摘要
目的本研究构建植酸—锌(PA-Zn^(2+))纳米颗粒负载毛蕊异黄酮(CAL),探究其体外成骨、成血管能力。方法以硝酸锌(4 mg/mL)和植酸(PA,15 mg/mL)为前驱体,通过界面自组装形成纳米颗粒(NPs),制备负载毛蕊异黄酮的纳米颗粒(CAL-PZ NPs),并评价其体外促成骨与促成血管能力。选用人成骨细胞(HOCs)和人脐静脉内皮细胞(HUVECs)建立双细胞模型,设空白对照组、游离CAL组及CAL-PZ NPs组;通过活/死细胞染色评估生物相容性;采用碱性磷酸酶(ALP)活性检测、茜素红S(ARS)染色、成骨标志物骨桥蛋白(OPN)免疫荧光染色及成骨相关基因(OPN/COL1)的qPCR分析评价成骨分化能力;通过管腔形成实验及血管生成标志物血管内皮生长因子(VEGF)和血小板—内皮细胞黏附分子(CD31)免疫荧光染色和qPCR分析评估促血管生成效应。结果透射电镜显示,CAL-PZ NPs呈均匀球形,平均粒径在100 nm左右。活/死细胞染色提示各组之间无显著差异(P>0.05),展现了良好的生物相容性。CAL-PZ NPs组的ALP和ARS染色、OPN免疫荧光染色均显示成骨活性高于空白对照组(P<0.05)和CAL组(P<0.05)。qPCR结果显示,CAL-PZ NPs组的COL1表达量显著高于游离CAL组(P<0.05)及空白对照组(P<0.0001),而OPN在CAL-PZ NPs组与游离CAL组间无统计学差异(P>0.05)。管腔形成实验、CD31和VEGF染色结果表明CAL-PZ NPs组血管生成均显著优于其他两组(P<0.05)。同时,qPCR结果显示CAL-PZ NPs组的CD31和VEGF mRNA表达水平相较于其他两组呈现显著上调(P<0.05)。结论本研究成功构建了CAL-PZ NPs,显著改善了CAL的生物利用度。体外实验证实,CAL-PZ NPs具有优异的生物相容性,并显著增强游离CAL的促成骨与促血管生成活性。
Objective To construct calycosin(CAL)-loaded phytic acid-zinc(PA-Zn^(2+))nanoparticles(NPs)and investigate their osteogenic and angiogenic capabilities in vitro.Methods Calycosin-loaded zinc phytate nanoparticles(CAL-PZ NPs)were fabricated via interfacial self-assembly using zinc nitrate(4 mg/mL)and phytic acid(PA,15 mg/mL)as precursors.That in vitro osteogenic and angiogenic potential was subsequently evaluated.A co-culture system comprising human osteoblast cells(HOCs)and human umbilical vein endothelial cells(HUVECs)was established.Experimental groups included blank control,Free CAL,and CAL-PZ NPs.Biocompatibility was evaluated using live/dead cell staining.Osteogenic differentiation capacity was assessed through alkaline phosphatase(ALP)activity assays,alizarin red S(ARS)staining,immunofluorescence staining of the osteogenic marker osteopontin(OPN),and qPCR analysis of osteogenesis-related genes(OPN/COL1).The pro-angiogenic effect was assessed through tube formation assays and by analyzing angiogenesis markers-vascular endothelial growth factor(VEGF)and platelet endothelial cell adhesion molecule-1(CD31),as well as immunofluorescence staining and qPCR analysis.Results Transmission electron microscopy revealed that CAL-PZ NPs exhibited uniform spherical morphology with particle sizes averaging approximately 100 nm.Live/dead cell staining indicated there were no statistically significant differences(P>0.05)in viable cell rates in each group,demonstrating favorable biocompatibility.In the CAL-PZ NPs group,both ALP and ARS staining,as well as OPN immunofluorescence staining,demonstrated significantly higher osteogenic activity compared to the blank control group(P<0.05)and the Free CAL group(P<0.05).qPCR results revealed that the expression level of COL1 in the CAL-PZ NPs group was significantly higher than that in the Free CAL group(P<0.05)and the blank control group(P<0.0001).However,there were no statistically significant differences in OPN expression was observed between the CAL-PZ NPs group and the Free CAL group(P>0.05).The tube formation assay,along with CD31 and VEGF immunofluorescence staining,indicated that angiogenesis was significantly enhanced in the CAL-PZ NPs group compared to both other groups(P<0.05).Furthermore,qPCR analysis showed that the mRNA expression levels of CD31 and VEGF in the CAL-PZ NPs group were significantly upregulated relative to the other two groups(P<0.05).Conclusion This study successfully constructed CAL-PZ NPs,which significantly improved the bioavailability of CAL.In vitro experiments confirmed that CAL-PZ NPs exhibited excellent biocompatibility and markedly enhanced the osteogenic and angiogenic activities of free CAL.
作者
孙泽宇
滕建祥
唐铭宏
李波
Sun Zeyu;Teng Jianxiang;Tang Minghong;Li Bo(Department of Orthopedics,Guizhou Provincial People's Hospital,Guiyang 550002,Guizhou,China;Guizhou Medical University,Guiyang 550001,Guizhou,China)
出处
《贵州医药》
2025年第9期1347-1352,共6页
基金
国家自然科学基金地区项目(82160419)
贵州省中医药管理局中医药、民族医药科学技术研究项目(QZYY-2024-114)
贵州省人民医院青年基金项目(GZSYQN202104号)。
