可降解含氟聚氨酯胶束体外生物安全性评价^*

In Vitro Biosafety Evaluation of Biodegradable Fluorinated Polyurethane Micelles

摘要

目的 基于国际标准化方法，系统评估含氟聚氨酯（fluorinated polyurethane, FPU）胶束的体外细胞毒性及血液相容性，为其临床应用提供安全性依据。方法 采用两步聚合法合成生物可降解FPU，采用动态光散射法测定胶束的水合粒径及Zeta电位，并通过透射电子显微镜观察胶束外观形貌。依据ISO 10993-5:2009、GB/T 16886.5-2017和GB/T 16886.4-2022，选取NIH/3T3细胞和SKOV3细胞，检测不同浓度FPU胶束处理24、48、72 h后对细胞增殖、膜通透性及细胞凋亡的影响。采用流式细胞术分析FPU胶束对NIH/3T3细胞和SKOV3细胞周期的影响。此外，采用体外溶血实验评估FPU胶束的血液相容性。结果 FPU胶束的粒径约为132.5 nm，多分散指数（polydispersity index，PDI）为0.096，低于0.150，粒径均匀。FPU胶束的Zeta电位为-21.5 mV。不同浓度的FPU胶束处理后，NIH/3T3细胞和SKOV3细胞的增殖活跃，未表现出明显的毒性，且未显著影响细胞膜通透性，也未诱导明显细胞凋亡。细胞周期分析显示，72 h处理后G1期细胞比例增加，而S期和G2期细胞比例减少，提示可能存在G1期阻滞，但未观察到显著毒性。此外，血液相容性实验表明，FPU胶束未引发红细胞破裂或溶血现象，进一步证实其良好的生物相容性。结论 FPU胶束在体外未表现出明显的细胞毒性和溶血现象，且细胞周期分析提示无显著毒性。FPU胶束具有良好的生物安全性。

Abstract

Objective To systematically evaluate the in vitro cytotoxicity and hemocompatibility of fluorinated polyurethane (FPU) micelles using international standardized methods, thereby providing a safety basis for their clinical application. Methods Biodegradable FPU was synthesized using a two-step polymerization method. The hydrated particle size and the zeta potential of the micelles were measured by dynamic light scattering, and their morphology was observed by transmission electron microscopy. In accordance with the relevant standards for in vitro cytotoxicity testing specified in the biological evaluation guidelines for medical devices (ISO 10993-5:2009, GB/T 16886.5-2017, and GB/T 16886.4-2022) NIH/3T3 and SKOV3 cells were used to assess the effects of FPU micelles at various concentrations on cell proliferation, membrane permeability, and apoptosis after 24, 48, and 72 hours of treatment. Flow cytometry was used to analyze the effects of FPU micelles on the cell cycle of NIH/3T3 and SKOV3 cells. Additionally, an in vitro hemolysis assay was performed to evaluate the hemocompatibility of FPU micelles. Results The particle size of FPU micelles was approximately 132.5 nm, with a polydispersity index (PDI) of 0.096, which is below 0.150, indicating uniform particle size. The zeta potential of FPU micelles was -21.5 mV. After treatment with FPU micelles at various concentrations, the proliferation activity of NIH/3T3 and SKOV3 cells was not affected, and no significant toxicity was observed. Furthermore, cell membrane permeability was not significantly altered, and no significant apoptosis was induced. Cell cycle analysis showed that after 72 hours of treatment, the proportion of cells in the G1 phase increased, while the proportions of cells in the S and G2 phases decreased, suggesting potential G1 phase arrest. However, no significant toxicity was observed. Furthermore, hemocompatibility experiments demonstrated that FPU micelles did not cause erythrocyte rupture or hemolysis, further confirming their favorable biocompatibility. Conclusion FPU micelles did not exhibit significant cytotoxicity or hemolysis in vitro, and cell cycle analysis indicated no notable toxicity, demonstrating good biosafety.

关键词

含氟聚氨酯胶束 / 生物相容性 / 细胞毒性 / 细胞周期 / 血液相容性

Key words

Fluorinated polyurethane micelles / Biocompatibility / Cytotoxicity / Cell cycle / Hemocompatibility

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基金资助

*国家自然科学基金重点项目(52433015)

四川省科技厅重点研发计划项目(2024YFFK0354)

四川省科技厅重点研发计划项目(2024YFFK0355)