穿膜肽钴原卟啉药物复合体对人晶状体上皮细胞的保护作用实验

刘雅婷; 马天驹; 叶子; 李朝辉

doi:10.3969/j.issn.2095-5227.2022.06.014

穿膜肽钴原卟啉药物复合体对人晶状体上皮细胞的保护作用实验

Protective effects of an arginine rich peptide cobalt protoporphyrinin complex on human lens epithelial cells

摘要

摘要:
背景细胞穿膜肽(cell-penetrating peptides，CPPs)是一类具有细胞膜穿透能力的多肽，可以作为药物载体携带大分子物质进入细胞，其因低毒性和多组织普适性广泛运用于医学成像和治疗的研究中。
目的构建穿膜肽钴原卟啉药物复合体(R6-CoPP)，研究其生物相容性和减轻晶状体上皮细胞氧化应激损伤的作用。
方法采用Fmoc固相合成法，根据设计的序列合成寡聚精氨酸穿膜肽(R6)，以脱水缩合法将其与钴原卟啉(cobalt protoporphyrin，CoPP)连接，使用异硫氰酸荧光素(fluorescein isothiocyanate，FITC)进行标记。应用高效液相色谱仪和质谱仪测定其纯度和相对分子质量，利用扫描透射电镜(STEM)能谱面/线扫和激光纳米粒度电位测量仪观测其表征形貌、元素分布、粒子直径和表面电位；细胞流式仪和共聚焦显微镜观察R6-CoPP对体外培养的人晶状体上皮细胞系的穿膜效果；CCK-8法检测R6-CoPP对人晶状体上皮细胞系细胞活性的影响。利用H₂O₂构建晶状体上皮细胞氧化损伤模型，比较R6-CoPP与CoPP预处理对细胞凋亡的影响。
结果经高效液相色谱法鉴定R6-CoPP纯度为94.56%，质谱鉴定相对分子质量为2109.75，与预计相符，粒径为227.8 nm，表面电位为 + 13.9 mV，STEM能谱面扫显示R6-CoPP内有钴元素分布。R6-CoPP对细胞毒性较小，生物相容性好。细胞流式结果显示穿膜肽药物孵育细胞2 h后荧光阳性细胞比例可达80%以上。共聚焦显微镜荧光显示孵育15 min后R6-CoPP已经穿越细胞膜进入细胞质，细胞荧光强度随孵育时间的增加而下降。与氧化损伤模型组相比，CoPP和R6-CoPP预处理后的细胞存活率明显提高(P＜0.05)。
结论穿膜肽药物复合体R6-CoPP生物相容性较好，具有较强的细胞膜穿透能力，体外实验显示其可减轻氧化损伤所致的晶状体上皮细胞凋亡。

Abstract:
Background Cell-penetrating peptides (CPPs) are a class of peptides with cell membrane penetrating ability, which can be used as drug carriers to transport macromolecules into cells. They have been widely used in medical imaging and therapeutic research because of their low cytotoxicity and stable penetrating ability in different tissue.
Objective To design an arginine-rich peptide cobalt protoporphyrin complex and evaluate its biocompatibility and effect on lens epithelial cells under oxidative stress damage.
Methods The cell-penetrating peptide was synthesized by the Fmoc solid-phase synthesis method and then linked with cobalt protoporphyrin (CoPP), the fluorescein isothiocyanate (FITC) was added as the label. The purity and relative molecular mass of the complex (R6-CoPP) was evaluated by high-performance liquid chromatography and mass spectrometry. The characterization, elemental distribution, particle diameter, and surface potential of R6-CoPP were evaluated by scanning transmission electron microscopy (STEM) surface/line scan and laser nanoparticle size potentiometry. The penetrating ability of R6-CoPP was evaluated by confocal microscopy and flow cytometry. R6-CoPP toxicity was investigated by the CCK-8 method using human lens epithelial cells. The oxidative damage model of lens epithelial cells was created by H₂O₂ to compare the antioxidant effects of R6-CoPP and CoPP.
Results The purity of R6-CoPP was 94.56% by high-performance liquid chromatography (HLPC), and the relative molecular weight was 2109.75 by mass spectrometry (MS). The particle size was 227.8 nm and the Zeta potential was + 13.9mV. STEM energy spectrum surface scan showed the distribution of cobalt elements within the complex. R6-CoPP was nontoxic in vitro with good biocompatibility. Cellular flow cytometry showed that the percentage of fluorescent positive cells could reach more than 80% after 2 h incubation. Confocal fluorescence microscopy showed that R6-CoPP could be detected inside the lens cell within 15 minutes of culturing, and the fluorescence intensity decreased with time. Compared to the oxidative damage model group, the cell survival rate was significantly higher after pretreatment with CoPP and R6-CoPP (P<0.05).
Conclusion The cell-penetrating peptide drug complex (R6-CoPP) is biocompatible and has strong cell membrane penetration ability, which also can attenuate the apoptosis of lens epithelial cells caused by oxidative injury in vitro.

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