Construction Of Tumor Microenvironment-regulated Nano-drug Delivery Systems And Their Application In Tumor Therapy(1)

Tumor cells,cancer stem cells(CSCs),a variety of stromal cells,extracellular mediators secreted by cells and the biological characteristics of oxygen content,highly expressed enzymes,p H value and redox environment together constitute a special and complex tumor microenvironment(TME).A large number of studies have shown that the non-tumor cells and biological characteristics in TME directly participate in the development of tumors,and affect anti-tumor efficacy.By regulating or eradicating related cells and components in TME,it has been regarded as a highly potential anti-cancer treatment strategy.A large number of studies have shown that tumor tissues have higher redox levels than normal tissues.The high level of hydrogen peroxide(H₂O₂)in tumor cells has been confirmed to be related to mutations and gene instability in the cells to maintain the rapid proliferation of cancer cells,angiogenesis and resistance to apoptosis.Correspondingly,tumor cells increase the level of glutathione(GSH)by enhancing the activation of the antioxidant system to reduce excess H₂O₂ to maintain a high degree of cellular homeostasis.This redox level in tumor cells allows tumor cells to maintain malignant proliferation,invasion and metastasis,while avoiding cell damage caused by oxidative stress.Higher concentrations of H₂O₂ can cause strong cytotoxicity,and it reacts with free Fe²⁺in cells to produce highly oxidized and hypertoxic hydroxyl radicals(·OH),thereby inducing tumor cell apoptosis.Therefore,by regulating the redox level of the tumor microenvironment,amplifying oxidative stress is expected to improve the anti-tumor efficacy.As an important cellular component of the tumor microenvironment,CSCs have the potential to transform into vascular cells,participate in the remodeling of the tumor microenvironment,and accelerate the growth and deterioration of tumors;drug resistance is one of the key reasons that limit the current tumor curative effect.However,CSCs express a variety of multidrug resistance-related proteins and have strong DNA damage repair capabilities,showing strong anti-apoptotic capabilities and multidrug resistance;in addition,CSCs also are regarded as the initiating factors of tumor recurrence and metastasis.After treatment,on the one hand,it can cause tumor recurrence by transforming into cancer cells,and on the other hand,they can induce tumor metastasis by their characteristics of epithelial-mesenchymal transformation.To sum up,the redox environment and CSCs components in the TME,as the specific environmental characteristics and key cell components of TME,play a key role in the occurrence,development,treatment tolerance,metastasis,and prognostic recurrence etc.of tumors.They are also currently approved one of the main reasons for the failure of traditional treatment methods such as radiotherapy,chemotherapy and surgery etc.Therefore,the treatment strategy of regulating the redox level in TME or using combination therapy to simultaneously eradicate tumor cells and CSCs in tumor is expected to improve anti-tumor efficacy.Due to the lack of targeting,specificity and complete eradication ability of traditional clinical treatment methods include chemotherapy,radiotherapy or surgical treatment,it has caused great irreversible damage and negative prognosis to patients.Based on the advantages of nanocarriers such as good biocompatibility,improved blood stability of the drug,excellent targeting,small side effects and controllable drug release,this article rationally designed different treatment plans based on the redox characteristics of TME and CSCs in order to improve the efficacy of anti-tumor treatment.The main research content and results of this research are as follows:(1)Preparation of magnetic targeting iron oxide drug delivery system and evaluation of its anti-tumor effectAmplifying intracellular oxidative stress can cause irreversible oxidative damage to tumor cells and effectively kill cancer cells.Based on the fact that the concentration of H₂O₂ in the tumor tissue is not enough to trigger an efficient Fenton reaction to produce sufficient high oxidative and highly toxic·OH,and the over-expressed GSH in the tumor can effectively remove·OH.In order to overcome this challenge,enhance the oxidative stress in the tumor cell,and improve the efficiency of targeted drug delivery,we used magnetic iron oxide(Fe₃O₄)as a drug carrier to construct a nanocarrier(Fe₃O₄-HSA@Lapa)loaded with the chemotherapeutic drug?