Evaluation Of A Cisplatin And Disulfiram Co-Loaded Inclusion Complex For Reversing Drug Resistance In Non-Small Cell Lung Cancer

Lung cancer is a malignant tumor originating from the lung glands or bronchial mucosa and is one of the deadliest tumors in the world.About 2.20 million cases of lung cancer were diagnosed worldwide in 2022.In recent years,the incidence of lung cancer has rose rapidly in both women and men,posing a great threat to human health.In terms of mortality,lung cancer is the leading results of cancer deaths,accounting for about 20%of all cancer deaths.There are two main subtypes of lung cancer:small cell lung cancer and non-small cell lung cancer(NSCLC),which account for 15%and 85%,respectively.NSCLC is further divided into three subtypes:adenocarcinoma,squamous cell carcinoma and large cell carcinoma.The etiology of NSCLC is currently unknown.Smoking and air pollution are two important risk factors.Compared to most countries,China has the relatively higher lung cancer mortality rate.It is projected that the mortality rate of lung cancer in China may increase by about 40%between 2015 and 2030.Platinum-based drugs are used to treat a wide range of solid malignancies.Among them,cisplatin(DDP)is considered as one of the first-line drugs in the treatment of NSCLC,but it has strong light instability.Although the initial treatment is effective,DDP treatment often leads to chemotherapy resistance.The main resistance mechanisms include abnormal DNA damage repair,reduced drug accumulation,intracellular drug inactivation,autophagy,changes in drug-resistant genes,and apoptosis.Therefore,it is urgent to study the mechanism of drug resistance and find ways to reverse DDP resistance.Disulfiram(DSF),an anti-alcohol drug approved by the Food and Drug Administration(FDA),has shown good anti-tumor activities in vivo and in vitro in recent years.However,its low solubility limits its effect.DSF has been found to exert anticancer effects through various mechanisms:inhibition of p97-NPL4-UFDI pathway,NF-κB pathway,induction of parasitism and apoptosis of cancer cells,and so on.In addition,DSF can effectively overcome tumor drug resistance caused by various mechanisms,including inhibition of tumor cell stemness,inhibition of drug resistance transporter overexpression,and reversal of drug resistance in combination with other antitumor drugs.Therefore,combining DSF with DDP may be an effective strategy to reverse drug resistance in NSCLC.Although a small amount of studies have been performed in this area,there is currently a lack of preparations for co-delivery of DDP and DSF,and the effect of the combination of DDP and DSF in drug-resistant NSCLC is unknown.In order to improve the water solubility of DSF and the photostability of DDP,and to enable DDP and DSF to enter cells in the optimal combination ratio to better play the therapeutic effect and reverse drug resistance,the co-loaded inclusion complex of DDP and DSF was found to be an excellent preparation through the screening of the combination dosage forms of DDP and DSF.The main research content of this work includes the following three sections:(1)Preparation and characterization of the coloaded inclusion complexes;(2)Evaluation of anti-resistant effects and mechanism of DDP-DSF/HP-β-CD on NSCLC A549/DDP cells;(3)Study of in vivo efficacy and mechanism of DDP-DSF/HP-β-CD on NSCLC.(1)Preparation and characterization of the co-loaded inclusion complexes:The MTT experiment was used to screen the optimal combination ratio of DDP and DSF.The optimal mass ratio of 1:1 was obtained based upon the combination index(CI).The inclusion material cyclodextrins were screened by using the equilibrant solubility method,and hydroxypropyl-β-cyclodextrin(HP-β-CD)was selected as the ideal inclusion material.The optimal inclusion conditions of DDP/HP-β-CD and DSF/HP-β-CD were screened by the single factor experiment.The optimal inclusion conditions of DDP/HP-β-CD were as follows:the molar ratio of DDP and HP-β-CD was 1:10,the inclusion temperature was 50℃ the inclusion time was 2 h,and the rotation speed was 600 r/min.The optimal inclusion conditions of DSF/HP-β-CD were as follows:the molar ratio of DSF and HP-β-CD was 1:5,the inclusion temperature was 40℃ the inclusion time was 2 h and the rotation speed was 800 r/min.Based on the optimal