Application Of Functional Nanoplatforms In Chemodynamic Therapy-Induced Synergistic Cancer Therapy

Currently,cancer remains the second most common cause of death in the world.Owing to poor prognosis and easy metastasis,the incidence and mortality rate of cancer have been rising over the past few decades.Studies have shown that tumor cells are more susceptible to oxidative damage than normal cells.Therefore,reactive oxygen species（ROS）-mediated therapy has become a promising approach for efficient cancer treatment.Compared with other ROS-mediated therapies,chemodynamic therapy（CDT）with unique advantages of independence from external stimuli,deep tissue treatment capability,and non-multidrug resistance has attracted widespread attention in cancer research.However,insufficient concentration of hydrogen peroxide（H₂O₂）and overexpression of reducing substances（such as glutathione（GSH））in tumor microenvironment（TME）can significantly reduce the therapeutic efficacy of CDT.In addition,due to the complexity and high heterogeneity of the TME,CDT alone cannot completely eliminate the tumor.To address these issues,this article designs a series of multifunctional nanoplatforms for enhancing CDT and achieving CDT-based synergistic cancer treatment,aiming to provide new methods and strategies for constructing multifunctional nanotherapeutic drugs with excellent therapeutic effects.Part One:Copper sulfide engineered covalent organic frameworks for acid-responsive chemo/photothermal/chemodynamic synergistic therapy against cancerObjective:Considering the insufficient H₂O₂ concentration in tumor cells and unsatisfactory therapeutic effect of single CDT,a multifunctional nanoplatform combined with chemotherapy,photothermal therapy（PTT）,and CDT was constructed.The PTT and chemotherapeutics could raise the local reaction temperature and intracellular H₂O₂ concentration,respectively,which could markedly improve the therapeutic effect of CDT and achieve efficient cancer treatment.Methods:The copper sulfide engineered covalent organic frameworks（Cu S@COFs）were prepared via the general encapsulation method.Followed by loading DOX and coating bovine serum albumin-folic acid（BSA-FA）layer on the surface of Cu S@COFs,a therapeutic nanoplatform based on Cu S@COFs-BSA-FA/DOX was obtained.The nanocomposites were characterized by transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,and X-ray diffraction（XRD）.The drug loading and release behavior were evaluated using fluorescence spectrophotometer.The catalytic ability of the system was estimated by fluorescence spectrophotometer and ultraviolet spectrophotometer.4T1 cells were chose for monitoring the cytotoxicity,the behavior of cell uptake,and ROS production ability of the nanoplatform.BALB/c tumor bearing mice were used to evaluate in vivo antitumor efficiency of Cu S@COFs-BSA-FA/DOX.And the biological safety of the nanoplatform was analyzed via hemolysis experiments and blood biochemical analysis.Results:The successful synthesis of Cu S@COFs-BSA-FA/DOX was characterized by TEM,SEM,XRD and other means.The in vitro drug release test indicated that Cu S@COFs-BSA-FA/DOX could achieve p H/near infrared light（NIR）dual stimuli-responsive drug release.The in vitro studies proved that Cu S@COFs-BSA-FA displayed excellent photothermal effect and catalytic performance.And compared with pure Cu S NPs,Cu S@COFs possessed higher catalytic activity.Cell uptake experiments demonstrated that the layer of BSA-FA endowed the nanoplatform with the ability to target cancer cells.Both cellular and animal experiments showed that Cu S@COFs-BSA-FA/DOX exhibited higher toxicity under 808 nm laser irradiation over other treatment group,which demonstrated this nanoplatform could achieve effective cancer treatment with chemotherapy/PTT/CDT synergistic therapy.Conclusions:A multifunctional therapeutic system based on Cu S@COFs-BSA-FA/DOX was proposed.And the nanoplatform possessed multi-amplified antitumor efficacy with negligible systemic toxicity both in vitro and in vivo.This work would provide a promising strategy for building CDT-based multifunctional nanoplatforms,and expand the biological application of COFs.Part Two:A multifunctional nanoplatform based on covalent organic frameworks-derivedcarbonnanomaterialsfor chemodynamic/photodynamic/photothermal synergetic cancer therapyObjective:Localized hyperthermia caused by nanomaterial-mediated PTT can enhance CDT performance.A facile strategy was developed to synthesize N-doped carbon nanomaterials with high photothermal conversion efficiency.Based on the N-doped carbon nanomaterials,a nanoplatform was constructed for CDT/PTT synergistic