Study On The Construction And Performance Of Cross-linked Polymer Nano-cooperative Prodrugs In Response To Tumor Microenvironment

Chemotherapy is an effective way to treat middle and late period cancer.However,traditional small-molecule chemotherapeutics cannot achieve ideal therapeutic effects and are accompanied by serious side effects due to their poor water solubility,short half-life,and poor tumor selectivity.In recent years,polymer nanomedicine has attracted much attention because of its ability to improve pharmacokinetics and tumor enrichment ability.Several nanometer drugs have been clinically marketed.Although they can significantly reduce virulence and side effects,they do not improve the overall survival rate of patients.The reasons are as follows:1)the drug composition cannot be accurately controlled,and there are large differences between different batches;2)the stability of the systemic circulation is poor,and the drug is easy to release early;3)it is impossible for drugs to target to tumor site,and a single drug is prone to lead to drug resistance.Stimulus-responsive polymer nanoprodrugs have precise structure and drug composition and can respond to the tumor microenvironment,which is expected to be used in the design of ideal nanomedicine.However,most of them can only respond to tumor intracellular stimulating factors and cannot respond to the tumor extracellular microenvironment,which limits the ability of nanomedicine to be enriched in tumor sites.Because the changes in the physical and chemical properties of the nanomedicine outside the tumor,such as the dynamic change of particle size,charge reversal,and shape change,can enhance the ability of nanomedicine to penetrate,retain and be easily taken up by cancer cells.Therefore,it is necessary to develop polymer nanoprodrugs that are ultra-sensitive to the tumor’s intracellular and extracellular microenvironment.Literature reports have confirmed that as an acid-sensitive biological material,polyorthoesters are closely related to the surrounding hydrophilic and hydrophobic environment.Through structural adjustment,polyorthoesters can respond to the micro-acid environment inside and outside tumor cells.In addition,cisplatin（Pt）and norcantharidin（DMC）,as first-line chemotherapy drugs,have different mechanisms of action and can achieve the best therapeutic effect when combined with a molar ratio of 1:2.Therefore,based on the above research background,this study is planned to be based on the gradient distribution of tumor site p H（blood vessel:p H～7.4,extracellular matrix:p H～6.5-7.0,intracellular:p H～4.0-6.0）and the higher concentration of GSH,used to construct a polymer nanoprodrug coordinated delivery system that is ultra-sensitive to the tumor microenvironment.Synthesize the small cisplatin-norcantharidin prodrug molecule in the optimal ratio,then cross-link the main chain polyorthoester to obtain the amphiphilic cross-linked polymer prodrug,and further self-assembly form a nano-prodrug coordinated delivery system.In the structure,orthoester and amide bond（formed by norcantharidin and amino group）perform acid-sensitive function,and tetravalent cisplatin performs reduction-sensitive function.So as to achieve:（i）stable existence in the blood circulation;（ii）response to the extracellular micro-acid environment at the tumor site,enhancing tumor enrichment and cancer cell uptake ability through changes in size and surface potential;（iii）after reaching the cell,further in response to the intracellular microenvironment,the drug is released efficiently to enhance the ability to synergistically kill cancer cells.The specific research content is as follows:First,divalent cisplatin was oxidized to tetravalent cisplatin Pt（IV）by hydrogen peroxide,then Pt（IV）was coupled with norcantharidin to form a small molecule crosslinker（Pt（IV）-1）.Finally,Pt（IV）-1 was combined with the poly orthoester（POEAd-NH₂）by amide reaction to prepare cross-linked polymer prodrug（PPD）.The product structure was confirmed by ¹H NMR and the drug loading（Pt:7.4%,DMC:12.7%）.Using inductively coupled plasma mass spectrometer（ICP-MS）to verify the platinum content（7.9%±0.8%）,and convert the content of DMC（13.6%±1.38%）by molar ratio.This result is similar with¹H NMR calculated results.The crosslinking degree was obtained by TNBSA（52%）.The cross-linked polymer nanoprodrug cooperative delivery system was prepared by solvent exchange method,and the average particle size was 204.7 nm and the average zeta potential was-23.7 m V as measured by dynamic light scattering（DLS）.Using Nile Red as the fluorescent molecule,the critical micelle concentration（CMC）of the nanoprodrug measured by a fluorescence spectrophotometer was 1×10^-3mg/m L.The above results indicate that the nanoprodrugs have suitable particle size and potential,as well as a small CMC,which is conducive to the stability of blood circulation.The ¹H NMR tracking experiment was used to determine the degradation rate of orthoester in the polymer backbone.The degradation rate of orthoester bond at p H=7.4,6.8,and 5.0 within 24 hours was 0%,40%,and 100%,respectively,suggesting that nanoprodrugs can exist stably in the blood circulation and can respond to the micro-acid environment inside and outside the tumor cell with p H hypersensitivity.Furthermore,phosphate buffer and glutathione solution were used to simulate the particle size and potential change behavior of nanoprodrugs in different physiological environments in vivo.The results show that the particle size and potential can remain unchanged for a long time under the condition of p H=7.4,the particle size and potential gradually increase under the condition of p H=6.8,and the nano-prodrug disintegrates rapidly under the condition of p H=5.0.The above-mentioned particle size change behavior is beneficial to maintain stable blood circulation,enhance tumor target enrichment,cancer cell uptake,drug release ability in the cell.The dialysis method was used to determine the drug release behavior under different physiological conditions.Within24 hours,the nanoprodrug released 15%at p H=7.4,30%at p H=6.8,and about 80%at p H=5.0/GSH.The results of drug release show that the drug release behavior of nanoprodrugs is closely related to the behavior of particle size changes,and the higher tumor intracellular drug release is beneficial to killing cancer cells efficiently.Through MTT method and apoptosis experiments of HepG2 and H22,it is found that the cytotoxicity and pro-apoptosis ability of cross-linked polymer nanoprodrugs is similar to that of Pt/DMC.Confocal laser microscope and flow cytometry were used to investigate the qualitative/quantitative uptake capacity of cross-linked polymer nanoprodrugs.The results show that nanoprodrugs can be effectively taken up by cancer cells and the uptake is more significant at p H=6.8.The charge reversal triggered by the protonation of the amino group promotes cellular uptake.Using H22 cells to establish a mouse tumor model and detect the content of the drug in the blood and tumor.The results show that the cross-linked polymer nanoprodrug has better blood circulation stability,and the tumor tissue targeting enrichment ability better than the free drug.The results of further anti-tumor experiments showed that,compared with other control groups,the cross-linked polymer nanoprodrugs can significantly inhibit tumor growth.In addition,it can be seen from the weight change and pathological tissue slices of mice that the cross-linked polymer nanoprodrug can significantly reduce the toxic side effects and enhance the tumor tissue destruction ability.In summary,a precise collaborative nano-drug delivery system constructed by synergizing small molecule prodrug cross-linked polyorthoesters,on the one hand,it can exist stably in the blood circulation,and on the other hand,it can efficiently respond to the intracellular and extracellular physiological microenvironment of the tumor.Through the dynamic changes of particle size and potential,it can enhance the ability of tumor targeting enrichment,cell uptake and synergistic killing of cancer cells.Show a high-efficiency and low-toxicity anti-tumor effect.Through further structural optimization and function,nano-prodrugs with ultra-sensitive response to the tumor microenvironment are expected to be used in the targeted and precise treatment of clinical cancer.