【作者】 李正荣；

【导师】 徐宇虹；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2010， 博士

【摘要】 生物大分子药物如蛋白、核酸等口服给药的生物利用度相当低,其原因之一在于蛋白或核酸等分子在苛刻的胃肠道环境中很不稳定,极易被胃肠道的酸性环境,多种蛋白酶,以及胆汁盐所降解。为了避免蛋白或核酸药物遭受胃肠道中的攻击,研究人员选用不同载体来保护这些生物活性药物。脂质体由于大小、表面电荷以及膜流动性的可控,作为口服载体的研究备受青睐。以酯键构成的普通脂质体,能够装载多肽或核酸药物,并对它们具有一定的保护作用。然而,普通脂质体作为口服输送载体,仍然面临的最大障碍之一就是:它们在肠道胆汁盐的作用下极不稳定,尤其是胆汁盐与肠道酶协同作用时。因此,我们期望寻求更加稳定的脂质体以口服输送生物活性药物。古细菌脂质体是脂质体的一种,是由古细菌细胞膜上提取的脂质组成。古细菌脂质包括二醚脂质和四醚脂质。四醚脂质的核心部分就是由40个碳原子构成的饱和类异戊二烯侧链并通过醚键与甘油骨架的2、3位碳原子相连接。而普通脂质体中的磷脂侧链是由不饱和脂肪酸链组成,并以酯键与甘油骨架的1、2位碳原子相连接。另外,由四醚脂质形成的脂质体的膜是由多个具有单分子层跨度的醚脂组成,这种独特的结构给予四醚脂质体稳定的特性。本研究就是从古细菌Sulfolobus acidocaldarius提取四醚脂质PLFE,考察由PLFE制备的四醚脂质体的体外稳定性特征,并着重评价了四醚脂质体作为口服疫苗载体在诱导粘膜免疫、系统免疫和细胞免疫中的作用以及作为多肽药物(胰岛素)的口服输送载体在糖尿病小鼠中的降血糖效果。最后结合小动物成像等技术初步分析了四醚脂质体口服后的在胃肠道中的滞留情况。本论文研究的主要内容及结果总结如下:收集生长成熟的古细菌S. acidocaldarius,成功的分离纯化得到醚脂,并通过薄层层析分析、质谱分析、红外光谱分析以及核磁共振分析进行表征。分析结果均表明我们提取、分离和纯化得到的脂质为极性醚脂—PLFE。我们以薄膜分散法制备四醚脂质体,并通过原子力显微镜对其进行形态表征,结果显示四醚脂质体外形成球形或近球形,分散效果好;颗粒边缘清晰且平均直径大小为210 nm左右。这个结果也与激光粒度测定仪测定的结果一致。而且,Zeta电位测定仪测得四醚脂质体的表面电荷为-35.1 mV,这样的电位使四醚脂质体具有好的分散性,不容易聚集。作为多肽药物和疫苗的口服载体,具有良好的稳定性特征是极其重要的。本研究中我们以钙黄绿素为渗透标记物,并模拟人的肠道环境在体外考察了普通脂质体和四醚脂质体对钙黄绿素的释放情况。结果表明:与普通脂质体相比,无论在模拟的肠道环境溶液中,由PLFE制备的四醚脂质体是相当稳定的。基于醚脂的独特结构和四醚脂质体的稳定特性,我们着重评估了四醚脂质体作为口服载体在疫苗中的作用。结果表明,与普通脂质体相比,四醚脂质体包裹的卵清蛋白(OVA)溶液经口服后,BALB/c小鼠体内不但产生了持久的、更强的系统免疫反应,而且也利于粘膜免疫反应的产生。而且,四醚脂质体在诱导体外CD8+ T细胞增殖反应方面也有着较普通脂质体超越的能力。为了初步探索脂质体经小鼠口服后的吸收或分布情况,我们首先借助于荧光标记脂质(Rhodamine-DHPE)来考察不同脂质体吸收后的情况,结果表明相对于普通脂质体,罗丹明标记的四醚脂质体被小肠吸收的总量更高,而且持续吸收的时间更长。其次我们合成了Cy5.5-OVA作为水相的荧光标记蛋白,并通过小鼠体内成像系统考察不同脂质体口服后的滞留情况,结果同样表明:相对于普通脂质体,装载Cy5.5-OVA的四醚脂质体被小肠吸收的荧光强度更强,而且在胃肠中能持续更长的时间。这两个实验中,然而无论是标记的荧光脂质,还是标记的荧光蛋白,它们的吸收以及持续特性的结果都是一致的。而且,在罗丹明标记的脂质体吸收实验中,小肠各部位对颗粒吸收的程度也是不一样的。相对于普通脂质体,罗丹明标记的四醚脂质体更容易被小肠回肠的尾部吸收;而且从离体小肠的荧光影像实验也得到类似的结果:四醚脂质体包裹的Cy5.5-OVA更多的被回肠下部分所吸收。这些也部分解释了四醚脂质体作为口服疫苗载体的体液免疫能力作用。然而真正的作用机理还需要进一步研究。尽管如此,我们的实验数据还是说明了四醚脂质体在发展成口服疫苗载体方面具有很大潜力。同时,我们也着重考察了四醚脂质体作为载体在口服胰岛素中的应用。首先,我们考察了普通脂质体和四醚脂质体在人工模拟的胃肠环境中对胰岛素的释放情况,结果表明相对于普通脂质体,无论是在胃环境还是肠道环境缓冲液中,由PLFE制备的四醚脂质体都是比较稳定的。其次,在体内试验中,我们建立的糖尿病小鼠口服不同的胰岛素溶液后体内降血糖的效果是不一样的。即相比较胰岛素的PBS溶液和普通脂质体包裹的胰岛素溶液,四醚脂质体包裹的胰岛素溶液经口服后有着较大的降血糖效果而且这种降血糖作用也会持续更长的时间。我们推测这也可能与脂质体的稳定性有关。因为进一步的体内荧光影像实验结果表明:在口服传递过程中,与普通脂质体相比,四醚脂质体包裹的Cy5.5-Insulin在胃肠道中能够滞留更长的时间,有利于药物的吸收。第三,我们在体外建立了人小肠上皮细胞的体外模型—Caco-2细胞模型,并应用此模型评估了脂质体包裹胰岛素的吸收情况。结果表明:与普通脂质体相比,四醚脂质体促进胰岛素经单层Caco-2细胞的转运量更低些。我们推测这可能归结于组成普通脂质体的磷脂与细胞膜磷脂具有类似性有关,虽然具体的吸收机制还有待于进一步证明,但提示我们:在设计药物的口服载体系统中,我们不但要考虑载体的稳定性,还要考虑载体促进小肠上皮细胞对药物的吸收能力。因此,我们也期待以四醚脂质体为基础结合靶向来改进口服载体,增加肠道对四醚脂质体胰岛素的吸收,提高生物利用度。更多还原

【Abstract】 Administering drugs orally is by far the most widely used route of administration, although it is generally not feasible for peptide and protein drugs. The main reasons for the low oral bioavailability of biologicals are presystemic enzymatic degradation. Various strategies have been pursued to overcome such barriers and to develop safe and effective oral delivery systems for biologicals.The potential usefulness of liposomes as drug carriers has attracted considerable interest due to their controlled size, charge and membrane fluidity.These phospholipid vesicles are capable of encapsulating both hydrophobic and hydrophilic drugs. The