Preparation Of Tumor Microenvironment-Responsive Nanomedicine Loaded With Metformin Or Disulfiram And Their Application In Tumor Synergistic Therapy

Malignant tumor seriously harms human health and brings enormous economic pressure to patients and society,therefore,tumor treatment has become a key and difficult problem for research in the medical field.Although tremendous advances in tumor therapy have been made in the past decades,the diversity and complexity of the tumor microenvironment（TME）remains an important factor limiting the effectiveness of treatment.On the other hand,the TME is characterized by low p H,high glutathione（GSH）concentration,matrix metalloproteinase（MMP）overexpression,and tumor hypoxia,demonstrating good applications in controlled drug release,the transformation of non-toxic drugs to toxic drugs,etc.With the conti nuous development of nanotechnology,loading drugs with different therapeutic effects into the same nanomedicine and using the characteristics of TME to release the drugs carried in nanomedicines can not only improve the anti-cancer activity of drugs and reduce the systemic toxic side effects,but also overcome the defects of monotherapy,therefore,the combined treatment modality has become the current research hot spot in tumor treatment.In addition,the use of clinically approved“old”drugs to enhance the effect of oncology treatment has also been effective.For example,metformin（Met）,a widely used clinical drug against type II diabetes,can not only alleviate tumor hypoxia,thus improving the effect of chemotherapy,photodynamic therapy（PDT）and radiotherapy（RT）;it can also disrupt glucose metabolism,thus inhibiting tumor growth.Disulfiram（DSF）,an ancient antidote to alcohol,can be used in combination with copper ions to produce highly toxic bis（N,N-diethyldithiocarbamate）-Cu²⁺complexes（Cu L₂）and reactive oxygen species（ROS）,which can inhibit tumor growth.In this thesis,several kinds of p H-,GSH-and MMP-responsive nanodrugs was prepared for loading Met,and used Met to alleviate tumor hypoxia or disrupt cellular metabolism in combination with chemotherapy,PDT,photothermal therapy or starvation therapy to achieve improved tumor combination therapy;we also constructed a p H-responsive nanodrug for loading DSF,and the interaction of DSF with copper ions to generate ROS and Cu L₂ was used to combine with the chemotherapeutic drug adriamycin for the purpose of combination chemotherapy.The research contents are as follows:（1）Biodegradable copper-metformin nanoscale coordination polymer for enhanced chemo/chemodynamic synergistic therapy b y reducing oxygen consumption to promote H₂O₂accumulation:p H/GSH-responsive degraded copper-metformin nanoscale coordination polymer loading with doxorubicin（Dox）（Dox@Cu-Met NPs）is designed and synthesized.Under TME,Dox@Cu-Met NPs can degrade itself and promote the release of Dox,Cu²⁺and Met.The released Met can impair mitochondrial respiratory chain complex I to increase O₂content and alleviate tumor hypoxia.Dox can not only be used for chemotherapy,but also promote the conversion of O₂ into H₂O₂ through a cascade catalytic reaction,thus increasing the content of H₂O₂ in tumor sites.The Fenton-like reaction between Cu⁺/Cu²⁺and H₂O₂accelerates the accumulation of highly toxic ROS in tumor sites.The accumulation of O₂ also facilitates the acceleration of Dox into tumor cells,thus improving the effectiveness of chemotherapy.The successful preparation of Dox@Cu-Met NPs and their excellent in vitro and in vivo antitumor effects demonstrate the remarkable effect of Met in enhancing chemotherapy and CDT,providing new possibilities for the clinical use of Met for oncology treatment.（2）Metformin-based nanoreactor via alleviating hypoxia and reducing ATP for enhanced chemotherapy/photodynamic/photothermal therapy:Met,chlorin e6（Ce6）is loaded inside gelatin nanoparticles（Gel NPs）（MCG NPs）,and polydopamine（PDA）is used as a“gatekeeper”to prevent drug leakage（MCGP NPs）,while chemotherapeutic drugs（DOX）and target recognition（Arg-Gly-Asp（RGD））are modified on the surface of MCGP NPs byπ-πstacking and covalent bonding to obtain MCGPD～RGD NPs.MCGPD～RGD NPs can specifically recognize tumor cells and accumulate at the tumor site,mild acidic conditions promoted DOX release;after irradiation of 808 nm laser,the combined effect of heat generated by PDA coating and matrix metalloproteinase-2（MMP-2）triggered the rapid degradation of Gel NPs and the release of Ce6 and Met.Met improves PDT effect by improving tumor hypoxia;Met can also inhibit the expression of HSP90 and improve the effect of PTT by reducing the content of ATP in cells.The heat generated by the PDA coating also facilitates the acceleration of DOX into tumor cells and enhances the chemotherapeutic effect.The result of research shows that the Met in MCGPD～RGD NPs has the effect of significantly enhancing PDT and PTT,and this result further extends the application of Met in tumor combination therapy.（3）Tumor metabolism destruction via metformin-based glycolysis inhibition and glucose oxidase-mediated glucose deprivation for enhanced cancer therapy:the p H-responsive histidine modified zeolite imidazole framework（His/ZIF-8）was loaded with Met and glucose oxidase（GOx）and surface-modified with RGD polypeptide（Met/GOx@His/ZIF-8-RGD）.In tumor sites,Met/GOx@His/ZIF-8-RGD can liberate Met blocks the glycolysis process via suppressing the activity of HK2 and impairing ATP production,which activates the AMP-activated protein kinase（AMPK）pathway and p53 pathway and damages the Warburg effect.And the GOx boosts the glucose shortage for starvation therapy by depleting accumulated glucose.The prepared Met/GOx@His/ZIF-8 not only had good tumor cell killing effect,but also significantly inhibited tumor growth.Thus,this study provides a new idea for Met interference with tumor metabolism for cancer therapy.（4）Smart responsive nanoplatform via in situ forming disulfiram-copper ion chelation complex for cancer combination chemotherapy:a p H-responsive smart nanomedicines is constructed by encapsulating DSF and DOX into the ZIF-8 and then coating with copper ion(Cu²⁺)-tannic acid（TA）complex（denoted as DSF/DOX@ZIF-8@Cu-TA）.Cu²⁺,DSF and DOX can be released from DSF/DOX@ZIF-8@Cu-TA under mild acidity TME.The accumulation of DSF（non-toxic drugs）and Cu²⁺results in the rapid formation of high cytotoxic bis（N,N-diethyl dithiocarbamato）-Cu²⁺complexes（Cu L₂）in situ via DSF and Cu²⁺chelating reaction,which accompany the production of ROS.Besides,the anticancer effect of DOX is augmented by Cu L₂-modulated ROS-MAPK and NF-κB signal pathways.DSF/DOX@ZIF-8@Cu-TA can significantly inhibit tumor growth without off-target toxicity to surrounding normal tissues/cells.The successful preparation of DSF/DOX@ZIF-8@Cu-TA and its remarkable anti-tumor effects provide a new example of DSF combined with existing oncology therapies.