Construction And Performance Of Paclitaxel/siRNA Co-delivery Systems For Anti-tumor Activity In Non-small Cell Lung Cancer

The combination of chemotherapy and gene therapy could inhibit tumor growth by different pathways or different mechanisms of action.When the two therapies work together on tumors,they could effectively improve the therapeutic effect.Therefore,the combination therapy by using co-delivery systems with drugs and genes has attracted great attention.However,unlike the loading of a single drug or gene,the interactions of hydrophobic drugs and negatively charged genes in the co-delivery systems have been found to have a significant impact on the ultimate synergistic effect.Current co-delivery systems always have some disadvantages,such as short blood circulation time and poor tumor targeting,which limit their clinical application.In this study,three novel co-delivery systems,PTX/PSur/QLD,PTX/siVEGF/FAPL and PTX/siVEGF/FAPLS,were designed and constructed based on the cationic lipids,and their anti-tumor activities against cancer were studied.1.Construction and performance of PTX/PSur/QLD for NSCLC therapy.In this part,PTX/PSur/QLD was constructed based on cationic lipids with the quaternary ammonium salt head(QLip)and helper lipids(DOPE,D)for co-delivery of PTX and survivin siRNA(siSur).siSur was compressed by the protamine(P)to form PSur.The results showed that the cellular uptake rate of PTX/PSur/QLD(68%)was higher than that of PTX/siSur/QLD(48%),which suggested that the co-delivery system with protamine could improve the delivery efficiency of siRNA.In vitro survivin protein expression assays and tumor cell proliferation inhibition results showed that PTX/PSur/QLD could significantly downregulate survivin protein expression by siSur,which increased the sensitivity of tumor cells to PTX by inhibiting the anti-apoptosis effect of tumor cells,resulting in a more efficient inhibition effect for NCI-H460 cells.2.Construction of PTX/siVEGF/FAPL and PTX/siVEGF/FAPLS for NSCLC therapy.In this part,two novel pH-sensitive delivery systems were constructed by using tripeptide lipids(PLip)without or with sucrose esters(S),and FA-PEG2000-DSPE(FA)for co-delivery of PTX and VEGF siRNA(siVEGF).They exhibited a pH-sensitive controlled release behavior.The cumulative release of PTX from co-delivery systems at pH 5.5 was 2-fold higher than that of pH 7.4,and their sustained release time could reach 120 h.Moreover,compared to PTX/siVEGF/FAPL,with the addition of sucrose esters,the sustained release ability of PTX/siVEGF/FAPLS was improved.In vitro analog release and CLSM images showed that both PTX/siVEGF/FAPL and PTX/siVEGF/FAPLS could deliver PTX and siVEGF to tumor cells,and release drugs in a controllable manner,leading to the synergistic effect of PTX and siVEGF for tumor therapy.SiVEGF could successfully escape from endosome and release in the cytosol to downregulate VEGF expression,and the silencing efficiency of VEGF protein reached up to 80%.The cell proliferation inhibition and apoptosis assays showed that both PTX/siVEGF/FAPL and PTX/siVEGF/FAPLS could co-deliver PTX and siVEGF to enhance apoptosis and inhibit the growth of NCI-H460 cells.3.In vivo study on anti-cancer activity of PTX/siVEGF/FAPL and PTX/siVEGF/FAPLS.It was found that the anti-tumor effects of PTX/siVEGF/FAPL and PTX/siVEGF/FAPLS were better than that of PTX delivery systems or siVEGF delivery systems,and the anti-tumor effect of PTX/siVEGF/FAPLS was better than that of PTX/siVEGF/FAPL.Ex vivo fluorescence imaging of organs and tumor tissues showed that both co-delivery systems could deliver PTX and siVEGF to tumor tissue at the same time,and the tumor-targeting ability of PTX/siVEGF/FAPLS with sucrose ester showed higher than that of PTX/siVEGF/FAPL.The results of VEGF protein expression showed that PTX/siVEGF/FAPLS could effectively deliver PTX and siVEGF to the tumor site,leading to a high gene silencing efficiency.The tumor sections assays demonstrated that PTX/siVEGF/FAPLS could lead to an apparent inhibition of angiogenesis in the tumors via the synergistic effect of PTX and siVEGF.And antiangiogenesis could enhance the sensitivity of tumor cells to PTX,resulting in enhanced tumor suppression effect via increased apoptosis.The average tumor size of PTX/siVEGF/FAPLS group was 8.68 times and 7.00 times smaller than that of the Saline group and Free PTX group,respectively.The tumor volume of PTX/siVEGF/FAPLS was 3.46 times smaller than that of PTX/siVEGF/FAPL,which fully demonstrated that the targeted delivery system PTX/siVEGF/FAPLS based on peptide lipids as carrier skeleton molecule and sucrose ester as helper lipid present good application prospects in NSCLC therapy.