pH敏感穿膜肽修饰的载α-半乳糖神经酰胺的脂质体免疫作用机制的初步研究

Preliminary study on immune mechanisms of pH-sensitive transmembrane peptide modified liposome loaded with α-galactosylceramides

通过制备pH敏感穿膜肽TH[AGYLLGHINLHHLAHL(Aib)HHIL-Cys]修饰的载免疫佐剂α-半乳糖神经酰胺的脂质体(TH modified liposome loaded with alpha-galactosylceramides,αGC-TH-Lip),并研究其对荷瘤小鼠免疫功能的影响及其作用机制。采用薄膜分散-探头超声法制备脂质体,测定TH修饰的脂质体(TH peptidemodified liposome,TH-Lip)体外被小鼠树突状细胞(dendritic cell,DC)系DC2.4细胞的摄取;给药后荷瘤小鼠体内抗肿瘤相关免疫细胞的激活、促进DC细胞成熟的程度以及辅助性T细胞(helper T cell,Th)的分化情况。结果显示,TH-Lip在体外被DC2.4细胞的摄取量是聚乙二醇修饰的脂质体(polyethylene glycols modified liposome,PEG-Lip)的1.48倍;给药后荷瘤小鼠体内自然杀伤性T细胞、自然杀伤细胞和巨噬细胞数量分别为(0.43±0.048)%、(12.80±0.50)%和(3.13±0.26)%,成熟DC细胞达到(2.30±0.22)%,细胞毒性T淋巴细胞的数量达到(32.30±0.80)%,较游离αGC组有显著性差异;αGC-TH-Lip对Th1细胞分化的诱导效应最强,对Th2细胞的分化无明显促进作用。此结果表明,该αGC-TH-Lip能够提高小鼠的免疫活性,增强α-半乳糖神经酰胺的作用效果,促进淋巴细胞更多地向细胞免疫的方向分化,从而达到更好的抗癌免疫治疗作用。

In this study, we aim to develop a pH-sensitive transmembrane peptide TH （AGYLLGHINLHH LAHL（Aib）HHIL-Cys） modified liposome loaded with immunoadjuvant a-galactosylceramides （aGC-TH- Lip） and then investigate its effect on the immune function in tumor-bearing mice and its immune mechanism of action. The liposomes were prepared by membrane dispersion-probe ultrasound method and the size and zeta potential of aGC-TH-Lip were also characterized. The uptake of TH modified liposomes （TH-Lip） and polyethylene glycols modified liposomes （PEG-Lip） in DC2.4 cells in vitro were analyzed and the activation of natural killer T （NKT）, natural killer （NK） and macrophages in tumor-bearing mice were also measured after systemic administrations of samples. Besides, the degree of maturation of dendritic cell （DC）, the number of cytotoxic T lymphocyte （CTL） and the differentiation of helper T cell （Th） were determined. The results showed the particle size of aGC-TH-Lip was about 117.9 nm and the zeta potential was about -8.37 mV under the neutral condition （pH 7.4） and the aGC-TH-Lip had high serum stability in 50% fetal bovine serum. The uptake of TH-Lip in DC2.4 cells in vitro was 1.48 times higher than that of PEG-Lip. After systemic administrations of the samples, the numbers of NKT cells, NK cells and macrophages in tumor-bearing mice were （0.43 ± 0.048） %, （12.80 ± 0.50） % and （3.13 ± 0.26） %, respectively, and the number of mature DCs and CTLs reached （2.30±0.22）% and （32.30±0.80）% separately, which was significantly different from the control group. Finally, we discovered the aGC-TH-Lip had the strongest induction effect on the differentiation of Thl cells, while barely promote the differentiation of Th2 cells. All the above results demonstrated that the aGC-TH-Lip can improve the immune the activity of mice, enhance the effect of a-galactosylceramide and promote the differentiation of lymphocytes toward the direction of cellular immunity, which consequently achieve a better anti-cancer immune activity.