Study Of The Effects And Mechanism Of Novel T7 Peptide-Modified Targeted Nanomedicine Co-loading Two Drugs For The Treatment Of Esophageal Carcinoma

Background:Esophageal cancer ranks third and the death rate is at the fourth place among malignancies in China.The five-year survival rate of esophageal cancer is only 10%-25%.Chemotherapy is the primary treatments of esophageal cancer,but the efficacy is not satisfying.Docetaxel has been widely used in advanced esophageal cancer.However,docetaxel has poor water solubility,high toxicity and adverse effects,and drug resistance,so its clinical application is still limited.It's a major challenge for chemotherapy to improve the efficacy of chemotherapeutic drugs and reduce their side-effects.Some proprietary Chinese medicines are often added as adjuvant drugs in clinical applications to improve the anti-tumor effect and reduce the toxicity of chemotherapy drugs.Curcumin(CUR)is a traditional Chinese medicine with diverse biological activities,it is safe,non-toxic and has certain anti-tumor effect.However,the drawbacks of insoluble in water,extreme degradation and metabolization limited clinical application of CUR.As reported in the literature,CUR improve the sensitivity of tumor cells to chemotherapy,however,the specific mechanism has not been clarified.The effect of curcumin and the combined effect of docetaxel on esophageal cancer have not been reported.Increasing attention has been paid to nanomedicines because of their good biological safety,targeting capabilities,and high-efficiency loading of multiple drugs.Therefore,this paper studied whether curcumin combined with docetaxel has anti-tumor effect on esophageal cancer,and found that the combination of these two drugs can induce apoptosis and autophagy of esophageal cancer cells,and further explored the related mechanisms.Then developed a novel T7 peptide-modified pH-responsive targeting nanosystem co-loaded with docetaxel and curcumin,and targeted delivery the drugs to esophageal cancer cells to enhanced the anti-tumor efficacy.Methods and Results(1)Effects of CUR combined with DTX on proliferation,migration and invation of ESCC.CUR inhibited the cell viability of ESCC according to CCK8 assays,which were concentration dependent.The inhibition of proliferation of ESCC was stronger for CUR combined with DTX than for the DTX monotherapy group.DTX and CUR weakened the migration and invasion capability of ESCC after sufficient treatment time with certain cancentrations,whereas CUR combined with DTX futher weakened the migration and invasion ability.(2)Effects of CUR combined with DTX on apoptosis and autophagy in ESCC.Flow cytometry indicated that the apoptosis rate of DC group was significantly higher than the monotherapy groups.After using Hoechst 33342 staining with ESCC,more pyknotic and hyperchromatic were observed in the DC group.The expression of anti-apoptotic protein Bcl-2 was lower and pro-apoptotic proteins BAX and cleaved-caspase3/9 increased in DC group than the control and monotherapy groups,the experiments demonstrated that CUR combined with DTX intensified apoptosis of ESCC.Transmission electron microscopy observed autophagic vacuoles and autolysosomes significant increases in the combined group,the expression of autophagy-related proteins LC3II/LC3I,Beclin-1?Atg7 were significantly higher and P62 expression was lower in the DC group,which indicated that CUR futher promotion of autophagy when combined with DTX in ESCC.(3)The mechanism of anti-tumor effect of CUR combined with DTX:Western blot test results showed that the expression of p-PI3K/PI3K,p-AKT/AKT,p-mTOR/mTOR in the combined treatment group was significantly lower than that in the DTX and CUR monotherapy groups,we hypothesized that the anti-tumor effect of CUR combined with DTX in ESCC by inhibiting activation of the PI3K/AKT/mTOR signaling pathway.(4)The relationship between autophagy and apoptosis induced by CUR and DTX:CCK8 and flow cytometry test results showed that the cell activity of the 3MA+DC group was significant lower than the DC group,the apoptosis rate was clearly higher in 3MA+DC group.Western blots showed the expression of the anti-apoptotic protein Bcl-2 and the ratio of LC3?/LC3? was downregulated in the 3MA+DC group Compared with the DC group,while BAX,cleaved caspase-3/9 were signifcantly upregulated(P<0.05).We hypothesized that autophagy induced by CUR and DTX had protective effects on the growth of ESCC.The autophagy inhibitors 3MA inhibited autophagy induced by DC and further increased antiproliferative and apoptosis-inducing effects of DC.(5)CUR enhances the antitumor effect of DTX in vivo.The tumor volume growth rate in the combined group was signifcantly slower than in the control,DTX and CUR groups,and tumor volume was signifcantly reduced.Immunohistochemistry demonstrated that expression of Ki-67 was significantly lower in the DC group than in the PBS,DTX and CUR groups.The results indicated that tumor proliferation was signifcantly decreased after combined DTX and CUR therapy.(6)Preparation and characterization of nanoparticles:Targeted nanocarriers T7-CM-?-CD-PEI-PEG was prepared,the lipid soluble drug DTX and CUR were loaded into the nanocarriers by the double emulsion method.The characterization of the nanomedicine indicated that the size of the nanoparticle was in the nano-scale and have a good drug loading rate.The nanomedicine was pH-sensitive that the drug-release rates were signifcantly enhanced in the slightly acidic environment(pH5.5)?(7)Tumor targeting and biocompatibility of nanomedicine:Cellular uptake of T7-NP,with T7-modifcation,was greater than the NP group.The T7-NP-D and T7-NP-DC groups showed enrichment of Cy5.5-labeled nanoparticles in tumors.In essence,nanoparticles with T7-modifcation have a good tumor targeting effect,indicating that the system is a good platform for delivery of anti-tumor drugs to tumor tissue.The nanocarriers T7-NP and NP have low toxicity to Het-lA.There was no deleterious effect on hematopoietic function and no obvious damage observed in the major organs in in esophageal cancer xenograft models after treated with the nanomedicines.(8)Anti-tumor effect of nanomedicine:The therapeutic efficacy of T7-NP-DC was better than T7-NP-D,NP-DC and DTX through MTT assays,apoptosis test,33-Dimensional tumorsphere and in esophageal cancer xenograft models.ConclusionThis study suggested that CUR and DTX improved the antitumor effect of DTX in ESCC,possibly by inhibiting activation of the PI3K/AKT/mTOR signaling pathway.DTX and CUR induced esophageal cancer cell autophagy,which may be protective for esophageal cancer cell.The combination of autophagy inhibitor 3MA,DTX and CUR may become a new strategy for esophageal cancer treatment.The findings demonstrated that T7-CM-?-CD-PEI-PEG-DTX/CUR is a promising,non-toxic,and controllable nanoparticle that is capable of simultaneous delivery of the chemotherapy drug,docetaxel,and the Chinese Medicine,curcumin,for treatment of esophageal cancer.This novel T7-modified targeting nanosystem releases loaded drugs when exposed to the acidic microenvironment of the tumor and exerts a synergistic anti-tumor effect.