基于原位聚合技术构建细胞内微环境响应型DNA递送系统

Fabrication of Multifunctional Gene Delivery Systems Responsible to Intracellular Microenvironments Through in situ Polymerization

传统的非病毒载体基于分子间静电自组装作用与核酸结合,组装的复合物在体内复杂的环境中容易发生结构解离,共价结合的交联聚合物载体有望成为解决传统非病毒载体结构稳定性差的有效方案。选择N-(3-氨丙基)甲基丙烯酰胺盐酸盐、1-乙烯基咪唑、2-甲基丙烯酰氧乙基磷酸胆碱与N,N′-双(丙稀酰)胱胺作为多功能性单体,采用原位聚合方法制备包载质粒DNA(pDNA)的交联聚合物-pDNA复合物。其中,共价键为载体提供优异的结构稳定性;1-乙烯基咪唑能够响应胞内溶酶体酸性微环境,触发质子海绵效应便于复合物的溶酶体逃逸;N,N′-双(丙稀酰)胱胺的二硫键可以响应胞内高水平的谷胱甘肽(GSH),实现复合物在细胞内部选择性解聚,释放内含pDNA。研究表明,该复合物平均水合半径约135 nm,ζ电势约−6.5 mV,形貌近似球形。该复合物可在10 mg/mL肝素环境中保持结构稳定性,具有响应细胞内GSH,触发释放包载核酸分子的功能。细胞实验证明该复合物细胞毒性低。细胞摄取、转染能力强。综上所述,基于原位聚合技术制备交联聚合物载体在基因递送领域具有重要应用前景,本研究为新型基因递送载体的开发提供了新思路。

The convention non-viral gene delivery systems are basically formulated based on electrostatic interactions between negatively charge nucleic acids and cationic materials,whose colloidal stabilities are not adequate in the harsh biological environment and subjected to structural disassembly.To resolve this drawback,we attempt to employ in situ polymerization onto plasmid DNA(pDNA)with a variety of monomers including N(‑3-aminopropyl)methacrylamide hydrochloride,1-vinylimidazole,2-methacryloyloxyethyl phosphorylcholine and N,N'-bis(acryloyl,cystamine)for overcoming sequential biological barriers encountered in the journey of transcellular gene delivery.Overall,our proposed in situ reaction of functional monomers into covalent network on top of pDNA could afford excellent structural stabilities.Of note,the appreciable proton buffering capacities of 1-vinylimidazole due to its responsiveness to the acidic and digestive endosomal environment could facilitate the escape of the pDNA payloads from endosomal entrapment.Redox-mediated cleavage of disulfide bond in N,N'-bis(acryloyl)cystamine allows selective breakage of covalent linked network in the cytosolic microenvironment due to the striking high intracellular level of glutathione,thereby promoting liberation of the pDNA payloads in the cell interiors.The subsequent investigations determined that the proposed polymerization strategies result in well-defined nanoscaled pDNA delivery systems(135 nm in hydrodynamic radius,−6.5 mV inζpotential and uniform spherical structures by TEM measurement).Excellent colloidal stabilities are validated despite incubation in presence of exceeding high concentration of heparin(10 mg/mL).In contrast,ready liberation of pDNA is confirmed when incubated in presence of glutathione by mimicking intracellular microenvironment.Cell transfection experiments verify its efficient cellular uptake and gene expression activities in the hard-transfected MCF-7 cells.To this end,the obtained results indicate the tempting potential of polymerization strategy in fabrication of covalent linked non-viral gene delivery systems,which could shed novel avenues in engineering a variety of high-performance gene delivery systems with diverse functionalities.