Bioinspired Tumor-homing Nanoplatform To Co-deliver Paclitaxel And SiRNA-E7 For Cervical Cancer Therapy

Cervical cancer is a prevalent gynecological malignancy.As a front-line chemotherapeutic agent,paclitaxel(PTX)plays a pivotal role in the treatment of cervical cancer.However,because of the complexity of cancer,the single anti-tumor mechanism of conventional mono-chemotherapy is usually insufficient to combat the complex alteration of signaling pathways that regulate cancer progression.Therefore,combination therapies of two or more agents aimed at different targets or acting by different anticancer mechanisms hasbeen a focus of attention.Among the various drug combinations,chemo-genecombined therapy represents a more promising approach.Through preciselyregulating crucial proteins involved in cancer progression and drug resistance at the level of biosynthesis,gene therapy effectively enhances the anti-tumor effect of chemotherapy,without exerting toxicity on normal cells.The serum instability,polyanion macromolecular characteristics and poor cell uptake of gene therapy tools remain main obstacles in its systematic administration.Thus,to realize the full potential of this chemo-gene combined strategy,a smart,highly biocompatible nanosystem that enables the precise co-delivery of small-molecule anticancer drugs and gene therapy tools is urgently needed.This nanosystem should protect the gene therapy tools fromdegration while assisting them to be efficiently taken up by tumor cells.In this study,by camouflaging He La cell membranes onto the si RNA-E7/PTX co-loaded(lactic-co-glycolic acid)(PLGA)nanoparticles,wedeveloped a biomimetic dual-drug delivery system to simultaneously deliver PTX and si RNA-E7,and applied it for cervical cancer therapy.Objective1.Design,preparation and characterization of He La cell membrane camouflaged PLGA nanoparticles co-loaded with si RNA and PTX(Si/PNPs@He La);2.To investigate the effects of membrane camouflaging on nanoparticles in terms of cell uptake behavior,knockdown efficiency and cytotoxicity,as well as to explore the mechanism by which si RNA-E7 sensitized He La cells to PTX;3.To explore the effects of cell membrane camouflaging on nanoparticles in terms of circulation time and biodistribution,as well as to evaluate the tumor suppressive effect and biosafety of the delivery system.MethodsThe double emulsion method was applied for the preparation of si RNAand PTX co-loaded nanoparticles(Si/PNPs).The resulting si RNA/PTX co-loaded PLGA-polymeric cores were subsequently fused with the He La membrane through physical extrusion to obtain the Si/PNPs@He La.The particle size and surface charge of nanoparticles(NPs)were determined by Dynamic light scattering(DLS),also the morphology of NPs was revealed by transmission electron microscope(TEM).Theencapsulation efficiency(EE%)and in vitro drug release profile of NPs were calculatd based on the concentration of si RNA measured by fluorescence spectrometerand the concentration of PTX analysed by high performance liquid chromatography(HPLC),respectively.The retention of membrane proteins on the surface of NPs was further characterized by protein gel electrophoresis,Western blot,immunogold staining analysis,and Confocal laser scanning microscope(CLSM).The influence of membrane camouflaging on the cell uptake behavior of NPs in different cell lines including He La cells was qualitatively observed by fluorescence microscopy,and quantitatively ananlyzed by flow cytometry.We next designed and screened effective si RNA sequences which could knockdown the target gene E7.The highly efficient si RNA was then loaded into different formulations and their gene knockdown abilities were evaluated by Real-time PCR(RT-PCR)and Western blot.The cytotoxicity of different formulations was determined by CCK-8 method.Cell apoptosis rate was further analysedby flow cytometry.To explore by which si RNA-E7 sensitized He La cells to PTX,Western blot was used to detect the alteration of signaling pathway and drug-resistant relevant proteins in He La cells treated with PTX.The biodistribution patterns of different formulations were examined in He La tumor-bearing mice by NIR-fluorescence whole body imaging.And the biodistribution of the two agents was visualized in frozen sections of tumor tissues using fluorescence microscopy.We further detected pharmacokinetics of NPs in ICR mice.The subcutaneous He La xenograft model was used to investigate antitumor effect and biosafety of various PTX-based formulations.ResultsSi/PNPs and Si/PNPs@He La were successfully prepared.Dynamic light scattering(DLS)data showed that the size of Si/PNPs increased by about 15 nm after fusing with the He La membrane,and the surface charge of Si/PNPs changed from-14.4 m V to-30 m V.The EE% of si RNA and PTX in Si/PNPs were 88.4 ± 0.22% and90.2± 0.43%,respectively.The releasing study in vitro suggested a sustained-release profile of NPs.Protein gel electrophoresis and Western blot confirmed that most He La cell membrane proteins,especially those related to cell adhesion and immune escape,were successfully transferred to the surface of NPs.The immunogold staining indicated that the surface membrane of the nanoparticles was coated in the right direction.And the CLSM indicated that the core-shell structure of NPs remained intact for a period even after they were ingested by cells.Compared with naked NPs,He La cell membrane camouflaged NPs were more efficiently internalized by He La cells,while were more likely to avoid phagocytosis of mouse macrophages RAW264.7.Three si RNA sequences were designed through si RNA prediction software.Si RNA-E7-93 was screened out by detecting the expression of E7 in the transfected cells with RT-PCR and Western blot and used in further study.RT-PCR data showed that the knockdown efficiency of Si/PNPs@He La at 24 and 48 hours was 26.8 ± 5.7 %and 75.2 ± 0.6 %,respectively,higher than that of Si/PNPs.The cell viability displayed a gradual decrease in the sequence of free PTX,PNPs,Si/PNPs,and Si/PNPs@He La.The apoptosis rate induced by Si/ PNPs@He La was 71.51 ± 4.88 % at 48 hours.Time-course experiment showedthat PTX can increase the expression level of phosphorylated AKT(p-akt)and MDR1 in cervical cancer cells,while this effect was partially abolished by silencing E7 via Si/PNPs and Si/PNPs@He La.The results of in vivo imaging showed that Si/PNPs@He Labegan to accumulate in tumor site in 8 hours after injection,significantly earlier than Si/PNPs.Ever-increasing fluorescence signals in the tumor were observed over 48 h.The fluorescence intensity of Si/PNPs@He La was stronger than that of Si/PNPs at each indicated time point.Frozen sections of tumor tissues suggested that the co-loaded NPs could deliver simultaneously two agents to the tumor site.We observed a proloned blood circulation time in the cell membrane coating group.The tumor inhibition rate of Si/PNPs@He La was up to 83.6% without causing side effects in major organs.Immunohistochemical(IHC)results showed that the expression of E7 protein decreased and the expression of Rb protein increased in the tumor tissue of Si/PNPs@ He La group.ConclusionsIn this study,we successfully constructed a biomimetic nanoplatform for precise co-delivery of si RNA and PTX(Si/PNPs@He La).Benefiting from the recognition and adhesion molecules on the surface of He La cells,Si/PNPs@He La exhibited an improved immune escape ability and an increased tumor region accumulation.The nano system efficientlytransfered si RNA-E7 to the tumor cells,down-regulated the expression of E7,increased the sensitivity of He La cells to PTX by inhibiting theactivation of AKTpathway and the expression of MDR.As a resulte,antitumor effect was improved without causing side effects in major organs.