Oude Blenke E, Sleszynska M, Evers M J W, Storm G, Martin N I, Mastrobattista E
Department of Pharmaceutics and ‡Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University , 3512 JE Utrecht, Netherlands.
Bioconjug Chem. 2017 Feb 15;28(2):574-582. doi: 10.1021/acs.bioconjchem.6b00677. Epub 2017 Jan 13.
Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.
溶酶体裂解肽通常与药物递送系统偶联，以增强溶酶体逃逸，这对于递送在内溶酶体途径中易降解的大分子药物至关重要。蜂毒肽是一种源自蜂毒的26个氨基酸的肽，具有非常高的膜溶解活性。然而，当这些裂解肽在体内应用时，也会带来重大的安全风险，因为它们通常对红细胞和其他非靶细胞膜具有类似的活性。我们的目标是通过将这些肽物理限制在纳米载体表面，从而在时间和空间上控制它们的膜破坏能力，使它们仅在被递送至酸性溶酶体内时才被激活。为此，探索了多种通过功能化聚乙二醇脂质将裂解肽偶联到脂质体上的化学方法，包括马来酰亚胺-硫醇化学、点击化学和醛-酰肼化学。后者能够通过腙键进行可逆共轭，使肽在溶酶体条件下释放。通过仔细选择共轭位点并使用蜂毒肽的pH激活类似物，在生理pH下对模型膜的裂解活性被完全抑制。在溶酶体pH下，酸不稳定的腙键水解恢复活性，以其最活跃的游离形式释放肽。此外，使用含有不可水解键的类似物作为对照，结果表明观察到的活性可完全归因于肽的释放，验证了动态共价共轭是一种在循环过程中维持安全性的合适策略。