therapy.Moreover,photodynamic therapy（PDT）as an alternative ROS-mediated cancer therapy was also introduced into this multifunctional nanoplatform to achieve synergistic CDT/PDT/PTT anticancer effects for efficient tumor treatment.Methods:After prepared the COFs-drived carbon nanomaterials（C-COF）by pyrolyzing,the manganese dioxide was coated on surface of the C-COF（C-COF@Mn O₂）using hydrothermal method.Followed by loading dihydroporphyrin（Ce6）and coating BSA-FA layer on the surface of C-COF@Mn O₂,C-COF@Mn O₂-BSA-FA/Ce6 were obtained.This nanoplatform was characterized by TEM,FTIR,XRD,and other means.The loading and in vitro release behavior of Ce6 were investigated using ultraviolet spectrophotometer.4T1 cells were selected to evaluate the cellular cytotoxicity,cell uptake,the cellular ROS and oxygen production ability of the nanoplatform.The in vivo antitumor effect of C-COF@Mn O₂-BSA-FA/Ce6 was evaluated using BALB/c tumor bearing mice.Results:The successful construction of multifunctional nanoplatforms has been demonstrated through various characterization methods,such as TEM,FTIR,and XRD.In vitro drug release experiments indicated that C-COF@Mn O₂-BSA-FA/Ce6 possessed acidic/GSH dual stimuli-responsive drug release characteristics.The in vitro studies demonstrated that the nanoplatform exhibited excellent photothermal conversion performance,oxygen and ROS production performance.Cell uptake experiments proved that C-COF@Mn O₂-BSA-FA/Ce6 had the ability to target FA-positive cells.RDPP and DCFH-DA as the indicators were used to verify the excellent oxygen and ROS production capabilities of the nanoplatform.In addition,C-COF@Mn O₂-BSA-FA/Ce6could also reduce intracellular GSH levels and amplify oxidative stress.The cell and animal experiments indicated that compared with single-mode therapies,the multifunctional nanoplatform possessed excellent antitumor effect and could achieve CDT/PTT/PDT synergistic cancer treatment.Conclusions:A multifunctional therapeutic system based on C-COF@Mn O₂-BSA-FA/Ce6 was designed.This nanoplatform not only exhibited excellent photothermal performance and catalytic ability,but also could generate O₂ for alleviating tumor hypoxia and improving the efficacy of Ce6-mediated PDT.Furthermore,C-COF@Mn O₂-BSA-FA/Ce6 could also reduce GSH levels,amplify oxidative stress,and achieve synergistic PDT/PTT/CDT therapeutic effect.C-COF@Mn O₂-BSA-FA/Ce6 with excellent antitumor activity and good biocompatibility exhibited great potential in the field of cancer therapy.Part Three:Ion channel-targeting near-infrared photothermal switch with chemo/photothermal/chemodynamic synergistic effect for specific cancer therapyObjective:Considering the heterogeneity of tumors,precise cancer treatment is an important factor should be considered in construction of nanoplatform for cancer treatment.Therefore,we combined precision therapy with CDT to achieve efficient tumor treatment.Capsaicin receptor（TRPV1）as a nonselective cation channel possesses the ability to regulate Ca²⁺influx,causing Ca²⁺-overloading.Exogenous ROS can destroy the Ca²⁺buffering capacity of mitochondria,which would improve the therapeutic effect of Ca²⁺-overloading.A TRPV1 channel-targeted multifunctional therapeutic platform combining CDT,chemotherapy,and TRPV1 channel light activation was constructed.It could not only specifically target TRPV1-positive tumor cells,but also achieve the spatiotemporally specific regulation of cancer collaborative therapy.Methods:Hollow mesoporous Prussian blue nanocages（h PBNCs）were synthesized by hydrothermal method.h PBNCs as nanocarriers were used to encapsulate chemotherapeutic agents（DOX）.The self-coating polydopamine layer was coated on the surface of the h PBNCs（h PBNCs@PDA）.Then the monoclonal antibody TRPV1 was conjugated to h PBNCs@PDA（h PBNCs@PDA-TRPV1）to make nanocomposites specifically target the overexpressed TRPV1 channels on cell membrane of cancer cells.The nanocomposites were characterized by TEM,XRD,FTIR,as well as N₂adsorption and desorption experiments.Ultraviolet spectrophotometer was used to investigate the loading and in vitro release behavior of DOX.The catalytic properties of nanomaterials were studied using EPR spectroscopy.The targeting capability of h PBNCs@PDA-TRPV1 was investigated by TEM and fluorescence imaging.U373 cells were chosen for evaluating the behavior of cellular cytotoxicity,cell uptake,and the ROS production ability of the nanocomposites.Finally,BALB/c nude mice were used to evaluate the in vivo antitumor effect of DOX-h PBNCs@PDA-TRPV1,and the tissue morphology was observed using hematoxylin-eosin staining（H&E staining）.Results:The characterization