Research On The Mechanism Of Biological Response-modulated Drug Delivery System Constructed By The Effective Ingredients Of Traditional Chinese Medicine Ecklonia Kurome In Breast Cancer Therapy

Background:Sodium alginate（ALG）is a sodium salt of alginate,a polysaccharide carbohydrate extracted from the traditional Chinese medicine Ecklonia Kurome.ALG has the characteristics of low toxicity,high stability,high water solubility,good biocompatibility and degradability.It is often used as a material for drug delivery and has been widely used in anti-tumor therapy.This topic uses ALG as drug delivery material to construct drug delivery system and studies the therapeutic effect of breast cancer by adopting different biological response modulated modes.The first modulation of the biological response of the drug delivery system is based on improved tumor microenvironment triggering gas therapy combined with chemotherapy for breast tumors.The second modulation of the biological response of the drug delivery system is the photothermally induced free radical and pyroptosis reaction to inhibit the growth of breast tumors.By verifying the role of two different biological response regulation modes and different mechanisms of inhibiting tumor growth of ALG drug delivery system in the treatment of breast cancer,it provides new ideas and schemes for the treatment of breast cancer,and provides more references for the drug delivery system of cancer treatment.Objective:1.A compound hydrogel drug delivery system based on sodium alginate,an effective component of Chinese herbal medicine Ecklonia Kurome,was established.Hydrogel with unique biological characteristics was used as drug carrier.Intratumor injection of complex hydrogel drug delivery system avoided the poor efficacy caused by the early degradation of drugs,and ultimately achieved the reduction of drug dose,precise targeted drug delivery and slow released function.2.Through two different biological response regulation modes,the role of different anti-cancer mechanisms ALG drug delivery system in inhibiting the growth of breast tumor was discussed,providing new ideas and schemes for the treatment of breast cancer,and providing more references for the drug delivery system of cancer treatment.Method:1.Study on improving tumor microenvironment triggered gas therapy combined with chemotherapy for tumor treatment:Establish an improved tumor microenvironment-triggered gas therapy combined with chemotherapy for tumor treatment.ALG could rapidly form a network structure by reacting with the normal concentration of Ca²⁺in the tumor,which can be uniformly dispersed within the tumor and slowly released to achieve accurate targeted drug delivery at the same time and reduce drug dose.Local inflammation of lipopolysaccharide（LPS）was used to induce changes in the microenvironment of breast cancer tumor,and promote the production of nitric oxide（NO）from nitric oxide donor L-arginine（L-Arg）,so as to sensitized the chemotherapy effect of doxorubicin（DOX）and achieve the purpose of gas therapy combined with chemotherapy for tumor.1.1.Detection of functional properties of hydrogel delivery system:The synthesis,characterization,drug release and functional properties of the composite hydrogel delivery system were tested by SEM scanning electron microscopy,ultraviolet spectrophotometry,NO detection kit,DCFH-DA fluorescence probe and other methods.In vitro efficacy study1.2.In vitro efficacy of LPS+DOX+L-Arg+ALG compound hydrogel administration system:The compatibility of 4T1 breast cancer cells and the toxicity of 4T1 breast cancer cells were tested by CCK8 detection,DCFH-DA fluorescent probe,AM-PI live and dead cell detection kit and confocal detection methods.1.3.Efficacy of LPS+DOX+L-Arg+ALG compound hydrogel administration system in tumor-bearing mice:After different administration methods,subcutaneous tumor volume and tumor inhibition rate of 4T1 breast cancer cell bearing mouse models were evaluated for efficacy in vivo.In vivo safety was evaluated by body weight and H&E staining of important tissues 15 days after treatment.2.Photothermally induced free radical and pyroptosis synergies inhibit breast tumor growth:Using ALG as the carrier,Hu-Kaiwen Ink（Ink）,2,2′-azobis[2-（2-imidazolin-2-yl）propane]-dihydrochloride（AIPH）and decitabine（DCT）were minimally injected into the tumor to react with the normal concentration of Ca²⁺in the tumor to form a network hydrogel structure.NIR-Ⅱlaser stimulation of tumor,Ink converts light stimulation into heat energy to achieve low temperature hyperthermia,and heat