Synergistic Anti-tumor Effect Of Dual SiRNA And Paclitaxel Co-loaded Nanosystem

Cancer remains a leading cause of human mortality worldwide,with an estimated 15 million new cases annually.Despite efforts to combat this disease,the number of deaths caused by lung and breast carcinomas has remained stubbornly high since 2010.Consequently,these two types of tumors remain the primary focus of antitumor therapy research.Paclitaxel(PTX),a potent chemotherapeutic agent originally derived from Taxus brevifolia,has shown efficacy against various types of tumors.However,chemoresistance presents considerable obstacles for cancer treatment.One mechanism driving PTX resistance is the overexpression of anti-apoptotic proteins.To address this issue,small interfering RNA(siRNA)can be introduced into tumor cells to inhibit the expression of anti-apoptotic proteins and potentially alleviate PTX-induced chemoresistance.Bcl-xL and Mcl-1,two anti-apoptotic proteins from the Bcl-2 protein family,are crucial in the development of treatment resistance in malignancies.These two proteins are located in the mitochondria.Interfering with the expression of these two proteins using siRNA has the potential to impact mitochondrial function and reverse chemoresistance in tumor cells.Combined interference with the expression of the two anti-apoptotic proteins may result in more complex anti-tumor effects on tumor cells.For siRNA combination therapy to be effective,they must be delivered into tumor cells.However,effective delivery remains difficult because of their high molecular weights,vulnerability to destruction by endonucleases in vivo,and incapacity to be internalized by cells.Another challenge encountered in PTX therapy is its limited solubility and poor bioavailability.These issues can be addressed by utilizing nanocarriers,which can effectively encapsulate siRNA and PTX to protect these medications during delivery to tumor cells.To address the aforementioned difficulties,we have developed a drug-loading system that facilitates the internalization of siRNA and PTX into cells and effectively protects them against degradation.The organic polymer carrier with hydroxypropyl methacrylate(2-Hydroxypropyl methacrylate,HPMA)as the hydrophilic shell offers advantages such as biocompatibility,low toxicity,low immunogenicity,and the ability to effectively prolong in vivo circulation time.The addition of positively charged groups at the hydrophilic end facilitates the adsorption of negatively charged siRNAs.A positively charged group was introduced at the hydrophilic end to adsorb negatively charged siRNA,thereby achieving the co-delivery effect of loading PTX inside the carrier and adsorbing siRNA outside the carrier.This approach further leverages the carrier’s ability to load multiple drugs.The details of the study are as follows:(1)Preparation and applicability study of drug-carrying nanosystems: The structure of Rhodamine B was modified to obtain the reduced Rhodamine B product Rhob,and then the methacryl modified Rho B-MA,resulting in a p H-independent fluorescence intensity.A copolymer named PPEMMRA,comprising Poly(HPMAAlt-TMAEMC)-b-Poly(MMA-Alt-(Rhob-MA))with Rhob-MA fluorescence,was successfully synthesized and characterized.The applicability of the carriers was investigated,and the self-assembly properties were examined using transmission electron microscopy and a Malvern particle sizer.The stability of the carrier was assessed under various media and storage conditions while its biosafety was assessed using different cell models.The drug loading of PTX-loaded nanoparticles and their release behavior were determined using high-performance liquid chromatography(HPLC).The adsorption of siRNA and the release properties of the carriers were investigated through gel retardation experiments.The results showed that PPEMMRA,a fluorescent drug carrier,exhibited non-hemolytic properties,low toxicity,and efficient cellular internalization.In terms of drug-carrying capacity,PPEMMRA can load PTX and siRNA and release them at different times.It releases PTX slowly after interfering with the expression of target proteins by siRNA.Additionally,it exhibits excellent stability and can serve as a carrier for both in vitro and in vivo delivery studies.