Polyoxometalate Supramolecular Complexes For Tumor Therapy

Polyoxometalates（POMs）are nanoscale anionic clusters composed of one or more pre-transition metals and oxygen atoms.Due to their rich structural composition,POMs have shown great potential in the fields of optoelectronics,catalysis and biological agents.In particular,POMs have received extensive attention as antibacterial,antitumor,anti-alzheimer’s disease,and multifunctional imaging contrast agents.POMs have been gradually developed in the fields of photothermal therapy,chemodynamic therapy and radiotherapy due to their flexible redox properties.The reduced POMs in the heteropoly blue state exhibit typical d-d transition and/or intervalence charge transfer absorption bands in the near infrared（NIR）region,which can convert light energy into thermal energy to ablate solid tumors,especially for photothermal therapy in the NIR-II region with deeper tissue penetration and lower energy dissipation.The reduced clusters can catalyze the overexpressed hydrogen peroxide（H₂O₂）in the tumor region to generate more toxic reactive oxygen species,and induce the apoptosis of tumor cells,so as to be used for chemodynamic therapy.However,safety issues arising from the p H sensitivity of bare clusters and their non-specific binding to biomolecules during physiological cycle still need to be considered.Therefore,choosing an appropriate cationic surfactant to coat on the surface of POMs is of great significance to improve the structural stability,regulate the morphology,and enrich the function.In this thesis,based on the unique redox properties of POMs,a series of nanocomposites were prepared by supramolecular methods such as electrostatic,π-π,and host-guest methods,with the purpose of building a universal multifunctional platform and achieving efficient tumor treatment.In order to enrich the function of the complexes,cationic drug and organic donor were selected to combine with POMs,respectively,to achieve the combined effect of in situ activated chemotherapy,NIR-II photothermal and chemodynamic therapy,as well as enhanced NIR-II photothermal therapy for intelligent response of tumor tissue.By changing the polarity of the solvent to control the morphology transition of the POM-based charge-transfer complex,the nucleolus-targeted NIR-II photothermal therapy was further realized.These studies have gradually promoted the application of POM complexes in the field of tumor therapy.First,a three-component electrostatically bound host-guest supramolecular complex with POM as the core,doxorubicin as the intermediate layer and hyaluronic acid as the protective layer was developed,and its antitumor properties were verified.The reduced POM cluster with strong absorption bands in NIR-II region and catalyzing H₂O₂ to ¹O₂was prepared by chemical reduction.In order to enhance the structural stability and biosafety of the reduced clusters during the physiological cycle,the positively charged chemotherapeutic drug doxorubicin was electrostatically bound to the reduced cluster as a structural stabilizer to form the complex.β-cyclodextrin grafted hyaluronic acid was modified on the surface of the complex through host-guest interaction betweenβ-cyclodextrin and drug,and a nanocomposite with strong structural stability,biocompatibility and drug selectivity was constructed.The chemical composition of the nanocomposite was characterized in detail by means of IR,elemental analysis,UV-vis-NIR,TG analysis,¹H,³¹P NMR,XPS and other means.The assembled morphology and stability of the nanocomposite were characterized by TEM and DLS.The ability of the reduced clusters to catalyze H₂O₂ to generate singlet oxygen was verified by ESR spectroscopy.The nanocomposite demonstrates a high photothermal conversion efficiency（43.8%）in the NIR-II region,and the performance is not affected by the concentration.The controlled release of the drug is realized under the dual stimulation of acidity and photothermal effect.The free doxorubicin and the production of singlet oxygen were observed by fluorescence microscopy in vitro.Under the triple effect of photothermal,chemodynamic and chemotherapeutic drugs,the nanocomposite show a strong killing effect on tumor cells.The nanocomposite shows high blood compatibility,the metabolic cycle in vivo is about 20 days with no obvious adverse effects on the health of mice.After intravenous injection,the nanocomposite was effectively enriched in the tumor site of mice,and the growth of the tumor was effectively inhibited under the synergy of the triple anti-tumor effect.The results of blood routine,liver and kidney function and H&E results of mice after treatment clarified the physiological safety of the nanocomposite.Second,in order to simplify the structural design,an organic-inorganic complex of organic NIR-II photothermal materials electrostatically combined with POMs was developed for NIR-II photothermal therapy.POMs are a class of excellent acceptors,which can form supramolecular complexes by charge transfer with organic electron donors.Based on this principle,a dark green ionic complex