Preparation Of PH-Responsive And Tumor-Targeting Nanoparticle-Mediated SiRNA Delivery And Anti-Tumor Research

Background and objectivesAs a promising gene-targeting drug,Small interfering RNAs(si RNA)can be widely used in the treatment of genetic diseases such as inherited disorders,viral infections,and cancers.However,how to maintain the stability and tissue targeting of si RNA is still a major problem limiting the clinical transformation and large-scale application of si RNA.With the progress of bioengineering and nanotechnology,various nanoparticles have been produced for the targeted delivery of drugs,including liposomes,polymers,peptides,and inorganic nanoparticles.However,these nanoparticles can only break through one or a couple of biological barriers,not all biological barriers(various opsonin proteins,reticuloendothelial system,dense extracellular matrix,cell membrane,intracellular lysosomal escape,etc.),the effectiveness of treatment for various diseases is still not satisfactory.Therefore,given various biological environments and barrier mechanisms in vivo,it is necessary to design a novel multifunctional platform to improve the delivery efficiency of si RNA.In this study,we used cholesterol,histidine,EGFR monoclonal antibody,and carboxymethyl chitosan as raw materials to synthesize a carboxymethyl chitosan polymer compound modified by histidine cholesteryl ester and EGFR monoclonal antibody(CHCE).CHCE polymer compounds could form a new type of nanocarrier through self-assembly at a lower critical micelle concentration.This nanocarrier had the dual characteristics of tumor targeting and pH-responsive protonation,which could meet the requirements of simultaneously overcoming various levels of biological obstacles in the body,and realized the efficient delivery of si RNA in the body.In order to verify the advantages of the nanocarrier system and better exert the interference effect of si RNA,we have adopted different strategies for anti-tumor therapy.The first strategy was to achieve the goal of anti-tumor by using nanocarriers loaded with a single VEGFA-si RNA to block blood vessel growth in tumor tissue.The second strategy,based on the verification in the first strategy,tried to overcome the shortcomings of using the single si RNA to treat a tumor.We used nanocarriers to co-encapsulate doxorubicin,MVP-si RNA,and BCL2-si RNA to achieve the goal of eliminating tumor multi-drug resistance and anti-tumor.Therefore,the main research contents of this thesis were divided into the following two parts:Part 1.CHCE nanoparticles loaded with VEGFA-si RNA inhibit angiogenesis and anti-tumor researchMethodsIn this research,CHCE polymer compound and VEGFA-si RNA self-assembled to form a novel CHCE/VEGFA-si RNA nanocarrier with the dual capability of tumor targeting and pH-responsive protonation.The nanocarrier system silenced the VEGFA-m RNA gene as a therapeutic target,inhibited the production of vascular endothelial growth factor,prevented the occurrence of blood vessels at the tumor site,and achieved the purpose of inhibiting the growth of tumor cells.During the nanocarrier assembly process,(1)we checked the morphology and structure of CHCE/VEGFA-si RNA nanocarriers by scanning electron microscopy.(2)The particle size distribution and surface Zeta charge of CHCE/VEGFA-si RNA nanocarriers were measured by particle size spectrophotometer.(3)The stability of CHCE/VEGFA-si RNA nanocarriers was tested by gel blocking experiment.At the cell level in vitro,(1)the specific adhesion capability,cell uptake efficiency and intracellular lysosome escape capability of CHCE/VEGFA-si RNA nanocarriers were detected by flow cytometry and laser confocal microscopy respectively.(2)The VEGFA-m RNA gene silencing efficiency of CHCE/VEGFA-si RNA nanocarriers in vitro was detected by q PCR and Western blot respectively.(3)The therapeutic effect of CHCE/VEGFA-si RNA nanocarriers was tested by anti-tumor experiments in vitro.In vivo experiments at the animal level,(1)the in vivo circulation time and tumor targeting accumulation ability of CHCE/VEGFA-si RNA nanocarriers were detected by a mouse live imaging instrument.(2)The therapeutic effect of CHCE/VEGFA-si RNA nanocarriers was tested by anti-tumor experiments in vivo.