新型光敏纳米粒子的制备及其在肿瘤光动力治疗中的应用

Preparation and application of novel photosensitive nanoparticles in tumor photodynamic therapy

目的制备一种新型光敏纳米粒子,并评价其理化性质及探讨其对肿瘤光动力治疗疗效的影响。方法将5,15-二溴-10,20-二苯基卟啉(5,15-dibromo-10,20-diphenylporphine,DBN)、四氟对苯二腈(tetrafluoroterephthalonitrile,TFN)、两亲性聚合物甲基-PEG-DSPE(DSPE-MPEG2000,PEG)混合溶解于四氢呋喃(tetrahydrofuran,THF)中,通过纳米共沉淀法制备新型光敏纳米粒子(DBN/TFN@PEG),并检测其理化性质。采用660 nm激光持续照射新型及传统纳米粒子,通过荧光光谱仪检测不同照射时间下纳米粒子的荧光发光强度即产生活性氧(reactive oxygen species,ROS)的水平。将新型及传统纳米粒子与肿瘤细胞共孵育并通过660 nm激光照射,使用免疫荧光法检测肿瘤细胞内ROS水平,采用PI/Calcein-AM染色法检测死/活肿瘤细胞比例。结果制备后的DBN/TFN@PEG水合粒径约为107.8 nm,分散均匀。DBN/TFN@PEG较传统纳米粒子具备更强的ROS产生能力(P<0.01)。免疫荧光结果显示,在激光照射条件下,DBN/TFN@PEG较传统纳米粒子更能诱导肿瘤细胞产生ROS(P<0.01);PI/Calcein-AM染色结果显示,DBN/TFN@PEG组死肿瘤细胞比例显著高于传统纳米粒子组(P<0.01)。结论DBN/TFN@PEG理化性质稳定,分布均匀,作为光敏剂具备更强的诱导肿瘤细胞产生ROS的能力,可提高光动力治疗的抗肿瘤疗效。

Objective To prepare a novel photosensitive nanoparticle and to evaluate its physicochemical properties, and effect on the efficacy of photodynamic therapy. Methods 5,15-dibromo-10,20-diphenylporphine(DBN), tetrafluoroterephthalonitrile(TFN), and the amphiphilic polymer methoxy-polyethylene glycol-distearoylphosphatidylethanolamine(DSPE-MPEG2000, PEG)were dissolved in tetrahydrofuran(THF) by co-precipitation method to prepare novel photosensitive nanoparticles, named DBN/TFN@PEG. The physicochemical properties of DBN/TFN@PEG were characterized. Both novel and conventional nanoparticles were continuously irradiated with a 660 nm laser, and the fluorescence intensity of nanoparticles, representing reactive oxygen species(ROS) production levels, was measured using a fluorescence spectrophotometer at different irradiation times.Tumor cells were co-incubated with the nanoparticles and irradiated with a 660 nm laser. ROS levels within the tumor cells were detected using immunofluorescence, and the ratio of dead to live tumor cells was determined using PI/Calcein-AM staining.Results Prepared DBN/TFN@PEG nanoparticles with hydrated particle size of approximately 107.8 nm were uniformly distributed in the solution. Compared to conventional nanoparticles, the ROS production capacity of DBN/TFN@PEG was significantly higher(P<0.01). Immunofluorescence results showed that the generation of ROS levels in the tumor cells of DBN/TFN@PEG group were significantly higher than in the conventional nanoparticles group under laser irradiation(P<0.01). PI/Calcein-AM staining results indicated a significantly higher ratio of dead tumor cells in the DBN/TFN@PEG group compared to the conventional nanoparticle group(P<0.01).Conclusions DBN/TFN@PEG has stable physicochemical properties and uniform distribution in the solution. As effective photosensitizers, DBN/TFN@PEG can exhibit stronger ability to induce ROS generation in tumor cells, and may enhance the efficacy of photodynamic therapy in cancer.