drugs encapsulated in liposomes are sufficiently protected from enzymatic attack to some extent. In this context, nevertheless, a major drawback of liposome formulations that have been studied extensively to date is their poor stability at low pH, susceptibility to attack by bile salts and GI tract enzymes. Therefore, it was desirable to develop more stable liposome formulations to protect peptide and protein drugs.Archaeosomes are liposomes which are made from structurally typical of archaeobacterial membrane lipids. Archaeobacterial lipids consist of archaeol (diether) and caldarchaeol (tetraether) core structures. wherein tetraether lipids regularly branched and usually fully saturated phytanyl chains (40 carbons in length), are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone. In contrast, conventional phospholipids have fatty acyl chains of variable length, which may be unsaturated, and which are attached via ester bonds to the sn-1,2 carbons of the glycerol backbone. Moreover, the lipid moieties were composed of membrane-spanning caldarchaeol lipids, known to form a monomolecular thick membrane by span the entire lamellar structure, and the unique lipid structures in general confer considerable stability to archaeosomes.In the present study, PLFE (polar lipid fraction E) was distilled from S. acidocaldarius, the stability of archaeosomes composed of PLFE has been examined in stimulated GI environment. The objective of our study was to evaluate the potential of archaeosomes to induce humoral and CD8+ T cell mediated immune responses to entrapped antigen via the oral immunization. As oral carrier of insulin, the hypoglycemic effect of the archaeosomes in diabetic mice was also evaluated. Finally, we examined the intestinal transit of archaeosomes through In Vivo Fluorescence Imaging. The detailed results were summarized as following:PLFE lipids were purified as a single fraction from the crude lipid extract of S. acidocaldarius. The isolated PLFE lipids were characterized through TLC, mass spectral, infrared spectroscopy and NMR, and these results provided conclusive support that the final product was PLFE. In order to investigate the shape and surface morphology of the archaeosomes, atomic force microscopy (AFM) was employed. The AFM images showed that the form of archaeosomes was nearly spherical, and that there was no aggregation or adhesion among the particles, and the AFM micrograph of the archaeosomes revealed that the mean diameter was about 210 nm, which was in good agreement with data obtained by PCS. In addition, the mean surface potential archaeosomes was measured to be -35.27 mV, which was responsible for the good poly.index. One of the requirements of a successful oral delivery system is reasonable stability in the environment of GI tract. In the present study, to determine the stability of carriers during passage through GI via oral route, our study was performed with the release of calcein from vesicles in mimic intestinal condition, and these results indicated that the archaeosomes composed of PLFE are essentially stable in these