Tumor Microenvironment Triggered Metal-organic Coordination Particles For Photo/Chemodynamic Therapy Of Cancers

Background:In China,the incidence of cancer is increasing year by year.At this stage,clinical surgery combined with radiotherapy and chemotherapy strategies are still difficult to suppress the tumor growth completely and tumor replace resulting in intermediate and advanced solid tumors.Therefore,the development of safe and effective therapeutic strategies has become a hot topic at the present stage.In recent years,phototherapy（photothermal therapy,PTT,and photodynamic therapy,PDT）,as a new treatment approach,has attracted much attention in cancer therapy because of its obvious advantages such as controllability,none-invasive,non-drug resistance and repeated administration.Both of PTT and PDT always require the photosensitizers to convert the light to heat or chemical energy.Traditional photosensitizers always suffers from poor stability,high dark toxicity in the skin,low biocompatibility,fast metabolism,lower intracellular uptake,and oxygen-dependent therapy.Besides,Hypoxia-inducible factor-1（HIF-1）or Heat sock potein70（HSP70）upregulation after PDT or PTT treatment could induce the resistance of tumors.The metal-organic coordination particles that response to tumor microenvironment have the advantages of structural diversity,function adjustability,and high porosity,and have broad application prospects in the multimodal diagnosis and treatment strategies.In this paper,we designed and constructed a class of tumor microenvironment sensitive metal-organic coordination particles for photo-chemodynamic therapy and photothermal/gene therapy.After downregulation of HSP70 protein expression by DNAzyme.The enhancement of synergistic anti-tumor effects of photo-chemodynamicand photothermal/gene therapy are achieved.Purposes:Herein,we constructed various metal-organic coordination particles to investigate their photodynamic,photothermal,chemodynamic and gene therapyproperties in vitro,and explore their synergy anti-tumor therapeutic effects in vivo.It may providing a new sight for cancer therapy.Methods:（1）Januscoordinationnanoparticles（J-MOPs）and DNAzyme@Mn-CONASHs were prepared by coordination self-assembly.Transmission electron microscopy,atomic force microscopy,X-ray powder diffractometer,infrared spectrometer and other methods were used to characterize the two metal organic coordination nanoparticles.The ultraviolet-visible spectrophotometer evaluates the stability of metal J-MOPs in different solutions.DCFH-DA active oxygen probe detects the generation efficiency of J-MOPs in singlet oxygen in vitro;The photothermal conversion ability of DNAzyme@Mn-CONASHs was evaluated;The in vivo and in vitro MRI of DNAzyme@Mn-CONASHs were performed.（2）By using laser confocal microscopy,the cellular upake of J-MOPs and DNAzyme@Mn-CONASHs by Hep G2 cells were investigated;the photodynamic/chemodynamic synergistic killing effect of J-MOPs,DNAzyme@Mn-CONASHs photothermal/gene synergistic treatment of anti-tumor cells were evaluated by CCK-8,Calcein-AM&PI;The down regulation of HSP 70 protein expression after treated with DNAzyme@Mn-CONASHs was analyzed by western blot and Q-PCR..DCFH-DA as a ROS detector was used to evaluate the singlet oxygen generation of our prepared metal-organic coordination particles in Hep G2 tumor cells.（3）Hep G2 tumor-bearing subcutaneous nude model was built.After injection of J-MOPs or DNAzyme@Mn-CONASHs the tumor volume and bodyweight change of nude mice in each group were measured every other day after irradiated by 670 or 808 nm laser.The synergy anti-tumor effect of J-MOPs or DNAzyme@Mn-CONASHs were investigated by H&E staining,TUNEL staining and immunohistochemical analysis.Results:1.Janus coordination nanoparticles for photodynamic therapy and chemodynamic therapy（1）Preparation and characterization of Janus-coordinated nanoparticles（J-MOPs）:Cu²⁺was used as the molecular bridge to coordinate with the hydroxyl group and carbonyl group in the tetrahydroanthraquinone organic fluorescent dye（THQ）,and then co-coordinating with the P of the black phosphorus quantum dots（BPQDs）.The new Janus coordination nanoparticles（J-MOPs）were prepared.Transmission electron microscopy results show that the average particle size of J-MOPs is 25±6.9 nm.Raman spectroscopy,X-ray photoelectron spectroscopy,and infrared spectroscopy prove that the synthesized J-MOPs have specific peaks of BPQDs and THQ.J-MOPs were dissolved in water,PBS and cell culture medium for 48 h,and the absorption peaks of each group showed no significant change.Under NIR laser irradiation（670 nm,0.1 W/cm²,6 min）,the singlet oxygen yield of J-MOPs in vitro was significantly higher than that of the control group（BPQDs）.