| The aim of this work was to prepare the large unilamellar liposomes containing the model antigen BSA. Then we linked UEAI to surface of liposomes in order to target M cells and induce systemic and mucosal immune response. In a word, we hoped that UEAI-modified BSA-encapsulating liposomes could be used the carriers of oral vaccine.The Bradford assay has become the preferred method for quantifying protein in many laboratories. We adoped two-colorimetric coomassie brilliant blue method, standard Bradford assay and directly detected method at 280nm to measure BSA solutions of low (0.2-20.0μg/ml), middle (20.24-202.4μg/ml) and high (201-1507.5μg/ml) concentrations.Firstly, we prepared and optimized LIP. By using centrifuge-reference method offered the first time, EE% of optimization formulation was 69.74%±3.41%, the mean diameter was 4.796±0.416μm and Zeta potential was -15.4±2.6mv. Then we synthesized and characterized a modified cholesterol, just cholesterol succinate (CHS) and used it to prepare liposomes which afforded carboxy group to link with Ulex europaeus agglutinin I (UEA I), a lectin binding exclusively to M cells of small intestine to strongly stimulate the immune response to protein, and most important this conjunction method could remain the activity of lectin. The conjugation efficiency (CE%) of UEAI-LIP calculated through Sepharose 4B was 70.80%±2.28%, the mean diameter was 5.129±1.671μm and Zeta potential was -21.4 mv±1.5 mv. From the release curve of BSA released from LIP and UEAI-LIP in vitro, we could see the latter was slower and steadier. Through detecting the stability in artificial GI fluid, UEAI-LIP could protect BSA against degradation by digestive enzymes in vitro to some extent and the stabilizing effect was increasing with concentration of UEAI solution.Bradford’s method and RBC agglutination test were established to determine the content and bioactivity of UEAI, respectively. It was observed that at 4℃-80℃, UEAI was stable, but above 100℃, the agglutination activity decreased obviously. Meantime, UEAI solution was found to be stable in solution at pH 2.0-10.0. The safety evaluation proved that concentration of UEAI solution between 104.6-1046.5μg·mL-1, UEAI was safe for gastro-intestinal tract. Furthermore, cytotoxicity research suggested UEAI had some effect of promoting cell growth.The specific bioadhesion on mice gastro-intestinal mucosa was studied ex vivo and in vivo. An important increase of interaction between UEAI-LIP and the intestinal segments with PPs was observed compared with the unconjugated one (P<0.01). However, under the presence ofα-L-fucose, which is reported specific sugar for UEAI, specifically inhibited the activity of conjugates.We used ELISA method to detect IgG and IgA levels in biological samples of mice. Firstly, we determined liposomes with different formulations to elicit systemic and local immunity ability, the results proved the ability production of anti-BSA IgG and IgA antibody was the highest for UEAI-modified liposomes, next for unmodified ones, and the lowest for BSA solution. And liposomes with small size of 137±18nm could induce high systemic immune response and those with large size of 5.657±1.924μm could induce high mucosal immune response. Antigen loaded in liposomes increased could produce higher IgA levels and similar IgG levels, therefore made better mucosal protection effect. When BSA solution was given by i.m, it could make high IgG level and high-UEAI-LIP given by i.g could produce high IgA level.In vivo immune-stimulating results in KM mice showed that HPRS antigen given by i.m generated systemic response, on 35th day, the IgG level reached the maximum, and maintained high level continuously. The IgA level did on 21th day, and decreased slowly. But groups of HPRS-loaded UEAI-LIP administered by i.g could induce simultaneously systemic and mucosal immune response. In conclusion, this study demonstrated high potential of lectin-modified liposomes containing antigen as carriers of oral vaccine. |
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