目的 制备 DiR(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)脂质体荧光探针(DiR-LP),研究柴胡作为引经药对其在小鼠体内分布的影响,初步验证柴胡引药入肝性能。方法 薄膜分散法制备 DiR-LP,以包封率为主要指标,通过单因素试验确立 DiR-LP 最佳处方与制备工艺,并进行表征。30只SPF级昆明种小鼠随机分为5组:对照组、DiR-LP组和柴胡水提液低、中、高剂量(0.5、1.0、2.0 mg·kg^-1)+DiR-LP组,柴胡水提液+DiR-LP组小鼠预先连续3 d 按10mL·kg^-1体积ig相应的柴胡水提液,对照组和DiR-LP组小鼠 ig 相同体积的生理盐水;除对照组外,各组小鼠以 10 mL·kg^-1尾iv DiR-LP,对照组小鼠尾 iv 相同剂量的生理盐水;利用小动物活体成像仪在不同时间点(4、5、6、8 h)拍照,示踪小鼠体内DiR-LP 的分布情况;8 h 时经解剖取出心、肝、脾、肺、肾等器官进行拍照,观察各器官的荧光强度。结果 DiR-LP 最佳处方与制备工艺:精密称取大豆卵磷脂 120 mg、胆固醇 12 mg、DiR 0.375 mg 于 100 mL 茄形瓶中,加入 10 mL 无水乙醇溶解,于40℃恒温水浴中,30 r·min^-1旋转减压蒸发1h,形成均匀透明薄膜;再加入聚山梨酯80 12mg、溶于纯净水10mL,于150r·min^-1旋转常压水化 1 h 后,置于冰水浴中超声 10 min(功率 100 W),分别过 0.45、0.22 μm 微孔滤膜整粒 3 次,即得 DiRLP。制得的 DiR-LP 包封率为 95.2%,粒径为 254.1 nm,聚合物分散性指数(PDI)为 0.196,Zeta 电位-5.21 mV,14 d 内无沉淀或浑浊现象。DiR-LP 经尾 iv 后主要在肝脏中分布,柴胡水提液+DiR-LP 组肝脏部位荧光信号明显比 DiR-LP 组强,且荧光信号强度与柴胡水提液剂量呈正相关。结论 使用薄膜分散法制备的 DiR-LP 包封率高、粒径适宜、稳定性较好;在小鼠体内,柴胡对 DiR-LP 具有肝脏导引作用,且与剂量相关,初步验证了柴胡"引药入肝"的肝靶向性。

Objective The DiR liposome fluorescent probe (DiR-LP) was prepared to study the effect of Bupleurum chinense as a primer on its distribution in mice and verify the liver function of B. chinense. Methods DiR-LP was prepared by thin film dispersion method, and the optimum formulation and preparation process of DiR-LP were determined by single factor test with the encapsulation rate as the main index. Thirty SPF Kunming mice were randomly divided into five groups: Control group, DiR-LP group and B. chinense water extract (BCWE) at low, medium and high doses (0.5, 1.0, 2.0 mg·kg^-1)+DiR-LP group. Mice in BCWE+ DiR-LP group were ig with the corresponding BCWE at 10 mL·kg^-1 volume for three consecutive days in advance. Mice in control group and DiR-LP group were given the same volume of normal saline. Except the control group, mice in each group were given 10 mL·kg^-1 DiR-LP by tail iv and the control group were given the same dose of normal saline. The distribution of DiR-LP in mice was traced by using small animal imager at different time points (4, 5, 6, 8 h). At 8 h, organs such as heart, liver, spleen, lung and kidney were dissected and photographed to observe the fluorescence intensity of each organ. Results The optimum formulation and preparation process of DiR-LP were as follows: 120 mg of soy lecithin, 12 mg of cholesterol and DiR 0.375 mg were accurately weighed in 100 mL nightshade bottle, dissolved in 10 mL of anhydrous ethanol, and then evaporated in a constant temperature water bath at 40℃ for 1 h at 30 r·min^-1 to form uniform transparent films. Then polysorbate 80 12 mg was added, 10 mL was dissolved in pure water, and the water was hydrated at 150 r·min^-1 under atmospheric pressure for 1 h, then ultrasonic was placed in ice water bath for 10 min (power 100 W), and the whole grain was passed through 0.45 and 0.22 μm microporous filter membrane three times, to obtain DiR-LP. The entrapment efficiency of DiR liposomes was 95.2%, the particle size was 254.1 nm, the PDI was 0.196, the Zeta potential was -5.21 mV, and there was no precipitation or turbidity within 14 days. DiR-LP was mainly distributed in liver after tailing iv. The fluorescence signal in liver of BCWE + DiR-LP group was significantly stronger than that of DiR-LP group, and the intensity of fluorescence signal was positively correlated with the dose of BCWE. Conclusion DiR-LP prepared by thin film dispersion method has high encapsulation efficiency, suitable particle size and good stability. In mice, BCWE has liver guiding effect on DiR-LP, which is related to the dose. It verifies the liver targeting of B. chinense "introducing drugs into the liver".

R943;R969.1

云南省科技厅-云南中医学院应用基础研究联合专项面上项目(2018FF001(-030));云南省高校外用给药系统与制剂技术研究重点实验室(2019YGZ03)