Construction of miRNA-126 stearylamine cationic liposome delivery system and cellular uptake investigation
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 LPS-stimulated 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° C 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 SDS-PAGE 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.
cationic liposomemiR-126film dispersion methodEDC/NHS methodmodified by monoclonal antibody to vascular cell adhesion molecule-1
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