还原响应型透明质酸抗肿瘤胶束的构建及其体外性质研究
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摘要
目的合成透明质酸-胱胺-硬脂酸(HA-CYS-SA)两亲性聚合物,并研究其所制备胶束的理化性质、还原敏感性以及体外抑瘤效果。方法以碳二亚胺作为偶联剂,使胱胺、硬脂酸先后与透明质酸发生酰胺偶联反应,制得载体聚合物并通过核磁共振波谱法确定其结构。采用改进的探头超声法制备载多西他赛的聚合物胶束,以芘荧光法测定其临界胶束浓度,动态光散射法测定胶束的粒径分布,透射电镜观察胶束的微观形态;通过对载药胶束在不同谷胱甘肽浓度下的粒径变化与药物释放行为的考察,验证胶束的还原敏感性;通过激光共聚焦显微镜研究载药胶束的细胞摄取能力,并对载药胶束的体外抑瘤效果进行考察。结果成功制得两亲性聚合物材料,胶束粒子呈规则的球形,粒径为(186.7±5.3)nm;制备的载药胶束具有良好的还原敏感响应性,在无谷胱甘肽或10μmol·L~(-1)谷胱甘肽存在时,胶束的粒径在24 h内无明显变化;而在10及20 mmol·L~(-1)谷胱甘肽存在时,24 h后胶束的粒径分别增大了1.53倍和1.89倍,多西他赛的释放量分别提升了约43%及84%。包载荧光染料香豆素6的HA-CYS-SA胶束组的荧光强度明显高于游离香豆素6组,证明其具有更好的细胞摄取能力;载药胶束对4T1细胞的半数抑制浓度为0.304μg·m L~(-1),说明其具有显著的体外抗肿瘤效果。结论所构建的还原敏感型胶束具有良好的理化性质、还原敏感响应性、细胞摄取能力以及抑瘤效果,是极具应用潜力与发展前景的药物载体。
Objective To synthesize an amphiphilic polymer of hyaluronic acid-cystamine-stearic acid(HA-CYS-SA), and to determine the physicochemical properties, reduction sensitivity and antitumor effect of micelles prepared in vitro. Methods Cystamine and stearic acid were amide-coupled with hyaluronic acid by carbodiimide as coupling agent to prepare carrier polymers whose structure was confirmed by NMR spectroscopy. The amphiphilic polymer micelles loaded with docetaxel were prepared by modified probe ultrasonic method. The critical micelle concentration of the polymers was determined by pyrene fluorescence method. The particle size distribution of micelles was determined by dynamic light scattering method. Transmission electron microscopy was used to observe the microscopic morphology of micelles. Moreover, the drug release behaviors of the drug-loaded micelles in the presence of glutathione was examined to verify the reduction sensitivity of the micelles. Finally, the in vitro anti-tumor effect of drug-loaded micelles was investigated. Results The amphiphilic polymer material was successfully prepared, and the micelle had a low critical micelle concentration at 14.1 mg·L~(-1). The micelle particles showed a regular spherical shape with a particle size of(186.7±5.3) nm and uniform distribution. The prepared drug-loaded micelles showed good reductionsensitive responsiveness, and the release of docetaxel at 24 h was increased about 43% with 10 mmol·L~(-1) glutathione and increased about 84% with 20 mmol·L~(-1) glutathione. Moreover, the half-inhibitory concentration of drug-loaded micelles on 4 T1 cells was 0.304 μg·m L~(-1), indicating significant vitro anti-tumor effect. Conclusion The reductionsensitive micelles constructed in this study have good physicalchemical properties, excellent reduction-sensitivity and significant anti-tumor effect, and exhibit great application potential and development prospects.
Objective To synthesize an amphiphilic polymer of hyaluronic acid-cystamine-stearic acid(HA-CYS-SA), and to determine the physicochemical properties, reduction sensitivity and antitumor effect of micelles prepared in vitro. Methods Cystamine and stearic acid were amide-coupled with hyaluronic acid by carbodiimide as coupling agent to prepare carrier polymers whose structure was confirmed by NMR spectroscopy. The amphiphilic polymer micelles loaded with docetaxel were prepared by modified probe ultrasonic method. The critical micelle concentration of the polymers was determined by pyrene fluorescence method. The particle size distribution of micelles was determined by dynamic light scattering method. Transmission electron microscopy was used to observe the microscopic morphology of micelles. Moreover, the drug release behaviors of the drug-loaded micelles in the presence of glutathione was examined to verify the reduction sensitivity of the micelles. Finally, the in vitro anti-tumor effect of drug-loaded micelles was investigated. Results The amphiphilic polymer material was successfully prepared, and the micelle had a low critical micelle concentration at 14.1 mg·L~(-1). The micelle particles showed a regular spherical shape with a particle size of(186.7±5.3) nm and uniform distribution. The prepared drug-loaded micelles showed good reductionsensitive responsiveness, and the release of docetaxel at 24 h was increased about 43% with 10 mmol·L~(-1) glutathione and increased about 84% with 20 mmol·L~(-1) glutathione. Moreover, the half-inhibitory concentration of drug-loaded micelles on 4 T1 cells was 0.304 μg·m L~(-1), indicating significant vitro anti-tumor effect. Conclusion The reductionsensitive micelles constructed in this study have good physicalchemical properties, excellent reduction-sensitivity and significant anti-tumor effect, and exhibit great application potential and development prospects.
