Folate Acid Modified Chitosan Nanoparticles Loaded With Ligustrazine Enhance The Inhibition Of Tumor Metastasis In Vitro

Objective:To investigate the mechanisms of folate acid modified chitosan nanoparticles loaded with ligustrazine?FA-CS-LZ-NPs?enhance the inhibition of tumor metastasis in vitro.Methods:1.Preparation and characterization of FA-CS-LZ-NPs.Ionic gelation method was put to use in preparing the FA-CS-LZ-NPs.HPLC assay was used to determine loading capacity?LC?,encapsulation efficiency?EE?of FA-CS-LZ-NPs.The characterization of FA-CS-LZ-NPs were determined by transmission electron microscopy.2.Determine the non-toxic dosage of FA-CS-LZ-NPs by MTT assay.Human cervical carcinoma cell Hela and human lung adenocarcinoma epithelial cell A549 which cultured in logarithmic phase were treated with different concentration of FA-CS-LZ-NPs and the nanoparticles without drugs of several concentrations? range.Calculated the dosage for 95% cells viability of FA-CS-LZ-NPs.3.Experimental groups and dosing regimenControl group: Blank medium group;Experimental Group: the cells were treated with active targeting nanoparticles?FA-CS-LZ-NPs?;Experimental control group: the cells were treated with passive targeting nanoparticles?CS-LZ-NPs??ligustrazine?LZ?solution group and empty nanoparticles;4.Evaluate the effects of FA-CS-LZ-NPs on cancer cell migration and invasion.Effects of FA-CS-LZ-NPs on cancer cell migration and invasion were evaluated in vitro with transwell assays.5.Determination of MMP-9 and VEGF level by Western Blotting.Hela and A549 were divided into groups.After the cells were treated with the drug for 24 h,the whole protein was extracted,electrophoresed,electrotransfered,incubated by different antibody,then stained by ECL,photographed.The expression of MMP-9 and VEGF was detected by gray analysis.6.Determination of the MMP-9 and VEGF level by Real Time PCR.After treated Hela and A549 to the drug for 24 h,the whole mRNA was extracted,MMP-9mRNA and VEGFmRNA was determined by Real Time PCR.Results:1.Preparation and characterization of FA-CS-LZ-NPs.Characterization of FA-CS-LZ-NPs was that the average size were 133.9±4.1nm,the EE were 38.61±0.79% LC were 9.65±0.20%,PDI were 0.249±0.0030,were presented with uniform spherical morphology.2.The cytotoxicity of FA-CS-LZ-NPs for Hela?A549 cellThe non-toxic dose of active targeting nanoparticles for Hela?A549 cell is LZ 42.4?g·mL^-1.In the concentrations? range,the nanoparticles without drugs were non-toxic for the two cell lines.3.The effects of FA-CS-LZ-NPs on Hela?A549 cell migration and invasion.With transwell assays,migration and invasion of Hela and A549 cells could be significantly suppressed by the treatment of active and passive targeting nanoparticles.For Hela cell line,It indicates statistical significant between active and passive targeting nanoparticles.4.The effects of FA-CS-LZ-NPs on the MMP-9 and VEGF levelWith Western Blotting assays,Both active and passive targeting nanoparticles could obviously suppressed the MMP-9 and VEGF level except the VEGF level of Hela cell line.For the MMP-9 level of Hela cell line,it indicates statistical significant between active and passive targeting nanoparticles.5.The effects of FA-CS-LZ-NPs on the MMP-9mRNA and VEGF mRNA levelWith Real Time PCR assays,Both active and passive targeting nanoparticles could obviously suppressed the MMP-9 mRNA and VEGF mRNA level.Further more,for Hela cell line,it indicates statistical significant between active and passive targeting nanoparticles.Conclusions:In this research,all the results indicate that compared with CS-LZ-NPs and LZ solution,FA-CS-LZ-NPs can suppresse migration and invasion of Hela cell line which the folate receptor is overexpressed by suppress the expression of MMP-9 protein and it?s gene level.What?s more,compared with CS-LZ-NPs and LZ solution,FA-CS-LZ-NPs can inhibit the expression of VEGF gene level.