海藻酸钠-角豆胶互穿聚合物网络水凝胶药物缓释体系的制备及评价

Preparation and evaluation of sodium alginate-locust bean gum interpenetrating polymer network hydrogel drug sustained-release system

目的:探讨不同配比的海藻酸钠(SA)-角豆胶(LBG)互穿聚合物网络(IPN)水凝胶的载药和释药性能,评价包裹顺铂(DDP)的IPN水凝胶对口腔鳞状细胞癌CAL-27细胞的持续性杀伤作用及其机制。方法:将不同配比SA和LBG溶于去离子水,以戊二醛(GA)和氯化钙(CaCl_(2))为交联剂,采用离子凝胶法制备不同配方的IPN水凝胶,扫描电镜(SEM)和傅里叶红外光谱(FTIR)表征水凝胶的物理化学结构,紫外分光光度分析法测定水凝胶的包封率(EE)并绘制药物释放曲线。三维柱状图展示不同配比LBG和GA条件下水凝胶珠的粒径及药物EE。结合EE和药物释放曲线选用优化配方包裹DDP制备DDP凝胶珠。CAL-27细胞分为对照组、DDP药液组和DDP凝胶珠组,MTS法和AO/PI荧光染色法观察不同时间点各组CAL-27细胞的存活率和凋亡情况。结果:SEM观察,干燥的凝胶珠表面粗糙甚至凹凸不平,出现褶皱现象。三维柱状图观察,粒径随LBG水平的升高而增大,随GA水平的升高而减小。随着LBG和GA水平的升高,水凝胶的EE均升高。FTIR检测,IPN水凝胶成功包封药物DDP。当SA和LBG的配比为2.25∶0.75,GA水平为3%,CaCl_(2)浓度为3%时,水凝胶药物EE为76.8%,药物缓释时间达48 h。MTS法检测,6和12 h后,DDP凝胶珠组CAL-27细胞存活率高于DDP药液组(P<0.05);在24和48 h时,DDP凝胶珠组细胞存活率低于DDP药液组(P<0.05)。6~48 h,对照组CAL-27细胞生长状态良好,增殖后的CAL-27细胞被染色为绿色;在6和12 h,DDP药液组和DDP凝胶珠组CAL-27细胞数量均少于对照组,DDP凝胶珠组存活细胞数略多于药物组;在24 h后,DDP凝胶珠组细胞生长速度变缓,CAL-27存活细胞数少于DDP药液组。结论:成功制备并优化了可缓释DDP的DDP-IPN水凝胶,其对口腔鳞状细胞癌CAL-27细胞具有持续杀伤作用,有望开发成一种新型药物缓释系统。

Objective:To investigate the drug loading and release properties of different ratios of sodium alginate(SA)-carob gum(LBG)interpenetrating polymer network(IPN)hydrogels,and to evaluate the persistent killing effect of IPN hydrogels coated with cisplatin(DDP)on the oral squamous cell carcinoma CAL-27 cells and its mechanism.Methods:Different ratios of SA and LBG were dissolved in the deionized water.The IPN hydrogels of different formulations were prepared by ion gel method with glutaraldehyde(GA)and calcium chloride(CaCl_(2))as the cross-linking agents.The physicochemical structures of the hydrogels were characterized with scanning electron microscope(SEM)and Fourier transform infrared spectroscopy(FTIR).The entrapment efficiency(EE)of the hydrogels was determined by ultraviolet spectrophotometry and the drug release curves were drawn.The particle sizes and drug EE of the hydrogel beads under different ratios of LBG and GA were shown by three-dimensional histogram.Combined with the EE and drug release curve,the optimized formula was selected to wrap DDP to prepare DDP hydrogel beads.The CAL-27 cells were divided into control group,DDP solution group and DDP hydrogel beads group.The survival rates and apoptotic state of the CAL-27 cells in various groups at different time points were observed by MTS and AO/PI fluorescence staining.Results:The SEM results showed that the surface of the dried gel beads was rough or even uneven and wrinkled.The three-dimensional histogram results showed that the particle size was increased with the increasing of LBG level and was decreased with the increasing of GA level.With the increasing of LBG and GA levels,the EE of the hydrogel was increased.The FTIR results showed that DDP was encapsulated successfully by the IPN hydrogel.When the ratio of SA and LBG was 2.25∶0.75,the level of GA was 3%,and the concentration of CaCl_(2) was 3%,the EE of hydrogel was 76.8%,and the drug sustained release time was 48 h.The MTS assay results showed that after treated for 6 and 12 h,the survival rates of the CAL-27 cells in DDP hydrogel beads group were slightly higher than those in DDP solution group(P<0.05).At 24 and 48 h,the survival rates of the CAL-27 cells in DDP hydrogel beads group were lower than those in DDP solution group(P<0.05).The cells in control group grew well from 6 to 48 h,and the proliferated cells were dyed green.At 6 and 12 h,the number of CAL-27 cells in DDP solution group and DDP hydrogel beads group was less than that in control group,and the number of survival cells in DDP hydrogel beads group was slightly more than that in DDP solution group.After 24 h,the growth rate of the CAL-27 cells in DDP hydrogel beads group became slower,and the number of survival cells in DDP hydrogel beads group was less than that in DDP solution group at 24 and 48 h.Conclusion:DDP-IPN hydrogel with sustained-release DDP is successfully prepared and optimized,which has a sustained killing effect on the oral squamous cell carcinoma CAL-27 cells,and it is expected to develop into a new drug sustained-release system.