mimic intestinal conditions.Based on the unique structure from PLFE and stability of archaeosomes, Our studies performed in mice have indicated that archaeosomes are superior in eliciting humoral immune response, as well as CD8+ T cell mediated immune response to encapsulated antigen, compared with conventional liposomal formulation. In order to explore primarily the distribution and absorption of various liposomal formulation post vaccination orally, one hand, Rhodamine-DHPE was employed, and the results showed that Rhodamine-DHPE associated with intestine when administered archaeosomes increased and attained a higher level than that of conventional liposomes, especially in the lower ileum. In the other hand, Cy5.5-labeled OVA was used as optical tracer, and the corresponding results showed archaeosomes prolonged intestinal residence time of optical tracer in GI tract, which reversibly increased the permeability of the mucosal epithelium. Further optical imaging results from incised small intestinal suggested that archaeosomes containing optical tracer were preferentially taken up in the lower ileum. These results may also be corrected with enhanced immunity. However, the mechanisms of action require further study. Nevertheless, these available data from our study indicate that archaeosomes may hold great promise in developing oral vaccines for weak antigens.Subsequent, we investigated the possibility of archaeosomes as a tool for the oral delivery of insulin. Firstly, the results from stability in vitro showed the release of insulin from archaeosomes was markedly reduced not only in the acidly solution but also in the bile salts. Secondly, when insulin was orally administered to diabetic mice as either a solution or conventional liposomes, no hypoglycemic effect was observed. Whereas administration of insulin encapsulated in archaeosomes caused the rapid decrease in the plasma glucose level and the hypoglycemic effect lasted for much longer duration than that of conventional liposomes, which, we speculated, was contributed to the stability of various carriers. Further optical imaging results from in vivo showed that the slow release of insulin from archaeosomes achieved the longer duration in the small intestine, which was benefit for drug absorption. Thirdly, we established Caco-2 cell monolayer model, and evaluated the absorption characteristics of insulin as various formulations. The results showed the cumulative amount of insulin transported through Caco-2 cell monolayer for archaeosomes was lower than that of conventional liposomes, which, we speculated, was ascribed to composition prepared for liposomes. Despite, the mechanisms of action require further study, these results suggested that an ideal delivery system for oral administration should not only protect drug or antigen from the harsh environment in the GI, but also enhance the absorption of them by small intestine. Therefore, we expect to optimize the delivery system through combination the stable archaeosomes with target strategy to increase the uptake of drug and improve the bioavailability.更多还原