| Oligonucleotides(OGN),as a new class of therapeutic agents,have broad application prospects in the treatment of various diseases.However,OGN has limited clinical applications due to its poor stability,rapid clearance,nuclease degradation,poor cell targeting and uptake,and inefficient intracellular delivery.At the same time,single OGN drug is difficult to achieve the desired therapeutic effect in the treatment of diseases,especially for diseases that are difficult to cure with complex mechanisms such as tumors and rheumatoid arthritis(RA).Therefore,it is necessary to rationally design and develop a vector design for OGN targeted delivery and to enhance its therapeutic effect,which is of great significance.Polyethylenimine(PEI)is widely used for OGN delivery due to its unique proton sponge effect and excellent electrostatic recombination ability.However,the clinical application of PEI is limited due to its high toxicity,high surface charge,and phagocytosis by the endothelial system.Thus,it needs to be modified for the application.The hydrophobic modification of PEI can reduce the surface charge of PEI and reduce toxicity.Simultaneously,the hydrophobic part can enhance the transfection efficiency of PEI through hydrophobic interaction with the cell membrane.Targeted modification of PEI can increase the aggregation of OGN at the target site,improve its delivery efficiency,and further reduce toxicity.Methotrexate(MTX)is a dihydrofolate reductase(DHFR)inhibitor and widely used clinically to treat a variety of tumors and autoimmune and inflammatory diseases.As a folic acid analog,MTX has potential folate receptor(FR)targeting.In recent years,it has been found that,through rational design,MTX covalently linked to a polymer has the dual functions of a therapeutic agent and an FR ligand.Polyethylene glycol(PEG)is the most widely used polymer for the hydrophilic modification of carriers.The addition of PEG can significantly prolong the in vivo circulation time,reduce interaction with blood proteins,avoid rapid phagocytosis of the endothelial system,and increase the biocompatibility and stability of the carriers.In order to improve the delivery efficiency of OGN,reduce the toxicity of the vector,prolong the circulation time in the body and enhance its cell-targeted uptake and therapeutic effect,we designed and synthesized a MTX-linked linoleic acid modified PEI with targeted and therapeutic dual functions(MTX-PEI-LA)cationic copolymer and linoleic acid modified methoxypolyethylene glycol(mPEG-LA)copolymer for extending the in vivo circulation time,increasing biocompatibility,and reducing toxicity of the carriers.By optimizing the ratio of mPEG-LA to MTX-PEI-LA,MTX conjugated polymeric hybrid micelle(M-PHMs)with dual functions of targeting and therapeutic agent and good biocompatibility was constructed for OGN delivery.Through the dual function of MTX in M-PHMs,M-PHMs can improve the delivery efficiency of OGN in FR high expression diseases,increase its cell-targeted uptake and enhance its therapeutic effect.The research content of this paper mainly includes the following parts:1.Synthesis of MTX-PEI-LA and mPEG-LA copolymersFirst,hydrophobic LA was attached to PEI and mPEG through an amide bond,respectively and PEI-LA and mPEG-LA was obtained.MTX was then attached to PEI-LA by amide condensation to obtain MTX-PEI-LA copolymer.The characterization of nuclear magnetic and infrared spectra indicated that mPEG-LA and MTX-PEI-LA were successfully synthesized and the purity of mPEG-LA and MTX-PEI-LA was 91.0% and 95.6%.The drug loading of MTX in MTX-PEI-LA was calculated to be 15.6%.2.Preparation and characterization of M-PHMsM-PHMs were formed by the self-assembly of two copolymers of MTX-PEI-LA and mPEG-LA.Firstly,the influence of different mass ratios of MTX-PEI-LA on the particle size,zeta potential,colloidal stability and hemolytic properties of M-PHMs were first investigated.The optimized mass ratio of MTX-PEI-LA for M-PHMs was 5%.The M-PHMs prepared at this ratio had good colloidal stability and blood compatibility and the particle size,zeta potential and critical micelle concentration(CMC)of the carrier was 141.9 ± 2.4 nm,7.57 ± 0.24 mV,and 6.28 ?g/ml,respectively.3.M-PHMs/miR-124 mediates OGN targeted delivery and its therapeutic effect in RA with high expression of FRActivated macrophages in RA express high FR,which is often used as a target for RA drug targeted therapy.miR-124 has been found to significantly inhibit joint damage and bone destruction in the progress of RA disease in recent years and thought to be a novel OGN molecule for the treatment of RA.Therefore,miR-124 was selected as an OGN drug for the therapy of RA,and the targeted delivery and therapeutic effect of M-PHMs/miR-124 on RA with FR high expression was studied.M-PHMs could successfully complex miR-124 when nitrogen/phosphorus(N/P)was 16/1,and their particle size and potential values are 116.6±1.1 nm and-4.66±0.21 mV,respectively.The fluorescence intensity of M-PHMs/Cy3-miR-124 in activated macrophages was 2.78 folds than the fluorescence intensity in unactivated macrophages,and the fluorescence intensity of M-PHMs/Cy3-miR-124 was gradually decreased with increasing FA