Chapter seven - Chitosan-Based Nanoparticles as a Hepatitis B Antigen Delivery System
Introduction
At present, the term “vaccination” is generally considered to be identical to “injection.” This conception is due to the fact that vaccines are typically given by intramuscular injection. In the new era of vaccine development, with the emergence of subunit vaccines, the formulation of needle-free vaccines is undoubtedly more challenging. Novel vaccines obtained by recombinant technology are, in principle, safer with regard to toxicity; however, they are also less immunogenic, making it mandatory to include adjuvants in the formulation of such vaccines. Numerous efforts made by the scientific community to develop needle-free vaccine formulations are justifiable by several distinct advantages. An obvious one is the possibility of painless self-administration of the vaccine. Moreover, vaccine delivery via mucosal surfaces elicits mucosal immune responses at the site of pathogen entry and enhances cellular immunity through stimulation of Toll-like receptors (Bessa and Bachmann, 2010), thus improving overall effectiveness. Taking these facts into account, needle-free vaccination could have a big impact on the efficacy of immunization against mucosal transmitted diseases such as hepatitis B. In 1981, FDA approved the first hepatitis B vaccine which consisted of the surface antigen of the hepatitis B (HBsAg) virus present in the blood of human carriers of the infection, replaced in 1986 by the currently available vaccine which represents the world's first subunit vaccine and the world's first recombinant expressed vaccine. Since the hepatitis B virus can be transmitted perinatally or by exchange of body fluids (e.g., blood, semen, and vaginal fluid), the design of new hepatitis B vaccines with the additional possibility to induce mucosal antibodies (e.g., secretory IgA) is particularly attractive. The only available hepatitis B vaccines to date are injectable formulations, adjuvanted with aluminum salts, which are evidently not appropriate for oral or intranasal administration owing to two main reasons. One, mucosally administered antigens will be exposed to enzymatic degradation, and second, the adjuvant is not adequate for application at mucosal surfaces. Therefore, formulations with enhanced adjuvant properties are needed for the application at mucosal surfaces to reduce the high antigen doses normally required to increase the low immune response and decrease the variability of the individual immune responses frequently observed. Preclinical investigation of new needle-free hepatitis B vaccines relies on the development of adjuvants/new formulations with additional capability to increase the immunogenicity of the antigen. To achieve this goal, several strategies are currently being discussed (Lebre et al., 2011, Thanavala et al., 2009). A good example is the development of nanosized carrier systems that adsorb or encapsulate antigens, protect them from proteolytic enzymes, allow the increase of antigen retention time at the nasal mucosa, and finally target antigens to M-cells present on the mucosa (Jabbal-Gill, 2010). Lastly, the loading of particles not only with antigens but also with immunopotentiators such as combinations of Toll-like receptor ligands (Kasturi et al., 2011) may modulate the quantity and the quality of the immune response.
Chitosan is a cationic polymer consisting of β-(1-4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit) monomers that can be obtained by deacetylation of chitin (Illum, 1998). It has been considered a nontoxic, biodegradable, and biocompatible polymer (Baldrick, 2010), thus extensive research has been directed toward its use in medical applications such as drug and vaccine delivery (Lebre et al., 2011, Panos et al., 2008, van der Lubben et al., 2001b). One major advantage of this polymer is its ability to easily produce nanoparticles under mild conditions without the application of harmful organic solvents. This has been one of the main reasons for its wide applicability to the encapsulation of different molecules such as therapeutic proteins, DNA, and antigens. Chitosan is also known to be mucoadhesive, and its ability to stimulate cells of the immune system has been shown in many studies (Borges et al., 2007a). These unique features make chitosan an attractive polymer to act as an adjuvant.
Section snippets
Alginate-coated chitosan particles
Chitosan nanoparticle preparation can be achieved by several techniques. One of the most common is the precipitation/coacervation method, which is a process of spontaneous phase separation that occurs when two oppositely charged polyelectrolytes are mixed in an aqueous solution.
The protocol used in our laboratory results from an adaptation and optimization of a previously described method (Berthold et al., 1996). The preparation of this delivery system contains three main steps: manufacturing
Size measurement
It is generally accepted that the size and size distribution of the particles are important for their adjuvant activity. Therefore, size characterization is an important step in vaccine formulation development even if the attempts to correlate particle size and the resultant immune responses lead to conflicting findings (Oyewumi et al., 2010).
The size of particles can be measured by Dynamic Light Scattering techniques. Among those techniques, Photon Correlation Spectroscopy (PCS) has been
Antigen Adsorption Studies
Polymeric particles can be used as delivery systems for molecules with biological interest such as proteins or more specifically antigens. The adsorption of the antigens onto particles is a mild process since can be performed simply by the incubation of the particles with the solution of the antigens at RT. Furthermore, given that the antigens are located at the surface of the particles, it is expected that they are more available to be presented by antigen-presenting cells. However, protein
In vitro Release Studies
To evaluate the suitability of nanoparticles as a delivery system for proteins with biological interest such as the HBsAg, an in vitro release study should be performed. These studies are performed with different buffers which mimetize the physiological conditions. To mimic the digestive tract, simulated gastric fluid and simulated intestinal fluid, both described by United States Pharmacopeia, are normally used as release buffers, by our group to study oral antigen delivery systems. The
Evaluation of the Bioactivity of the Antigen
The preliminary evaluation of the bioactivity of the antigen, after their association with particles, is normally performed using the Western blotting technique. The integrity and bioactivity of hepatitis B antigen are done after antigen being released from nanoparticles. Therefore, the samples resulting from the release studies mentioned above need to be centrifuged at 14,000rpm in order to separate the released antigen from the particles and then an aliquot is solubilized with the SDS-PAGE
Cell Viability Studies with Spleen Cells
There are different methods for assessing the in vitro cytotoxicity. Alterations in plasma membrane permeability can be evaluated both by the release of cytoplasmic enzymes (e.g., lactate dehydrogenase) or by the uptake of dyes (e.g., trypan blue, propidium iodide). Alternatively, cytotoxicity can be evaluated by changes in cell metabolic activity. Tetrazolium salts are widely used in these metabolic assays (e.g., MTT, XTT, WST-1). In our laboratory, the MTT assay (Sigma-Aldrich, St. Louis, MO,
Studies on Uptake into Peyer's Patches
Oral vaccination presents advantages over parenteral injection, nevertheless the degradation of the vaccine and the low uptake by the gut-associated lymphoid tissue are determinant factors that limit the success of this strategy (Jung et al., 2000, Van Der Lubben et al., 2001a). The uptake of inert particles across the GI tract is known to occur mainly transcellularly through normal enterocytes and Peyer's patches via M-cells (Hussain et al., 2001). Considering nanoparticulate vaccines for oral
Concluding Remarks
The development of novel vaccine adjuvants is becoming as important as the development of novel antigens itself. Presently, most of the vaccines are given by intramuscular injection, which requires the use of needles that are painful, are potentially dangerous, requires trained medical personnel, and are therefore unsuitable for mass vaccination campaigns, especially in developing countries. Recently, many researchers have focused their interest on needle-free technologies for immunization,
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