[關(guān)鍵詞]
[摘要]
目的 優(yōu)化甘草次酸(GA)和葉酸(FA)共修飾雙靶向pH敏感黃芩苷/姜黃素共載脂質(zhì)體(GA/FA-pH-Lip@Bai/Cur)的處方工藝,并對(duì)其進(jìn)行評(píng)價(jià)。方法 采用薄膜分散超聲法制備GA/FA-pH-Lip@Bai/Cur,以包封率、不同pH釋放介質(zhì)中溶出曲線相似因子(f2)、粒徑和多分散指數(shù)(PDI)為評(píng)價(jià)指標(biāo),使用單因素考察和Box-Behnken設(shè)計(jì)-響應(yīng)面法篩選出最佳處方工藝;對(duì)GA/FA-pH-Lip@Bai/Cur的外觀形態(tài)、粒徑、不同pH濃度的體外釋放度等進(jìn)行評(píng)價(jià);通過溶血性實(shí)驗(yàn)評(píng)估GA/FA-pH-Lip@Bai/Cur的生物相容性;采用細(xì)胞CCK-8法考察黃芩苷、姜黃素、黃芩苷/姜黃素(質(zhì)量比為5∶1)混合溶液、GA/FA-pH-Lip@Bai/Cur對(duì)肝癌細(xì)胞HepG2的體外增殖抑制作用;通過小鼠組織藥物分布實(shí)驗(yàn),考察GA/FA-pH-Lip@Bai/Cur體內(nèi)的肝靶向性;探究GA/FA-pH-Lip@Bai/Cur (120、60、30 mg·kg-1)對(duì)H22荷瘤小鼠的抑瘤率、HE染色后腫瘤組織病理學(xué)、血清腫瘤壞死因子-α(TNF-α)和白細(xì)胞介素-6(IL-6)水平的影響。結(jié)果 GA/FA-pH-Lip@Bai/Cur的最佳制備工藝為:黃芩苷與姜黃素質(zhì)量比為5∶1,琥珀酸膽固醇單酯(CHEMS)與磷脂質(zhì)量比為2∶10,DSPE-PEG 2000與磷脂質(zhì)量比為4∶100,DSPE-PEG-GA與磷脂質(zhì)量比為2∶100,水化體積為10 mL,磷脂與藥物質(zhì)量比7.95∶1,磷脂與膽固醇質(zhì)量比6.53∶1,超聲破碎時(shí)間為69 s。經(jīng)響應(yīng)面優(yōu)化制得的GA/FA-pH-Lip@Bai/Cur黃芩苷包封率為90.90%,姜黃素包封率為90.97%,平均粒徑82.5 nm,釋放行為具有緩釋特性。黃芩苷對(duì)HepG2細(xì)胞的半數(shù)抑制濃度(IC50)大約是姜黃素的5倍,GA/FA-pH-Lip@Bai/Cur的IC50較混合溶液低。體內(nèi)藥效評(píng)價(jià)結(jié)果表明,GA/FA-pH-Lip@Bai/Cur抑瘤率與5-氟脲嘧啶(5-FU)相當(dāng);給藥組的腫瘤壞死細(xì)胞數(shù)量變少,腫瘤細(xì)胞的排列疏松,細(xì)胞呈現(xiàn)不同程度變??;血清中TNF-α和IL-6水平均較模型組顯著降低(P<0.01)。結(jié)論 制備的GA/FA-pH-Lip@Bai/Cur安全性好,肝靶向性高,具有良好的抑制肝腫瘤作用。
[Key word]
[Abstract]
Objective To optimize the formulation process of dual-targeted pH-sensitive baicalin/curcumin co-loaded liposomes (GA/ FA-pH-Lip@Bai/Cur) and evaluate their properties. Methods GA/FA-pH-Lip@Bai/Cur was prepared using a thin film dispersion ultrasonication method. The evaluation criteria included encapsulation efficiency, similarity factor (f2) of release curves in different pH media, particle size, and polydispersity index (PDI). The optimal formulation process was determined through single-factor testing and Box-Behnken design-response surface methodology to evaluate the liposomes' morphology, particle size, and in vitro release profiles at different pH levels. The biocompatibility of GA/FA-pH-Lip@Bai/Cur was assessed through a hemolysis test. The in vitro inhibitory effect of baicalin, curcumin, baicalin/curcumin (mass ratio 5∶ 1) mixed solution, GA/FA-pH-Lip@Bai/Cur on liver cancer cells (HepG2) was examined using the CCK-8 assay. The liver targeting of GA/FA-pH-Lip@Bai/Cur in vivo was investigated through a mouse tissue distribution experiment. Subsequently, pharmacological experiments were conducted to study the antitumor efficacy of GA/FA-pH-Lip@Bai/Cur on H22 tumor-bearing mice and its impact on histopathological changes of tumor tissue after HE staining, serum TNF-α and IL-6 levels. Results The optimal preparation method for GA/FA-pH-Lip@Bai/Cur was as follows: The ratio of baicalin to curcumin was 5∶1, the ratio of cholesteryl succinate (CHEMS) to phospholipids was 2∶10, the ratio of DSPE-PEG 2000 to phospholipids was 4∶ 100, the ratio of DSPE-PEG-GA to phospholipids was 2∶ 100, the hydration volume was 10 mL, the ratio of phospholipids to drugs was 7.95∶ 1, and the ratio of phospholipids to cholesterol was 6.53∶ 1, and the ultrasonic crushing time was 69 s. The optimized GA/FA-pH-Lip@Bai/Cur exhibited an encapsulation efficiency of 90.90% for baicalin and 90.97% for curcumin, with an average particle size of 82.5 nm and sustained-release behavior. The IC50 of baicalin for HepG2 cells was approximately five times that of curcumin, and the IC50 of GA/FA-pH-Lip@Bai/Cur was lower than that of the mixed solution. The in vivo pharmacodynamic evaluation results show that the tumor inhibition rate of GA/FA-pH-Lip@Bai/Cur was comparable to that of 5-fluorouracil (5-FU). The number of necrotic cells in the treated group decreased, the arrangement of tumor cells becomes loose, and the cells showed varying degrees of shrinkage, and the levels of TNF-α and IL-6 in the serum were significantly lower than those in the model group (P <0.01). Conclusion The GA/FA-pH-Lip@Bai/Cur prepared had good safety and high liver targeting, and has good inhibitory effects on liver tumors.
[中圖分類號(hào)]
R943
[基金項(xiàng)目]
2021年江蘇省高校自然科學(xué)基金項(xiàng)目(21KJB360021);2024年江蘇高校青藍(lán)工程優(yōu)秀教學(xué)團(tuán)隊(duì)資助項(xiàng)目;2021年國(guó)家中醫(yī)藥管理局領(lǐng)軍人才基金項(xiàng)目;泰州市科技支撐項(xiàng)目(TS202232、TS202325);2023年江蘇省大學(xué)生創(chuàng)新創(chuàng)業(yè)項(xiàng)目(202313981004Y、202313981011Y、202313981002Y、202313981003Y)