<i>Rhodotorula glutinis</i> as a living cell liposome to deliver polypeptide drugs <i>in vivo</i>

The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living cell liposomes to carry polypeptide drugs may be an ideal polypeptide oral drug delivery system. GM4-ΔTS was constructed by LFH-PCR from <i>Rhodotorula glutinis</i> GM4, which was screened and preserved in our laboratory, and then transferred into choline-phosphate cytidylyltransferase (CCT), which is a rate-limiting enzyme for lecithin synthesis. The results showed that the CCT gene was highly expressed in the GM4-ΔTS strain and could significantly increase fatty acid and lecithin contents in GM4-ΔTS-PGK1-CCT. Moreover, insulin, H22-LP, and α-MSH were successfully introduced into cells <i>in vitro</i>, and the strain no longer proliferated <i>in vivo</i>, for safe and controllable polypeptide drug delivery. <i>In vivo</i>, normal mice were intragastrically administered with recombinant strains carrying insulin and α-MSH, and different levels of polypeptide drugs were detected in serum and tissue, respectively. Then, recombinant strains carrying insulin were administered to type II diabetes mellitus mice. The results showed that the strains could effectively reduce blood glucose levels in mice, which indicated that the recombinant strains could carry insulin into the body, and the drug effect was remarkable. Therefore, recombinant GM4-ΔTS-PGK1-CCT strains were successfully used as living cell liposomes to carry insulin, H22-LP, and α-MSH peptides into the body for the first time; additionally, these strains have enhanced safety, controllability, and efficacy.

重组微生物作为疾病治疗用口服药物载体的潜在优势已受到广泛关注。以重组油脂微生物作为活细胞脂质体来递送多肽药物，或可成为理想的多肽口服给药系统。本研究从本实验室筛选保藏的粘红酵母（Rhodotorula glutinis）GM4出发，通过长片段同源PCR（LFH-PCR）构建得到GM4-ΔTS菌株，随后将编码磷脂酰胆碱（卵磷脂）合成限速酶的胆碱磷酸胞苷酰转移酶（choline-phosphate cytidylyltransferase, CCT）基因导入该菌株。结果显示，CCT基因在GM4-ΔTS菌株中实现高效表达，可显著提升GM4-ΔTS-PGK1-CCT菌株内的脂肪酸与磷脂酰胆碱含量。此外，本研究成功将胰岛素、H22-LP与α-促黑素细胞激素（α-MSH）导入该菌株的体外（in vitro）培养体系中，且该菌株在体内（in vivo）不再增殖，可实现安全可控的多肽药物递送。体内实验中，正常小鼠经灌胃给予携带胰岛素与α-MSH的重组菌株后，分别在其血清与组织中检测到不同水平的多肽药物。随后，研究团队将携带胰岛素的重组菌株施用于II型糖尿病小鼠，结果显示该菌株可有效降低小鼠血糖水平，表明重组菌株能够将胰岛素递送至体内，且药效显著。综上，本研究首次成功将重组GM4-ΔTS-PGK1-CCT菌株作为活细胞脂质体，用于递送胰岛素、H22-LP及α-MSH多肽至体内；该菌株同时具备更优异的安全性、可控性与药效。