生物人工肝用海藻酸钠/壳聚糖微囊的安全性和生物功能评价

Safety and biological function evaluation of sodium alginate/chitosan microcapsule used in bioartificial liver

目的:评价生物人工肝用海藻酸钠/壳聚糖微囊(AC微囊)的安全性和预期生物功能。方法:制备AC微囊,通过倒置电子显微镜、扫描电子显微镜(SEM)等对微囊的表面结构、密度、机械强度和物质通透性等进行综合表征;通过AC微囊的细胞毒性及溶血性能评价,考察其生物相容性;模拟其预期临床使用方式,用AC微囊包裹肝细胞,评价细胞的存活率和代谢活性:进一步考察微囊的预期生物功能。结果:SEM显示微囊表面具有微孔洞;微囊密度为(1.39±0.155)g·m L^(-1);180 r·min^(-1)振摇24 h后其完整率在95%以上;微囊通透性试验显示10 min时人血清白蛋白释放率约为50%,90 min左右达到平衡状态。细胞毒性评价(MTT、LDH)的结果显示微囊浸提液与空白组相比细胞的相对活性没有显著性差异。血液相容性评价直接法显示溶血率为(1.36±0.211)%;浸提法的溶血率为(2.69±0.219)%,均低于标准限值(≤5%)。包裹的肝细胞均匀分布在微囊内,并且与未包裹的肝细胞相比其白蛋白和尿素合成量均有所提高。结论:本研究制备的AC微囊机械强度好,可允许小分子物质透过。微囊无明显细胞毒性,溶血率低,生物相容性良好;微囊化培养的细胞能够维持并提高肝细胞生存活性和代谢功能。本研究建立的规范性评价技术和试验方法为制定生物人工肝用AC微囊质量控制标准提供了技术支持,并为合理的科学监管提供依据。

Objective： To evaluate the safety and biological function of sodium alginate/chitosan microcapsule （ AC microcapsule ） used in biological artificial liver.Methods： Inverted electron microscope and scanning electron microscope （ SEM ） were applied to characterize the structure, density, mechanical strength and molecular permeability of microeapsules.In addition, the in vitro cytotoxicity and hemolytic property of AC microcapsule was investigated, so as to assess its biocompatibility. To simulate the intended use in clinical, liver cells （ HepG2 ） were encapsulated into AC microcapsules, and subsequently cell viability and metabolic activity were studied to further evaluate the biological function of microcapsule.Results： Micro-holes were observed on the microcapsule surface by SEM. The density of microcapsules was （ 1.39 ± 0.155 ）g· mL^-1. The integrity percentage of microcapsules was more than 95% after 24 hours shaking at 180 r· min^-1. Permeability results showed that release rate of serum albumin was about 50% in 10 minutes, and the release rate reached equilibrium state at about 90 minutes. Cell toxicity assessment （ MTT and LDH assay ） showed that there was no significant difference in cell viability between microcapsule extracted solution exposure group and the control group. The direct method and extraction solution method showed that the hemolytic rate of AC microcapsule was （ 1.36 ± 0.211 ） % and （ 2.69 ± 0.219 ） %, respectively, which were significantly lower than the standard requirement （ ≤ 5% ）. When HepG2 cells were encapsulated into AC microcapsule, the cells were distributed uniformly in the microcapsules, and the cell growth, albumin synthesis and urea production were improved when compared with that in normal cell culture condition. Conclusion：In this study, AC microcapsule were prepared, which showed excellent mechanical strength and permeability allowing small molecules to pass through microcapsules.Cytotoxicity and hemolytic property assessments proved that the microcapsules had good biocompatibilities.Furthermore, the microencapsulation could improve the cell survival and metabolism.The assessment methods of AC microcapsules established in this study provide a scientific basis for developing quality control standard of AC microcapsule used in the biological artificial liver.