of empty (A) and pDNA-loaded (B) chitosan–sodium deoxycholate nanoparticles.

Relative cell viability of gastric adenocarcinoma cell lines N87 (A) and AGS (B) exposed to empty and pDNA-loaded chitosan–sodium deoxycholate nanoparticles.

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Introduction

Many of the currently approved vaccines are intended for parental routes, which induce strong systemic immune responses. However, most pathogens preferentially infect their hosts via the extended mucosal surfaces of the gastrointestinal, respiratory or genitourinary tracts. These mucosal surfaces serve as the first line of defence against infection, suggesting the mucosal route is ideal for vaccination purposes (Kang et al., 2009, Wang et al., 2004). In addition, studies have demonstrated that immunization through a combination of mucosal and systemic routes may increase mucosal immunity (Taylor et al., 2007). Although mucosal vaccines can be delivered through different routes, such as nasal, oral, vaginal, conjunctival or transdermal, vaccines specially administered via the oral and nasal routes are being actively studied (Kang et al., 2009). Mucosal vaccination via the oral and nasal routes usually favors the development of mucosal antibody and cell mediated immune responses, resulting in a strong protective effect. Other advantages include ease of self-administration and non-invasiveness which improve patient compliance, avoiding the use of needles and do not requiring trained personnel for administration (Chadwick et al., 2010). Despite all these advantages, oral vaccine delivery still represents a major challenge.

One of the current vaccination strategies for DNA vaccines utilizes naked plasmid DNA that encodes antigenic proteins and induces immune response after intramuscular. However, DNA vaccines may be administered at mucosal sites with improved efficiency upon association to polymeric nano- and microparticles as carrier systems able to target the specific mucosal region while protecting the DNA against degradation during delivery (Chadwick et al., 2010, Donnelly et al., 2005).

Chitosan is a natural polysaccharide derived through the partial deacetylation of chitin, the major compound of exoskeletons in crustaceans (Illum, 1988). With a pKa of approximately 6.5 on the amine groups, chitosan is pH-dependent (Chen et al., 2007) being soluble and positively charged at acidic pH (Dastan and Turan, 2004). It is one of the most promising polymers for drug delivery through the mucosal routes because of its polycationic, biocompatible, and biodegradable nature, as well as its mucoadhesive and permeation-enhancing properties (Kang et al., 2009). Positively charged chitosan binds to cell membranes and is reported to increase Tran cellular and paracellular permeability (Schipper et al., 1999, Bowman and Leong, 2006). These features make chitosan nanoparticles a suitable vehicle for oral vaccine delivery (Chadwick et al., 2010) capable of transfecting mammalian cells because of the ability to overcome the three steps of cellular transport: cell uptake, release from endosomes and nuclear transport (Ishii et al., 2001). Li et al. (2009) have investigated the effect of chitosan–DNA nanoparticles on immune response in mice by oral delivery of chitosan–DNA nanoparticles demonstrating that a high level of gene expression was found in the epithelial cells of both stomach and intestine. As no effect on cell viability was observed and due to the high transfection efficacy, chitosan is seen as a good candidate for the development of novel gastrointestinal drug and gene delivery systems. Nanoencapsulation of DNA into chitosan nanoparticles showed efficient binding of the nanoparticles to cellular membranes leading to transfection (Boyoglu et al., 2009).

Chitosan–DNA nanoparticles may be formed by ionic gelation, a mild process based on the complexation between oppositely charged macromolecules (Agnithotri et al., 2004). Nanoparticles made of low molecular weight chitosan with tetanus toxoid (TT) as a model protein could be produced by an ionic cross-linking technique and proving to be promising carriers for nasal vaccine delivery (Vila et al., 2004). Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response as compared to the soluble antigen.