concentration.At the same time,the uptake of M-PHMs/Cy3-miR-124 in activated macrophages with high FR expression was 2.98 folds than that of PHMs/Cy3-miR-124.There was no significant difference in M-PHMs/Cy3-miR-124 and PHMs/Cy3-miR-124 in unactivated macrophages with low FR expression.Laser scan confocal microscopy(LSCM)experiments further showed that M-PHMs/Cy3-miR-124 had the strongest intracellular fluorescence intensity,and Cy3-miR-124 was successfully delivered into cells and achieved lysosomal escape.Cellular uptake results indicated that M-PHMs were dependent on FR mediated cellular uptake and could significantly increase the uptake of miR-124 in activated macrophages with FR high expression and successfully release miR-124 into the cytoplasm.In vivo distribution results showed that M-PHMs/Cy5-miR-124 accumulated most at the inflamed joints compared to Cy5-miR-124 and PHMs/Cy5-miR-124,and its fluorescence intensity was 3.60 folds and 1.83 folds compared with Cy5-miR-124 and PHMs/Cy5-miR-124.The therapeutic effect of M-PHMs/miR-124 was evaluated by establishing an adjuvant-induced(AIA)rat arthritis model.Compared with the saline and MTX group,the swelling joint of M-PHMs/miR-124 was reduced by 24.7% and 21.5%,and had significant articular cartilage protection and anti-inflammatory effects.MTX in M-PHMs still had a much better therapeutic effect compared with the saline and MTX groups,and the joint swelling was reduced by 16.9% and 13.3%.The results of the concentration of proinflammatory cytokines in rat serum and joint histopathology further confirmed that M-PHMs/miR-124 group significantly inhibited the expression of proinflammatory cytokines,and had the lightest synovial hyperplasia,the most cartilage integrity and the least number of osteoclasts in joint.In vivo experiments showed that M-PHMs could deliver more miR-124 to the inflamed joints,and enhance the therapeutic effect of RA.4.M-PHMs mediated OGN targeted delivery and its therapeutic effect in tumor with FR high expressionHeLa cells highly expressed FR and Survivin protein,and Survivin-siRNA was selected as the OGN drug for tumor treatment.The FR-mediated cell uptake of M-PHMs/Survivin-siRNA was studied and applied in tumor therapy.The fluorescence intensity of M-PHMs/Cy3-siRNA in HeLa cells was 4.40 folds than that of the PHMs/Cy3-siRNA group and the cellular uptake was gradually decreased with increasing FA concentration.As shown in the confocal photographs,M-PHMs/Cy3-siRNA had the strongest fluorescence intensity and was inhibited by the addition of free FA to the cells.The lysosomal escape results demonstrated that FAM-siRNA could successfully escape from the lysosome and been released into the cytoplasm after internalization to the cells.In vitro cell cytotoxicity results showed that M-PHMs/Survivin-siRNA significantly inhibited the proliferation of tumor cells,and the cell death rate was 1.77 times and 1.79 times higher than that of PHMs/Survivin-siRNA group and M-PHMs group.After the addition of the free FA,the cell viability of M-PHMs/Survivin-siRNA was increased,and the cell death rate was decreased by 4.35 times.The results of western blotting were similar to those of cell cytotoxicity.The expression of Survivin protein was the lowest in M-PHMs/Survivin-siRNA group,and the protein expression was increased by adding free FA.M-PHMs/Survivin-siRNA enhanced the anti-tumor effect in vitro.The cell cytotoxicity mediated by M-PHMs was mainly achieved by inhibiting the activity of dihydrofolate reductase(DHFR)by MTX-PEI-LA.The results of tissue distribution in tumor-bearing mice showed that mice injected with M-PHMs/Cy5-siRNA had the highest aggregation at the tumor site and the reduced distribution in the kidney and liver.In vivo therapy showed that the average tumor weight of M-PHMs/Survivin-siRNA was reduced by 60.6%,60.3%,and 51.7% respectively,compared with the saline group,the MTX group,and the M-PHMs/Survivin-siRNA negative control group.The expression of Survivin in M-PHMs/Survivin-siRNA was decreased by 35.8%,32.5%,and 11.1% compared with the saline group,MTX group,and M-PHMs/Survivin-siRNA negative control group.Large areas of apoptosis occurred in tumor tissues of the M-PHMs/Survivin-siRNA group.The M-PHMs/Survivin-siRNA negative control group was more effective than saline and MTX group and indicated that MTX in M-PHMs had certain anti-tumor effects.The results of in vitro and in vivo experiments showed that M-PHMs/Survivin-siRNA had significantly increased uptake in tumor cells with high expression of FR,increased tumor tissue aggregation,and enhanced anti-tumor effect.M-PHMs had dual functions as targeting and therapeutic agents.In this paper,we designed and prepared multi-functional polymeric hybrid micelles with dual functions of targeting and therapeutic agents for efficient delivery of OGN.The delivery system could deliver OGN to the tumor and RA sites with high FR expression and be applied as a therapeutic agent together with OGN to enhance the therapeutic effect.Moreover,the delivery system was prepared with the simple method by self-assembly,convenient for application with the broad prospect. |
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