(2)Study on the anti-tumor effect of the combination of Bcl-xL and PTX: Bcl-xL,an anti-apoptotic protein situated within the mitochondria,functions to impede apoptosis by upholding the integrity of the mitochondrial membrane.The concurrent delivery of siBcl-x L and PTX was facilitated through the utilization of PPEMMRA.PPEMMRA@PTX/siBcl-x L was able to deliver PTX and siBcl-x L into the cells.The inhibitory impact of siBcl-x L on expression in vitro and the synergistic anti-tumor influence were explored using A549 cells as a model.The results showed that the combination application of siBcl-x L and PTX effectively suppressed the proliferation of A549 cells and induced apoptosis.Notably,transfection with siBcl-x L was shown to significantly impede cell migration,as evidenced by a scratch assay.(3)Study on the anti-tumor effect of Mcl-1 and PTX combination: Triplenegative breast cancer is characterized by its poor differentiation,propensity for invasion,resistance to chemotherapy,and a lack of viable therapeutic targets and treatments.Mcl-1 and Bcl-x L are both members of the anti-apoptotic proteins within the Bcl-2 proteins family.Given the high expression of Mcl-1 in breast cancer cells,targeting Mcl-1 expression is deemed essential for effective breast cancer treatment.The PPEMMRA was utilized for the co-delivery of siMcl-1 and PTX,resulting in the formulation of PPEMMRA@PTX/siMcl-1.The study analyzed the suppressed effect of siMcl-1 and the combined anti-tumor effects of siMcl-1 and PTX in human triplenegative breast cancer cell lines MDA-MB-231 as a model.The study also assessed the impact of this treatment on mitochondrial function and cell migration.The results showed that PPEMMRA@PTX/siMcl-1 effectively suppressed Mcl-1 protein expression,leading to inhibiting cell migration and promoting intracellular reactive oxygen species(ROS)release.Additionally,the synergistic effect of PTX and siMcl-1could further enhance the cytotoxicity against tumor cells.(4)Antitumor effects of the combined action of three drugs on drug-resistant cells: Chemoresistance in tumors presents a significant challenge to effective treatment,leading to a rapid decline in the efficacy of chemotherapeutic agents.Based on the effectiveness of combining siBcl-x L,siMcl-1,and PTX,with a focus on the enhanced internalization of PPEMMRA in MDA-MB-231 cells,as a promising strategy against drug-resistant tumor cells.The feasibility of targeting Bcl-x L and Mcl-1 expression simultaneously in breast carcinoma treatment was assessed using data from the TCGA database.The research investigated the therapeutic efficacy of PPEMMRA-mediated triple-combination therapy in resistant strains,utilizing the PTX-resistant MCF-7/PTX cell line as a model.In vivo,experiments using nude mice as a model organism demonstrated that the treatment with PPEMMRA@PTX/siBM exhibited potent anti-tumor efficacy and synergistic interactions among the three medications.Transfection of the two siRNAs in MCF-7/PTX cells led to a reduction in the mitochondrial membrane potential and an increase in the release of ROS.Flow cytometry analysis revealed a significant pro-apoptotic effect when combined with PTX,effectively overcoming the chemoresistance in the cells.Furthermore,experiments conducted in live organisms confirmed that the PPEMMRA@PTX/siBM displayed enhanced anti-tumor properties and a more advantageous safety profile.In this study,an organic polymer,PPEMMRA,was synthesized to co-load a variety of siBcl-x L and PTX.The polymer exhibited excellent drug delivery properties and demonstrated good biosafety.It was found that siBcl-x L and siMcl-1interfered with the expression of target proteins,promoted apoptosis,inhibited cell migration,and synergized with PTX.Co-administration of the three drugs(PPEMMRA@PTX/siBM)reversed drug resistance in drug-resistant cells.The synergistic effect of silencing two proteins simultaneously suggests that we can identify proteins that are inherently interconnected and execute precise gene regulation to enhance the impact of protein silencing,leading to more effective therapeutic outcomes.