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。

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.

R943

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）