Application And Mechanism Of PH-responsive Dual-loaded Injectable Hydrogel In The Treatment Of Esophageal Squamous Cell Carcinoma

Part I.Preparation,physicochemical properties,and safety studies of CS-BA-PVA@Cu/CDDPObjective:An injectable,p H-responsive hydrogel drug delivery system（CS-BA-PVA@Cu/CDDP）was designed and prepared for the simultaneous delivery of Cu²⁺and CDDP.The chemical composition,molecular structure,and three-dimensional network structure of CS-BA-PVA@Cu/CDDP were characterized;its physicochemical properties,such as rheological properties,swelling properties,porosity,in vivo and ex vivo degradation properties,and p H-responsiveness,were determined;and its in vitro drug-release behavior,in vivo drug distribution,and antimicrobial capacity were explored.The role of CS-BA-PVA@Cu/CDDP in the generation of hydroxyl radical（·OH）and the depletion of glutathione（GSH）was focused on to determine if it has the potential for achieving chemodynamic therapy.Finally,the biosafety of CS-BA-PVA@Cu/CDDP was evaluated through cellular and animal experiments.Methods:CS-BA-PVA@Cu/CDDP dual drug-loaded hydrogels,containing CDDP and Cu Cl₂,were synthesized using chitosan（CS）,poly（vinyl alcohol）（PVA）,and 3-carboxyphenylboronic acid（BA）as raw materials.The synthesis was carried out in a two-step process,with N-hydroxysuccinimide（NHS）and 1-（3-Dimethylaminopropyl）-3-ethylcarbodi-imide hydrochloride（EDC）used as coupling agents.The physicochemical properties and characterization of CS-BA-PVA@Cu/CDDP were investigated using various techniques.These included ¹H Nuclear Magnetic Resonance Spectra（¹H NMR）,Fourier Transform infrared spectroscopy（FT-IR）,Scanning Electron Microscope（SEM）,and rotational rheometer.The in vitro drug release and in vivo drug distribution of CS-BA-PVA@Cu/CDDP were determined using inductive coupled plasma-mass spectrometry（ICP-MS）.Additionally,the antimicrobial activity of CS-BA-PVA@Cu/CDDP was determined using plate counting.UV-Vis spectrophotometry was used to determine the production of·OH and consumption of GSH.The cytocompatibility and hemocompatibility of CS-BA-PVA@Cu/CDDP were evaluated using CCK-8 assay,live/dead cell staining,scratch assay,and hemolysis assay.The in vivo biocompatibility of CS-BA-PVA@Cu/CDDP was evaluated through routine blood and blood biochemistry tests,H&E staining of vital internal organs and skin tissues,and MASSON staining of skin tissues.Results:CS-BA-PVA@Cu/CDDP dual drug-carrying hydrogels with a pore size of approximately 60μm were successfully prepared.The hydrogels are cross-linked by borate bonds and have a typical three-dimensional network structure.The backbone of the hydrogels mainly consists of three elements:carbon,oxygen,and nitrogen.Platinum and copper are uniformly distributed in this three-dimensional network structure.CS-BA-PVA@Cu/CDDP hydrogels exhibit p H responsiveness,degrading faster in CBS and releasing a higher amount of drug compared to SBF.Upon injection into tumor tissues,CS-BA-PVA@Cu/CDDP rapidly transforms from a liquid to a gel,creating a reservoir for the drug and enhancing the accumulation of Cu²⁺and CDDP in the tumor tissues.CS-BA-PVA@Cu/CDDP generates·OH and consumes GSH,with its concentration directly proportional to the amount of·OH generated and GSH consumed.Finally,CS-BA-PVA@Cu/CDDP exhibits good biosafety and does not affect the activity or migration ability of normal cells in in vitro experiments.Additionally,it does not cause hemolysis or damage important organs or skin tissues in mice during animal experiments.Furthermore,it does not affect blood routine,liver and kidney functions,or other indicators.Conclusion:An injectable,p H-responsive hydrogel co-loaded with Cu²⁺and CDDP has been successfully synthesized.The hydrogel,known as CS-BA-PVA@Cu/CDDP,has tunable