| ObjectivePancreatic cancer is a malignant tumor of digestive system with high mortality.However,the efficacy of first-line clinical drugs such as gemcitabine,paclitaxel,platinum drugs and 5-fluorouracil is very limited.Some studies have found that the addition of platinum combination chemotherapy can improve the efficacy of chemotherapy.Studies on the nanodelivery system of chemotherapy drugs suggest that it can promote the distribution of chemotherapy drugs to tumor tissues and reduce the toxic and side effects of chemotherapy drugs.However,in vivo studies have shown that the efficacy of these treatment strategies is still limited.This is due to the abundant stroma and stromal cells in pancreatic cancer tumor tissue,which on the one hand promote the malignant proliferation of tumor,on the other hand hinder the entry of drugs into tumor tissue to kill tumor cells.Hyaluronan（HA）is one of the important components of tumor tissue matrix.Clinical trials have found that the degradation of HA by hyaluronidase can significantly improve the efficacy of chemotherapy drugs against pancreatic cancer despite systemic toxicity.Therefore,this project constructed a cisplatin and hyaluronidase drug delivery system BPEI-SS-Pt/HAase@Ca P in tumor microenvironment and redox response.This drug delivery system is formed by self-assembly of hyaluronidase（HAase）and cisplatin polymer prodrug BPEI-SS-Pt through amorphous calcium phosphate biological mineralization.In order to achieve responsive release of hyaluronidase in the tumor microenvironment to degrade hyaluronan in the matrix,penetrate the matrix barrier,promote the antitumor chemotherapy effect of cisplatin polymer precursor drug BPEI-SS-Pt into tumor cells,through rapid release of cisplatin in the intracellular redox response,so as to effectively kill tumor cells.It provides a new strategy for clinical treatment of pancreatic cancer.Methods1.The tumor microenvironment and redox responsive nanodrug delivery system BPEI-SS-Pt/HAase@Ca P was constructed using hyaluronidase as substrate degradation enzyme,BPEI-cisplatin as antitumor drug and amorphous calcium phosphate as precursor.The loading capacity of BPEI-SS-Pt to cisplatin was determined by ICP-MS.The particle size,PDI and Zeta potential of BPEI-SS-Pt/HAase@Ca P were measured by nano laser particle size analyzer.XRD was used to characterize the crystal structure of calcium phosphate nanoparticles.The morphology of BPEI-SS-Pt/HAase@Ca P was characterized by scanning electron microscope.The acid-sensitive response and redox response of BPEI-SS-Pt/HAase@Ca P nanoparticles were experimentally observed.2.The biocompatibility of BPEI-SS-Pt/HAase@Ca P nanoparticles was detected by hemolysis assay.MTT assay was used to observe the antitumor effect of BPEI-SS-Pt and BPEI-SS-Pt/HAase@Ca P on Panc02 cells.BPEI-SS-Pt was labeled with 5-FAM fluorescein,and the effect of specific inhibitors of different endocytosis pathways on BPEI-SS-Pt-5-FAM endocytosis pathway was determined by fluorescence microscopy.Transwell model by hyaluronan agarose gel was constructed to observe the effect of matrix breakthrough and drug penetration.3.C57 mice were used to establish Panc02 subcutaneous graft tumor model of pancreatic cancer,and the in vivo antitumor activity of BPEI-SS-Pt/HAase@Ca P was evaluated.TUNEL and H&E staining was used to observe the apoptosis in each group.Molecular levels of Caspase3 and Ki67 were detected to evaluate tumor apoptosis and proliferation.The changes of hyaluronan in tumor tissues of BPEI-SS-Pt group and BPEI-SS-Pt/HAase@Ca P group were observed by hyaluronan staining and drug penetration of the two groups were observed.The in vivo safety of BPEI-SS-Pt/HAase@Ca P nanoparticles was evaluated by H&E staining each organ of mice after administration.Results1.The synthetic branched polyethyleneimine（BPEI）-cisplatin precursor drug linked by disulfide bond had a loading capacity of 33.4%to cisplatin.The particle size of BPEI-SS-Pt/HAase@Ca P nanodelivery system optimized by prescription was 143±14nm.The results of SEM showed that the nanoparticles were round and regular in shape with good uniformity.The stability results showed that BPEI-SS-Pt/HAase@Ca P nanoparticles could be stored at 4℃for 28 days,and the average size and PDI changed a little The X-ray diffraction pattern of BPEI-SS-Pt/HAase@Ca P nanoparticles shows a wide peak near 2θ=30°,which proves that they are amorphous calcium phosphate.The results of acid sensitivity of BPEI-SS-Pt/HAase@Ca P nanoparticles showed that the