Department of Interventional Radiology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China.
Department of Radiology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China.
J Nanosci Nanotechnol. 2021 Feb 1;21(2):977-986. doi: 10.1166/jnn.2021.18627.
Poly[lactic-co-glycolic] acid (PLGA) targeting nanoparticles AFP/PLGA/Dt, loaded with Dt plasmid of diphtheria toxin gene, modified by Alpha fetoprotein (AFP) monoclonal antibody, is prepared. Its physical and chemical properties and its effect on HepG2 cells are studied. Firstly, Dt expression plasmid pET11a/Dt is constructed and PLGA nanoparticles are prepared by emulsion solvent evaporation (ESE). Scanning electron microscope (SEM) is used to observe its morphology. Laser Particle Sizer is used to measure the particle size. In addition, the encapsulation efficiency, drug loading and release rate of PLGA nanoparticles are measured. Carboxy fluorescein and rhodamine fluorescein are used to double label IgG/PLGA/Dt and AFP/PLGA/Dt nanospheres, respectively, the entry of nanospheres into HepG2 cells are observed at 3 h and 12 h. The effect of AFP/PLGA/Dt nanospheres on the migration of HepG2 cells is examined by wounding healing assay. Transwell chamber experiment is used to detect the effect of AFP/PLGA/Dt nanospheres on the invasion of HepG2 cells. MTT method is utilized to determine the inhibitory activity of nanoparticles on HepG2 cell proliferation. After treated with IgG/PLGA/Dt and AFP/PLGA/Dt nanoparticles for 48 hours, flow cytometry is used to detect the apoptosis rate and cell cycle of HepG2 cells in each group. The results show that the prepared nanospheres have regular morphology, flat surface, average particle size of 265.72±12.46 nm, zeta potential of -18.15 mV. The average entrapment efficiency and drug loading are 78.48±1.71% and 3.16±0.35%, respectively. The nanoparticles release slowly and stably . At the 10th day, the release rate reaches 75.13%. PLGA nanospheres can effectively protect DNA from nuclease degradation. The results show that AFP/PLGA/Dt nanospheres have biological targeting effect and can be enriched in cells. AFP/PLGA/Dt nanoparticles can significantly inhibit the migration, invasion and proliferation of HepG2 cells, and promote apoptosis.
聚（乳酸-共-乙醇酸）（PLGA）靶向纳米粒 AFP/PLGA/Dt，负载白喉毒素基因的 Dt 质粒，由甲胎蛋白（AFP）单克隆抗体修饰，被制备。研究了其物理化学性质及其对 HepG2 细胞的作用。首先，构建白喉毒素表达质粒 pET11a/Dt，采用乳液溶剂蒸发（ESE）法制备 PLGA 纳米粒。扫描电子显微镜（SEM）观察其形态。激光粒度仪测定粒径。此外，还测定了 PLGA 纳米粒的包封率、载药量和释放率。用羧基荧光素和罗丹明荧光素分别对 IgG/PLGA/Dt 和 AFP/PLGA/Dt 纳米球进行双标记，观察 3 h 和 12 h 纳米球进入 HepG2 细胞的情况。划痕愈合试验检测 AFP/PLGA/Dt 纳米球对 HepG2 细胞迁移的影响。Transwell 室实验检测 AFP/PLGA/Dt 纳米球对 HepG2 细胞侵袭的影响。MTT 法测定纳米粒对 HepG2 细胞增殖的抑制活性。经 IgG/PLGA/Dt 和 AFP/PLGA/Dt 纳米粒处理 48 h 后，流式细胞术检测各组 HepG2 细胞的凋亡率和细胞周期。结果表明，所制备的纳米球形态规则，表面平整，平均粒径为 265.72±12.46nm，Zeta 电位为-18.15 mV。平均包封率和载药量分别为 78.48±1.71%和 3.16±0.35%。纳米粒释放缓慢稳定，第 10 天释放率达到 75.13%。PLGA 纳米球能有效保护 DNA 免受核酸酶降解。结果表明，AFP/PLGA/Dt 纳米球具有生物靶向作用，能在细胞内富集。AFP/PLGA/Dt 纳米粒能显著抑制 HepG2 细胞的迁移、侵袭和增殖，促进凋亡。
