Study On Anti-mouse Colon Cancer Activity Of Delivering SiRNA Based On DMP Cationic Nanoparticles And Curcumin Nanoparticles

At present,tumor treatment is the focus of medical research all around of the word,and the study of anti-tumor drugs and their delivery systems is the key to cancer treatment.In antitumor grug research,for active ingredient of Chinise medicine and natural medicine,due to their poor stability,excessive metabolism and low bioavailability in vivo limit their application,and for gene drugs,they also have some disadvantages such as instability,vulnerability to degradation and low cell uptake rate,which make their clinical application limit.Therefore,establishing and seeking appropriate drug delivery system has become a common need to develop anti-tumor active ingredient of Chinise medicine,natural medicine and gene drugs to achieve safe and effective cancer therapy.In previous study,(2,3-Dioleoyloxy-propyl)-trimethylammonium(DOTAP)and monomethoxy poly(ethylene glycol)-poly(?-caprolactone)MPEG-PCL were combined to create the cationic self-assembled DOTAP and MPEG-PCL hybrid nanoparticles(DMP).However,DMP nanoparticles were limited to the delivery of plasmid DNA,DMP nanoparticles delivering for siRNA has never been reported.In this article,we obtained a new DMP nanoparticle preparation recipe by optimizing the preparation process of DMP,and this study utilized DMP delivered anti-MCL1 siRNA(DMP/siMCL1)and DMP delivered anti-Bcl-xl siRNA(DMP/siBcl-xl)on C26 cells for anti-mouse colon cancer activity study in vitro.Curcumin has significant anti-tumor activity,but due to its poor stability,rapid metabolism,and low bioavailability,which inhibits its anti-tumor effects in vivo.Therefore,this study aimed to prepare watersoluble curcumin nanoparticles by encapsulating the insoluble antitumor drugcurcumin into MPEG-PCL to improve the water solut.Firstly,through the related parameters of cationic nanoparticles DMP to evaluate the characteristics of its physical and chemical properties: particle size distribution,size,ability to absorb siRNA,cytotoxicity,stability and delivery capacity under serum conditions.This study obtained the best preparation process by screening the preparation process of DMP.The prepared DMP had an average particle diameter of 46.4nm and a zeta potential of 44.1mV.The positively charged nanoparticles were uniformly dispersed in aqueous solution(PDI=0.215).It was found through gel retardation that DMP had the ability to load siRNA.Cytotoxicity experiments showed that DMP had low cytotoxicity than PEI25 K.DMP showed low cytotoxicity and high transfection efficiency in vitro transfection experiments.There was no significant difference in the transfection efficiency between serum transfection group and serumfree transfection group.Transfection efficiency of DMP nanoparticles was not affected by serum concentration.Second,we used MCL1 siRNA and Bcl-xl siRNA as therapeutic targets to study the anti-mouse colon cancer activity in vitro.In vitro gene silencing assays,C26 cells were treated with MCL1 siRNA and Bcl-xl siRNA,and their corresponding mRNA expression was measured by fluorescence quantitative PCR.The results showed that the expression level of mRNA in C26 cells of the experimental group transfected with DMP/siMCL1 and DMP/Bcl-xl was significantly reduced compared with control group.In the study of in vitro anti-tumor activity,cell proliferation ability was examined by MTT assay and colone formation assay.The results of MTT assay showed that the cell survival rates of DMP/siMCL1 and DMP/siBcl-xl were lower than that of the corresponding DMP group,indicating that DMP/siMCL1 and DMP/siBcl-xl can inhibit the growth and proliferation of C26 cells.In clone formation assays,DMP/siMcl group and DMP/siBcl-xl group had higher clone formation inhibition and had a stronger inhibitory effect on the proliferation of tumor cells.In apoptotic experiments,apoptotic cells were quantitatively determined by flow cytometry.The apoptosis rates of DMP/siMCL1 and DMP/siBcl-xl groups were significantly higher than that of the corresponding control group and other experimental groups,and DMP/siMCL1 and DMP/siBcl-xl had the effect of inducing apoptosis.Western Blot experiments showed that compared with control,DMP and DMP/Scramble siRNA groups,DMP/siBcl-xl group significantly reduced the expression of Bcl-xl protein levels,and the effect of high concentration of DMP/siBclxl group was more obvious.Curcumin has significant antitumor activity,but because of its poor water solubility,which limited the anti-tumor effect of curcumin in vivo.In order to achieve a combination of traditional drugs and gene drugs based on DMP nanoparticles to enhance the anti-tumor efficacy,we used MP nanoparticles to encapsulate curcumin to improve its water solubility.The prepared curcumin nanoparticles had an average particle size of 38.65 nm,and was electrically neutral,and had a polydispersity index(PDI)of 0.386.The curcumin nanoparticles could be completely dispersed in an aqueous solution,and the average entrapment efficiency was 92.8% and the average drug loading was 1.5%.In vitro anti-tumor experiments,it could inhibit the growth of tumor cells and showed significant anti-tumor activity.In this study,DMP/siMCL1 and DMP/siBcl-xl can inhibit the expression of MCL1 gene and Bcl-xl gene,inducing apoptosis of C26 cells,and thus achieve the therapeutic effect of inhibiting the growth of C26 cells.DMP has the ability to transport different siRNAs and can safely and efficiently deliver MCL1 siRNA and Bcl-xl siRNA to C26 cells.DMP/siMCL1 and DMP/siBcl-xl have anti-mouse colon cancer activity in vitro and can inhibit the growth and proliferation of C26 cells.Throughing the preliminary characterization of the prepared curcumin nanoparticles and the investigation of antitumor activity laid the foundation for the coordinated treatment of DMP-loaded curcumin-prepared nanoparticles and siRNA to improve the efficacy of anti-tumor therapy.In this study,DMP had the ability to transport different siRNAs and can safely and efficiently deliver MCL1 siRNA and Bcl-xl siRNA to C26 cells.DMP/siMCL1 and DMP/siBcl-xl had anti-mouse colon cancer activity in vitro and can inhibit the growth and proliferation of C26 cells.Curcumin nanoparticles had good physicochemical properties and anti-mouse colon cancer activity in vitro.Based on the DMP,evaluating the related activities of delivery for gene drug siRNA and the curcumin which provided the basis for the synergistic treatment of the DMP-loaded curcumin-prepared nanoparticles and the siRNA to enhance the anti-tumor therapeutic effect.