目的 基于陽離子脂質體制備血管細胞黏附分子-1（VCAM-1）單克隆抗體（VCAM-1 mAb）修飾的負載微小RNA-126（miR-126）的靶向遞送系統(tǒng)，并初步評價其穩(wěn)定性、細胞毒性及細胞攝取。方法 采用薄膜分散-擠出法制備硬脂胺陽離子脂質體（SCL），以脂質體粒徑、電位為指標，單因素法考察大豆磷脂與膽固醇質量比、硬脂胺用量、無水乙醇的用量、成膜溫度、水合介質用量、水合制備溫度、水合制備時間；并考察其稀釋穩(wěn)定性及儲存穩(wěn)定性。采用瓊脂糖凝膠阻滯實驗考察SCL對miR-126的負載能力及對酶解的保護效果。利用1-乙基-（3-二甲基氨基丙基）碳二亞胺鹽酸鹽（EDC）/N-羥基琥珀酰亞胺（NHS）交聯(lián)法制備VCAM-1 mAb修飾的SCL（Va-SCL），并通過SDS-PAGE法考察VCAM-1 mAb與SCL的耦連效率。MTT法考察其對人臍靜脈內皮細胞（HUVECs）的毒性，流式細胞術和熒光顯微鏡考察正常HUVECs及LPS（1 μg·mL^-1）刺激后HUVECs對miR-126、SCL/miR-126、Va-SCL/miR-126的攝取情況。結果 精密稱量大豆磷脂120 mg、膽固醇40 mg、硬脂胺10 mg于茄形瓶中，加入5 mL無水乙醇，50℃超聲（功率300 W、頻率40 kHz）使其充分溶解，肉眼觀察無可見顆粒物或者不溶物，水浴減壓蒸發(fā)除去有機溶劑，在瓶壁上形成均勻透明的脂質薄膜；加入12 mL DEPC水作為水合介質，水浴超聲30 min，擠出器過孔徑為200 nm聚碳酸酯膜7次，取續(xù)濾液，得SCL，穩(wěn)定性良好。當氮磷比為10∶1時，SCL能夠有效負載miR-126；miR-126通過靜電吸附于SCL表面，SCL一定程度上保護miR-126不被酶解。SDS-PAGE結果顯示，VCAM-1mAb與SCL成功偶聯(lián)，接枝率為（53.2±7.6）%。MTT結果顯示，Va-SCL、Va-SCL/miR-126對HUVECs的半數抑制濃度（IC₅₀）值分別為17.38、71.61 nmol·L^-1，SCL、SCL/miR-126的IC₅₀值分別為81.03、97.79 nmol·L^-1。熒光顯微鏡及流式結果均顯示Va-SCL/miR-126的細胞攝取效果更為明顯。結論 成功制備了Va-SCL/miR-126，能明顯增加miR-126的細胞攝取。

Objective To prepare a VCAM-1 monoclonal antibody (VCAM-1 mAb) modified miR-126 targeted delivery system based on cationic liposomes, and to preliminarily evaluate its stability, cytotoxicity and cellular uptake. Methods SCL was prepared by a thin film dispersion-extrusion method. With liposome size and zeta potential as the indicators, single factor method was used to investigate the effects of soybean phospholipid to cholesterol ratio, amount of stearic amine, amount of ethanol, film-forming temperature, amount of hydration medium, hydration preparation temperature, and hydration preparation time on the liposome. The dilution stability and storage stability were also examined. The loading capacity of miR-126 by SCL and the protection effect of SCL on the enzymatic hydrolysis were investigated by agarose gel retardation assay. The coupling efficiency of VCAM-1 mAb with SCL was examined by SDS-PAGE. The toxicity of Va-SCL, Va-SCL/miR-126 to human umbilical vein endothelial cells (HUVECs) was investigated by MTT assay, and the uptake of miR-126, SCL/miR-126, and Va-SCL/miR-126 by normal HUVECs and LPSstimulated HUVECs was examined by flow cytometry and fluorescence microscopy. Results Weigh out 120 mg of soybean phospholipids, 40 mg of cholesterol, and 10 mg of stearic amine into a funnel-shaped bottle, and add 5 mL of anhydrous ethanol. Ultrasonicate at 50 ℃ (power 300 W, frequency 40 kHz) to dissolve the ingredients completely, and visually inspect for any visible particles or insoluble substances. Remove the organic solvent by rotary evaporation at 50 ℃ under a vacuum, forming a uniform, transparent lipid film on the bottle wall. Add 12 mL of DEPC-treated water as a hydration medium, and ultrasonicate at 50℃ for 30 min. Pass the mixture through a 200 nm polycarbonate membrane using a syringe pump 7 times, collect the filtrate, and obtain SCL. The stability is good. When the nitrogen-to-phosphorus ratio is 10:1, SCL can effectively load miR-126. miR-126 adheres to the surface of SCL via electrostatic attraction, and SCL to some extent protects miR-126 from enzymatic degradation. The SDSPAGE results show that the Va-SCL and Va-SCL/miR-126 antibodies successfully coupled to SCL, with an attachment rate of (53.2 ±7.6)% . The MTT results show that the IC₅₀ values of Va-SCL, Va-SCL/miR-126, SCL, and SCL/miR-126 for inhibiting HUVECs are 17.38, 71.61, 81.03, and 97.79 nmol·L^-1, respectively. The fluorescence microscope and flow cytometry results show that Va-SCL/miR-126 has a more pronounced cell uptake effect. Conclusion Va-SCL/miR-126 was successfully prepared with a high safety profile and significantly increased the cellular uptake of miR-126.

R943.42;R979.1

國家自然科學基金資助項目（No.82260827和U1812403-4-4）;貴州省自然科學基金會（［2020］1Z069）;貴州醫(yī)科大學國家自然科學基金培育項目（No.20NSP050）;貴州省科技計劃項目（黔科合支撐［2020］4Y240號）;貴州省高層次創(chuàng)新型人才十層次人才黔科合平臺人才（GCC［2023］048）;黔科合中引地（［2023］003）