胰岛素/维生素B12-透明质酸纳米粒的制备及口服给药体内外性质的评价

Vitamin B12-conjugated Hyaluronic Nanoparticles Enhance Oral Delivery of Insulin

【目的】维生素B12修饰的透明质酸纳米粒口服递送胰岛素的体内外性质评价。【方法】采用二次乳化法制备载胰岛素/维生素B12修饰透明质酸纳米粒(INS/VB12-HA NP),激光粒度分析仪测定纳米粒粒径和分布,反相高效液相法测定纳米粒包封率和载药量;并用人结肠腺癌细胞(Caco-2)单层膜模型体外评价INS/VB12-HA NP的细胞摄取与跨膜转运;以糖尿病模型大鼠降血糖实验评价口服INS/VB12-HA NP的药效。【结果】所制备的INS/VB12-HA NP粒径在185～286 nm之间,PDI小于0.25,包封率在55%左右。细胞摄取实验表明在25～200μg/mL胰岛素浓度范围内和孵育0.5 h后Caco-2细胞对INS/VB12-HA NP的摄取量显著高于胰岛素溶液组。Caco-2细胞单层膜跨膜转运实验中,4 h内跨膜电阻没有明显变化,VB12-HA NP组比对照溶液组有更多的胰岛素跨膜量和更快的跨膜速率。糖尿病大鼠的降糖实验显示,与口服胰岛素溶液相比,纳米粒组均有显著的口服降血糖作用。【结论】VB12修饰的透明质酸纳米粒可促进胰岛素跨过Caco-2细胞单层膜,且对糖尿病大鼠的口服降糖作用优于胰岛素溶液。

[Objective] To investigate the effects of vitamin B12-conjugated hyaluronic nanoparticles on oral delivery of insulin. [ Methods ] Insulin loaded vitamin B 12-conjugated hyaluronic nanoparticles （INS/VB 12-HA NP） were prepared by a modified double- emulsion method. The particle size and Zeta potential measurements were carried out by photon correction spectroscopy. The insulin entrapment efficiency and loading efficiency were quantified by RP-HPLC. Caco-2 monolayer was applied as a cell model for the uptake and penetration experiment. Hypoglycemic effects of INS/VB12-HA NP on the blood glucose level of STZ- diabetic rats were evaluated. [Results] The particle size was in the range of 185-286 nm, and their PDI values were less than 0.25. The insulin entrapment efficiency was around 55%. The cellular uptake of INS/VB12-HA NP by Caco-2 cells was significantly higher than those of insulin solution after incubation 0.Sh and in the insulin concentration of 25-200 ~g/mL. The Caco-2 cell monolayer treating with INS/ VB12-HA NP for 4 h did not show any significant change in TEER. The VB12-HA NP transferred more insulin across the Caco-2 cell monolayer than insulin solution did. In vivo experiments, the INS/VB12-HA NP could reduce the blood glucose level of STZ- diabetic rats after oral administration. [Conclusion] Compared with insulin solution, VB12-HA NP enhanced the insulin through Caco-2 cell monolayer by transcellular pathway and showed strong hypoglycemic effects on STZ-diabetic rats.