Impact of PEGylation and hyaluronan functionalization on lipoplex-mediated mRNA delivery to the canine retina

Efficient messenger ribonucleic acid (mRNA) delivery to the retina remains challenging. This study investigated the effects of various polyethylene glycol (PEG) derivatives on the stability and uptake of cationic lipid-based mRNA lipoplexes <i>in vitro</i> and assessed the delivery of selected formulations to the canine retina. We present an optimized workflow for formulating mRNA lipoplexes in pure water, achieving high encapsulation efficiency. PEGylation enhanced stability of lipoplexes, particularly with PEG-DMG or hyaluronan conjugated to PEG-DPPE (HA-PEG-DPPE), maintaining size and zeta potential for 48 hours. RNA <i>in situ</i> hybridization (RNA-ISH) confirmed efficient internalization of PEGylated mRNA lipoplexes by cultured RAW264.7 and ARPE19 cells, though corresponding protein expression varied between cell lines. Analysis at 24 hours post-intravitreal injection of PEG-DMG- and HA-PEG-DPPE-stabilized enhanced green fluorescent protein (<i>eGFP</i>) mRNA lipoplexes revealed limited mRNA accumulation in inner retinal layers. In contrast, 24 hours after their subretinal administration, <i>eGFP</i> mRNA was detected in all retinal cell types, including photoreceptors, with accumulation comparable to endogenous rhodopsin (<i>RHO</i>) mRNA levels. eGFP protein expression, though, was limited to the retinal pigment epithelium (RPE). At 72 hours post-subretinal delivery, <i>eGFP</i> mRNA and protein persisted in the RPE. However, a marked reduction in <i>eGFP</i> levels was seen in other retinal layers, displaying a patchy pattern. Similarly, eGFP protein exhibited a patchy distribution across retinal layers outside the RPE. Furthermore, distinct differences in the cell types expressing the eGFP protein were observed between the two PEGylated mRNA lipoplex formulations. The data suggest that transfection efficiency in retinal cells is influenced by both intracellular processing of mRNA lipoplexes and their uptake, with the former playing a predominant role.

将信使核糖核酸（messenger ribonucleic acid, mRNA）高效递送至视网膜仍颇具挑战。本研究考察了多种聚乙二醇（polyethylene glycol, PEG）衍生物对基于阳离子脂质的mRNA脂质复合物的稳定性与摄取的体外（in vitro）影响，并评估了优选制剂在犬视网膜中的递送效果。我们开发了一套在纯水中配制mRNA脂质复合物的优化流程，可实现较高的包封率。聚乙二醇化修饰可增强脂质复合物的稳定性，尤以PEG-DMG或与透明质酸偶联的PEG-DPPE（HA-PEG-DPPE）效果显著，其可使脂质复合物的粒径与zeta电位维持48小时。RNA原位杂交（RNA in situ hybridization, RNA-ISH）结果证实，经聚乙二醇化修饰的mRNA脂质复合物可被培养的RAW264.7与ARPE19细胞高效内化，但不同细胞系的对应蛋白表达水平存在差异。向犬眼玻璃体腔注射经PEG-DMG与HA-PEG-DPPE稳定的增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）mRNA脂质复合物后24小时分析发现，其在视网膜内层的mRNA积累量有限。与之相反，经视网膜下腔给药后24小时，所有视网膜细胞类型（包括感光细胞）中均可检测到eGFP mRNA，其积累水平与内源性视紫红质（rhodopsin, RHO）mRNA相当。但eGFP蛋白的表达仅局限于视网膜色素上皮（retinal pigment epithelium, RPE）。视网膜下腔递送后72小时，eGFP mRNA与蛋白仍可在RPE中维持，但在其他视网膜层中eGFP水平显著降低，呈现斑驳分布模式。同理，eGFP蛋白在RPE以外的视网膜层中也呈现斑驳分布。此外，两种聚乙二醇化mRNA脂质复合物制剂所表达eGFP蛋白的细胞类型存在显著差异。本研究数据表明，视网膜细胞的转染效率同时受mRNA脂质复合物的胞内加工与摄取过程影响，其中胞内加工过程发挥主导作用。