was obtained by spontaneous redox and ionic complexation between oxidized POM and small organic molecule3,3’,5,5’-tetramethylbenzidine（TMB）in ethanol by one-pot reaction.During the redox process,TMB is oxidized to TMB⁺⁺in the diimine state,and then forms a positive charge-transfer complex with TMB,and the anion cluster is reduced to the heteropoly blue state.Both of them exhibit strong absorption bands in the NIR region,and form stable ionic complexes by ionic interaction.The structural composition of the ionic complex was analyzed in detail by IR,UV-vis-NIR,XPS,ESR,elemental analysis,TG analysis,and³¹P NMR spectroscopy.The aggregated morphology of the ionic complex,as well as the phenomenon of acid-induced aggregation,were observed by TEM microscopy and DLS.The combination of the reduced cluster and the charge-transfer complex endows the ionic complex with a high NIR-II photothermal conversion efficiency（48.4%）and a strong photothermal conversion stability.Compared with the charge-transfer complex alone,the additional hydrogen bonding and charge-transfer interaction greatly improve the structural stability and heat resistance of the ionic complex.The results of TEM showed that the ionic complex can be effectively enriched in the cytoplasm when co-cultured with tumor cells,presenting a state of aggregation after acid stimulation,and retained in the cells for a long time.The results of MTT,nuclear staining,flow cytometry,clone formation,and Western Blot showed that the ionic complex effectively promoted the apoptosis of tumor cells under 1064 nm laser irradiation.The biocompatibility of the ionic complex is good,and the metabolic cycle in vivo is about 20 days without threatening the health of mice.After intravenous injection,the ionic complex was efficiently enriched in the tumor site of mice,and the NIR-II photothermal performance was well maintained in vivo.Under the enhanced photothermal effect,the tumor was effectively ablated without adversely affecting the health of the mice after the treatment.Third,a nanostructure-controlled nucleolus-targeted electrostatic complex combining cationic charge-transfer complexes with POMs was constructed.The rod-shaped material has a larger contact area with the cell and is more likely to break through the cell barrier and enter the nucleus.The specific binding of rod-like materials to organelles is expected to achieve precise treatment targeting organelles.Utilizing the redox reaction and ionic combination between POM and TMB,we increased the solvent polarity to obtain the nanorods in the mixed solvent of water and ethanol,which is expected to achieve nucleolus-targeted therapy.First,the composition of the nanorods was characterized by means of IR,ESR,UV-vis-NIR,XPS,³¹P NMR,elemental analysis and TG analysis.The rod-like morphology was observed by TEM,AFM and DLS.The assembly mechanism of the nanorods was analyzed by XRD and selected area electron diffraction patterns,and the packing model was given.The nanorods showed a broad and strong absorption band in the NIR region,and the photothermal conversion efficiency in the NIR-II region reached 57.2%with good photothermal conversion stability.The structural stability of nanorods was greatly affected by p H,which was relatively stable in the slightly acidic environment of tumors,and degraded slowly in the normal physiological environment,which helps to improve the physiological safety.There are a large number of basic proteins with strong affinity for negatively charged POMs in the nucleus,especially in the nucleolus.TEM results showed that the nanorods were stable in the nucleoli in a coherent state after 1.5 h of co-culture with Hep G2 cells.In the neutral environment of the nucleolus,the nanorods gradually dissociated,and the monomers further bound to the nucleolar components,interfering with the phase of the cell cycle.Coomassie brilliant blue,¹H NMR spectroscopy and ITC experiments demonstrated the binding of nanorods to nucleolar proteins and their main monomers（Lysine and Arginine）.The results of MTT assay and nucleolus staining assay analysis proved the inhibition effect of nanorods on tumor cell growth.This study achieved nucleolus-targeted NIR-II photothermal synergistic therapy,which has not been reported before.In conclusion,we selected different types of positively charged organic molecules as structural stabilizers,and electrostatically bonded to the surface of POMs to construct a series of multifunctional complexes.From a tumor-activated multifunctional combined therapy system to an enhanced NIR-II photothermal therapy platform constructed by simplified structural design and intelligent response to the tumor microenvironment,and finally to the realization of nucleolus-targeted NIR-II photothermal therapy by adjusting the morphology of the POM-based charge-transfer complexes.These studies provide references for the application of POM complexes in the field of tumor therapy.We have constructed a series of simple and universal systems based on POMs,which provides new ideas and methods for the development of such materials.