morphology of the nanoparticles was complete and spherical when incubated at p H7.4,but the morphology was incomplete and obvious rupture when incubated at p H6.5.The cumulative release of HAase in the p H6.5 buffer was significantly higher than that in the p H7.4 buffer,indicating that BPEI-SS-Pt/HAase@Ca P nanoparticles can respond to drug release in the weak acid environment of tumor microenvironment about p H6.5.The results of GSH response experiment of the released BPEI-SS-Pt prodrug showed that Pt was rapidly released under the condition of high concentration of GSH,indicating that the BPEI-SS-Pt disulphide bond could be broken under the condition of intracellular glutathione reduction and platinum could be rapidly released to play an antitumor role.2.Hemolysis test results showed that the hemolysis rates of BPEI-SS-Pt and BPEI-SS-Pt/HAase@Ca P were both less than 5%,which suggested that BPEI-SS-Pt/HAase@Ca P had good biocompatibility.MTT results showed that the IC50of BPEI-SS-Pt was 2.87±0.49μg/m L.The IC50of BPEI-SS-Pt/HAase@Ca P was 3.12±0.21μg/m L,both of which had good antitumor effect.BPEI-SS-Pt/HAase@Ca P endocytosis pathway showed that chlorpromazine had a significant inhibitory effect on cell uptake,the uptake rate was greatly reduced to 8.12±2.7%,while amiloride and Aureus isoflavone had little effect on cell uptake.The uptake rates maintained at 95.34±3.7%and 87.25±1.9%,respectively,and chlorpromazine significantly inhibited the uptake,suggesting that clathrin-mediated endocytosis was the main uptake pathway in the transcellular transport of nanoparticles.The results of transwell experiment showed that BPEI-SS-Pt-5-FAM/Haese@Ca P nanoparticles could penetrate the matrix barrier more effectivelythan BPEI-SS-Pt-5-FAM.These results indicated that BPEI-SS-Pt/HAase@Ca P nanoparticles can break the stromal barrier of tumor tissue and promote the drug penetration and uptake into tumor cells.3.Compared with the control group,BPEI-SS-Pt/HAase@Ca P,BPEI-SS-Pt and free cisplatin significantly inhibited tumor growth in Panc02 tumor bearing C57 mice.Compared with the BPEI-SS-Pt group and cisplatin group,the BPEI-SS-Pt/HAase@Ca P nanodelivery system had the best antitumor effect.The weight of cisplatin declined significantly,but the other groups had little change in body weight.These results indicate that the BPEI-SS-Pt/HAase@Ca P nanodelivery system can promote the tumor killing ability of cisplatin and reduce the systemic toxicity of free cisplatin.TUNEL and H&E staining results of tumor tissue showed that the death rate of tumor cells in BPEI-SS-Pt/HAase@Ca P group was higher than that in cisplatin and BPEI-SS-Pt group.The detection results of Caspase3 and Ki67 molecules representing apoptosis and proliferation in tumor tissues showed that the activation of Caspase3 representing apoptosis was significantly increased in BPEI-SS-Pt/HAase@Ca P group compared with other groups.However,Ki67 representing tumor proliferation was significantly reduced in BPEI-SS-Pt/HAase@Ca P group,which further verified that BPEI-SS-Pt/HAase@Ca P increased tumor tissue apoptosis and reduced tumor proliferation from molecular level.The results of hyaluronan staining and drug penetration experiments showed that hyaluronan could be degraded by BPEI-SS-Pt/HAase@Ca P,and the fluorescence nanoparticles of BPEI-SS-Pt-5-FAM/HAase@Ca P could penetrate deep into the tumor,thus enhancing the chemotherapy effect of pancreatic cancer.H&E staining results of mouse organs showed that BPEI-SS-Pt/HAase@Ca P nanoparticles had good safety and biocompatibility.ConclusionsIn this study,the BPEI skeleton was used to achieve high load of cisplatin,and the tumor microenvironment and redox responsive BPEI-SS-Pt/HAase@Ca P nanodelivery system were constructed jointly with HAase.A large amount of hyaluronan exists in the HAase degradable matrix released by the nanoparticles in response to the tumor microenvironment,which enhances the drug penetration ability in the tumor tissue,enabling BPEI-SS-Pt,the polymer precursor of cisplatin,to penetrate deep into the tumor.After entering the tumor cells,BPEI-SS-Pt releases cisplatin in response to intracellular glutathione,thus killing the tumor.Thus,the BPEI-SS-Pt/HAase@Ca P nanoparticles constructed by improving the chemotherapy effect of cisplatin on pancreatic cancer have more effective antitumor effect in vivo,laying a foundation for the application of cisplatin in clinical effective chemotherapy for pancreatic cancer. |
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