大多数视网膜疾病涉及脉络膜视网膜色素上皮(RPE)细胞的变性。由于血液-视网膜屏障(紧密连接形成),RPE细胞限制亲水性大分子的进入(例如,小干扰RNA(siRNA))通过血流和滴眼液。细胞质响应性硬脂酰化(STR)肽,STR-CH2R4H2C(CH2R4)能够实现稳定的siRNA复合,细胞渗透,和细胞内动力学控制。我们先前证明了CH2R4修饰的脂质体如何促进siRNA功效。我们研究了功能肽的氨基酸序列对细胞摄取途径的影响,siRNA转染功效,和肽修饰脂质体在大鼠RPE-J细胞中的渗透。四种STR肽,由精氨酸(R)组成,半胱氨酸(C),组氨酸(H),赖氨酸(K)或丝氨酸(S),设计了基于CH2R4。我们准备了装载siRNA,肽修饰的阳离子脂质体(CH2R4-,CH2K4-,CH2S4-,SH2R4-,和SH2S4-脂质体复合物)。CH2R4-,CH2K4-,和SH2R4-脂质复合物通过激活细胞骨架F-肌动蛋白,可能是由于阳离子氨基酸(精氨酸,赖氨酸)。SH2R4-脂质复合物被困在内体中,而CH2R4-和CH2K4-脂质体复合物增强了内体siRNA的释放,表明半胱氨酸有助于内体逃逸。虽然阳离子脂质体为基础,CH2S4-和SH2S4-脂质复合物(不包括精氨酸和赖氨酸)显示较低的siRNA转染效率。这种差异可能是因为siRNA保留在CH2R4-中的肽部分和阳离子脂质体上,CH2K4-和SH2R4-脂质体复合物,而在CH2S4-和SH2S4-脂质体复合物中,将siRNA加载到阳离子脂质体中,但不是肽。在三维球体中,CH2R4-和CH2K4-修饰的脂质体促进通过紧密连接的渗透。因此,CH2R4的肽序列中的阳离子氨基酸和半胱氨酸对于使用功能性肽修饰的脂质体将siRNA递送至视网膜可能是有效的。
Most retinal diseases involve the degeneration of choroidal retinal pigment epithelial (RPE) cells. Because of a blood-retina barrier (tight junction formation), RPE cells restrict the entry of hydrophilic macromolecules (e.g., small interfering RNA (siRNA)) through blood stream and eye drops. A cytoplasm-responsive stearylated (STR) peptide, STR-CH2R4H2C (CH2R4) enables stable siRNA complexation, cell permeation, and intracellular dynamics control. We previously demonstrated how CH2R4-modified liposomes promoted siRNA efficacy. We investigated the influence of amino acid sequences of functional peptides on cellular uptake pathways, siRNA transfection efficacy, and the permeation of peptide-modified liposomes in rat RPE-J cells. Four STR-peptides, consisting of arginine (R), cysteine (C), histidine (H), lysine (K) or serine (S), were designed based on CH2R4. We prepared siRNA-loaded, peptide-modified cationic liposomes (CH2R4-, CH2K4-, CH2S4-, SH2R4-, and SH2S4-lipoplexes). CH2R4-, CH2K4-, and SH2R4-lipoplexes induced cellular uptake by macropinocytosis by activating cytoskeletal F-actin, possibly due to cationic amino acids (arginine, lysine). SH2R4-lipoplexes were trapped in endosomes, whereas CH2R4- and CH2K4-lipoplexes enhanced endosomal siRNA release suggesting cysteine contributes to endosomal escape. Although cationic liposome-based, CH2S4- and SH2S4-lipoplexes (not including arginine and lysine) showed lower siRNA transfection efficiency. This difference may be because siRNAs were retained on both peptide moieties and cationic liposomes in CH2R4-, CH2K4- and SH2R4-lipoplexes, whereas in CH2S4- and SH2S4-lipoplexes, siRNAs were loaded to the cationic liposomes, but not on peptides. In three-dimensional spheroids, CH2R4- and CH2K4-modified liposomes promoted permeation through tight junctions. Thus, cationic amino acids and cysteine within peptide sequences of CH2R4 could be effective for siRNA delivery to the retina using functional peptide-modified liposomes.