基质金属蛋白酶响应的紫杉醇-二氢卟吩e6纳米粒子诱导结肠癌细胞凋亡
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摘要
合成基质金属蛋白酶(Matrix metalloproteinases,MMPs)响应的m PEG-CPLGLAGG(PDDC)聚合物,与紫杉醇、二氢卟吩e6(Ce6)共组装形成纳米胶束,评价其对结肠癌细胞HCT-116增殖和凋亡的影响。以碳化二亚胺/N-羟基琥珀酰亚胺为缩合剂,将MMPs响应的多肽CPLGLAGG通过酰胺化反应接支到m PEG上,采用核磁共振氢谱和红外光谱对其结构进行表征。将PDDC与紫杉醇、Ce6共组装制备成纳米胶束,动态光散射和透射电镜测试粒径分布和形态,荧光光谱和HPLC检测载药量、包封率及体外释放。以纳米粒子刺激结肠癌细胞HCT-116,CCK-8法检测细胞毒性,流式检测细胞摄取和凋亡诱导作用,荧光法观察对ROS生成的影响。结果显示:PDDC聚合物对HCT-116增殖基本无影响,具有良好的安全性,可与紫杉醇、Ce6共组装形成粒径100 nm左右的纳米胶束。该纳米粒子具有良好的稳定性和MMP-2响应性,能在肿瘤微环境中特异性的释放药物;易被HCT-116细胞摄取,并可显著抑制HCT-116细胞的增殖、诱导细胞凋亡和ROS生成,且作用优于紫杉醇、Ce6单药及联合应用。PDDC包埋的紫杉醇与Ce6能有效促进HCT-116细胞凋亡,且表现出明显的协同效应,表明该聚合物可作为潜在的药物载体用于纳米药物输送及恶性肿瘤的治疗。
To design and synthesize a matrix metalloproteinases( MMPs) responsive m PEG-CPLGLAGG( PDDC) polymer,which can be assembled with paclitaxel and Chlorin e6( Ce6) to form nanoparticles,and preliminarily to evaluate its effects on cell viability and apoptosis of HCT-116 cells in vitro,PDDC was synthesized via amide bond,with EDC/NHS as condensing agents,and its chemical structure was characterized by1 H-NMR and FT-IR. PDDC was then assembled with paclitaxel and Ce6 to form nanoparticles. For characteristics of the nanoparticles,DLS and TEM were used to examine morphology,HPLC and fluorescence spectrum were used to assess drug loading capacity,entrapment efficiency and in vitro drug release. Then HCT-116 cells were treated with the nanoparticles and cytotoxicity was detected by CCK-8 Kit,cellular uptake and cell apoptosis were detected under flow cytometry,ROS production was observed under fluorescence microscope. Results showed that PDDC polymer had barely no influence on the proliferation of HCT-116 cells,indicating its good performance on safety. PDDC polymer could be assembled with paclitaxel and Ce6 to form drug-loaded particles with average particle size being about 100 nm. The drug-loaded particles were stable and had good MMP-2 responsiveness,which could specifically release in tumor microenvironment. The nanoparticles could be easily absorbed by HCT-116 cells,significantly inhibiting cell viability and inducing apoptosis and ROS production. Its effects were better than those of individual or combined treatment of paclitaxel and Ce6. In conclusion,the MMPs responsive nanoparticles,which were assembled with paclitaxel and Ce6,exhibited synergistic effect in induction of HCT-116 cells apoptosis,indicating PDDC could be used as a potential drug carrier for cancer therapy in the future.
