Research On ShMDR1 And Gefitinib Co-loaded Chitosan Nanoparticles In Overcoming Tumor Resistance

ObjectiveIn China,Cervical cancer is one of the most malignant tumor in the gynaecology,which seriously affects women's health.It has strong metastasis and high recurrence rate of prognosis.At present,drug therapy is often used as a conservative treatment in clinics.However,with the prolonged treatment cycle,it is easy to develop drug resistance.Therefore,it is very important to find ways to suppress the drug resistance and restore the sensitivity of drugs.A large number of reports have explained that the expression of P-glycoprotein(P-gp)on the surface of cervical cancer cells is much higher than normal cells,and the cervical cancer patients with high P-gp content are more likely to develop resistance.It has been shown that reduced the overexpression of P-glycoprotein may be the key to overcome drug resistance.Geftinib,as the frst selective inhibitor of epidermal growth factor receptor(EGFR)tyrosine kinase domain,is widely used in the chemical therapy of many human cancers,but the drug resistance limits antitumor effect.Therefore,this paper intends to use the small interference RNA(shMDR1)to silence MDR1.It reduces the expression of P-gp and inhibits the occurrence of drug resistance.So it can restore the sensitivity of Gefitinib-resistant Hela cell line to Gefitinib.In this study,we designed and prepared chitosan nanoparticles co-encapsulated shMDR1 and Gefitinib to achieve the co-delivery of gene and anti-tumor drugs.The stability and characterization of co-loaded nanoparticles were investigated.Analyzing the effect of co-loaded nanoparticles on the proliferation and apoptosis of Gefitinib-resistant cell lines.The results demonstrated that chitosan nanoparticles entrapping Geftinib and shMDR1 had the potential to overcome the multidrug resistance and improve cancer treatment effcacy.MethodsIn this experiment,the determination method of Gefitinib was established by ultraviolet absorption spectrophotometry,and the linearity,repeatability,stability and recovery were investigated.The co-loaded nanoparticles was prepared by ion-gel method.The prepared loaded nanoparticles were characterized and analyzed,including appearance,morphology and particle size.The in vitro release and long-term stability of the nanoparticles were investigated.We investigated the protective effect of chitosan on the encapsulated shMDR1 by gel retardation,serum protection and enzymolysis experiments.Laser confocal microscopy was used to observe the intracellular distribution of co-loaded nanoparticles.We analyzed the ingestion of co-loaded nanoparticles by uptake experiments.We investigated the antitumor activity of co-loaded nanoparticles loaded in vitro.MTT assay was used to detect the inhibitory effects of co-loaded nanoparticles on the proliferation of cervical cancer cells.Flow cytometry was used to analyze the effect of co-loaded nanoparticles on cell apoptosis.Western blot assay was used to detect the effect of the co-loaded nanoparticles on apoptosis and autophagy-associated proteins.ResultsThe linear,precision,repeatability,stability and recovery of Gefitinib and shMDR1 were determined by UV-Vis,which were in accordance with the requirements of in vitro assay.So it could be used for Gefitinib and shMDR1 content determination in vitro.Geftinib and shMDR1-encapsulating chitosan nanoparticles with sustained release,small particle size,and high encapsulation effciency were prepared.The entrapment efficiency results showed that the selected formulation was able to encapsulate the two in the nanoparticles.Among them,the entrapment efficiency of shMDR1 was 88.3%±7.2%,and the Gefitinib was 89.8%±7.1%.The release of nanoparticles exhibits pH dependence,Gefitinib released rapidly at pH=5.8,but slowed down at pH=7.4.Likewise,the release of shMDR1 nanoparticles was comparable to Gefitinib.At 48 h,the cumulative release rate of shMDR1 was approximately 75% at both pH conditions.Laser confocal microscopy showed that co-loaded nanoparticles could be concentrated around the nucleus,and the enrichment behavior was time and dose-dependent.The uptake experiments could prove that the uptake of the nanoparticles by the cells was related to clathrin-mediated effects.The addition of inhibitors initially revealed that the transport of nanoparticles within the cells were energy-dependent.The results of MTT and flow cytometry showed that co-loaded nanoparticles could inhibit the proliferation of cervical cancer cells,and could induce apoptosis.The results of Western blot showed that co-loaded nanoparticles could significantly increase the expression of Cleaved caspase-3.It could reduce the content of MDR1.The results demonstrated that co-loaded nanoparticles could reverse MDR1-mediated drug resistance.ConclusionsIn this study,chitosan nanoparticles co-loaded with shMDR1 and Gefitinib were successfully prepared.The characteristics of co-loaded nanoparticles were with uniform size,spherical shape,good dispersion,pH-dependent release.Chitosan as a nanocarrier could effectively protect shMDR1 from degradation by serum and enzymes.At the same time,nanoparticles loaded with Gefitinib could significantly inhibit the proliferation of Hela cells.The possible mechanism was that shMDR1 entered the cells,blocked the expression of MDR1.The co-loaded nanoparticles could reverse drug resistance and restore the resistance of Hela-resistant cell line to Gefitinib.These fndings indicated that co-encapsulation of the anticancer drug Gefitinib and shMDR1 could be more effective in reversing MDR and a nano drug-delivery system could contribute greatly to reversing MDR.