(4)The secretion of IL-1,IL-6,IFN-γ and TNF-α cytokines in mice after injection of CHCE/VEGFA-si RNA nanocarriers were tested by ELISA.ResultsThe physical and chemical characteristics of CHCE/VEGFA-si RNA nanocarriers are as follows: CHCE/VEGFA-si RNA nanocarriers have a smaller particle size(80 nm),a narrower particle size distribution(DPI=0.025)and a complete capsule structure.The results of pH responsiveness experiments showed that the surface Zeta charges of CHCE/VEGFA-si RNA nanocarriers at different pH values(pH 7.4,6.5,5.4)were-5mv,9mv and 19 mv,indicating CHCE/VEGFA-si RNA nanocarriers have the ability of sensitive pH-responsive protonation.The particle size distributions at different pH values(pH 7.4,6.5,5.4)are 72 nm,91 nm,114 nm,respectively,and the Pd I(particle dispersion index)is 0.045,0.025,0.035,respectively,indicating that the CHCE/VEGFA-si RNA nanocarrier had uniform particle size distribution and good stability in different pH environments.Gel blocking experiments showed that the 50% dissociation time of CHCE/VEGFA-si RNA nanocarriers in serum was 12 h,indicating that the nanocarriers have good serum stability and can better protect si RNA from nuclease degradation.The results of cell experiments in vitro are as follows: Targeted adhesion results showed that CHCE/VEGFA-si RNA nanocarriers have a higher tumor cell adhesion efficiency(92%,p<0.05).Targeting inhibitor experiments showed that as the concentration of inhibitor increased,the adhesion efficiency of CHCE/VEGFA-si RNA nanocarriers to tumor cells decreased from 93.1% to 5.59%,indicating that CHCE/VEGFA-si RNA nanocarriers have specific tumor-targeting properties.The cell uptake results showed that the cellular uptake efficiency of CHCE/VEGFA-si RNA nanocarriers increased from 0.5% to 95% over time and as the concentration of CHCE/VEGFA-si RNA nanocarriers increased,the cell uptake efficiency increased from 7.3% To 94.2%,Indicating that the cellular uptake of CHCE/VEGFA-si RNA nanocarriers was time-dependent and concentration-dependent.The lysosomal escape results showed that the binding rate of CHCE/VEGFA-si RNA nanocarriers to lysosomes increased from 39% to 93.2% in the first 2 hours,and the binding rate dropped to 45% at 4 hours,Indicating that in the early stage of cell uptake,the CHCE/VEGFA-si RNA nanocarrier will gradually bind to the lysosome,and then begin to escape in the later stage and have excellent lysosomal escape ability.The results of in vitro silencing showed that compared with the PBS group,the CHCE/VEGFA-si RNA nanocarrier can effectively silence the VEGFA-m RNA gene and inhibit the expression of vascular endothelial factor by 75%(p<0.05).The anti-tumor results in vitro showed that compared with the PBS group,CHCE/VEGFA-si RNA nanocarriers can induce 48.3% of tumor cells to undergo apoptosis(p<0.05).The cell viability results show that the CHCE/VEGFA-si RNA nanocarrier can better inhibit cell growth.The results of animal experiments in vivo are as follows: The results of in vivo distribution show that CHCE/VEGFA-si RNA nanocarriers not only have a good tumor-targeting and accumulation ability,but also prolong the circulation time of si RNA in the body.The results of in vivo tumor treatment showed that compared with other treatment groups,CHCE/VEGFA-si RNA nanocarriers can effectively silence VEGFA gene and inhibit tumor growth(p<0.05),and did not cause the weight loss of mice during the treatment period.The immunotoxicity test results show that compared with simple si RNA and PEI/si RNA,the secretion of IL-1,IL-6,IFN-γ and TNF-α cytokines in the body is lower after CHCE/VEGFA-si RNA nanocarrier injection(P<0.05),indicating that CHCE/VEGFA-si RNA nanocarriers have lower immunotoxicity.Part 2.CHCE nanocarriers co-loaded with doxorubicin,MVP-si RNA and BCL2-si RNA to overcome tumor resistance in chemotherapy and enhance anti-tumor effectMethodsMulti-drug resistance(MDR)has become the biggest obstacle to tumor chemotherapy treatment.Therefore,the multi-drug resistance of tumor cells needs to be eliminated to improve the anti-tumor effects of various drugs.In this research,we adopt the strategy of using RNA interference technology to silence genes related to drug efflux and anti-apoptosis to achieve the goal of eliminating tumor cells with multi-drug resistance.We used CHCE polymer compound and Adriamycin,MVP-si RNA and BCL2-si RNA through self-assembly to prepare a novel of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles with the dual capability of targeting and pH-responsive protonation.This nanocarrier system took MVP-m RNA and BCL2-m RNA genes as the silencing targets,which could simultaneously inhibit the cell’s drug efflux and anti-apoptotic ability;thereby promoting the better anti-tumor therapeutic effect of doxorubicin.In this research,same as the first part,at the nanocarrier assembly level,(1)we checked the morphology and structure of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles by scanning electron microscopy.(2)The particle size distribution and surface Zeta charge of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles were measured by particle size spectrophotometer.(3)The stability of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles was tested by gel blocking experiment.At the cell level in vitro,(1)the specific adhesion ability,cell uptake efficiency and intracellular lysosome escape ability of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles were detected by flow cytometry and laser confocal microscopy respectively.(2)The MVP-m RNA and BCL2-m RNA genes silencing efficiency of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles in vitro were detected by q PCR and Western blot respectively.