文献：Cell-Penetrating Peptide Enhanced Insulin Buccal Absorption

文献链接：https://xueshu.baidu.com/usercenter/paper/show?paperid=1g2s0pk0rs7g00s0m61e0cr0j5489666&site=xueshu_se

作者：You Xu, Xiaojuan Zhang, Nana Wang, Xing Pei, Yiyue Guo, Jianxin Wang,Stefan Barth, Fei Yu, Seung Jin Lee, Huining He, Victor C. Yang

相关产品：RB-insulin

原文摘要：Non-injectable delivery of peptides and proteins is not feasible due to the limitations of large molecular mass, high hydrophilic properties, and gastrointestinal degradation. Therefore,

proposing a new method to solve this problem is a burning issue. The objective of this study was to propose a novel protein delivery strategy to overcome the poor efficacy and irritation of buccal insulin delivery. In this study, we applied a conjugate of cell-penetrating peptides (LMWP) and insulin (INS-PEG-LMWP) for buccal delivery. INS-PEG-LMWP was prepared using insulin solution and mixture as references. The transport behaviour, in vivo bioactivity, hypoglycaemic effect, and safety of INS-PEG-LMWP were systematically characterised. An in vitro study demonstrated that the uptake and transportation of INS-PEG-LMWP across buccal mucosal multilayers significantly increased. By comparing the effects of different endocytic inhibitors on INS-PEG-LMWP uptake, the conjugate might be delivered via an energy independent, electrostatically adsorbed pathway. INS-PEG-LMWP’s relative pharmacological bioavailability was high and its relative bioavailability was up to 26.86%, demonstrating no visible mucosal irritation. Cell-penetrating peptides are likely to become a reliable and safe tool for overcoming insulin’s low permeability through the epithelial multilayers, the major barrier to buccal delivery.

罗丹明 B 标记胰岛素是一种生物分子标记物。由于罗丹明 B 染料的存在，它呈现出微弱的粉色或红色色调。该产品具有特异性和稳定性。罗丹明 B 标记的胰岛素保留了胰岛素本身调节血糖的生物活性，同时通过荧光标记使得在实验研究中能够更方便地追踪和观察其作用过程。它为研究胰岛素的代谢途径、作用机制以及与细胞的相互作用提供了工具。为克服口腔胰岛素传递的效果和刺激。应用一种细胞穿透肽（LMWP）和胰岛素（INS-PEG-LMWP）的偶联物用于输送。以胰岛素溶液和混合物为参考，制备INS-PEG-LMWP。过程如下：

图：INS-PEG-LMWP共轭物的制备过程。

胰岛素的DMMA保护

溶解的胰岛素与DMMA轻微旋转反应，溶液的pH值保持在6.8-6.9之间。然后通过透析过夜去除未反应的DMMA.

INS-PEG-MAL的制备

DMMA保护胰岛素与NHS-PEG-MAL在室温下反应，在胰岛素的氨基和PEG的-NHS基之间形成酰胺键生成INS-PEG-MAL

INS-PEG-LMWP的制备

将LMWP-SH加入INS-PEG-MAL中，室温孵育2h，通过马来酰亚胺基团生成INS-PEG-LMWP。LMWP也与INS-PEG-MAL孵育2小时，以产生混合的INS-PEG-MAL/LMWP作为对照。罗丹明标记的胰岛素INS-PEG-LMWP使用罗丹明标记的胰岛素制备，其他步骤与INS-PEG-LMWP相同。

图：INS-PEG-LMWP共轭物制备过程

结论：体外研究表明，使用罗丹明标记的胰岛素制备的INS-PEG-LMWP在颊部黏膜多层膜上的摄取和运输增加。通过比较不同的内吞抑制剂对INS-PEG-LMWP摄取的影响，偶联物可能通过一个能量独立的、静电吸附的途径传递。INS-PEG-LMWP的相对药理生物利用度较高，其相对生物利用度高达26.86%，显示无明显的黏膜刺激。细胞穿透肽很可能成为一种可靠和安全的工具，以克服胰岛素通过上皮多层膜的低渗透性，这是口腔传递的主要障碍。