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亚微米Pickering乳液的制备和生物应用研究
屈艳玲
Thesis Advisor吴颉
2020-07-01
Degree Grantor中国科学院大学
Degree Name硕士
Degree Discipline生物化工
Keyword亚微米pickering乳液,乳液稳定性,疫苗佐剂,纤维素纳米晶(Cncs),生物相容性
Abstract

Pickering乳液是由固体颗粒代替表面活性剂稳定的一种乳液剂型。固体颗粒的使用,不仅大大提高了乳液的稳定性,还赋予了乳液许多特殊的性质,使得Pickering乳液在生物医药领域内的应用受到广泛关注。但目前仍存在Pickering乳液尺寸较大、生物相容性欠佳等问题,限制了其实际应用。针对上述问题,本论文的研究目的是制备一种具有良好生物相容性的亚微米Pickering乳液,并初步探究其生物应用潜力。具体研究内容如下:(1)亚微米Pickering乳液的制备:以制备稳定的亚微米Pickering乳液为研究目标,采用角鲨烯作为油相,筛选多种纳米颗粒作为稳定剂,考察了颗粒浓度、油水比例、水相成分、超声时间及频率对Pickering乳液粒径分布及稳定性的影响,最终成功制备得到了由纤维素纳米晶(CNCs)纳米颗粒稳定的亚微米Pickering乳液。结果表明,该亚微米Pickering乳液尺寸在600 nm左右,并且粒径均一、分散性良好,具有良好的抗离心稳定性。乳液在4ºC、25ºC和37ºC等不同储存温度下能稳定保存30天以上,粒径和电位基本保持不变,具备良好的储存稳定性。激光共聚焦显微镜照片证实了CNCs吸附在油水界面,形成了Pickering乳液结构。(2)亚微米Pickering乳液作为疫苗佐剂的生物应用初步评价:以鸡卵清白蛋白(OVA)为模型抗原,考察CNCs稳定的亚微米Pickering乳液在OVA预防性疫苗中的佐剂作用和机制。通过细胞实验和动物实验对体系的安全性和有效性进行评估,并对其作用机制进行初步探究。细胞毒性实验以及注射部位的肌肉组织切片和主要脏器切片结果,均证明该亚微米Pickering乳液具备良好的生物安全性。在动物免疫实验中,与3 μm左右的Pickering乳液组相比,亚微米Pickering乳液组虽未能显著提高抗体滴度水平,但却能够提高脾细胞因子IFN-γ的分泌,倾向于促进Th1型免疫响应。综上所述,成功制备了亚微米Pickering乳液,作为疫苗佐剂可提高细胞免疫效果,但其作为疫苗佐剂的应用效果还有待进一步研究。;Pickering emulsions are special emulsion formulations using solid particles instead of surfactants. The use of solid particles as emulsion stabilizers not only improve the stability of the Pickering emulsions, but also endue Pickering emulsions with many special properties, making Pickering emulsions been used as promising materials for biomedical applications. However, there are still many problems such as poor biocompatibility and the droplet size of Pickering emulsions is in micron, which limit their wide applications in the biomedical field. The purpose of this paper is to prepare a submicron Pickering emulsion with good biocompatibility and preliminarily explore its potential biological applications. The contents of this thesis are as follows:(1) To achieve the goal of preparation of stable submicron Pickering emulsion, the Pickering emulsions were prepared by sonification using squalene as oil phase and screened nanoparticles as emulsifier. The effects of preparation conditions such as particle concentration, oil-water ratio, aqueous phase, ultrasonic time and frequency on size distribution and stability of Pickering emulsions were investigated. Finally, the stable submicron-sized Pickering emulsion with uniform size, good dispersibility and centrifugal resistance was prepared using cellulose nanocrystals (CNCs) as particle emulsifier, and the average size was about 600 nm. And it can be stably stored more than 30 days. The size and zeta potential of the submicron Pickering emulsion remain basically unchanged under different storage temperatures, such as 4ºC、25ºC and 37ºC, which means that the submicron Pickering emulsion with good storage stability. The confocal laser scanning microscopic (CLSM) images revealed that Pickering emulsion was formed successfully by the adsorption of CNCs on the oil-water interface.(2) To initially evaluate the biological application potential of submicron Pickering emulsion as a vaccine adjuvant, the effect and mechanism of CNCs stabilized submicron Pickering emulsion in OVA prophylactic vaccine were investigated by using chicken egg ovalbumin (OVA) as a model antigen. The safety and efficacy of this adjuvant system were evaluated by cell and animal experiments, and then the potential mechanism was further explored. The cellular safety of the submicron Pickering emulsion was demonstrated by cytotoxicity experiments, and the results of muscle tissue section at the injection site and main organ section proved that the submicron Pickering emulsion had good injection safety. In the immunoassay of OVA model antigen, although the submicron Pickering emulsion did not significantly increase the antibody titer level compared with Pickering emulsion groups of which size was about 3 μm , the secretion of spleen cytokine IFN-γ was improved, which means that there was the incline of the Th1-type immune response.In summary, the submicron Pickering emulsion was successfully prepared, and it improved the cellular immunity as a vaccine adjuvant. But its application effect as a vaccine adjuvant remains to be further studied.

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/49644
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
屈艳玲. 亚微米Pickering乳液的制备和生物应用研究[D]. 中国科学院大学,2020.
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