引文
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[13]Wang X,Chen JT,Zhu HM,et al. One-step solvothermal synthesis of targetable optomagnetic upconversion nanoparticles for in vivo bimodal imaging[J]. Anal Chem,2013,85(21):10225-10231.
[14]Kalyanasundaram K. Pyrene fluorescence as a probe of fluorocarbon micelles and their mixed micelles with hydrocarbon surfactants[J]. Langmuir,1988,4(4):885-886.
[15]Li J,Huo M,Wang J,et al. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel[J]. Biomaterials,2012,33(7):2310-2320.
[16]Zhu Z,Li D,Li Y,et al. In vitro-in vivo evaluation of hyaluronic acid-based amphiphilic copolymers for tumour targeted delivery:the role of hydrophobic groups[J]. Rsc Advances,2017,7(39):23942-23953.
[17]Zhou R,Zhang F,He PL,et al.(5R)-5-hydroxytriptolide(LLDT-8),a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo[J]. Int Immunopharmacol,2005,5(13-14):1895-1903.
[18]harada h,takahashi m. cd44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1and-2[J]. J Biol Chem,2007,282(8):5597-5607.
[19]Lee JY,Termsarasab U,Park JH,et al. Dual CD44 and folate receptor-targeted nanoparticles for cancer diagnosis and anticancer drug delivery[J]. J Control Release,2016,236:38-46.
[2]Cajot S,Schol D,Danhier F,et al. In vitro investigations of smart drug delivery systems based on redox-sensitive cross-linked micelles[J]. Macromolecular Bioscience,2013,13(12):1661-1670.
[3]Chen F,Zhang J,Fang X,et al. Reversal of paclitaxel resistance in human ovarian cancer cells with redox-responsive micelles consisting ofα-tocopheryl succinate-based polyphosphoester copolymers[J]. Acta Pharmacologica Sinica,2017,38(6):859-873.
[4]Dosio F,Arpicco S,Stella B,et al. Hyaluronic acid for anticancer drug and nucleic acid delivery[J]. Adv Drug Deliv Rev,2016,97:204-236.
[5]Lyle DB,Breger JC,Baeva LF,et al. Low molecular weight hyaluronic acid effects on murine macrophage nitric oxide production[J]. J Biomed Mater Res A,2010,94(3):893-904.
[6]Ganesh S,Iyer AK,Morrissey DV,et al. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors[J]. Biomaterials,2013,34(13):3489-3502.
[7]Hu K,Zhou H,Liu Y,et al. Hyaluronic acid functional amphipathic and redox-responsive polymer particles for the co-delivery of doxorubicin and cyclopamine to eradicate breast cancer cells and cancer stem cells[J]. Nanoscale,2015,7(18):8607-8618.
[8]Raza A,Hayat U,Rasheed T,et al. Redox-responsive nano-carriers as tumor-targeted drug delivery systems[J].Eur J Med Chem,2018,157:705-715.
[9]Tang Z,Zhang L,Wang Y,et al. Redox-responsive starshaped magnetic micelles with active-targeted and magnetic-guided functions for cancer therapy[J]. Acta Biomaterialia,2016,42:232-246.
[10]Yan Z,Wang C,Huang Y,et al. Core-crosslinked polymeric micelles with high doxorubicin loading capacity and intracellular pH-and redox-triggered payload release[J]. Eur Polym J,2015,68:104-114.
[11]Liu Y,Sun J,Cao W,et al. Dual targeting folate-conjugated hyaluronic acid polymeric micelles for paclitaxel delivery[J]. Int J Pharm,2011,421(1):160-169.
[12]Hamid ZA,Blencowe A,Ozcelik B,et al. Epoxy-amine synthesised hydrogel scaffolds for soft-tissue engineering[J].Biomaterials,2010,31(25):6454-6467.
[13]Wang X,Chen JT,Zhu HM,et al. One-step solvothermal synthesis of targetable optomagnetic upconversion nanoparticles for in vivo bimodal imaging[J]. Anal Chem,2013,85(21):10225-10231.
[14]Kalyanasundaram K. Pyrene fluorescence as a probe of fluorocarbon micelles and their mixed micelles with hydrocarbon surfactants[J]. Langmuir,1988,4(4):885-886.
[15]Li J,Huo M,Wang J,et al. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel[J]. Biomaterials,2012,33(7):2310-2320.
[16]Zhu Z,Li D,Li Y,et al. In vitro-in vivo evaluation of hyaluronic acid-based amphiphilic copolymers for tumour targeted delivery:the role of hydrophobic groups[J]. Rsc Advances,2017,7(39):23942-23953.
[17]Zhou R,Zhang F,He PL,et al.(5R)-5-hydroxytriptolide(LLDT-8),a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo[J]. Int Immunopharmacol,2005,5(13-14):1895-1903.
[18]harada h,takahashi m. cd44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1and-2[J]. J Biol Chem,2007,282(8):5597-5607.
[19]Lee JY,Termsarasab U,Park JH,et al. Dual CD44 and folate receptor-targeted nanoparticles for cancer diagnosis and anticancer drug delivery[J]. J Control Release,2016,236:38-46.