ABSTRACT
Oral vaccine delivery is an attractive mode of immunization because of its ease of administration, low manufacturing cost, and patient compliance. However, the major hurdle in oral vaccine delivery is the protection of the antigen from acidic and enzymatic degradation in the gastrointestinal tract. One of the ways to avoid these issues is to formulate microparticles by using enteric coating polymers. Intestinal Peyer’s patches are the predominant sites for uptake of such particles upon oral administration [6]. The particle uptake depends on various factors such as size, charge, and hydrophobicity [7,8]. For oral delivery, it has been reported that particles of size less than 5 µm with positive charge and hydrophobic nature can preferentially enter Peyer’s patch of small intestine [9]. Orally delivered vaccines, especially particulate antigens are recognized and sampled by microfold (M) cells in Peyer’s patches. This is followed by transport of the particles to underlying follicles and to professional antigen presenting cells (APCs) such as dendritic cells and macrophages. These APCs can phagocytose the particles, process them and present them on both MHC Class I, through cross priming, and MHC Class II molecules due to which both T and B cells can be triggered [10,11].Several studies have been performed involving enteric coating polymers such as Eudragit L 100, S 100, L 100-55, cellulose acetate phthalate, and hydroxyl propyl methyl cellulose phthalate/acetate succinate as a polymer of choice for a particulate delivery vehicle [12-15]. Microparticles containing these enteric polymers are protected from gastric pH and can be delivered to the intestine for further uptake. Oral delivery of vaccine antigens using such
polymeric microparticles offers remarkable advantages over others, such as induction of mucosal as well as systemic immune response, protection of antigen from gastric degradation, prolonged presentation of antigen to immune system, and obviation of the need of vaccine adjuvants because microparticles themselves can act as self-adjuvants [16,17].The present work explores the formulation and evaluation of oral murine breast cancer vaccine, which can prevent the proteolytic degradation of antigens and retain the particles intact until they reach the M cells in the small intestine. For this purpose, enteric polymers such as Eudragit® FS 30 D and hydroxyl propyl methyl cellulose acetate succinate (HPMCAS) were used. In an attempt to enhance the targeting capability of our vaccine formulation to the M cells, the M cell targeting agent, aleuria aurantia lectin (AAL) was added to the particulate matrix. These microparticles were formulated by using a single-step spray drying process. Spray drying involves atomization of aqueous antigen containing polymeric suspension, followed by short drying period. The process limits the exposure of the antigens to high temperatures thus causing minimal or no degradation of proteins [18-21]. Our group investigated the potential of these particles to target M cells in Peyer’s patches upon oral administration, leading to activation of underlying immune cells. Thus, we aim to trigger both humoral and cell-mediated immune response through this prophylactic breast cancer vaccine, which can impart resistance against tumor challenge later. 9.3 Methods
9.3.1.1 Whole-cell lysate preparationThe murine breast cancer cell line 4T07 was used for preparation of whole-cell lysate. The cells were lysed using hypotonic lysis buffer (10 mM Tris and 10 mM NaCl) and further subjected to five freeze thaw cycles at –80 and 37°C for 10 min each. At the end of last freeze thaw cycle, cell lysis was confirmed using trypan blue
dye exclusion assay; presence of dead cells confirmed the end point. The whole-cell lysate (WCL) thus obtained was stored at –80°C for further use. The total protein content of WCL was quantified by Bio-Rad DC protein assay. 9.3.1.2 Vaccine microparticle preparationThe 4T07 antigen-loaded vaccine particles were formulated using the WCL, b-cyclodextrin, ethyl cellulose, albumin, trehalose, hydroxyl-propyl methylcellulose acetate succinate (HPMCAS) and targeting agent AAL dissolved in de-ionized water. The formulation also contained trehalose to increase protein stability. AAL was added to target the particles to M cells in the intestinal lumen. The resulting microparticles were stored at –20°C in a desiccant chamber until further use. 9.3.2 Physical Characterization of Microparticles
Particle size analysis using Spectrex laser counter indicated an average particle size of 1.5 μm, with particles of size range of 1-4 μm as shown in Fig. 9.1b. The zeta potential was neutral, ranging from +4 to +7 mV. 9.3.2.2 In vitro antigen release from microparticles Antigen release from microparticles was studied to evaluate antigen protection in gastric pH conditions. Amount of antigen released was plotted against square root of time. The linearity for Higuchi release profile of these microparticles shows 10.0 ± 6.9% antigen release in first 2 h in pH 1.2 (gastric simulated fluid) and 51.6 ± 12.8% release at the end of 24 h in pH 6.8 (intestinal simulated fluid) (Fig. 9.1c). Amount of antigen remaining in the microparticles post release study was evaluated to confirm the results. The antigen content of remaining particles was also analyzed to confirm these results.
