Eudragit® L100-coated mannosylated chitosan nanoparticles for oral protein vaccine delivery

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Abstract

The aim of this study was to develop a novel biodegradable polymeric carrier for the delivery of protein vaccine orally in order to target the antigen presenting cells (APCs) in the region of Peyer's patches (PPs). Here, bovine serum albumin (BSA) was chosen as a model protein vaccine and was loaded into the mannosylated chitosan nanoparticles (MCS NPs) by ionic gelation method with tripolyphosphate (TPP), followed by coating MCS NPs with Eudragit® L100 (Eud) by electrostatic interaction. The spherical NPs were successfully prepared with appropriate particle size around 558.2 ± 35.6 nm, high entrapment efficiency about 90.38 ± 9.12%, good stability and reasonable release behavior in the simulated gastrointestinal fluid, meanwhile high resistance of enzymatic and acid degradations were verified. The targeting ability of the NPs to PPs in rat was investigated by a closed ileal loop assay, where fluorescence visualization was performed. MCS NPs were accumulated more specifically into PPs after Eudragit® L100 dissolved in intestinal juices. Oral immunization using BSA-loaded Eudragit® L100-coated MCS NPs was found to elicit strong systemic IgG antibody and mucosal IgA responses. These results suggested that enteric-coated MCS NPs could serve as a promising carrier for oral protein vaccine delivery.

Introduction

Mucosal surfaces, such as nasal cavity, respiratory tract, gastrointestinal tract and reproductive tract establish a local first-line defense against viral and bacterial infections [1]. Compared with the injection of vaccines, administration of vaccines at mucosal surfaces has a unique advantage that inducing an adequate local immune response at administration site and systemic immune response at distant mucosal site with the generation of both mucosal IgA antibodies and serum IgG antibodies [2,3].

Among various kinds of mucosal immune way, oral immunization has unique advantages. The intestine mucosa comprises the largest surface area of the body, where the gut associated lymphoid tissue (GALT) represents the most extensive immune organ and the inductive sites for immune responses in the gut are Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) [4]. Since oral immunization is able to induce protective immunity in mucosal area, it has been considered ideal for combating intestinal infection and autoimmune diseases such as rheumatoid arthritis, type I diabetes, multiple sclerosis and so on [[5], [6], [7], [8]]. Several studies have indicated that oral delivery vaccines have unique effects on intestinal infection, because the primary immune cells tend to be home to effective site where antigen presenting cells (APCs) are first initiated [[9], [10], [11]]. In addition, oral administration, as the most common natural route for drug delivery, could significantly improve patients' compliance and has the capacity for mass immunization with needle-associated risks [12].

Nevertheless, the effectiveness of oral vaccines is compromised by the physiological and immunological environment of the gastrointestinal tract. The oral vaccine is not only exposed to the extremely acidic environment in stomach, various proteolytic enzymes and as well as bile salts, but also reluctantly overcomes the mucus layer and the tight intestinal epithelial cellular junctions before reaching the GALT. One of the most promising strategies to enhance oral vaccine delivery is the application of biodegradable polymeric NPs, which can protect vaccine from degradation and increase higher uptake efficiency of M cells which are specialized epithelial cells responsible for antigen sampling and possess a relatively high transcytotic capacity at the interface of mucosal surfaces [[13], [14], [15]]. Vaccines-loaded NPs tend to transport through M cells to reach the sub-epithelial dome (SED) of the PPs, where lymphocytes and APCs are aggregated, and release the cargoes to induce immune response. Moreover, the effective uptake of the vaccine by APCs which is crucial to the immune response is still a challenge. Therefore, a crucial work for developing oral protein vaccines vectors is to achieve good protection of protein structure in the gastrointestinal tract and target delivery to APCs underlying PPs.

