Department of Neurosurgery, Chang Gung Memorial Hospital, Chia-Yi Branch, Chia-Yi 61363, Taiwan.
Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan.
Int J Mol Sci. 2021 Aug 14;22(16):8741. doi: 10.3390/ijms22168741.
Gene transfection is a valuable tool for analyzing gene regulation and function, and providing an avenue for the genetic engineering of cells for therapeutic purposes. Though efficient, the potential concerns over viral vectors for gene transfection has led to research in non-viral alternatives. Cationic polyplexes such as those synthesized from chitosan offer distinct advantages such as enhanced polyplex stability, cellular uptake, endo-lysosomal escape, and release, but are limited by the poor solubility and viscosity of chitosan. In this study, the easily synthesized biocompatible and biodegradable polymeric polysorbate 80 polybutylcyanoacrylate nanoparticles (PS80 PBCA NP) are utilized as the backbone for surface modification with chitosan, in order to address the synthetic issues faced when using chitosan alone as a carrier. Plasmid DNA (DNA) containing the brain-derived neurotrophic factor (BDNF) gene coupled to a hypoxia-responsive element and the cytomegalovirus promotor gene was selected as the genetic cargo for the in vitro transfection-guided neural-lineage specification of mouse induced pluripotent stem cells (iPSCs), which were assessed by immunofluorescence staining. The chitosan-coated PS80 PBCA NP/BDNF DNA polyplex measured 163.8 ± 1.8 nm and zeta potential measured -34.8 ± 1.8 mV with 0.01% (/) high molecular weight chitosan (HMWC); the DNA loading efficiency reached 90% at a nanoparticle to DNA weight ratio of 15, which also corresponded to enhanced polyplex stability on the DNA stability assay. The HMWC-PS80 PBCA NP/BDNF DNA polyplex was non-toxic to mouse iPSCs for up to 80 μg/mL (weight ratio = 40) and enhanced the expression of BDNF when compared with PS80 PBCA NP/BDNF DNA polyplex. Evidence for neural-lineage specification of mouse iPSCs was observed by an increased expression of nestin, neurofilament heavy polypeptide, and beta III tubulin, and the effects appeared superior when transfection was performed with the chitosan-coated formulation. This study illustrates the versatility of the PS80 PBCA NP and that surface decoration with chitosan enabled this delivery platform to be used for the transfection-guided differentiation of mouse iPSCs.
基因转染是分析基因调控和功能的一种有价值的工具，并且为细胞的基因工程治疗提供了途径。尽管病毒载体在基因转染方面效率很高，但人们对其潜在的担忧也促使研究人员转而寻找非病毒替代物。壳聚糖等阳离子多聚物如合成的多聚物具有增强的多聚物稳定性、细胞摄取、内体-溶酶体逃逸和释放等优势，但受到壳聚糖较差的溶解度和粘度的限制。在这项研究中，利用容易合成的生物相容性和可生物降解的聚山梨醇酯 80 聚氰基丙烯酸丁酯纳米粒子（PS80 PBCA NP）作为壳聚糖表面修饰的骨架，以解决单独使用壳聚糖作为载体时所面临的合成问题。选择含有脑源性神经营养因子（BDNF）基因的质粒 DNA（DNA），该基因与缺氧反应元件和巨细胞病毒启动子基因相连，作为遗传货物，用于体外转染指导的小鼠诱导多能干细胞（iPSC）向神经谱系的特化，通过免疫荧光染色进行评估。用 0.01%（/）高分子量壳聚糖（HMWC）测量壳聚糖涂覆的 PS80 PBCA NP/BDNF DNA 多聚物的粒径为 163.8±1.8nm，测得的 Zeta 电位为-34.8±1.8mV；当纳米粒子与 DNA 的重量比为 15 时，DNA 负载效率达到 90%，这也对应于 DNA 稳定性测定中多聚物稳定性的增强。对于高达 80μg/mL（重量比=40）的 HMWC-PS80 PBCA NP/BDNF DNA 多聚物，对小鼠 iPSC 没有毒性，并且与 PS80 PBCA NP/BDNF DNA 多聚物相比，增强了 BDNF 的表达。通过 nestin、神经丝重链多肽和β III 微管蛋白表达的增加观察到对小鼠 iPSC 向神经谱系的特化，并且当用壳聚糖涂覆的制剂进行转染时，效果更为明显。这项研究说明了 PS80 PBCA NP 的多功能性，并且壳聚糖的表面修饰使这种递药平台能够用于指导小鼠 iPSC 的转染分化。
