壳聚糖的纳米载体应用于口服蛋白类药物/疫苗递送的研究

The Application of Chitosan-Based Nanocarrier for Oral Protein Drug/Vaccine Delivery

近年来,细菌性和病毒性的传染性疾病已成为制约水产鱼类养殖业发展的瓶颈。口服免疫因其对鱼体操作方便、无损失、不受时间地点及鱼体大小的限制近年来得到了广泛的关注。然而口服免疫因疫苗在通过鱼的胃肠道时易被消化酶降解,导致免疫效率低下。因此,随着纳米技术的迅速发展,基于壳聚糖的纳米载体与水产疫苗的有效结合在鱼类口服免疫领域越来越受到重视。本文利用离子交联法和聚电解质凝聚法分别合成了壳聚糖纳米粒(CS-NPs)和壳聚糖/羧甲基壳聚糖复合纳米粒(CS/CMCS-NPs),其平均粒径分别为(178±6.27)和(255±7.54)nm,zeta电位分别为(+16.4±0.51)和(+15.9±0.37)mV。纳米粒对模式蛋白药物牛血清白蛋白(BSA)的包封率分别为(42.9±1.2)%和(59.4±3.2)%。红细胞溶血试验及MTT试验表明相较于CS-NPs,CS/CMCS-NPs具有更好的生物相容性。以荧光染料Cy5.5对纳米载体进行了荧光标记来评价Caco-2细胞对荧光纳米载体的摄取,结果表明,在一定浓度和时间范围内,Caco-2细胞对纳米粒的摄取存在一定的浓度依赖性和时间依赖性。为了研究纳米载体所包载的蛋白类药物在大菱鲆体内的分布情况,以FITC对BSA进行荧光标记,口服灌胃大菱鲆36h后,对于FITC-BSA:CS-NPs组,荧光药物主要分布在肝脏和前肠中;对于FITC-BSA:CS/CMCS-NPs组,荧光药物主要分布在后肠、肝脏和脾脏中;作为对照组的FITC-BSA组,在肝脏中的荧光分布最强,由此可见,CS/CMCS-NPs能够更有效的保护药物到达大菱鲆的后肠,有望成为一种安全有效的口服蛋白疫苗运送载体。

In recent years, infectious bacterial and viral diseases have restricted the development of fish aquaculture. Oral immunization of fish has caused extensive attention because of its convenient operation with no restricted time, place, the sizes and no injury to fish. But oral vaccine may be degraded by digestive enzymes in the gastrointestinal tract （GI）, which lead to the low immune efficiency. Therefore, with the rapid development of nanotechnology, the potent combination of chitosan （CS） based-nanocarriers with aquatic vaccines has caused much attention in the field of fish oral vaccination. Chitosan-nanoparticles （CS-NPs） and chitosan/carboxymethyl chitosan-nanoparticles （CS/CMCS-NPs） were prepared by ionic gelation and polyelectrolyte complexation method, respectively. Their average particle sizes were （178 ± 6.27）and （255 ± 7. 54） nm, respectively. The zeta potential was （＋15.9±0. 37） and （＋16.4 ± 0. 51） mV, respectively. The model protein of bovine serum albumin （BSA） was encapsulated in nanoparticles with the encapsulation efficiency （EE） of （42.9 ± 1.2） % and （59.4 ± 3.2） %, respectively. The hemolysis and MTT assay showed that CS/CMCS-NPs exhibited better biocompatibility compared with CS-NPs. In order to evaluate cellular uptake efficiency of nanoparticles by Caco-2 cells, the nanocarriers were labeled with CyS. 5. The results showed that the cellular uptake efficiency revealed concentration-dependence and time-dependence in the certain range of concentration and time. For the sake of tracing the biodistribution of BSA encapsulated in nanoparticles, BSA was labeled with FITC. After oral administration in turbot with nanoparticles 36 hours later, the fluorescent proteins were mainly distributed in liver and foregut with the group of FITC-BSA： CS-NPs, while in hindgut, liver and spleen with the group of FITC-BSA. CS/CMCS-NPs. As for the control group, there were mainly distributed in liver. Thus, CS/CMCS-NPs could effectively protect the protein to reach the hindgut of turbot and had great potential to be applied as safe and effective oral vaccine delivery.