physicochemical properties that allow for drug release in response to a weakly acidic environment,leading to increased drug accumulation in tumor tissues.Additionally,it exhibits excellent antimicrobial properties and has the potential to enable chemodynamic therapy.CS-BA-PVA@Cu/CDDP demonstrates excellent biosafety,providing a strong basis for its use in ESCC therapy.Part II.Biochemical characterization and efficacy of CS-BA-PVA@Cu/CDDP in the treatment of ESCCObjective:A prerequisite for the therapeutic effect of CS-BA-PVA@Cu/CDDP in CDT in vivo is a high level of GSH in the tumor tissue and cells of ESCC.In the presence of GSH,Cu²⁺can generate·OH via a Fenton-like reaction after entering the cells,which in turn kills tumor cells.This part of the study aims to first investigate the biochemical properties of CS-BA-PVA@Cu/CDDP in the treatment of ESCC to clarify the effects of CS-BA-PVA@Cu/CDDP on intracellular GSH levels and·OH production specifically.In addition,the efficacy of CS-BA-PVA@Cu/CDDP in the treatment of ESCC was evaluated using cellular and animal experiments,and it was further investigated whether it could synergistically kill ESCC by chemotherapy and CDT.Methods:Firstly,CCK-8 assay was used to analyze the toxic effects of Cu²⁺,CDDP and CS-BA-PVA@CDDP on three normal cells,HEEC,HET-1A and L929;and to explore the optimal loading concentration of CS-BA-PVA@Cu/CDDP.Secondly,the chemodynamic therapeutic performance was evaluated by detecting the GSH content in ESCC tumor tissues and normal esophageal tissues,as well as detecting the changes in the levels of reactive oxygen species（ROS）and GSH in ESCC cells after CS-BA-PVA@Cu/CDDP stimulation.In addition,live/dead cell staining was utilized to evaluate the toxic effects of CS-BA-PVA@Cu/CDDP on ESCC cells,CCK-8 assay and clone formation assay to evaluate its effects on the proliferative ability of ESCC cells,Transwell and scratch assay to evaluate its effects on the migratory ability of ESCC cells,as well as flow cytometry and Western Blot to evaluate its effect on ESCC cell apoptosis.Finally,the in vivo anti-tumor effect of CS-BA-PVA@Cu/CDDP was evaluated using two animal models,K150-tumor-bearing nude mice,and m EC25--tumor-bearing C57BL/6J mice;and the disruption of tumor tissue structure was analyzed using H&E staining,and its effect on ESCC cell apoptosis and proliferation in tumor tissues was analyzed by TUNEL staining and Ki-67 histochemical staining.Results:The survival rates of HEEC,HET-1A,and L929 cells were over 90%when the concentration of Cu²⁺was not more than 20μg/m L.CDDP showed a strong toxic effect on all three normal cells,and this toxic effect was enhanced with the increase of CDDP concentration;CS-BA-PVA@CDDP,on the other hand,effectively reduced the damage of CDDP on normal cells.We found that the optimal Cu²⁺concentration in CS-BA-PVA@Cu/CDDP was 20μg/m L and the concentration of CDDP was 4μg/m L,at which time the two drugs had a synergistic therapeutic effect.Compared with normal esophageal tissue,ESCC tumor tissue had a higher GSH content,indicating that ESCC is prone to CDDP resistance.Meanwhile,CS-BA-PVA@Cu/CDDP significantly reduced GSH levels in K150and m EC25 cells,which was related to the depletion of GSH by Cu²⁺.The highest intracellular ROS levels were observed in ESCC cells after CS-BA-PVA@Cu/CDDP stimulation by flow cytometry and fluorescence microscopy,suggesting good therapeutic properties of CDT.Live/dead cell staining results showed that CS-BA-PVA@Cu/CDDP-treated ESCC cells exhibited the highest red/green fluorescence ratio and altered cell morphology,demonstrating that Cu²⁺and CDDP synergistically killed ESCC cells.CCK-8and clone formation assays showed that CS-BA-PVA@Cu/CDDP had the most significant ability to inhibit cell proliferation and