透明质酸微针经皮递送胰岛素智能给药系统用于糖尿病治疗

Application of hyaluronic acid microneedles in insulin intelligent delivery system for the treatment of diabetes

摘要:

本研究通过共沉淀法制备了胰岛素(insulin,INS)/Ca₃PO₄复合物和葡萄糖氧化酶(glucose oxidase,GOx)/Cu₃(PO₄)₂复合物，得到的矿化胰岛素(mineralized insulin,m-INS)呈现不规则结晶团簇状，矿化葡萄糖氧化酶(m-Gox)呈花球状形貌，直径约1–2 μm。体外模拟释放实验表明，m-INS会随介质pH值降低而释放出INS，pH为4.5时其释放量达到96.68%；酶活力检测实验表明m-GOx的酶活力稳定性高于游离的GOx，在室温放置10 d后仍保持较高活力，而GOx活力小于60%。通过配制葡萄糖溶液模拟正常血糖(5.6 mmol/L)和高血糖(22.2 mmol/L)状态，在葡萄糖溶液中加入m-INS和m-GOx，INS的释放量呈现显著的葡萄糖浓度依赖性，即葡萄糖浓度越高，INS释放量和释放速率越大。最后，将m-INS、m-GOx与透明质酸(hyaluronic acid，HA)溶液混合，制备负载m-INS和m-GOx的HA微针阵列，构建1型糖尿病模型鼠，通过微针给药的方式评估载药HA微针对糖尿病大鼠的血糖控制效果。结果表明：负载m-INS/m-GOx的HA微针能有效递送药物，糖尿病大鼠的平均血糖浓度在1 h内下降到约7 mmol/L，并能维持10 h的正常血糖，使血糖浓度低于给药前水平长达36 h。与仅负载INS的HA微针相比，m-INS微针具有更好的葡萄糖耐受性、更持久的控糖效果和更小的低血糖风险。相对于其他的缓释系统，本研究中的核心成分制备流程简单、效率高和安全有效，具备较大的商业化潜力。

Abstract:

In this study, insulin (insulin, INS)/Ca₃PO₄ complex and glucose oxidase (glucose oxidase, GOx)/Cu₃(PO₄)₂ complex were prepared by coprecipitation method. The mineralized insulin (mineralized insulin, m-INS) showed irregular crystalline clusters, and the mineralized glucose oxidase (m-GOx) showed flower spherical morphology, with a diameter of about 1-2 μm. In vitro simulated release experiment showed that m-INS released INS as the pH value of the medium decreased. When the pH value was 4.5, the release amount reached 96.68%. The enzyme activity detection experiment showed that the enzyme activity stability of m-GOx was higher than that of free GOx. It still maintained high activity after 10 days at room temperature, while the activity of GOx was less than 60%. The glucose solution was prepared to simulate the state of normal blood glucose (5.6 mmol/L) and hyperglycemia (22.2 mmol/L). When m-INS and m-GOx were added to the glucose solution, the release amount of INS showed a significant glucose concentration dependence. The higher the glucose concentration, the greater the release amount and release rate of INS. Finally, m-INS, m-GOx and hyaluronic acid (HA) solution were mixed to prepare HA microneedle arrays loaded with m-INS and m-GOx. Type 1 diabetes mice were constructed to evaluate the effect of drug-loaded HA microarray on blood glucose control in diabetic rats. The results show that the HA microneedles loaded with m-INS/m-GOx could deliver drugs effectively. The average blood glucose concentration in diabetic rats dropped to about 7 mmol/L within 1 h, normal blood glucose concentration could be maintained for 10 h, and the overall blood glucose concentration was lower than the level before administration for 36 hours. Compared with HA microneedles loaded with INS only, m-ins microneedles showed better glucose tolerance, longer-lasting glucose control effect and less risk of hypoglycemia. Compared with other sustained-release systems, the preparation process of the core components in this study is simple, efficient, safe and effective, and has great commercial potential.