energy induces AIPH to release alkyl free radicals to kill tumor cells.DCT can enhance the expression of GSDME in tumor cells,and the sensitive sequence of GSDME is cut off by photothermal treatment and caspase-3 activated by cytotoxic free radicals,resulting in pyroptosis.Pyroptosis releases mature pro-inflammatory cytokines and antigens associated with dead cells,causing inflammatory and immune responses,achieving systemic immunity and preventing metastasis and recurrence.2.1.Functional properties test of hydrogel delivery system:The synthesis,characterization,drug release,photothermal properties and photothermal stability of the composite hydrogel delivery system were tested by SEM scanning electron microscopy,ultraviolet spectrophotometry detection,infrared thermal imaging detection and other methods.2.2.In vitro efficacy study of DCT+AIPH+Ink+ALG composite hydrogel delivery system:The in vitro cytocompatibility,cytotoxicity and intracellular alkyl radical generation of the composite hydrogel delivery system were tested by CCK8 detection and confocal detection methods.2.3.Pyroptosis verification of DCT+AIPH+Ink+ALG compound hydrogel administration system:microscopic observation,LDH kit,Ca²⁺fluorescent probe,IL-18,IL-1βdetection kit and other detection methods were used to test the generation of pyroptosis.2.4.In vivo efficacy study of DCT+AIPH+Ink+ALG compound hydrogel administration system in tumor-bearing mice:After different administration methods,subcutaneous tumor volume and tumor inhibition rate of 4T1 breast cancer cell tumor-bearing mouse models were used to evaluate the in vivo efficacy.In vivo safety was evaluated by body weight and H&E staining of important tissues 15 days after treatment.Result:1.Study on improving tumor microenvironment triggered gas therapy combined with chemotherapy for tumor treatment.1.1.When the concentration of ALG is 5 mg/m L,moderate hardness hydrogel can be formed with Ca²⁺,and the drug can be released continuously.The release of NO at 24 h was 7.52 times of that at neutral condition with weak acidity PH6.5.After LPS stimulation,a large number of ROS were produced in both 4T1 cells and tumor sections.1.2.Each component of the compound hydrogel administration system has good biocompatibility,and the combination of gas therapy and chemotherapy can significantly enhance the effect of inhibiting the growth of tumor cells1.3.In the LPS+DOX+L-Arg+ALG compound hydrogel administration group,tumor growth was slow,and the tumor inhibitory rate was 85.10%.The body weight grew steadily,and H&E staining of major organs showed no obvious pathological changes.2.Photothermally induced free radical and pyroptosis synergies inhibit breast tumor growth.2.1.When the concentration of ALG is 5 mg/m L,moderate hardness hydrogel can be formed with Ca²⁺,and the drug can be released continuously.Ink has good photothermal effect in vitro and in vivo,and can still maintain photothermal stability after multiple laser irradiation.2.2.Each component of the compound hydrogel administration system had good biocompatibility.After the combination administration,the cell survival rate decreased gradually with the increase of AIPH concentration.The experimental group with AIPH and Ink simultaneously irradiated with laser for 10 min produced a large number of free radicals.2.3.In the compound hydrogel administration group,4T1 cells would bulge and produce bubbles after exposure to light,showing the typical morphological changes of pyrosis.At the same time,a large amount of Ca²⁺and LDH were released.IL-1β,IL-18 and other inflammatory factors were detected in the supernatant.2.4.The tumor growth of tumor bearing mice in the laser irradiation group of compound gel administration system was slow,and the tumor inhibitory rate was 85.81%.The body weight grew steadily,and H&E staining of major organs showed no obvious pathological changes.Conclusion:In this course,a compound hydrogel drug delivery system based on sodium alginate,an effective component of the traditional Chinese medicine Ecklonia Kurome was successfully established.The drug delivery system was constructed by means of different biological response regulation to study the therapeutic effect of breast cancer.Although the two ALG-based drug delivery systems have different biological response regulation and tumor growth inhibition mechanisms,both of them can play a good role in tumor growth inhibition.This study provides new ideas and plans for the treatment of breast cancer,and also provides more references for the delivery system of cancer treatment.