Preparation Of Bortezomib Hydrogel With Photothermal And PH Response And Its Anti-Colorectal Cancer Performance

Background Colorectal cancer(CRC)is one of the common malignant tumors of the digestive tract.It is difficult to detect early due to insidious onset.Most patients have advanced disease at the time of onset,and the prognosis is poor,which seriously threatens human life.The clinical CRC treatment program is mainly based on the combination of chemotherapy drugs,such as FOLFOX(Fluorouracil + Oxaliplatin),XELOX(Oxaliplatin + Capecitabine)and IROX(Oxaliplatin + Irinotecan,but the prognosis of patients with advanced CRC is poor.These treatments have entered the plateau,and problems such as side effects,multi-drug resistance and recurrence are becoming more and more serious.Therefore,it is imperative to find new therapeutic drugs and treatments.Studies have shown that the ubiquitin-proteasome pathway can regulate the proliferation and differentiation of CRC epithelial cells,and its composition and substrate molecules are important prognostic indicators of CRC.Therefore,proteasome inhibitors are a new hotspot of CRC targeted drugs.Bortezomib(BTZ)is the first proteasome inhibitor approved by the US Food and Drug Administration(FDA)for the treatment of relapsed,refractory multiple myeloma and mantle cell lymphoma.Domestic and foreign scholars have found that BTZ can effectively induce apoptosis of CRC cells through a variety of tumor cell signaling pathways.BTZ,like other chemotherapeutic drugs,has the disadvantages of non-specific killing and resistance to normal cells,which greatly limits its further clinical application.In recent years,photothermal therapy has attracted great attention from researchers.Photothermal therapy has a significant killing effect on primary tumors.Combined with chemotherapy,it can reduce the amount of chemotherapy and reduce the side effects of chemotherapy.Indocyanine green(ICG)is the only FDA-approved clinical contrast diagnostic agent for the primary diagnosis of liver function and cardiac function.In addition,ICG is a photothermal agent with high photothermal conversion rate,which is widely used in tumor treatment research.Based on the above research background,this paper proposes an experimental study of BTZ combined with ICG photothermal therapy for CRC.A nano-drug(m PEG-LUT-BTZ)with p H responsiveness was prepared by using an amphiphilic polymer polyethylene glycol-luteolin(m PEG-LUT)as a carrier material and BTZ by a borate ester bond.The ICG is then embedded in a supramolecular hydrogel formed by m PEG-LUT-BTZ and α-cyclodextrin(CDs)through host-guest interaction and hydrogen bonding to construct a BTZ nanosystem with photothermal and p H response.From the in vitro cell level and in vivo animal experiments,the therapeutic effect of m PEG-LUT-BTZ@ICG on colorectal cancer was evaluated,which provided a valuable experimental basis for improving the therapeutic efficacy of clinical colorectal cancer.Purpose The hydrogel drug-loading system with the combination of chemotherapy and photothermiotherapy was prepared,and the therapeutic effect on colorectal cancer was studied at the cellular and animal levels.Methods 1.Preparation and characterization of m PEG-LUT-BTZ@ICG hydrogel: The preparation of m PEG-LUT-BTZ@ICG by chemical reaction,detection of its nuclear magnetic spectrum。 2.m PEG-LUT-BTZ@ICG p H responsive release assay: The cumulative release rate of BTZ in m PEG-LUT-BTZ was determined by dialysis method at different p H values.3.Evaluation of photothermal effect: Different concentrations of m PEG-LUT-BTZ@ICG solution were prepared,and the laser was irradiated for 5 min to investigate the photothermal effect.4.In vitro experiments:1)Cell uptake assay: The uptake of m PEG-LUT-BTZ@ICG by HCT116 cells was examined by fluorescence microscopy.2)Cell proliferation activity experiment: The cell survival rate of each experimental group was measured by CCK-8 method.3)Acridine orange/ethidium bromide(AO/EB)double staining experiment: The toxic effects of each experimental group on HCT116 cells were detected by AO/EB double staining test.4)Apoptosis experiment: The apoptosis rate of each experimental group was detected by cell flow Annexin V-FITC/PI apoptosis double staining test.5.In vivo experiments:1)Establishment of tumor model,experimental grouping and treatment: Human colorectal cancer cells(HCT116)were injected into each nude mouse with 2×10~7 cells,and subcutaneously injected into the right upper limb of the nude mouse.When the tumor grew to about 100 mm~3,it was randomly divided into 6 groups,5 in each group.The experimental groups were: saline group,ICG group,ICG+laser irradiation group,laser irradiation group,m PEG-LUT-BTZ group,m PEG-LUT-BTZ@ICG+ laser irradiation group.2)Evaluation of anti-tumor effect: The body weight and tumor length of nude mice were measured every 2 days,the tumor volume was calculated,and the body weight and tumor volume curve of nude mice were plotted.3)Histological morphology evaluation: HE staining tablets of various organ tissues and tumor tissues were prepared to evaluate antitumor effects.Results 1.The BTZ hydgogel m PEG-LUT-BTZ@ICG with photothermal and p H response was successfully prepared.2.p H responsive release effect,photothermal effect: In 48 hours,the in vitro release rate of m PEG-LUT-BTZ was about 30% higher than that in the p H=7.4 group at p H=5.0;the in vitro cumulative release rate of BTZ in m PEG-LUT-BTZ@ICG was about 15% higher than that in the p H=7.4 at p H = 5.0;it shows that m PEG-LUT-BTZ has acidic responsiveness.The prepared m PEG-LUT-BTZ@ICG hydrogel has substantially the same photothermal effect as ICG at the same concentration.3.In vitro experiment: CCK-8 method for m PEG-LUT-BTZ@ICG was applied to HCT116 cells for 24 h and 48 h respectively,the survival rates were 25.19% and 17.14%,respectively;AO/EB double staining experiment,Large-area apoptosis was observed in HCT116 cells in the m PEG-LUT-BTZ@ICG +Laser group;the apoptosis rate of HCT116 cells treated with m PEG-LUT-BTZ@ICG for 24 hours was 52.97% by flow cytometry.4.In vivo experiments: After treatment,the weight of nude mice in each group remained stable,and there was no significant difference between the experimental groups,indicating that m PEG-LUT-BTZ@ICG has good biosafety.In the m PEG-LUT-BTZ@ICG+ laser irradiation group,tumors disappeared in nude mice,indicating that the combination of chemotherapy and photothermal therapy was effective.Conclusion 1.In this paper,the BTZ nano drug-loading system m PEG-LUT-BTZ@ICG with photothermal and p H response was successfully prepared.2.In vitro experiments have shown that BTZ and ICG synergistic treatment is significant.3.In vivo experiments confirmed that m PEG-LUT-BTZ@ICG had no obvious toxic side effects on normal organ tissues,while for tumor tissues,BTZ and ICG synergistic treatments were significant.