colony formation.Transwell and scratch assays showed the most significant decrease in the proportion of ESCC cells undergoing migration and the distance that ESCC cells migrated in the CS-BA-PVA@Cu/CDDP group,suggesting a strong inhibitory effect on ESCC cell migration ability had a strong inhibitory effect.Flow cytometry results showed that the apoptosis rate of K150 cells in the CS-BA-PVA@Cu/CDDP group was about 79%,and the apoptosis rate of m EC25 cells was about 27%.WB results showed that the expression of pro-apoptotic protein Bax was increased,that the expression of anti-apoptotic protein Bcl-2 was decreased,and that the expression of the active form of caspase-3was increased in the two ESCC cells.Finally,the results of subcutaneous tumor formation experiments showed that all mice did not lose significant weight after intratumor injection of hydrogel compared to the control group.Among them,the mice in the CS-BA-PVA@Cu/CDDP treatment group had the smallest tumor volume,and the tumor weight results were consistent with the volume results,with the lightest tumor weight.In addition,H&E staining of tumor tissues in this treatment group could observe obvious nuclear consolidation,rupture and nucleolysis,and the normal morphology of cell membrane and nucleus could not be seen.TUNEL staining results showed that the proportion of positive cells presenting green fluorescence was the highest in the tumor tissues of this treatment group,which indicated that a large number of ESCC cells underwent apoptosis;and the analysis of Ki-67 histochemical staining revealed that almost no Ki-67 staining showing brownish-yellow color could be seen in the tumor tissues.Conclusion:Compared with CDDP,CS-BA-PVA@Cu/CDDP has higher biosafety and less damaging effect on normal cells at the same concentration.CS-BA-PVA@Cu/CDDP has good biochemical properties,which can significantly reduce the content of GSH and accelerate the generation of ROS in tumor cells.In cell and animal experiments,CS-BA-PVA@Cu/CDDP can effectively eliminate ESCC cells and significantly inhibit the growth of ESCC tumors,and it shows obvious and good efficacy in the treatment of ESCC.Part III.Mechanistic Study of CS-BA-PVA@Cu/CDDP in the Treatment of ESCCObjective:In our previous study,we noted that the mechanism of tumor cell killing by CS-BA-PVA@Cu/CDDP in the treatment of ESCC may involve other modes of cell death in addition to the induction of apoptosis in ESCC cells.Therefore,in this part of the study,we delved into the possibility that CS-BA-PVA@Cu/CDDP induced cuproptosis in ESCC cells.In addition,considering the possibility that both chemotherapy and CDT have the potential to induce tumor cells to undergo immunogenic cell death（ICD）,we also preliminarily explored the role of CS-BA-PVA@Cu/CDDP in the anti-tumor immune response.Methods:First,the expression of 12 cuproptosis-related genes in esophageal cancer was analyzed using the UALCAN database.Further,q PCR was used to detect the expression of the four most common cuproptosis-related genes,FDX1,LIAS,DLAT,and ATP7B,in the tumor tissues and surrounding normal tissues of eight ESCC patients.Meanwhile,copper ion levels were measured in tumor tissues and surrounding normal tissues of eight ESCC patients to explore the relationship between copper ion levels and ESCC.Secondly,the changes in copper ion levels in K150 and m EC25 cells after CS-BA-PVA@Cu/CDDP treatment were determined.Changes in the expression of FDX1,LIAS,DLAT,and ATP7B in K150 and m EC25 cells after CS-BA-PVA@Cu/CDDP treatment were detected by q PCR and WB,and changes in the expression of FDX1 and DLAT in the tumor tissues of mice treated with CS-BA-PVA@Cu/CDDP were detected by immunohistochemistry.Finally,we evaluated the possibility that CS-BA-PVA@Cu/CDDP induced occurrence of