| BackgroundAt present,clinical treatment of malignant tumors adopts a comprehensive approach,including surgery,radiation therapy,and chemotherapy.In the treatment of cancer,drug treatment is a very important part,and the use of effective anti-cancer drugs can help patients to obtain a longer survival time and increase their survival rate.In the 1960s,the combination chemotherapy emerged.That was the simultaneous or sequential use of several chemotherapeutic agents in one chemotherapy course.It was widely used in many cancer treatments.At present,a variety of clinical cancer treatments have a combination chemotherapy programs.Based on the concept of combination chemotherapy,more and more research has been conducted on drug co-delivery in cancer treatment.From the structure of the used drug,it could be the co-delivery of two or more small-molecule drugs,or the co-delivery of small-molecule drugs and macromolecular drugs;From the mechanism of action of drug,it could be two or more anticancer drugs,which is the same as combination chemotherapy,or it also could be combined drug delivery of anticancer drugs and their sensitizers.In recent years,more and more studies have been conducted on the joint delivery of chemotherapeutic agents and their chemosensitizers in cancer treatment research.Chemosensitization or reversal of multidrug resistance means that the use of small doses of this substance in combination with chemotherapeutic agents can significantly increase the effect of chemotherapy.Current chemotherapy sensitizers mainly include:multidrug resistance(MDR)reversal agents,hypoxic cell killers,gene preparations,extracts of traditional Chinese medicines,etc.Among them,the study of P-gp inhibitors is relatively mature,and there have been third-generation P-pg inhibitors.Based on the principle of autophagy induction,the study of anticancer drugs in combination with autophagy inhibitors has also begun to attract attention in recent years.Induction of autophagy is one of the reasons for the resistance of tumor cells to chemotherapeutic drugs.When tumor cells are stimulated by chemotherapeutic drugs,the level of intracellular autophagy will significantly increase,which is an adaptive response of tumor cells to avoid chemotherapy stimulation.Therefore,autophagy can make tumor cells tolerant to chemotherapeutic drugs,and inhibition of autophagy will increase the sensitivity of tumors to chemotherapeutic drugs.Indeed,the combination therapy regimen with cytotoxic chemotherapy drugs and autophagy inhibitors has been extensively applied in clinical treatment.Combining the anti-cancer sensitization effect of autophagy inhibition and the advantages of nano drug delivery systems,the nano drug co-delivery system containing autophagy inhibitors and anti-cancer drugs can reduce the dose and side effects of anti-cancer drugs,and improve anti-cancer effect,reverse multidrug resistance.AimWe designed and prepared a single layer of dual drug-loaded nanoparticles named NPDOX+CQ.The nanoparticles load two drugs,one is an anticancer drug DOX and the other is an autophagy inhibitor CQ.We confirmed that NPDOX+CQ could prevent the drug from being recognized and effluxed by P-gp,release the drugs in the late endosomes of the cancer cells.CQ could block the autophagy process,protect DOX,and make it accumulate more to the nucleus.In addition,we also studied the specific transport process of nanoparticles in the cell and their effects on the expression of autophagy-related proteins.The nanoparticles provide an effective strategy for overcoming MDR.Methods1.Preparation and characterization:PLGA/TPGS NPs,NPDOX,NPCQ and NPDOX+CQ were prepared by emulsion solvent evaporation method.The particle size and surface potential of several nanoparticles were analyzed.Observed the surface morphology and dispersion of several nanoparticles respectively by scanning electron microscopy and transmission electron microscopy.The drug loading condition was measured by diffuse reflectance Fourier transform infrared spectroscopy(DR-FTIR)and X-ray diffraction(X-RD).The UV-Vis spectrophotometer was used to examine the encapsulation efficiency and drug loading efficiency of the nanoparticles,as well as the release in vitro.2.Biological effects:The cytotoxicity of PLGA/TPGS NPs to A549 and A549/Taxol cells was measured by WST-1 method to evaluate the safety of the materials.The cytotoxicity of DOX,DOX+CQ,NPDOX,and NPDOX+CQ on A549 and A549/Taxol cells was measured by the WST-1 kit,and IC50 values were calculated to evaluate the improvement of the anticancer effect of the nano drug co-delivery system.3.Cell Uptake and Migration:Prepare NPDOX+C6,and we used C6 to label nanoparticles as CQ had no fluorescence.Confocal laser microscopy was used to observe the efflux of free DOX in A549 and A549/Taxol cells,and the uptake and migration of NPDOX-C6 in A549/Taxol cells.4.Mechanism study:A549/Taxol cells transfected with DsRed-Rab5 and DsRed-Rab7 plasmids were treated with NPC6 respectively,and the transport of nanoparticles into the cytosol was observed using a laser confocal microscope,which was the positional relationship between the nanoparticles and the two fluorescent proteins.The expression condition of LC3-? and LC3-? in A549/Taxol cells and the ratio of LC3-?/LC3-? in each group were determined by Western Blot after treatment with different groups of drugs.Results1.The test results showed that the nanoparticles had a uniform particle size and good dispersion,and the dual-drug loaded nanoparticles were approximately 170 nm.Nanoparticles were negatively charged,facilitating their transport in the body.The results of infrared and X-ray diffraction showed that both drugs were successfully loaded into the nanoparticles.Nanoparticles had high encapsulation and drug loading efficiency for both drugs.In vitro drug release experiments showed that nanoparticles were pH-sensitive and could release drugs in weakly acidic late endosomes.2.The results of in vitro cytotoxicity experiments showed that the cytotoxicity of the materials on the A549 and A549/Taxol cells was' very low,demonstrating that the material was basically safe.The cytotoxicity results of drug formulations showed that the combined delivery of anticancer drugs DOX and its sensitizer CQ could improve the anti-cancer efficacy,reduce the dose of drugs,and reduce toxic and side effects.3.The results of drug efflux experiments showed that free DOX could enter the nucleus of A549 cells,but eventually there was also a small amount of efflux.For A549/Taxol cells,free DOX did not enter their cell nucleus and was only near the nucleus at 2h-6h but was eventually excreted.Prepared NPDOX+C6 for fluorescence imaging.The results showed that both drugs were delivered to A549/Taxol cells.From 6 h to 24 h,it showed that more DOX migrated and accumulated in the nucleus.These results showed that the nanoparticles could avoid the drug efflux which was P-gp substrate,and facilitates its accumulation in the nucleus.4.The results of the mechanistic study showed that nanoparticles entered the cells and were transported by autophagy process.CQ could play a role in the late stage of autophagy,protect DOX from degradation,make it more into the nucleus and play an anti-cancer effect.Western Blot results showed that compared with other drug formulations groups,the dual-drug loaded nanoparticles had the best autophagy inhibition effect,enabling DOX to maximize its efficacy and improve the anti-cancer effect.ConclusionIn this study,we designed and prepared single-layer double drug-loaded nanoparticles loaded with the anticancer drug DOX and the autophagy inhibitor CQ.As an anti-cancer sensitizer of DOX,CQ could prevent the binding of late endosomes to lysosomes and protect DOX from being degraded by cellular autophagy during nanoparticle transport through autophagy process.More DOX accumulated in cells and killed tumor cells.The dose of anticancer drugs was reduced,the side effects were reduced,the anti-cancer effect was increased,and multidrug resistance was reversed. |
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