Lin Xiaojie, Fukazawa Kyoko, Ishihara Kazuhiko
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Acta Biomater. 2016 Aug;40:226-234. doi: 10.1016/j.actbio.2016.03.040. Epub 2016 Mar 29.
Nile blue (NB)-tagged DNA helix-targeting amphiphilic photoreactive 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, poly(MPC-co-3-methacryloyloxy-2-hydroxypropyl-4-oxybenzophenone-co-2-trimethylammonium ethyl methacrylate chloride) (PMHT-NB), containing a cationic group to facilitate cell membrane penetration and a benzophenone (BP) group to promote photoinduced conjugation with DNA helix was synthesized using radical polymerization method. Ultraviolet light (UV)-visible light absorption spectra of PMHT-NB showed absorption peaks at wavelengths 254, 289, and 600nm, suggesting successful incorporation of BP and NB groups. PMHT-NB was highly sensitive to photoirradiation with UV irradiation at the second level, as confirmed based on the degradation spectra of UV absorption peaks for the BP group in PBS (pH=7.4). PMHT-NB showed good solubility in both aqueous solution and in ethanol. In a cell culture medium containing 10mg/mL PMHT-NB, the NB group showed fluorescence peaks at an emission wavelength of 650nm and excitation wavelength of 633nm. PMHT-NB also showed low cytotoxicity and good cell membrane permeability toward cancerous HeLa cells. Further, photoinduced PMHT-NB effectively inhibited the unwinding of a molecular beacon with a hairpin structure, indicating that synthetic photoreactive MPC polymers photoregulated the unwinding of DNA.
Natural and synthetic genetic hybrid biomaterials consisting of well-designed polymers loaded with oligonucleotide fragments are considered as an attractive alternative to conventional transgene systems and chemical methods for precisely and rapidly modulation of intracellular gene expression. Containing versatile functional moieties, the effectiveness of well-designed cytocompatible polymers themselves without oligonucleotide fragments on gene regulation is rarely investigated. In the present study, a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer composed of a tumor/DNA-targeting moiety and photo-controllable unit demonstrated low cytotoxicity, rapid cell membrane permeability and effective inhibitive ability on DNA unwinding under a light irradiation. The synthetic polymer was considered as promising material to effectively inhibit intracellular partial DNA unwinding for cancer/gene therapy.
采用自由基聚合法合成了尼罗蓝（NB）标记的靶向DNA螺旋的两亲性光反应性2-甲基丙烯酰氧乙基磷酰胆碱（MPC）聚合物，聚（MPC-co-3-甲基丙烯酰氧基-2-羟丙基-4-氧基二苯甲酮-co-2-甲基丙烯酰氯乙基三甲基氯化铵）（PMHT-NB），其含有促进细胞膜渗透的阳离子基团和促进与DNA螺旋光诱导共轭的二苯甲酮（BP）基团。PMHT-NB的紫外可见吸收光谱在波长254、289和600nm处显示吸收峰，表明BP和NB基团成功引入。基于PBS（pH = 7.4）中BP基团的紫外吸收峰降解光谱证实，PMHT-NB对二级紫外光照射高度敏感。PMHT-NB在水溶液和乙醇中均表现出良好的溶解性。在含有10mg/mL PMHT-NB的细胞培养基中，NB基团在发射波长650nm和激发波长633nm处显示荧光峰。PMHT-NB对癌细胞HeLa细胞也表现出低细胞毒性和良好的细胞膜通透性。此外，光诱导的PMHT-NB有效抑制了具有发夹结构的分子信标的解旋，表明合成的光反应性MPC聚合物对DNA解旋具有光调节作用。
由负载寡核苷酸片段的精心设计的聚合物组成的天然和合成基因杂交生物材料被认为是传统转基因系统和化学方法的有吸引力的替代方案，用于精确快速调节细胞内基因表达。含有多种功能部分的精心设计的无寡核苷酸片段的细胞相容性聚合物本身对基因调控的有效性很少被研究。在本研究中，一种由肿瘤/DNA靶向部分和光控单元组成的2-甲基丙烯酰氧乙基磷酰胆碱（MPC）聚合物在光照下表现出低细胞毒性、快速的细胞膜通透性和对DNA解旋的有效抑制能力。该合成聚合物被认为是有效抑制细胞内部分DNA解旋用于癌症/基因治疗的有前景的材料。
