Molecular Chelates That Regulate Lactate Levels For Hormone-Photodynamic Synergistic Antitumor Research

Objective:This study designed a Chlorin e6(Ce6)molecular chelate for photodynamic therapy of breast cancer based on lactate modulation in tumor tissues.GdCl3 was used to complex the photosensitizers Ce6 and FAD respectively(Ce6-GdCl3-FAD),which improved the solubility of Ce6 while encapsulating the estrogen receptor antagonist tamoxifen(Tam),resulting in the final preparation,the multifunctional hormone-photodynamic synergist Ce6-GdCl3-FAD/Tam,which can further exert a synergistic effect on lactate depletion by FAD while Estrogen receptor and Monocarboxylate Transporter(MCT)blockade,resulting in tumor lactate modulation and synergistic photodynamic therapy.Methods:(1)Ce6-GdCl3-FAD/Tam was synthesized using GdCl3 as the complexation center,complexed Ce6 as the photosensitizer,FAD as the lactate depleting agent and Tamoxifen(Tam,Tam)as the estrogen receptor antagonist.The synthesis of Ce6-GdCl3-FAD/Tam was carried out by ultraviolet-visible spectroscopy(UV-Vis),X-ray photoelectron spectroscopy(XPS),X-ray energy spectrometry(EDS)The targets were analyzed by UVVis,X-ray photoelectron spectroscopy(XPS)and X-ray energy spectrometry(EDS)to verify their successful synthesis,and characterized by TEM,SEM,Malvern particle size meter and other physicochemical properties such as morphology,particle size,potential and elements.(2)The effect of single component FAD,Tamoxifen,and Ce6-GdCl3-FAD and the final preparation Ce6-GdCl3-FAD/Tam on the cellular activity of estrogen-positive(ER+)MCF-7 was evaluated by CCK-8 assay;the effect of the formulation on lactate depletion in solution and cells and H2O2 The effect of MCF-7 on the uptake of the preparation and the expression of ROS was investigated by immunofluorescence staining.(3)The MCF-7 in situ breast cancer model was successfully established,and the therapeutic effect of Ce6-GdCl3-FAD/Tam in inhibiting the growth of in situ breast cancer was systematically evaluated,and the synergistic effect of tumor site lactate depletion and tamoxifen hormone therapy combined with photodynamic therapy was investigated.The anti-tumor efficacy of this formulation was evaluated by using tumor suppression curves,in vivo imaging,TUNEL,and Ki67 staining of tumor tissues;its lactic acid depletion effect was evaluated by lactic acid and H2O2 concentration at the tumor site;the expression levels of HIF-1α and CD31 in tumor tissues were investigated by immunofluorescence staining.The expression levels of MCT-4 and γ-H2A.X in tumor tissues were determined by Western Blot.The biological safety of the formulation was evaluated by monitoring the body weight of animals and the quality of vital organs,organ H&E staining,and routine blood and blood biochemical analysis,which provide implications for the design of new tumor-targeting formulations and the study of hormone-photodynamic synergistic treatment strategies.Results:The synthesized Ce6-GdCl3,Ce6-GdCl3-FAD,and Ce6-GdCl3-FAD/Tam are spherelike particles with an average hydrated particle size of～200 nm and potentials of-21.30 mV,-20.45 mV and-2.05 mV,respectively,and the final formulation Ce6-GdCl3FAD/Tam is nearly electrically neutral.The UV-Vis results showed that the preparations Ce6-GdCl3-FAD and Ce6-GdCl3-FAD/Tam possessed the characteristic peaks of Ce6(340-430 nm,640-660 nm)and FAD(300-500 nm double peaks),and XPS showed that the preparations contained the characteristic peaks of Gd 4d.represents the successful synthesis of the formulation.(2)In this study,the key lactate group FAD was used to deplete lactate,which is more stable than traditional lactate dehydrogenase/lactate oxidase and is beneficial for practical application;its effect on the concentration of lactate solution was detected,and both FAD and Ce6-GdCl3-FAD were effective in depleting lactate compared to the control group;meanwhile,tamoxifen interfered with lactate efflux and synergistically controlled the lactate concentration in the tumor microenvironment,and in MCF-7 Free Tamoxifen and FAD and the preparations Ce6-GdCl3-FAD and Ce6-GdCl3-FAD/Tam was effective in depleting lactate and promoting H2O2 production during the cell culture.The toxicity of the agents to cells was examined by CCK-8 cell viability assay,which verified that Tamoxifen was only toxic to estrogen-positive cells(MCF-7),but not to estrogennegative cells(MCF-7 ADR);Ce6-GdCl3-FAD was not significantly toxic to tumor cells under laser-free conditions;Ce6-GdCl3-FAD/Tam was more toxic to tumor cells and the toxicity was significantly enhanced under PDT interference;the confocal results showed that the formulation enhanced uptake compared to free Ce6;the immunofluorescence staining results showed that the formulation enhanced intracellular ROS production and effectively killed cells.(3)In vivo results showed that Ce6-GdCl3-FAD/Tam+PDT effectively inhibited tumor growth,and tissue immunofluorescence showed that it inhibited HIF-1α expression in vivo and effectively alleviated the tumor hypoxic microenvironment,and lactate concentration in tumor tissues showed that both Ce6-GdCl3-FAD and Ce6-GdCl3FAD/Tam effectively depleted intratumor lactate and generated The WB results showed that the expression of lactate transporter MCT-4 in the tumor tissues of Ce6-GdCl3FAD/Tam group was reduced,which regulated the lactate concentration in the tumor microenvironment,and the expression of γ-H2A.X was increased,which inhibited tumor growth;there were no significant differences in body weight monitoring,blood routine,and blood biochemical indexes compared with the control group,and the relative mass of vital organs and tissue H&E results showed no significant toxicity to vital organs.The relative mass of vital organs and tissue H&E results showed no significant toxicity to vital organs,demonstrating the good biosafety of Ce6-GdCl3-FAD/Tam.Conclusion:This project constructs a particle Ce6-GdCl3-FAD/Tam for combined photodynamic therapy and hormone therapy,in which FAD,as a key group in the lactate degradation process,was initially verified by us to have a lactate degradation effect in vitro and in vivo.Ce6 is widely used as a photosensitizer,but its poor solubility becomes a major limitation in the application,and the solubility of Ce6 was improved by complexation with GdCl3,FAD effectively depletes the tumor microenvironment lactate;meanwhile,the addition of the hormone drug Tamoxifen interferes with the tumor development process by interfering with the binding of ER to estrogen and inhibiting the expression of MCT.The successful construction of Ce6-GdCl3-FAD/Tam is physicochemically stable,effective in inhibiting tumor growth,and has good biosafety,which is expected to achieve hormone-photodynamic synergistic therapy,while Gd as a common contrast agent in MRI is expected to realize the integration of imaging treatment.