Institute of Pharmaceutical Science, King's College London, Franklin-Wilkins Building, 150 Stamford Street, Waterloo Campus, London SE1 9NH, UK.
Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H 0AJ, UK.
Biomater Sci. 2023 May 2;11(9):3335-3353. doi: 10.1039/d2bm01905a.
Lipopolyplexes (LPDs) are of considerable interest for use as gene delivery vehicles. Here LPDs have been prepared from cationic vesicles (composed of a 1 : 1 molar ratio of DOTMA with the neutral helper lipid, DOPE), singly branched cationic peptides and plasmid DNA. All peptides contained a linker sequence (cleaved by endosomal furin) attached to a targeting sequence selected to bind human airway epithelial cells and mediate gene delivery. The current study investigates the effects of novel Arg-containing cationic peptide sequences on the biophysical and transfection properties of LPDs. Mixed His/Arg cationic peptides were of particular interest, as these sequences have not been previously used in LPD formulations. Lengthening the number of cationic residues in a homopolymer from 6 to 12 in each branch reduced transfection using LPDs, most likely due to increased DNA compaction hindering the release of pDNA within the target cell. Furthermore, LPDs containing mixed Arg-containing peptides, particularly an alternating Arg/His sequence exhibited an increase in transfection, probably because of their optimal ability to complex and subsequently release pDNA. To confer stability in serum, LPDs were prepared in 0.12 M sodium chloride solution (as opposed to the more commonly used water) yielding multilamellar LPDs with very high levels of size reproducibility and DNA protection, especially when compared to the (unilamellar) LPDs formed in water. Significantly for the clinical applications of the LPDs, those prepared in the presence of sodium chloride retained high levels of transfection in the presence of media supplemented with fetal bovine serum. This work therefore represents a significant advance for the optimisation of LPD formulation for gene delivery, under physiologically relevant conditions, .
脂多聚体（LPD）作为基因传递载体具有重要的应用价值。本研究采用阳离子脂质体（由 DOTMA 与中性辅助脂质 DOPE 以 1:1 的摩尔比组成）、单支链阳离子肽和质粒 DNA 制备 LPD。所有肽均含有连接序列（被内体弗林蛋白酶切割），连接序列连接靶向序列，该靶向序列用于结合人呼吸道上皮细胞并介导基因传递。本研究考察了新型含精氨酸的阳离子肽序列对 LPD 的生物物理和转染特性的影响。具有特别兴趣的是混合 His/Arg 阳离子肽，因为这些序列以前未用于 LPD 制剂中。每条支链中的阳离子残基数目从 6 增加到 12 时，LPD 的转染效率降低，这很可能是由于 DNA 压缩增加，从而阻碍了靶细胞内 pDNA 的释放。此外，含有混合含精氨酸肽的 LPD，特别是交替的 Arg/His 序列，表现出转染效率提高，这可能是因为它们具有最佳的能力来复合随后释放 pDNA。为了在血清中赋予稳定性，将 LPD 制备在 0.12 M 氯化钠溶液中（而不是更常用的水），得到具有非常高的尺寸重现性和 DNA 保护水平的多层 LPD，特别是与在水中形成的（单分子层）LPD 相比。对于 LPD 的临床应用而言，重要的是，在含有氯化钠的条件下制备的 LPD 在含有补充有胎牛血清的培养基中保持高转染水平。因此，本工作代表了在生理相关条件下优化 LPD 制剂用于基因传递的重要进展。