（2）The best time for J-MOPs uptake in Hep G2 cells was 3 h.J-MOPs,and the toxicity of Hep G2 was the lowest when the concentration was below 40 mg/m L.When the concentration increased to more than 60 g/m L,Hep G2cells showed cytotoxicity.When J-MOPs concentration was 80μg/m L,the viability of Hep G2 cells was significantly reduced to 22%after the NIR laser irradiation（670 nm,0.1 g/m L 5min）.After laser irradiation,the survival rate of cells was maintained by 56.1%（**P<0.005）.PI staining.The results showed that under the irradiation of near infrared light（670 nm,0.1 W/cm²,5 min）,the dead cells in J-MOPs group were significantly more than those in BPQDs treatment group.（3）After tumor injection of J-MOPs 3 h,the tumor growth inhibition effect of nude mice treated with near infrared laser irradiation（670 nm,0.1 W/cm²,5 min）was the most significant.After treatment,pathological examination showed that a large number of typical cytoskeletal collapse and cell lysis were found.In group J-MOPs+Laser,there was a large number of tumor cells apoptosis and no obvious proliferation of tumor cells.2.Mn²⁺coordination nanosheets for antiheat endurance photothermal/gene synergy therapy（1）Preparation and characterization results of DNAzyme@Mn-CONASHs:The formation of two-dimensional Mn-CONASHs is composed of tetrahydroxyanthraquinone organic dyes（THAQ）and Mn²⁺ions throughπ-πstacking and C-OH/C=O of Mn²⁺and THAQ The coordination bond between is formed by self-assembly.Transmission electron microscopy observations showed that Mn-CONASHs had a sheet structure with an average diameter of about 78.7±15.9 nm.Atomic force microscopy analysis showed that the average thickness of the nanosheets was 3.64±0.8 nm.X-ray photoelectron spectroscopy and infrared spectroscopy proved the successful synthesis of Mn-CONASHs.Under near infrared laser irradiation（808 nm,2 W/cm²,5 min）,the temperature of the Mn-CONASHs solution increased from 27.1℃to 54.7℃,and the photothermal conversion efficiency（η）was 39.3%.In vitro MRI results show that within a certain concentration range,as the concentration of Mn-CONASHs nanomaterials increases,its T₁-MRI signal gradually increases.Polyethyleneimine（PEI）was adsorbed on the surface of Mn-CONASHs through electrostatic interaction,its Zeta potential changed from-11.47±0.47 m V to 31.67±2.36 m V,and finally adsorbed on DNAzyme to obtain DNAzyme@Mn-CONASHs,which The potential decreased from 31.67±2.36 m V to 13.57±1.01 m V.（2）After incubating Mn-CONASHs with Hep G2 cells for 4 h,under the near-infrared laser irradiation（808 nm,1 W/cm²,5 min）,CCK-8 results showed that the survival rate of Hep G2cells decreased to 21.8%;Calcein-AM/PI cell live/dead staining showed a large number of necrotic cells in the Mn-CONASHs+Laser group.In addition,in the absence of laser irradiation,as the concentration of Mn-CONASHs increases,the fluorescence intensity of the DCFH-DA probe gradually increases.DNAzyme@Mn-CONASHs and Hep G2 cells were incubated with Hep G2 cells for2h to observe the fluorescence superposition of THAQ and DNAzyme ^FAMto produce a yellow signal,indicating that Mn-CONASHs and DNAzyme ^FAMco-localized in lysosomes.Western Blot and Q-PCR experiments showed that the DNAzyme released in the weak acid tumor microenvironment cleaved HSP70m RNA,which can significantly eliminate the up-regulation of HSP70 m RNA transcription and protein expression in Hep G2 cells due to high fever（42℃）.In addition,under the same 808 nm laser irradiation,the survival rate of Hep G2 cells after DNAzyme@Mn-CONASHs treatment was significantly lower than that of the Mn-CONASHs treatment group with the same dose（***P<0.001）.（3）After injecting DNAzyme@Mn-CONASHs through the tail vein for 4 h,under the near-infrared laser irradiation（808 nm,1 W/cm²,5 min）,Mn-CONASHs and DNAzyme@Mn-CONASHs treated nude mice tumor The temperature of the site rose rapidly to 49.4±0.2℃and 49.7±0.4℃.The DNAzyme@Mn-CONASHs group showed more significant tumor growth inhibition after near-infrared laser irradiation;The results showed that the expression level of HSP70 had no significant change compared with the control group（PBS）.Histopathological examination showed a large number of apoptosis and necrosis of tissue cells.Conclusions:In summary,we discovered two kinds of tumor microenvironmentally responsive metal-organic coordination nanoparticles（J-MOPs and DNAzyme@Mn-CONASHs）,which have good photodynamic/chemodynamic,photothermal/gene therapy synergistic antitumor effects.It may provide research foundation for developing new cancer treatment strategies.