Study On Glutathione Response-Type “Carrier-Free” Self-Assembled Nanomedicen Based On Porphyrin Molecules For Breast Cancer Treatment And Imaging

Diagnosis and treatment integrated nanotechnology has received extensive attention in the field of anti-tumor drug design because of its good imaging capabilities,high efficiency delivery level and better disease treatment effects.However,the limitations of the temporal and spatial specific effects of the existing nano-theranostic systems on tumor tissues make it difficult to achieve the goals of efficient treatment and precise imaging.Therefore,designing a highly efficient theranostic system that can control time and space has become an urgent need for nanomedicine research.In order to solve such obstacles,we use the photodynamic therapy(PDT)properties of chlorin e6(Ce6),and the intelligent response and ferrous ion conversion properties of hemin,synthesized a brand new nanomedicines(HCNPs)by self-assembly technology.Preliminary realization of a new non-invasive tumor treatment strategy with controllable time and space.After administration to tumor-bearing mice,under the irradiation of a specific wavelength light source,on the one hand,HCNPs convert oxygen(Oxygen,O₂)into singlet oxygen(¹O₂)through the action of PDT;on the other hand,through the Fenton reaction of Chemodynamic therapy(CDT),converts H₂O₂ in the tumor microenvironment into highly cytotoxic hydroxyl radicals(·OH).With the production of O₂,O₂ enters the PDT cycle and is further transformed into¹O₂ for enhanced cytotoxicity.Interestingly,we have observed that hemin releases ferrous ions in response to the microenvironment,triggering ferroptosis.Therefore,the designed nano-theranostic agent enhances the local photospecific cytotoxicity on tumor tissues and synergistically enhances the therapeutic effect of tumors,it also reduces systemic side effects.ObjectiveDesign a tumor TME-responsive nano-theranostic system with adjustable particle size,good blood circulation stability and biocompatibility,explore its anti-tumor activity in vivo and in vitro as well as in vivo tumor imaging effect,and realize the integrated strategy of multi-modal therapy and imaging,built a theoretical and experimental foundation for the development of nano-diagnosis and treatment systems in the field of cancer.Methods1.Preparation and characterization of HCNPs:Through a simple one-step self-assembly strategy,Ce6 and hemin are combined to form self-assembled nanomedicines(HCNPs).The particle size and distribution of HCNPs were detected by transmission electron microscope and particle size analyzer.X-ray photoelectron spectrometer was used to analyze the element composition and iron valence state in HCNPs.Using ultraviolet spectrophotometry and molecular dynamics simulation technology,the self-assembly mechanism of HCNPs was deeply explored.The in vitro O₂production,GSH consumption,¹O₂ production,Fe²⁺release,and·OH production capacity,in vitro stability and hemolysis of HCNPs were investigated.2.In vitro anti-tumor activity of HCNPs:The internalization ability of HCNPs by 4T1 cells was evaluated by flow cytometry.The CCK-8 was used to verify the therapeutic effects of HCNPs monotherapy and multimodal therapy,and the ability of HCNPs to generate reactive oxygen species(ROS)was verified by fluorescence staining.The GSH detection kit and Western blot were used to evaluate the ability of HCNPs to consume intracellular GSH and induce ferroptosis.3.The anti-tumor activity and imaging ability of HCNPs in vivo:Taking female Balb/c mice bearing 4T1 tumor as an animal model,after injecting HCNPs through the tail vein,the tumor site accumulation effect,in vivo distribution and fluorescence imaging ability were evaluated.And determine the irradiation time point of the tumor site.Record mouse tumor volume changes,body weight changes,and pathological examination of tumor sections and important organ sections to evaluate the tumor growth inhibitory effect and biological safety of HCNPs.An automatic biochemical analyzer was used to analyze the blood biochemical indicators of the mouse blood after administration to further investigate the biological safety of HCNPs.Results1.The prepared HCNPs have the property of adjustable particle size.When the concentration ratio of precursor molecules is 2:1(Hemin:Ce6),the HCNPs present a uniform spherical morphology,uniform particle size(about 120nm),good stability and long-term storage ability.HCNPs are formed by hydrophobic force,?-?stacking force,coordination interaction force,and intermolecular and intramolecular hydrogen bond interaction force.HCNPs can catalyze H₂O₂ to produce O₂and improve the efficiency of ¹O₂ production.HCNPs can effectively consume GSH,release Fe²⁺,and promote·OH production;HCNPs does not cause hemolysis of red blood cells,and has good biocompatibility.2.Cell experiments show that HCNPs can be effectively internalized by4T1 cells,and the difference in expression of GSH between tumor cells and normal cells can be used to accurately identify tumor cells and play a killing effect;HCNPs can effectively produce large amounts in cells ROS,and deplete intracellular GSH,trigger a decrease in the activity of GPX4protein,and induce ferroptosis.Compared with monotherapy,HCNPs combined with PDT/CDT/Ferroptosis achieves the greatest inhibition of tumor activity at low doses.3.In vivo anti-tumor activity and imaging experiments have proved that compared with free Ce6,HCNPs can effectively improve the accumulation of drugs in tumor sites and have certain tumor imaging capabilities.In the4T1 tumor-bearing mouse model,HCNPs with multimodal treatment strategy has the most obvious tumor growth inhibitory effect on mouse tumors,and there is no obvious organic damage to important organs,and the mouse body weight has also no obvious change.There is no obvious abnormality in the blood biochemical indicators of mice,which proves that HCNPs has good biological safety under the guarantee of effective anti-tumor activity.ConclusionIn this work,a simple one-step self-assembly strategy is used to construct a nano-theranostic delivery system that responds to tumor TME.Through in-depth exploration of the self-assembly mechanism of HCNPs,the theoretical and experimental foundations for the study of self-assembled nanomedicine based on porphyrin molecules are laid.The newly synthesized HCNPs realize the"carrier-free"drug delivery for the first time,and enhance the accumulation of drugs in the tumor site.Through the PDT,CDT and Ferroptosis induction properties of HCNPs,a multi-modal diagnosis and treatment integrated treatment strategy that inhibits tumor growth can be realized.It provides new ideas for the implementation of precise and efficient coordinated treatment of solid tumors with multiple mechanisms.