还原响应型甲氨蝶呤/羟基喜树碱纳米粒的制备及体外评价

Preparation and in Vitro Evaluation of Reduction-responsive Methotrexate/Hydroxycamptothecin Nanoparticles

关键词:  透明质酸  甲氨蝶呤  药物-聚合物偶联物  纳米粒  羟基喜树碱

Key words:  hyaluronic acid  methotrexate  drug-polymer conjugate  nanoparticles  hydroxycamptothecin

摘要:

目的 合成药物-聚合物偶联物透明质酸-二硫键-甲氨蝶呤-亚油酸(hyaluronic acid-disulfide bond-methotrexate- linoleic acid，HA-SS-MTX-LA)，并以其为载体包载羟基喜树碱(hydroxycamptothecin，HCPT)制备纳米粒(nanoparticles，NPs)(HA-SS-MTX-LA@HCPT NPs)，考察该NPs的体外释药行为及体外抗肿瘤活性。方法 在1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、N-羟基琥珀酰亚胺的催化下，通过酰胺化反应合成药物-聚合物偶联物HA-SS-MTX-LA，并采用核磁共振氢谱、傅里叶变换红外光谱确证其化学结构。采用超声法制备HA-SS-MTX-LA NPs，以临界聚集浓度和粒径为指标，筛选获得LA和HA-SS-MTX的最佳投料比。以HCPT为模型药物，采用乳化溶剂挥发法包载药物，考察HA-SS- MTX-LA NPs的共载药性能。通过体外释放试验考察HA-SS-MTX-LA@HCPT NPs的还原响应性，采用MTT法考察其体外抗肿瘤活性。结果 成功制得HA-SS-MTX-LA NPs，LA和HA-SS-MTX的最佳摩尔投料比为1∶1，临界聚集浓度为60.50 mg×mL^-1，NPs粒径为(226.6±2.5)nm，PDI为0.180±0.036。HA-SS-MTX-LA@HCPT NPs的粒径为(257.59±1.41)nm，PDI为0.132±0.009，包封率为(72.46±0.73)%，载药量为(11.51±0.32)%。体外释放结果表明药物在高浓度谷胱甘肽条件下可快速释放，MTT试验结果表明HA-SS-MTX-LA@HCPT NPs对HepG2细胞和Bel-7402细胞生长具有显著的抑制作用。结论 制得的HA-SS-MTX-LA@HCPT NPs粒径均匀，包封率和载药量较高，具有良好的共载药性能、还原响应性和抗肿瘤活性，同时可进一步提高HCPT和MTX的体外抗肿瘤效果。

Abstract:

OBJECTIVE To synthesize drug-polymer conjugate hyaluronic acid-disulfide bond-methotrexate-linoleic acid (HA-SS-MTX-LA), prepare nanoparticles(NPs)(HA-SS-MTX-LA@HCPT NPs) with it as a carrier by encapsulating hydroxycamptothecin(HCPT), and investigate its in vitro drug release behavior and in vitro antitumor activity.METHODS The drug-polymer conjugate HA-SS-MTX-LA was synthesized by amidation reaction under the catalysis of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimde, and its structure was confirmed by hydrogen nuclear magnetic spectroscopy and Fourier transform infrared spectroscopy. HA-SS-MTX-LA NPs were prepared by ultrasound method. The critical aggregation concentration and particle size were used as indicators to optimize the feeding ratio of LA to HA-SS-MTX. Using HCPT as a model drug, HCPT-loaded HA-SS-MTX-LA NPs(HA-SS-MTX-LA@HCPT) were prepared by emulsifying solvent evaporation method to investigate the drug co-loading property. The reduction responsiveness of HA-SS-MTX-LA@HCPT NPs was evaluated by in vitro release test. The anti-tumor activity in vitro of drug-loaded nanoparticles was assessed by MTT assay. RESULTS HA-SS-MTX-LA NPs was prepared successfully. The optimal feeding ratio of LA to HA-SS-MTX was 1∶1, the critical aggregation concentration was 60.50 mg×mL^-1, and the particle size was (226.6±2.5)nm with PDI of 0.180±0.036. The particle size of HA-SS-MTX-LA@ HCPT NPs was (257.59±1.41)nm with PDI of 0.132±0.009. The encapsulation efficiency and drug loading of HCPT were (72.46±0.73)% and (11.51±0.32)%, respectively. The in vitro release results showed that the drug could be rapidly released under high concentration of glutathione. The results of MTT experiment indicated that HA-SS-MTX-LA@HCPT NPs had a significant inhibitory effect on the growth of HepG2 liver cancer cells and Bel-7402 liver cancer cells. CONCLUSION The HA-SS- MTX-LA@HCPT NPs have uniform particle size, with high encapsulation efficiency and drug loading, good drug co-loading ability, good reduction responsiveness and anti-cancer activity. The anti-tumor effect of HCPT and MTX in vitro is further improved.