[關(guān)鍵詞]
[摘要]
目的 研究甘草次酸修飾的姜黃素陽離子脂質(zhì)體(GAMCLCL)肝靶向性以及抗肝癌作用。方法 制備甘草次酸(GA)配體——GA和十八胺鹽(SGO),用紅外光譜和質(zhì)譜檢測;并進(jìn)一步利用SGO制備GAMCLCL,透射電鏡觀察脂質(zhì)體形態(tài),Nano ZS90納米粒度儀測定脂質(zhì)體粒徑與電位;采用活體成像系統(tǒng)觀察GAMCLCL小鼠體內(nèi)熒光分布。Wistar大鼠隨機分為對照組、模型組、阿霉素(陽性藥,2 mg·kg-1)組、姜黃素(20 mg·kg-1)組和GAMCLCL低、高劑量(2、4 mg·kg-1)組,除對照組外,采用Walker-256細(xì)胞種植法制備肝原位移植瘤模型,每天1次,尾iv給藥,連續(xù)7 d ;另設(shè)GAMCLCL (4 mg·kg-1)給藥14 d組;稱腫瘤質(zhì)量,計算肝臟系數(shù)、脾臟系數(shù);全自動生化分析儀測定血液紅細(xì)胞(RBC)、白細(xì)胞(WBC)、血小板(PLT)、丙氨酸氨基轉(zhuǎn)移酶(ALT)和肌酐(CRE)水平,試劑盒法檢測乳酸脫氫酶(LDH)水平; ELISA法檢測血清腫瘤壞死因子-α (TNF-α)和白細(xì)胞介素-6 (IL-6)水平; Western blotting法檢測腫瘤組織血管內(nèi)皮生長因子(VEGF)、cleaved Caspase-3、Bcl-2、p53、p-PI3K、p-Akt蛋白表達(dá)水平。結(jié)果 紅外光譜和質(zhì)譜的結(jié)果可以驗證GA與十八胺的反應(yīng)生成了SGO;GAMCLCL外觀呈球形,其粒徑為(194±0.25) nm,聚合物分散性指數(shù)(PDI)為0.21±0.02,電位為(31.9±0.31) mV;GAMCLCL在2個月內(nèi)呈黃色透明溶液,無沉淀析出; GAMCLCL與血清混合未出現(xiàn)任何聚集和沉淀現(xiàn)象;活體成像實驗顯示,給藥后各時間點熒光主要集中在肝臟,10、30、60 min時肝臟熒光較強,120 min時肝臟熒光明顯減弱,240 min時肝臟熒光基本消失。與模型組比較,各給藥組大鼠腫瘤質(zhì)量均明顯減輕(P<0.05、0.01);各給藥組肝臟系數(shù)顯著降低(P<0.05、0.01),游離姜黃素組、GAMCLCL 4 mg·kg-1(7、14 d)組脾臟系數(shù)顯著下降(P<0.01);阿霉素及GAMCLCL 4 mg·kg-1(7、14 d)組的RBC和PLT計數(shù)均顯著升高(P<0.01),WBC計數(shù)均顯著降低(P<0.05、0.01);各給藥組大鼠ALT、CRE均顯著降低(P<0.05、0.01),除游離姜黃素組外,各給藥組LDH顯著降低(P<0.05、0.01);各給藥組IL-6、TNF-α均顯著降低(P<0.01、0.05);各給藥組VEGF、Bcl-2、Akt、p-PI3K的蛋白表達(dá)均顯著下調(diào)(P<0.05、0.01),cleaved Caspase-3和P53蛋白表達(dá)均顯著上調(diào)(P<0.05、0.01); GAMCLCL (4 mg·kg-1)給藥7 d與14 d的抗腫瘤效果相似,明顯強于游離姜黃素。結(jié)論 GAMCLCL能顯著增強姜黃素的肝靶向性和抗肝癌作用,有利于提高荷瘤大鼠的無進(jìn)展生存期。
[Key word]
[Abstract]
Objective To investigate the liver targeting of glycyrrhizic acid-modified curcumin-loaded cationic liposomes (GAMCLC) and its anticancer effects. Methods Prepared glycyrrhetinic acid (GA) ligands-GA and octadecylamine salt (SGO) and detected them using infrared spectroscopy and mass spectrometry, and further used SGO to prepare GAMCLC, and observed the morphology of liposomes under transmission electron microscopy, and measured the particle size and potential of liposomes using Nano ZS90 nanoparticle size analyzer. Using a live imaging system to observe the fluorescence distribution in GAMCLC mice. Wistar rats were randomly divided into a control group, a model group, a doxorubicin (positive drug, 2 mg·kg?1) group, a curcumin (20 mg·kg?1) group, and a GAMCLCL low and high dose (2, 4 mg·kg?1) group. Except for the control group, a Walker-256 cell seeding method was used to prepare a liver orthotopic transplantation tumor model. The rats were administered once a day for 7 consecutive days, and set up a 14 day group of GAMCLCL (4 mg·kg?1) for administration. Weighed the tumor mass, calculated the liver coefficient and spleen coefficient. The levels of red blood cells (RBC), white blood cells (WBC), platelets (PLT), alanine aminotransferase (ALT), and creatinine (CRE) were measured using a fully automated biochemical analyzer, and lactate dehydrogenase (LDH) levels were detected using a reagent kit method. ELISA method was used for detecting serum tumor necrosis factor- α (TNF- α) and the level of interleukin-6 (IL-6). Western blotting was used to detect the expression levels of vascular endothelial growth factor (VEGF), cleaved Caspase-3, Bcl-2, p53, p-PI3K, and p-AKT proteins in tumor tissues. Results The results of infrared spectroscopy and mass spectrometry could verify that the reaction between GA and octadecylamine generated SGO. GAMCLCL had a spherical appearance with a particle size of (194±0.25) nm, a polymer dispersibility index (PDI) of 0.21±0.02, and a potential of (31.9±0.31) mV. GAMCLCL appeared as a yellow transparent solution within two months, with no precipitation. GAMCLCL did not exhibit any aggregation or precipitation when mixed with serum. Live imaging experiments showed that fluorescence was mainly concentrated in the liver at various time points after administration. The liver fluorescence was strong at 10, 30, and 60 min, significantly weakened at 120 min, and basically disappeared at 240 min. Compared with model group, the tumor mass of rats in each treatment group was significantly reduced (P < 0.05, 0.01). The liver coefficient significantly decreased in each treatment group (P < 0.05, 0.01), while the spleen coefficient significantly decreased in the free curcumin group and GAMCLCL 4 mg·kg?1 (7, 14 d) group (P < 0.01). The RBC and PLT counts were significantly increased (P < 0.01) and WBC counts were significantly decreased (P < 0.05, 0.01) in the groups of doxorubicin and GAMCLCL 4 mg·kg?1 (7, 14 d). ALT and CRE of rats in each treatment group were significantly reduced (P < 0.05, 0.01). Except for the free curcumin group, LDH in each treatment group was significantly reduced (P < 0.05, 0.01). IL-6 and TNF-α in each administration group significantly decreased (P < 0.01, 0.05). The protein expression of VEGF, Bcl-2, AKT, and p-PI3K in each treatment group was significantly downregulated (P < 0.05, 0.01), while the protein expression of cleaved Caspase-3 and p53 was significantly upregulated (P < 0.05, 0.01). The anti-tumor effects of GAMCLCL (4 mg·kg?1) administered for 7 d and 14 d were similar, significantly stronger than free curcumin. Conclusion GAMCLCL significantly enhanced the liver-targeting and anti-hepatocellular carcinoma effects of curcumin, and was beneficial to the progression-free survival of tumor-bearing rats.
[中圖分類號]
R285.5
[基金項目]
國家自然科學(xué)基金資助項目(81973669;81274147);廣西中醫(yī)藥跨學(xué)科創(chuàng)新團(tuán)隊項目(GZKJ2306)