目的 制備 DiR(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)脂質(zhì)體熒光探針(DiR-LP),研究柴胡作為引經(jīng)藥對(duì)其在小鼠體內(nèi)分布的影響,初步驗(yàn)證柴胡引藥入肝性能。方法 薄膜分散法制備 DiR-LP,以包封率為主要指標(biāo),通過(guò)單因素試驗(yàn)確立 DiR-LP 最佳處方與制備工藝,并進(jìn)行表征。30只SPF級(jí)昆明種小鼠隨機(jī)分為5組:對(duì)照組、DiR-LP組和柴胡水提液低、中、高劑量(0.5、1.0、2.0 mg·kg^-1)+DiR-LP組,柴胡水提液+DiR-LP組小鼠預(yù)先連續(xù)3 d 按10mL·kg^-1體積ig相應(yīng)的柴胡水提液,對(duì)照組和DiR-LP組小鼠 ig 相同體積的生理鹽水;除對(duì)照組外,各組小鼠以 10 mL·kg^-1尾iv DiR-LP,對(duì)照組小鼠尾 iv 相同劑量的生理鹽水;利用小動(dòng)物活體成像儀在不同時(shí)間點(diǎn)(4、5、6、8 h)拍照,示蹤小鼠體內(nèi)DiR-LP 的分布情況;8 h 時(shí)經(jīng)解剖取出心、肝、脾、肺、腎等器官進(jìn)行拍照,觀察各器官的熒光強(qiáng)度。結(jié)果 DiR-LP 最佳處方與制備工藝:精密稱(chēng)取大豆卵磷脂 120 mg、膽固醇 12 mg、DiR 0.375 mg 于 100 mL 茄形瓶中,加入 10 mL 無(wú)水乙醇溶解,于40℃恒溫水浴中,30 r·min^-1旋轉(zhuǎn)減壓蒸發(fā)1h,形成均勻透明薄膜;再加入聚山梨酯80 12mg、溶于純凈水10mL,于150r·min^-1旋轉(zhuǎn)常壓水化 1 h 后,置于冰水浴中超聲 10 min(功率 100 W),分別過(guò) 0.45、0.22 μm 微孔濾膜整粒 3 次,即得 DiRLP。制得的 DiR-LP 包封率為 95.2%,粒徑為 254.1 nm,聚合物分散性指數(shù)(PDI)為 0.196,Zeta 電位-5.21 mV,14 d 內(nèi)無(wú)沉淀或渾濁現(xiàn)象。DiR-LP 經(jīng)尾 iv 后主要在肝臟中分布,柴胡水提液+DiR-LP 組肝臟部位熒光信號(hào)明顯比 DiR-LP 組強(qiáng),且熒光信號(hào)強(qiáng)度與柴胡水提液劑量呈正相關(guān)。結(jié)論 使用薄膜分散法制備的 DiR-LP 包封率高、粒徑適宜、穩(wěn)定性較好;在小鼠體內(nèi),柴胡對(duì) DiR-LP 具有肝臟導(dǎo)引作用,且與劑量相關(guān),初步驗(yàn)證了柴胡"引藥入肝"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

云南省科技廳-云南中醫(yī)學(xué)院應(yīng)用基礎(chǔ)研究聯(lián)合專(zhuān)項(xiàng)面上項(xiàng)目(2018FF001(-030));云南省高校外用給藥系統(tǒng)與制劑技術(shù)研究重點(diǎn)實(shí)驗(yàn)室(2019YGZ03)