Greschner Andrea A, Brahiti Nadine, Auger Maud, Hu Lei, Soleymani Abyaneh Hoda, Barbeau Xavier, Parent Victor, Gaillet Bruno, Guay David, Soultan Al-Halifa, Gauthier Marc A
Institut National de la Recherche Scientifique (INRS),EMT Research Center, 1650 boulevard Lionel-Boulet, Varennes, QC J3X 1P7, Canada.
Feldan Therapeutics, 2666 boulevard du Parc Technologique Suite 290, Québec QC G1P 4S6, Canada.
Biomacromolecules. 2023 Nov 13;24(11):4890-4900. doi: 10.1021/acs.biomac.3c00603. Epub 2023 Oct 20.
The cell membrane is a restrictive biological barrier, especially for large, charged molecules, such as proteins. The use of cell-penetrating peptides (CPPs) can facilitate the delivery of proteins, protein complexes, and peptides across the membrane by a variety of mechanisms that are all limited by endosomal sequestration. To improve CPP-mediated delivery, we previously reported the rapid and effective cytosolic delivery of proteins in vitro and in vivo by their coadministration with the peptide S10, which combines a CPP and an endosomal leakage domain. Amphiphilic peptides with hydrophobic properties, such as S10, can interact with lipids to destabilize the cell membrane, thus promoting cargo internalization or escape from endosomal entrapment. However, acute membrane destabilization can result in a dose-limiting cytotoxicity. In this context, the partial or transient deactivation of S10 by modification with methoxy poly(ethylene glycol) (mPEG; i.e., PEGylation) may provide the means to alter membrane destabilization kinetics, thereby attenuating the impact of acute permeabilization on cell viability. This study investigates the influence of PEGylation parameters (molecular weight, architecture, and conjugation chemistry) on the delivery efficiency of a green fluorescent protein tagged with a nuclear localization signal (GFP-NLS) and cytotoxicity on cells in vitro. Results suggest that PEGylation mostly interferes with adsorption and secondary structure formation of S10 at the cell membrane, and this effect is exacerbated by the mPEG molecular weight. This effect can be compensated for by increasing the concentration of conjugates prepared with lower molecular weight mPEG (5 to ∼20 kDa) but not for conjugates prepared with higher molecular weight mPEG (40 kDa). For conjugates prepared with moderate-to-high molecular weight mPEG (10 to 20 kDa), partial compensation of inactivation could be achieved by the inclusion of a reducible disulfide bond, which provides a mechanism to liberate the S10 from the polymer. Grafting multiple copies of S10 to a high-molecular-weight multiarmed PEG (40 kDa) improved GFP-NLS delivery efficiency. However, these constructs were more cytotoxic than the native peptide. Considering that PEGylation could be harnessed for altering the pharmacokinetics and biodistribution profiles of peptide-based delivery agents in vivo, the trends observed herein provide new perspectives on how to manipulate the membrane permeabilization process, which is an important variable for achieving delivery.
细胞膜是一种具有限制作用的生物屏障，对于诸如蛋白质等大分子带电物质而言尤其如此。细胞穿透肽（CPP）的应用能够通过多种机制促进蛋白质、蛋白质复合物及肽跨膜转运，但这些机制均受内体截留的限制。为了改善CPP介导的转运，我们之前报道了通过将蛋白质与肽S10共同给药，可在体外和体内实现蛋白质快速有效的胞质转运，肽S10结合了一种CPP和一个内体渗漏结构域。具有疏水特性的两亲性肽，如S10，可与脂质相互作用使细胞膜不稳定，从而促进货物内化或从内体截留中逃逸。然而，急性膜不稳定可导致剂量限制性细胞毒性。在此背景下，用甲氧基聚（乙二醇）（mPEG；即聚乙二醇化）修饰使S10部分或暂时失活，可能提供改变膜不稳定动力学的方法，从而减轻急性通透性改变对细胞活力的影响。本研究调查了聚乙二醇化参数（分子量、结构和偶联化学）对体外标记有核定位信号的绿色荧光蛋白（GFP-NLS）递送效率及细胞毒性的影响。结果表明，聚乙二醇化主要干扰S10在细胞膜上的吸附和二级结构形成，且这种效应会因mPEG分子量的增加而加剧。通过增加用较低分子量mPEG（5至约20 kDa）制备的偶联物浓度可弥补这种效应，但对于用较高分子量mPEG（40 kDa）制备的偶联物则无法弥补。对于用中等到高分子量mPEG（10至20 kDa）制备的偶联物，通过引入可还原的二硫键可实现部分失活补偿，该二硫键提供了一种从聚合物中释放S10的机制。将多个S10拷贝嫁接到高分子量多臂PEG（40 kDa）上可提高GFP-NLS的递送效率。然而，这些构建体比天然肽的细胞毒性更大。考虑到聚乙二醇化可用于改变基于肽的递送剂在体内的药代动力学和生物分布情况，本文观察到的趋势为如何操控膜通透化过程提供了新视角，而膜通透化过程是实现递送的一个重要变量。