In order to improve the efficacy of mucosal vaccines and their ability to prolong drug delivery, chitosan nanoparticles could be chemically modified or used together with a potent immunological adjuvant (Alves and Mano, 2008, Baudner et al., 2004). The use of chitosan delivery systems containing a bile salt, such as sodium deoxycholate, could be an effective strategy for improving the efficacy of an oral vaccine (Chae et al., 2005). Bile salts are the most widely used surfactants for nasal absorption optimization and at relatively low concentrations, 10–20 mM; they are able to improve the absorption of peptides and other drugs. These molecules are known to interact with lipid membranes, increasing their permeability and promoting the membrane transport of peptides and proteins through intercellular junctions (Behl et al., 1998, Chae et al., 2005). Samstein et al (2008) have studied the potential of poly(lactide-co-glicolide) (PLGA) nanoparticles non-covalently associated to deoxycholic acid (DCA) and concluded that the system had improved the bioavailability of encapsulated rhodamine B. Furthermore, the addition of bile salts to chitosan matrices may also improve the delivery characteristics of the system, making it suitable for mucosal administration of bioactive substances (Chae et al., 2005). In a concentration range between 0.5–1% DS did not demonstrate any notable toxic effects and it was found to markedly improve the humoral responses to nasally delivered TT and diphtheria toxoid, two poor immunogens following nasal delivery (Alpar et al., 2001). A few publications have described the association of chitosan to sodium deoxycholate to produce microparticles and also nanoparticles prepared by covalent attachment of deoxycholic acid to chitosan (Kim et al., 2005). Chae et al. (2005) have developed chitosan oligosaccharides (COS) chemically modified with DCA (COSD). When compared to unmodified COS, the COSD nanoparticles showed high levels of gene transfection on HEK293 cells, which make it a potential system for local mucosal gene delivery.

To the best of our knowledge, there are no published data whereby chitosan/sodium deoxycholate (CS/DS) nanoparticles are produced by ionic gelation. The aim of this study was the development of chitosan nanoparticles produced by ionic gelation with sodium deoxycholate as counter-ion and evaluate their potential as gene delivery systems. The physicochemical properties and the loading plasmid DNA capacity of the particles were evaluated. Transfection studies, using a plasmid that codify for the enzyme luciferase, were evaluated in human cell lines, with different differentiated states, derived from stomach. Cell viability was also evaluated using the same cell lines and for the different formulations of nanoparticles.

Section snippets

Materials

Chitosan low molecular weight (LMW) with degree of deacetylation (DD) 75–85%, sodium deoxycholate, cibacron brilliant red 3B-A, glycine, sodium chloride and hydrochloric acid were obtained from Sigma–Aldrich (Dorset, UK). All cell culture reagents and LipofectamineTM were from Invitrogen (Scotland, UK). Plasmid DNA (pDNA) expressing a “humanized” secreted Gaussia Luciferase as reporter gene (pCMV-GLuc, 5.7 Kbp (pGLuc)) that possesses a natural secretor signal and upon expression is secreted into

Preparation and physicochemical characterization of CS/DS nanoparticles

The formation of CS/DS nanoparticles is a process based on the complexation of oppositely charged molecules by electrostatic interactions (Calvo et al., 1997a, Calvo et al., 1997b, Csaba et al., 2009). Nanoparticles were produced using an ionic gelation process that involves sodium deoxycholate as a negatively charged molecule and chitosan as a polycation. This method presents an advantage when compared to the covalent attachment of CS to DS or chemically modified CS because, after

Conclusions

We investigated the potential of CS nanoparticles incorporating deoxycholic acid for the specific delivery of gene to the mucosa. The advantages of this system in gene delivery include their ability to interact intimately with the cell surfaces, thereby increasing delivery by the added properties presented by deoxycholic acid without the need of reactions involving chemicals or solvents. The nanoparticles showed a high capacity for DNA condensation allowing the delivery of high amounts on a

Acknowledgements

This work was supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/BIO/69242/2006). The support of Andreas Bergner (LOT-Oriel, Germany) on the AFM analysis is also acknowledged.

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