基于DiR脂质体荧光探针验证柴胡“引药入肝”靶向性研究

Verification of Bupleurum chinense"drug introduction into liver"based on DiR liposome fluorescent probe

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10 mL·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,30r·min^(-1)旋转减压蒸发1h,形成均匀透明薄膜;再加入聚山梨酯8012mg、溶于纯净水10mL,于150 r·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、0.22μm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 8012 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 0.22μm 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".