results proved the successful preparation of DOX-h PBNCs@PDA-TRPV1.The nanoplatform possessed p H/NIR dual-responsive drug release capability in in vitro drug release experiments.In vitro property studies demonstrated that DOX-h PBNCs@PDA-TRPV1 exhibited excellent photothermal properties and catalytic ability.TEM and fluorescence imaging results suggested that h PBNCs@PDA-TRPV1 could target and strongly bind with the TRPV1 channels on plasma membrane of U373 cells.Using Fluo-3 AM probe as the indicator,we observed that h PBNCs@PDA-TRPV1 could specifically activate TRPV1 channels under 808 nm laser irradiation,leading to Ca²⁺influx.Compared with h PBNCs@PDA-TRPV1 and free DOX,the DOX-h PBNCs@PDA-TRPV1 showed higher cytotoxicity under808 nm laser irradiation.In addition,the multifunctional nanoplatform could also specifically inhibit tumor growth in in vivo experiments.Conclusions:DOX-h PBNCs@PDA-TRPV1 as a TRPV1 channel targeted therapeutic platform was constructed and could achieve amplified anti-tumor efficacy without any systemic toxicity by combined CDT,chemotherapy,and photothermal activation of TRPV1 channels.Our research provides a powerful idea for designing multifunctional therapeutic nanoplatforms to achieve site-specific cancer synergetic therapy via precisely controlled activation of ion channel signaling.Part Four:NIR-responsive NO nanogenerator with targeted regulation of TRPV1 channels for chemodynamic therapy-based precise tumor therapyObjective:Although the TRPV1 channels could be remotely activated by localized heating induced by nanomaterial with photothermal behavior,relatively high temperature（＞43℃）would cause the inevitable injury to surrounding normal tissues.Achieving effective cancer therapy under relatively low temperatures has a great importance for its future clinical transformation.A nanocomposite was constructed to manipulate TRPV1 signaling without adverse effects by NIR-triggered nitric-oxide（NO）release.It could achieve specific tumor collaborative treatment by combining with CDT and controlled activation of TRPV1 channel.Methods:Hollow copper sulfide nanoparticles（HCu S NPs）coated with polydopamine（HCu S@PDA）with excellent catalytic activity were used in our study.At the same time,the NIR sensitive NO donors（N,N’-di-sec-butyl-N,N’-dinitroso-1,4-phenylenediamine,BNN6）were loaded throughπ-πstacking.The HCu S@PDA could serve as a substrate to conjugate with monoclonal antibody TRPV1（HCu S@PDA-TRPV1/BNN6）.The nanocomposites were characterized by TEM,XRD,FTIR,and other characterization methods.The catalytic performance of nanomaterials was studied by ultraviolet spectrophotometer.The in vitro NO release behavior of HCu S@PDA1/BNN6was investigated using NO assay kit.TEM and fluorescence imaging were used to evaluate the targeting capability of h PBNCs@PDA-TRPV1.U373 cells were selected for investigating the cellular cytotoxicity,cell uptake,the ROS production ability,and the damage to mitochondrial membrane potential changes of nanocomposites.Finally,BALB/c nude mice were used to evaluate the in vivo antitumor effect of HCu S@PDA-TRPV1/BNN6,and the tissue morphology was observed using H&E staining.Results:The successful preparation of HCu S@PDA-TRPV1/BNN6 was proved by the characterization results.The in vitro NO release experiments demonstrated that HCu S@PDA/BNN6 could achieve NIR-responsive NO release.In vitro experiments indicated the nanoplatform possessed excellent photothermal properties and catalytic performance.TEM and fluorescence microscopy images illustrated that HCu S@PDA-TRPV1 could specifically target and bind with the overexpressed TRPV1 channels on plasma membrane of U373 cells.Under carefully control of irradiation conditions to make sure local temperatures below 43℃,the HCu S@PDA-TRPV1/BNN6 could specifically activate TRPV1 channels under 808 nm laser irradiation and cause Ca²⁺influx,which indicated by the probe of Fluo-3 AM.MTT experiment showed the HCu S@PDA-TRPV1/BNN6 exhibited highest cytotoxicity under808 nm laser irradiation over the groups of HCu S@PDA-TRPV1/BNN6,HCu S@PDA-TRPV1,and HCu S@PDA-TRPV1+808 nm laser.In addition,the multifunctional nanoplatform could also specifically inhibit tumor growth in in vivo experiments.Conclusions:The multifunctional nanoplatform based on HCu S@PDA-TRPV1/BNN6 was constructed in this study.HCu S@PDA-TRPV1/BNN6could not only remotely and specifically activate TRPV1 channels by NIR-responsive on-demand release of NO,but also catalyze endogenous cellular H₂O₂ for CDT.Excellent anticancer activity and biosafety endowed this multifunctional nanoplatform with great application potential in cancer treatment.