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载胰岛素口服固体脂质纳米粒的构建及其跨肠上皮细胞转运的研究

Construction of Oral Insulin-Loaded Solid Lipid Nanoparticles and Their Intestinal Epithelial Cell Transcytosis Study

    摘要
  • 摘要:
      目的  采用混合溶剂构建载多肽蛋白类药物的固体脂质纳米递药系统（solid lipid nanoparticles, SNPs），探究SNPs跨肠上皮细胞的转运机理，提高多肽蛋白类药物的细胞摄取和跨膜转运效率。
      方法  采用甲醇-氯仿混合溶剂制备载胰岛素的水包油包水型固体脂质纳米粒（INS-SNPs）。通过单因素筛选法优化INS-SNPs的处方，并表征最优纳米粒的形态、体外稳定性和药物释放等性质。通过Caco-2细胞考察INS-SNPs的细胞毒性、细胞摄取和入胞机制。采用Transwell®单层细胞模型进一步评价纳米粒的跨膜转运效率。
      结果  筛选得到的最优处方INS-SNPs的粒径为（145.4±0.5） nm，电位为（−12.9±0.2） mV，载药量为（7.93±0.02）%。INS-SNPs在模拟肠液中展现出良好的胶体稳定性，能持续缓慢释放药物。细胞摄取结果显示，INS-SNPs在与细胞孵育2 h时达到最大的摄取量，较游离胰岛素提高了1.53倍。摄取机制表明，INS-SNPs主要通过网格蛋白和小窝蛋白介导的内吞途径进入肠上皮细胞。进一步考察纳米粒跨膜转运发现，跨膜转运效率较游离胰岛素提高了1.54倍，与细胞摄取的提高程度（1.53倍）相当，提示纳米粒具有较好的跨膜转运效率。
      结论  本研究采用混合溶剂构建的INS-SNPs能显著提高多肽蛋白类药物跨膜转运效率，展现出较大的口服应用潜力，可为后续口服纳米递药系统的设计提供参考。

     

    Abstract:
      Objective  To construct solid lipid nanoparticle (SNPs) drug delivery system loaded with peptide and protein drugs by using mixedsolvents, to study the transcytosis mechanisms of SNPs across intestinal epithelial cells, and to improve the endocytosis and transcytosis efficiency of peptide and protein drugs.
      Methods  The formulation of insulin-loaded water-in-oil-in-water solid lipid nanoparticles (INS-SNPs) was prepared by using a methanol-chloroform mixed solvent. The formulation was optimized with the single factor screening method. The optimized INS-SNPs were then characterized in terms of their morphology, stability and drug release properties. The cytotoxicity, cellular uptake and endocytosis mechanisms of INS-SNPs were then assessed on Caco-2 cells. The transcytosis efficiency of INS-SNPs was finally evaluated by using cellular monolayer in Transwell® insert.
      Results  The size, zeta potentials and drug loading efficiency of the optimized INS-SNPs were observed to be (145.4±0.5) nm, (−12.9±0.2) mV and (7.93±0.02)%, respectively. INS-SNPs were then shown to maintain desirable colloidal stability and sustained release of insulin in the simulated intestinal fluid. It was revealed that the cellular uptake of INS-SNPs reached its maximum after cellular incubation for 2 hours and was 1.53-fold higher than that of free insulin. Investigation of the endocytic mechanism revealed that INS-SNPs enter intestinal epithelial cells mainly through the clathrin-mediated and caveolae-mediated endocytosis pathways. Further investigation revealed that the amount of INS-SNPs permeating the cell monolayers was 1.54-fold higher than that of free insulin, which was comparable to the increase in their cellular uptake efficiency, indicating that INS-SNPs displayed enhanced absorption across the intestinal epithelium.
      Conclusion  The INS-SNPs prepared with mixed solvents in this study could significantly enhance the transcytosis efficiency of peptide and protein drugs, displaying great potentials in the application of oral drug delivery. This study may provide information and reference for the designing of efficient oral nano-drug delivery system in the future.

     

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