To design and synthesize a matrix metalloproteinases( MMPs) responsive m PEG-CPLGLAGG( PDDC) polymer,which can be assembled with paclitaxel and Chlorin e6( Ce6) to form nanoparticles,and preliminarily to evaluate its effects on cell viability and apoptosis of HCT-116 cells in vitro,PDDC was synthesized via amide bond,with EDC/NHS as condensing agents,and its chemical structure was characterized by1 H-NMR and FT-IR. PDDC was then assembled with paclitaxel and Ce6 to form nanoparticles. For characteristics of the nanoparticles,DLS and TEM were used to examine morphology,HPLC and fluorescence spectrum were used to assess drug loading capacity,entrapment efficiency and in vitro drug release. Then HCT-116 cells were treated with the nanoparticles and cytotoxicity was detected by CCK-8 Kit,cellular uptake and cell apoptosis were detected under flow cytometry,ROS production was observed under fluorescence microscope. Results showed that PDDC polymer had barely no influence on the proliferation of HCT-116 cells,indicating its good performance on safety. PDDC polymer could be assembled with paclitaxel and Ce6 to form drug-loaded particles with average particle size being about 100 nm. The drug-loaded particles were stable and had good MMP-2 responsiveness,which could specifically release in tumor microenvironment. The nanoparticles could be easily absorbed by HCT-116 cells,significantly inhibiting cell viability and inducing apoptosis and ROS production. Its effects were better than those of individual or combined treatment of paclitaxel and Ce6. In conclusion,the MMPs responsive nanoparticles,which were assembled with paclitaxel and Ce6,exhibited synergistic effect in induction of HCT-116 cells apoptosis,indicating PDDC could be used as a potential drug carrier for cancer therapy in the future.
引文
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[16]康玲,徐隽,张杰,等.二氢卟吩e6光动力对结肠癌SW620细胞周期和凋亡的影响[J].中国现代医学杂志,2015,31:10-16.
[2]王凯苑,甘雅玲,周岩,等.结直肠癌治疗药物的纳米化及临床转化研究进展[J].药学进展,2017,11:824-829.
[3]Mishra B,Chaurasia S.Design of novel chemotherapeutic delivery system for colon cancer therapy based on oral polymeric nanoparticles[J].Ther Deliv,2017,8(1):29-47.
[4]Hui L,Chen Y.Tumor microenvironment:sanctuary of the devil[J].Cancer Lett,2015,368(1):7-13.
[5]于小越,张娜.肿瘤微环境敏感型纳米载药系统[J].药物生物技术,2015,5(22):446-451.
[6]何益平,龚建平,刘川.基质金属蛋白酶与结肠癌侵袭性转移关系概述[J].实用中医药杂志,2017,23(12):814-815.
[7]LinS,Jiang T,Yu Y,et al.Secernin-1 contributes to colon cancer progression through enhancing matrix metalloproteinase-2/9 exocytosis[J].Dis Markers,2015:230703.
[8]Hensley H,Cooper HS,Chang WL,et al.Imaging matrix metalloproteinase in spontaneous colon tumors:validation by correlation with histopathology[J].Methods Mol Biol,2017,1579:245-255.
[9]Inafuku Y,Furuhata T,Tayama M,et al.Matrix metalloproteinase-2 expression in stromal tissues is a consistent prognostic factor in stageⅡcancer[J].Cancer Sci,2009,100(5):852-858.
[10]皇甫铭一.MMP-2酶刺激响应性纳米给药系统的构建及其体内外评价[D].杭州:浙江大学,2017.
[11]王鹏,熊力,雷振东,等.结直肠癌的光动力治疗进展[J].激光生物学报,2015,6:495-499.
[12]Yang L,He J,Wen Y,et al.Nanoscale photodynamic agents for colorectal cancer treatment:a review[J].J Biomed Nanotechnol,2016,12(7):1348-1373.
[13]郑锐年,张为民,王晓怀,等.紫杉醇对Chlorin e6声动力抑制人肺腺癌SPCA-1细胞生长增敏作用[J].激光生物学报,2010,12:1064-1067.
[14]何耀明.光动力疗法联合紫杉醇对结直肠癌患者淋巴细胞转移增殖的影响[J].实用药物与临床,2016,5:561-664.
[15]赵相轩,温锋,任莹,等.细胞凋亡在结肠癌治疗中作用的研究进展[J].肿瘤学杂志,2016,11:946-950.
[16]康玲,徐隽,张杰,等.二氢卟吩e6光动力对结肠癌SW620细胞周期和凋亡的影响[J].中国现代医学杂志,2015,31:10-16.