MicroRNA delivery by arginine-rich cell-penetrating peptides: An investigation on expression and the cellular uptake mechanisms

被引:0
作者
Mahmoudi, Ali [1 ,2 ]
Jalili, Amin [1 ]
Aghaee-Bakhtiari, Seyed Hamid [1 ,2 ]
Oskuee, Reza Kazemi [3 ,4 ]
Mostafapour, Asma [1 ]
Kesharwani, Prashant [5 ]
Sahebkar, Amirhossein [6 ,7 ]
机构
[1] Mashhad Univ Med Sci, Fac Med, Dept Med Biotechnol & Nanotechnol, Mashhad, Iran
[2] Mashhad Univ Med Sci, Bioinformat Res Ctr, Mashhad, Iran
[3] Mashhad Univ Med Sci, Appl Biomed Res Ctr, Mashhad, Iran
[4] Mashhad Univ Med Sci, Targeted Drug Delivery Res Ctr, Mashhad, Iran
[5] Jamia Hamdard, Sch Pharmaceut Educ & Res, Dept Pharmaceut, New Delhi 110062, India
[6] Saveetha Univ, Saveetha Med Coll & Hosp, Saveetha Inst Med & Tech Sci, Ctr Global Hlth Res, Chennai, India
[7] Mashhad Univ Med Sci, Pharmaceut Technol Inst, Biotechnol Res Ctr, Mashhad, Iran
关键词
MicroRNA delivery; Cellular Uptake mechanism; Gene therapy; CPPs; NANOPARTICLES; ENDOCYTOSIS;
D O I
10.1016/j.colsurfa.2024.134749
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
Developing safe and effective methods for intracellular miRNA delivery remains a challenge. This study used the MR peptide-based carrier for miRNA delivery into liver cells (Huh7) followed by assessment of gene expression and cellular uptake mechanisms. The MR peptide is a fusion of R9 with two membrane-active peptides, namely cecropin and melittin. This fusion peptide possesses cell-penetrating properties. Huh7 cells were cultured and transfected with pLenti-III-miR-124-GFP plasmid containing pre-miR-124. MR- and R9-formulated complexes with plasmid were added to cells and GFP and miR-124 expression were assessed using fluorescence microscopy, flow cytometry and Real time-PCR (RT-PCR). Fluorescence microscopy confirmed the transfection of plasmid by R9 and MR carrier peptides. RT-PCR also showed the overexpression of miR-124 expression by R9 (2.5-fold) and MR (1.7-fold) respectively. However, no difference between the peptides was found. Hemolysis assays showed that MR and R9 induced less lysis compared with Triton X-100, demonstrating their low cytotoxicity. In flow cytometry assay we observed higher transfection of MR mediated delivery than R9. The cellular uptake mechanism studies found that both MR and R9 peptides rely on energy-dependent pathways, as transfection was significantly reduced at low temperatures. Macropinocytosis inhibition substantially decreased transfection for both peptides, suggesting it represents an important route of cellular entry. Clathrin- and caveolin-dependent endocytosis inhibitions also reduced transfection levels to a significant degree for each peptide. This implies that, in addition to macropinocytosis, these receptor-mediated pathways also contribute substantially to intracellular delivery. No clear predominant mechanism could be identified, as no single inhibitor entirely blocked transfection. Overall, the results point to the peptides having flexibility to utilize multiple co-existing endocytic pathways rather than solely relying on one mechanism. Further studies are needed to more precisely define each pathway ' s relative contribution to peptide-mediated miRNA delivery.
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页数:12
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