Chitosan (CS) is obtained by alkaline deacetylation of chitin, which distributed in nature as the principal component of exoskeletons of crustaceans and insects, as well as of cell walls of some microorganisms, bacteria and fungi. Chitosan with a degree of deacetylation over 80% possess abundant of free amino groups that are active sites for many chemical reactions [16]. Owing to its biocompatibility, biodegradability and bioadhesion, chitosan has been extensively studied for mucosal delivery of vaccines as well as for its immunogenic activities properties and mucosal permeation ability [17]. The positive surface charge of chitosan can be used as vaccine vectors loading protein or gene vaccines with negative charges. Also, its positive charge allows it to interact with negatively charged mucosal surfaces and cell membrane, which facilitate the absorption of the vaccines [18]. In recent years, chitosan NPs have been widely used to deliver peptides, proteins and vaccines, and reported to improve immune system stimulation by enhancing paracellular absorption and allowing increased access of antigens to improve the contact of antigens with lymphocytes [[19], [20], [21], [22]]. However, chitosan based NPs were easily dissolved in the acidic gastric juice resulting in the exposure of vaccines to harsh gastric environment. Moreover, the effective uptake of vaccine loaded NPs by APCs which is crucial to antigen sampling and immune response is also a challenge.

APCs, including macrophages and dendritic cells (DCs), are over-express mannose receptors on the surface of cell membranes, which play a critical role in recognizing environmental antigens, as well as initiating and regulating adaptive immune responses [23]. Mannose can recognize the mannose receptors located in the cell surface by the specific ligand-receptor interactions. Many studies have confirmed that mannosylated vaccine carriers can induce the receptor-mediated endocytosis for targeting into APCs, leading to high transfection efficiency and enhanced immune responses [[24], [25], [26]].

In this study, mannosylated chitosan (MCS) NPs were prepared as a novel oral protein vaccine vector to improve the antigen presentation ability and the immune response efficiency. MCS alone isn't able to provide sufficient protection for antigens, because the polymer matrix was dissolvable in gastric juice by protonation of the amino groups at low pH values, which results in the immediate burst release of antigens. Therefore, MCS NPs were enteric-coated by Eudragit® L100 (Eud) to improve the stability of the loaded vaccines in acidic environment. Bovine serum albumin (BSA) was chosen as a model antigen to evaluate the mucosal immune effect of MCS/Eud NPs for oral vaccine delivery.

Section snippets

Materials and animals

Chitosan (Mw of 115 kDa, deacetylation degree of 95%) was supplied by Shanghai KABO Trading Co. Ltd. MCS was synthesized in-house. Sodium tripolyphosphate (TPP) was purchased from Aladdin Industrial Inc. BSA was purchased from Biosharp Co. Ltd. Eudragit® L100 was obtained from Evonik Industries. Fluorescein isothiocyanate (FITC) and Rhodamine B Isothiocyanate (RITC) were purchased from Sigma-Aldrich. All other chemicals and reagents were of analytical grade.

Sprague Dawley rats (SD rats,

Preparation and characterization of the MCS/Eud nanoparticle

The MCS NPs were prepared by ionic gelation method using TPP as cross-linking agent, which is a poly-anion and widely used on the coacervation of CS NPs because of high gelation character and low toxicity. After BSA was loaded into the MCS NPs, the particle size increased from 313.1 nm to 355.6 nm and zeta potential decreased from 40.8 mV to 35.6 mV. The near spherical morphologies of MCS/BSA NPs were observed by TEM, as shown in Fig. 1.A. In order to protect the stability of MCS/BSA NPs in the

Conclusion

This research enabled us to establish an efficient method for non-invasive delivery of protein vaccine to the intestinal immune system by constructing a novel oral vaccine carrier. The results confirmed that encapsulation of BSA, a model protein antigen, in enteric-coated MCS NPs could efficiently protect the antigen against the gastric acid and enzymes in alimentary tract, enhance its accumulation at the PPs mainly through M cells and favor its targeting to APCs, thereby inducing strong

Acknowledgments

This work was supported by the Natural Science Research Project of Universities in Jiangsu Province of China (15KJD350002) and the Science and Technology Projects of Nantong (MS12015064).

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1

Bohui Xu and Wenjing Zhang contributed equally to this work.

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