向日葵花粉微胶囊作为药物载体用于膀胱癌膀胱灌注化疗的研究

Sunflower pollen microcapsules as drug carriers for bladder perfusion chemotherapy of bladder cancer

目的探究向日葵花粉微胶囊作为膀胱灌注药物载体的安全性以及有效性。方法天然向日葵花粉经脱脂、空心化、涂覆海藻酸盐等处理后最终制备出向日葵花粉微胶囊,使用扫描电子显微镜观察花粉的形态特征。体外实验中将小鼠膀胱癌MB49细胞分为花粉共培养组与对照组,使用细胞计数试剂盒(CCK-8)试验测定细胞活力,并使用酶联免疫吸附试验(ELISA)检测细胞分泌的细胞因子变化。使用异硫氰酸荧光素-牛血清白蛋白(FITC-BSA)作为模拟药物装载进入花粉,测试花粉微胶囊的缓释效果。体内实验选用BALB/c-Nu裸鼠用MB49细胞建立原位膀胱癌模型,用吉西他滨或花粉微胶囊装载吉西他滨进行膀胱灌注化疗,观察治疗效果。两组间差异比较采用单样本t检验。结果CCK-8试验结果表示花粉在不同的浓度与时间上对MB49细胞没有显著细胞毒性(P>0.05);ELISA实验结果显示花粉微胶囊促进MB49细胞细胞因子[白细胞介素(IL)-2(1.230±0.208)pg/ml比(0.670±0.208)pg/ml(t=3.297,P<0.05);IL-6(2027.00±64.29)pg/ml比(1550.00±70.00)pg/ml(t=8.693,P<0.05);IL-8(26.330±1.528)ng/ml比(22.900±1.054)ng/ml(t=3.200,P<0.05);肿瘤坏死因子-α(TNF-α)(99.670±3.512)pg/ml比(75.670±7.024)pg/ml,t=5.293,P<0.05]分泌,而海藻酸钠涂层减弱这一作用[IL-2(0.830±0.153)pg/ml比(1.230±0.208)pg/ml(t=3.354,P<0.05);IL-6(1770.00±62.45)pg/ml比(2027.00±64.29)pg/ml(t=4.966,P<0.05);IL-8(27.700±1.852)ng/ml比(26.330±1.528)ng/ml(t=0.990,P>0.05);TNF-α(88.670±8.505)pg/ml比(99.670±3.512)pg/ml,t=3.200,P<0.05]。药物释放曲线显示海藻酸盐涂层显著延长向日葵花粉微胶囊的药物释放时间[80%(600 min比5 min),t=70.730,P<0.05]。体内实验结果证实,向日葵花粉组小鼠膀胱肿瘤病变数量显著低于直接灌注组(2.20±0.83比5.60±1.14,t=4.176,P<0.05)。结论本研究证明天然向日葵花粉粒在膀胱癌膀胱灌注疗法中作为药物输送载体具有良好的安全性和有效性,是膀胱灌注中一种有希望的新材料。

Objective To investigate the safety as well as efficacy of sunflower pollen microcapsules as drug carriers for bladder instillation.MethodsNatural sunflower pollen was defatted,hollowed out and coated with alginate to finally prepare sunflower pollen microcapsules,and the morphological characteristics of the pollen were observed using scanning electron microscopy.In the in vitro experiments,mouse bladder cancer MB49 cells were divided into pollen co-culture and control groups,and cell viability was determined using the cell counting kit-8(CCK-8)assay,and changes in cytokines secreted by the cells were detected using enzyme linked immunosorbent assay(ELISA).The slow release effect of pollen microcapsules was tested using fluoresceine isothiocyanate-bovine serum albumin(FITC-BSA)as a simulated drug loading into pollen.For in vivo experiments,BALB/c-Nu nude mice were used to establish in situ bladder cancer model with MB49 cells,and bladder perfusion chemotherapy with gemcitabine or pollen microcapsules loaded with gemcitabine was performed to observe the therapeutic effect.All results were expressed as mean±standard deviation,and one-sample t-test was used to compare the differences between the two groups.ResultsThe results of CCK-8 assay indicated that pollen was not significantly cytotoxic to MB49 cells at different concentrations and time points(P>0.05).ELISA assay showed that pollen microcapsules promoted the secretion of cytokines in MB49 cells[IL-2(1.230±0.208)vs.(0.670±0.208)pg/ml,t=3.297,P<0.05;IL-6(2027.00±64.29)vs.(1550.00±70.00)pg/ml,t=8.693,P<0.05;IL-8(26.330±1.528)vs.(22.900±1.054)ng/ml,t=3.200,P<0.05;TNF-α(99.670±3.512)vs.(75.670±7.024)pg/ml,t=5.293,P<0.05,P<0.05],which was attenuated by sodium alginate coating[IL-2(0.830±0.153)vs.(1.230±0.208)pg/ml,t=3.354,P<0.05;IL-6(1770.00±62.45)vs.(2027.00±64.29)pg/ml,t=4.966,P<0.05;IL-8(27.700±1.852)vs.(26.330±1.528)ng/ml,t=0.990,P>0.05;TNF-α(82.670±8.505)vs.(99.670±3.512)pg/ml,t=3.200,P<0.05].The drug release profile showed that alginate coating significantly extended the drug release time of sunflower pollen microcapsules(t=70.730,P<0.05).The results of in vivo experiments confirmed that the number of bladder tumour lesions in mice was significantly less in the sunflower pollen group than the direct infusion group(2.20±0.83 vs.5.60±1.14,t=5.391,P<0.05).ConclusionThis study demonstrated that natural sunflower pollen granules have good safety and efficacy as a drug delivery vehicle in bladder cancer bladder perfusion therapy,and are a promising new material in bladder perfusion.