ICD.First,the expression of endoplasmic reticulum stress-related proteins such as CHOP,Bip（Grp78）,and ATF-4 was detected using WB to analyze the effect of CS-BA-PVA@Cu/CDDP on endoplasmic reticulum stress in ESCC cells.Secondly,flow cytometry and confocal microscopy were used to detect the exposure of CRT on the cell membrane surface,WB and ELISA were used to detect the release of HMGB1,and chemiluminescence was used to detect ATP secretion,so as to analyze the effect of CS-BA-PVA@Cu/CDDP on the release of damage-associated molecular patterns from K150 and m EC25 cells.The maturation of DC cells was evaluated using flow cytometry and ELISA.Finally,by using the C57BL/6J mouse tail vein lung metastasis model,we evaluated the inhibitory effect of CS-BA-PVA@Cu/CDDP on distant lung metastases,and preliminarily explored the tumor immunotherapeutic effect of CS-BA-PVA@Cu/CDDP.Results:In the UALCAN database,except for DLD,ATP7B,ATP7A and MTF,the remaining eight cuproptosis-related genes were highly expressed in esophageal cancer tumor tissues compared with normal tissues,and the difference was statistically significant.Consistent with the results of the UALCAN database analysis,FDX1,LIAS,DLAT and ATP7B were expressed at higher levels in tumor tissues of ESCC patients.Meanwhile,ESCC tumor tissues had higher levels of copper ions,and the levels of copper ions in the tumor tissues of five patients were significantly higher than those in the surrounding normal tissues,and the difference was statistically significant.We found that the copper ion content in the two ESCC cells was significantly higher after CS-BA-PVA@Cu/CDDP treatment,the expression levels of FDX1,LIAS,and ATP7B were decreased,and the expression level of DLAT was increased.FDX1 protein levels decreased and DLAT protein levels increased in tumor tissues of CS-BA-PVA@Cu/CDDP-treated mice.In addition,the expression levels of endoplasmic reticulum stress-related proteins such as CHOP,Bip,and ATF-4 were increased in K150 and m EC25 cells after treatment with CS-BA-PVA@Cu/CDDP.In the CS-BA-PVA@Cu/CDDP group,the number of CRT-positive K150 and m EC25 cells was significantly increased,and the intensity of fluorescence was significantly enhanced.In the CS-BA-PVA@Cu/CDDP group,the proportion of CD11c⁺CD80⁺CD86⁺DC cells was increased,and the secretion of pro-inflammatory inflammatory factors TNF-α,INF-γ,IL-1β,IL-2,IL-6,and IL-12p70 was increased,and the secretion of anti-inflammatory inflammatory factor IL-10was decreased.Finally,the results of the tail vein lung metastatic tumor model in C57BL/6J mice showed that significant lung metastatic tumor foci were visible in the lung tissue of m EC25-bearing mice receiving intratumoral injections of PBS and CS-BA-PVA hydrogel,with approximately 33 and 26 foci,respectively.The number of lung metastatic tumor foci was approximately 12 and 15 in the lungs of the hormonal mice treated with CS-BA-PVA@Cu and CS-BA-PVA@CDDP,respectively.In contrast,the number of lung metastatic foci in mice receiving CS-BA-PVA@Cu/CDDP intratumorally was only about 3.Conclusion:ESCC is sensitive to the cuproptosis pathway,and treatment with CS-BA-PVA@Cu/CDDP induced the onset of cuproptosis in ESCC.In addition,CS-BA-PVA@Cu/CDDP induced ICD in ESCC cells via endoplasmic reticulum stress,which promoted CRT translocation from the endoplasmic reticulum to the cell membrane surface,as well as HMGB1 and ATP secretion.The cumulative release of these DAMPs further induced maturation of immature DC cells,increased expression of CD80 and CD86 on the DC cell surface,increased secretion of pro-inflammatory factors and decreased secretion of anti-inflammatory factors in the cell supernatants,thereby promoting anti-tumor immune responses and inhibiting the development of distant lung metastases.