Department of Internal Medicine, Section of Infectious Diseases, Yale University School of Medicine, New Haven, CT, 06520, USA; Department of Bioengineering and Institute of Nano Science and Technology, Hanyang University, Seoul, 133-791, Korea.
Small. 2015 May 6;11(17):2069-79. doi: 10.1002/smll.201402933. Epub 2014 Dec 16.
Stem cells are poorly permissive to non-viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell-binding ligand with a polymer that releases nucleic acids in a cytoplasm-responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9-derived hESC. Conjugating RVG to a redox-sensitive biodegradable dendrimer-type arginine-grafted polymer (PAM-ABP) enabled nanoparticle formation with plasmid DNA without altering the environment-sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG-PAM-ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. ∼60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG-PAM-ABP is thus a novel bioreducible, biocompatible, non-toxic, synthetic gene delivery system for nAchR-expressing stem cells. Our data also demonstrates that a cell-binding ligand like RVG can cooperate with a gene delivery system like PAM-ABP to enable transfection of poorly-permissive cells.
干细胞对非病毒基因转染试剂的通透性较差。在这项研究中，我们探索了通过协同细胞结合配体与一种以细胞质反应方式释放核酸的聚合物来提高人胚胎（hESC）和间充质（hMSC）干细胞中基因传递的可能性。一种 29 个氨基酸长的肽 RVG 被鉴定为可以结合 hMSC 和源自 H9 的 hESC 的烟碱型乙酰胆碱受体（nAchR）。将 RVG 与氧化还原敏感的可生物降解的树枝状聚电解质型精氨酸接枝聚合物（PAM-ABP）偶联，能够在不改变环境敏感 DNA 释放特性和母体聚合物良好毒性特征的情况下，形成与质粒 DNA 的纳米颗粒。重要的是，与商业转染试剂如 Lipofectamine 2000 和 Fugene 相比，RVG-PAM-ABP 定量增强了对 hMSC 和 hESC 的转染。分别有 60%和 50%的 hMSC 和 hESC 被转染，并且在每个细胞的基础上增加了转染水平，而不会影响多能性标记物的表达。因此，RVG-PAM-ABP 是一种新型的生物还原、生物相容、无毒的合成基因转染系统，适用于表达 nAchR 的干细胞。我们的数据还表明，像 RVG 这样的细胞结合配体可以与像 PAM-ABP 这样的基因传递系统合作，从而实现对低通透性细胞的转染。