-Lapachone(Lapa),which not only have Fenton-like effects,but also have GSH depletion characteristics,thereby amplifying oxidative stress in tumor cells.Relevant characterization data confirmed that we successfully prepared Fe₃O₄-HSA@Lapa nanocarriers.Both in vivo and in vitro data show that Lapa selectively increases the level of H₂O₂ in the tumor and depletes the intracellular GSH through the catalysis of the highly expressed NAD(P)H:quinone oxidoreductase 1(NQO1)in tumor cells.The in vitro test results show that Fe₃O₄ can effectively dissociate and release iron ions in an acidic environment,and can convert H₂O₂ into highly toxic·OH through the Fenton reaction.Live and dead staining,cell viability(CCK-8 assay),and flow cytometry apoptosis test data show that the constructed Fe₃O₄-HSA@Lapa has a better effect of promoting tumor cell apoptosis under the action of a magnetic field.In addition,in vivo data indicate that the designed Fe₃O₄-HSA@Lapa nanoparticles exhibit excellent targeting capabilities,prolong blood circulation and increase tumor accumulation,and show outstanding drug delivery efficiency.Fe₃O₄-HSA@Lapa under the action of a magnetic field effectively enhances the inhibitory effect on tumor growth and reduces the side effects of anti-cancer drugs.This nanocarrier effectively overcomes the problems of insufficient production of·OH by the Fenton reaction and elimination of·OH,and at the same time shows targeted and specifically high-efficiency anti-tumor therapy and good biological safety.It provides a promising drug delivery system for exploring efficient and safe anti-tumor treatment strategies.(2)Preparation of Zeolitic imidazolate frameworks-90(ZIF-90)drug delivery system modified by penetrating peptide and evaluation of its anti-tumor effectBased on the regional differences in the distribution of CSCs in tumor tissues and the limitation of the tissue penetration of pharmaceutical preparations:that is,in addition to the perivascular area,CSCs are also located in the center of the cancer tissue away from the blood vessel.Due to the dense and complex extracellular matrix network,the accessibility of pharmaceutical preparations to CSCs still faces major challenges.Therefore,we designed and constructed a metal-organic framework ZIF-90nano-pharmaceutical formulation(ZIF-90@DOX-HSA@DAPT-PEG-tLyP-1)which is modified by tumor penetrating peptide(tLyP-1)and simultaneously loads the chemotherapeutic drug doxorubicin(DOX)and the Notch signaling pathway inhibitor(DAPT)that can eradicate CSCs.Multiple data confirm that we have successfully prepared the ZIF-90@DOX-HSA@DAPT-PEG-tLyP-1 nanocarrier.In addition,nanocarrier can achieve p H-responsive drug release.Both in vivo and in vitro data show that it has good tissue penetration ability.Live cell staining,flow cytometric apoptosis,cell viability assay,and the self-renewal ability and stemness inhibition evaluation of CSCs respectively indicate that ZIF-90@DOX-HSA@DAPT-PEG-tLyP-1 has excellent ability to promote tumor cell apoptosis and CSCs eradication effect.Performing relevant in vivo evaluations on tumor-bearing mice found that the nanocarriers can effectively accumulate in tumor tissues through long-time blood circulation,and penetrate deeply into the tissues,effectively reaching the core area away from blood vessels in the tumor tissues.The combination of the two drugs kills differentiated tumor cells and CSCs in tumor tissues,effectively inhibits tumor growth,while reducing the side effects of chemotherapy drugs,and ultimately enhancing the therapeutic effect of liver cancer.The nano drug-carrying system improves the permeability of drug preparations,and at the same time effectively eliminates CSCs with self-renewal ability and differentiation potential distributed in different areas in the tumor microenvironment,thereby improving the anti-tumor effect.Based on the above results,the constructed iron oxide nanodrug delivery system disrupts the redox balance of the tumor microenvironment,expands oxidative stress,enhances the CDT efficacy,and specifically targets and enhances the anti-tumor effect.In addition,the penetrating peptide-modified nanodrug co-transport system effectively enhances the accessibility to CSCs located at the distal end of blood vessels in tumor tissues,and achieves simultaneous eradication of tumor cells and CSCs in tumor tissues,greatly improving the anti-tumor effect.The results of this study provide a methodological reference for exploring efficient,safe and specific anti-tumor drug delivery systems,and have potential clinical application value.