(3)The therapeutic effect of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanoparticles was tested by anti-tumor experiments in vitro.In vivo experiments at the animal level,(1)the in vivo tumor targeting accumulation ability of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers were detected by a mouse live imaging instrument.(2)The therapeutic effect of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers was tested by anti-tumor experiments in vivo.ResultsThe physical and chemical characteristics of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarrier were as follows: Scanning electron microscopy results show that the CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers had a smaller particle size and a complete capsule structure;The results of pH responsiveness experiments showed that the surface Zeta charges of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers at different pH values(pH 7.4,6.5,5.4)were-8.6mv,17 mv and 30 mv,respectively,indicating that CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers have the ability of sensitive pH-responsive protonation;The particle size distributions at different pH values(pH 7.4,6.5,5.4)are 90.26 nm,100.2 nm and 129.1 nm,respectively,and Pd I is 0.197,0.128 and 0.037,respectively,indicating that CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers had uniform particle size distribution good stability in different pH environments;Scanning electron microscopy and particle size distribution results show that the time for CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers to completely dissociate in serum is about 12 hours,and gel blocking experiments also show that the time for CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers to completely dissociate in serum is 12 h,indicating that the nanocarriers have good serum stability and can better protect si RNA from nuclease degradation.The results of cell experiments in vitro are as follows: Targeted adhesion results show that compared to CHC/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers,CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers have higher tumor cell adhesion efficiency;The results of cell uptake showed that with the increase in the concentration of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers,the efficiency of cell uptake of si RNA and doxorubicin increased to more than 90%,indicating that the uptake of CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers is concentration-dependent;The results of intracellular delivery experiments showed that with the extension of intracellular delivery time,the intracellular co-localization rate of si RNA and doxorubicin decreased from 90±5% to 42±4%,indicating that the nanocarrier can effectively release si RNA and doxorubicin Into the cytoplasm;The lysosomal escape results showed that the binding rate of si RNA and lysosome increased from 42±5% to 91±6%,and then dropped to 39±5%;the binding rate of doxorubicin and lysosome increased from 42±5% Rose to 92±5%,and then dropped to 45±6%;The results of in vitro silencing showed that CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers have the ability to simultaneously silence MVP and BCL2 genes,compared with the PBS group,the silencing efficiency reached 75% and 70%,respectively(p< 0.05);Apoptosis experiments showed that compared to CHCE/Adriamycin/BCL2-si RNA nanocarriers or CHCE/Adriamycin/MVP-si RNA nanocarriers,CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers could induce tumor cell apoptosis more effectively;The cell viability results showed the same results as the apoptosis experiment,compared with CHCE/Adriamycin/BCL2-si RNA nanocarriers or CHCE/Adriamycin/MVP-si RNA nanocarriers,CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers could better inhibit the growth of tumor cells.The results of animal experiments in vivo are as follows: The results of in vivo distribution show that CHCE/Adriamycin/MVP-si RNA/BCL2-si RNA nanocarriers have a good tumor-targeting and accumulation ability in the body.The results of in vivo tumor treatment showed that compared with other treatment groups,CHCE/VEGFA-si RNA nanocarriers have the ability to simultaneously silence MVP and BCL2 genes and can effectively inhibit tumor growth(p<0.05).The anti-tumor treatment results showed the dual sensitization strategy could more effectively eliminate the multidrug resistance of tumor cells and promote the anti-tumor effect of Adriamyci.ConclusionIn summary,we have synthesized a new type of nanocarrier for si RNA delivery.This nanocarrier has the dual characteristics of tumor targeting and pH-responsive protonation,which can meet the requirements of simultaneously overcoming all levels of biological obstacles and realize the efficient delivery of si RNA to achieve better anti-tumor effect in vivo.This nanocarrier will provide a usable platform for the clinical application of RNA interference technology and other therapeutic technologies.