Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
Biomaterials. 2013 Oct;34(31):7733-43. doi: 10.1016/j.biomaterials.2013.06.047. Epub 2013 Jul 14.
Although oral delivery of insulin offers a number of unmatched advantages, it nevertheless is beset by the poor permeability of insulin molecules through the epithelial cell membranes of the intestinal mucosal layer. We previously reported the development of low molecular weight protamine (LMWP) as a non-toxic yet potent cell-penetrating peptide, of which via covalent linkage was capable of translocating protein cargos through the membranes of almost all cell types. It is therefore hypothesized that LMWP could be practically employed as a safe and effective tool to deliver insulin across the intestinal mucosal membrane, thereby augmenting its absorption through the GI tract. However, formulating 1:1 monomeric insulin/LMWP conjugate presents a tall order of challenge, as the acidic insulin and basic LMWP would automatically form tight aggregates through electrostatic interactions. In this paper, we developed an innovative conjugation strategy to solve this problem, by using succinimidyl-[(N-maleimidopropionamido)-polyethyleneglycol] ester (NHS-PEG-MAL) as an intermediate cross-linker during the coupling process. Both SDS-PAGE and MALDI-TOF mass spectroscopy confirmed the formation of a homogenous, monomeric (1:1 ratio) insulin/LMWP conjugate without encountering the conventional problem of substrate aggregation. Cell culture studies demonstrated that transport of the Insulin-PEG-LMWP conjugate across the intestinal mucosal monolayer was augmented by almost five-folds compared to native insulin. Furthermore, results from the in situ loop absorption tests in rats showed that systemic pharmacological bioavailability of insulin was significantly enhanced after its conjugation with LMWP. Overall, the presented chemical conjugation with LMWP could offer a reliable and safe means to improve the intestinal permeability of therapeutic peptides/proteins, shedding light of the possibility for their effective oral delivery.
虽然胰岛素的口服给药具有许多无与伦比的优势，但它仍然存在胰岛素分子通过肠黏膜层上皮细胞膜的通透性差的问题。我们之前报道了低分子量鱼精蛋白（LMWP）的开发作为一种无毒但有效的细胞穿透肽，通过共价键连接能够将蛋白质货物穿过几乎所有细胞类型的细胞膜。因此，假设 LMWP 可以实际用作安全有效的工具，将胰岛素递送到肠黏膜膜中，从而增加其通过胃肠道的吸收。然而，将 1:1 单体胰岛素/LMWP 缀合物配方化是一个巨大的挑战，因为酸性胰岛素和碱性 LMWP 会自动通过静电相互作用形成紧密的聚集体。在本文中，我们开发了一种创新的缀合策略来解决这个问题，使用琥珀酰亚胺基-[(N-马来酰亚胺丙酰胺基)-聚乙二醇]酯（NHS-PEG-MAL）作为偶联过程中的中间交联剂。SDS-PAGE 和 MALDI-TOF 质谱分析均证实了均一的、单体（1:1 比）胰岛素/LMWP 缀合物的形成，而不会遇到传统的基质聚集问题。细胞培养研究表明，与天然胰岛素相比，胰岛素-PEG-LMWP 缀合物穿过肠黏膜单层的转运增加了近五倍。此外，在大鼠体内环吸收试验中的结果表明，胰岛素与 LMWP 缀合后，其系统药代动力学生物利用度显著提高。总体而言，与 LMWP 的化学缀合可以提供一种可靠和安全的方法来提高治疗性肽/蛋白质的肠通透性，为它们的有效口服递送提供了可能性。