preparation processes of the two drugs,the co-load inclusion conditions were determined as follows:the ratio was 1:10,the inclusion temperature was 50℃,the inclusion time was 2 h,the rotation speed was 600 r/min.The results show that the encapsulation rates of DDP and DSF were 78.7%and 80.8%,respectively.The successful preparation of DDP-DSF/HP-β-CD was demonstrated by differential scanning calorimetry,infrared spectroscopy and scanning electron microscopy.The solubility,stability and in vitro release experiments proved that DDP-DSF/HP-β-CD significantly improved the solubility,stability and dissolution of the co-loaded drugs.The uptake experiments showed that DDP-DSF/HP-β-CD was easier to enter cells than the bulk drug.(2)Evaluation of anti-resistant effects and mechanism of DDP-DSF/HP-β-CD on NSCLC A549/DDP cells:The scratch and transwell assays showed that DDPDSF/HP-β-CD inhibited the migration and invasion of A549/DDP cells.Flow cytometry analysis indicated that the apoptosis rate of A549/DDP cells was significantly increased after the treatment of DDP-DSF/HP-β-CD.The western blot assay detected the decrease of anti-apoptotic protein Bcl2 and increase of pro-apoptotic protein Bax,showing that DDP-DSF/HP-β-CD promoted apoptosis of A549/DDP cells.The western blot and qRT-PCR assays demonstrated that Nanog,Oct4,and MRP1 were highly expressed in A549/DDP cells,suggesting that the drug resistance of A549/DDP cells was related to the stemness and drug-resistant protein of the tumor cells.The expression levels of Nanog,Oct4,and MRP1 in A549/DDP cells decreased significantly after the treatment with the different doses of DDP-DSF/HP-β-CD in a concentration-dependent manner,suggesting that DDP-DSF/HP-β-CD inhibited the stemness and drug resistance of A549/DDP cells.In addition,the inhibitory capacity of DDP-DSF/HP-β-CD on MRP1 detected by Rhodamine 123(RH123)uptake assay was decreased in A549/DDP cells by DDP-DSF/HP-β-CD.Thus,the drug resistance of A549/DDP cells was reduced.(3)Study of in vivo anti-resistant efficacy and mechanism of DDP-DSF/HP-βCD on NSCLC:Subcutaneous injection of A549/DDP cells into nude mice was used to establish a subcutaneous drug-resistant lung cancer model.Tumor volume and body weight of nude mice were recorded.The H&E staining and immunohistochemistry assay showed that the relative tumor growth rates of 87.1%,71.0%and 35.5%for the DSF/HP-β-CD,DDP/HP-β-CD,DDP-DSF/HP-β-CD groups,respectively,indicating that the tumor volume of the DDP-DSF/HP-β-CD group was the smallest and achieved the effective therapeutic level.In addition,DDP-DSF/HP-β-CD also decreased the dose of DDP compared with the known DDP dose commonly used in the literature.The Ki67 staining also showed the lowest tumor proliferation ability in the DDP-DSF/HP-β-CD group,which was consistent with the TUNEL apoptosis test.In addition,immunohistochemical analyses of Nanog and MRP1 also confirmed that DDPDSF/HP-β-CD inhibited the stemness and drug resistance of tumor cells in vivo.The H&E staining of major organs demonstrated the relative lower toxicity in the DDPDSF/HP-β-CD group.In conclusion,of the final inclusion complex DDP-DSF/HP-β-CD for the coloading of DDP and DSF was successfully prepared through the screening of the drug ratios and preparation.DDP-DSF/HP-β-CD improved the water solubility of DSF and the photostability of DDP,and allowed DDP and DSF to enter cells in the optimal combination ratio,so as to better play the synergistic effect of drugs and reverse DDP resistance.The stem cell transcriptional regulatory genes Nanog,Oct4,and drugresistant protein MRP1 in A549 and A549/DDP cells were tested,proving that the DDP resistance of NSCLC A549 cells was closely related to the overexpression of stemness and drug-resistant proteins in tumor cells.DDP-DSF/HP-β-CD inhibited the expressions of these genes in vitro.In addition,the measurement of the tumor mass and relative tumor growth rate of each group in a subcutaneous resistant tumor model in nude mice demonstrated that DDP-DSF/HP-β-CD could inhibit the growth of drugresistant NSCLC in vivo,reduce the dose of administration,and decrease the toxicity of DDP.The expressions of Nanog and MRP1 by immunohistochemistry proved that DDP-DSF/HP-β-CD reversed the resistance of DDP to NSCLC by inhibiting the stemness and drug resistant protein.