Construction Of Bi-metallic Hybrid Nano-based Drug Delivery System And Its Application In Tumor-sensitive Chemo-chemodynamic Synergistic Therapy

Part I Construction and characterization of bi-metallic hybrid nano-based drug delivery systemObjectives: The clinical use of conventional Pt-based chemical drugs is constrained owing to the serious systemic toxicity and non-specific drug delivery.Mesoporous organic silica（MS）nanoparticles possess a great drug loading capacity,but accompanied with the premature drug leakage from the pore channels,which is leading to a significantly decreased delivery efficiency.The aim of this study is to construct a kind of novel drug delivery system that is able to improve the delivery efficiency through preventing drug leakage of MS-based nanoparticles,as well as to realize the specific drug release to kill cancer cells.Methods: Metal organic framework（MOF）with copper doping was used to coat the exterior surface of HMOS to construct the bi-metallic hybrid nanoparticles.MOF could block the pore channels of HMOS and prevent the drug leakage.The thickness of MOF on HMOS and morphology of final synthesized nanoparticles（denoted as HMOS@ZC）were altered through changing the mass ratio of HMOS and 2-methylimidazole zinc salt with copper doping（denoted as ZC-MOF）.Transmission electron microscope（TEM）,dynamic light scattering（DLS）were used to measure the size of nanoparticles.Zeta potential,X-ray diffraction（XRD）,X-ray photoelectron spectroscopy（XPS）,TEM-based energy dispersive X-ray Sspectroscopy（EDS）,element mapping,Brunauer-Emmett-Teller surface area and pore size analysis（BET）were used to investigate the structure and composition of HMOS@ZC.Results: The results showed that with the increase of ZC-MOF precursor mass,the corresponding thickness of MOF layer on the exterior surface of HMOS could be observed by TEM,accompanied with changed dispersity of nanoparticles.TEM results indicated that the size of HMOS@ZC was about 45 nm,and the hydrodynamic size was about 100 nm,which could be altered by changing the mass of ZC-MOF precursor during the synthesizing process.Other results including zeta potential,XRD,XPS,EDS,element mapping,and BET provided strong evidence that ZC-MOF could be coated on the surface of HMOS.Conclusion: ZC-MOF can be coated on the exterior surface of HMOS successfully.The thickness of ZC-MOF layer can be regulated by altering the mass ratio of HMOS and ZC-MOF precursor during the synthesizing process.Part II Responsive drug release and functional test of bi-metallic hybrid nano-based drug delivery systemObjectives: Owing to the pore channels,premature drug leakage will be observed when using HMOS as the drug carrier,which will significantly reduce the drug delivery efficiency.MOF can be coated on the exterior surface of HMOS and prevent the drug leakage.Besides,the acidresponsive degradation of MOF enables the loading drugs to release under acid conditions smartly.Glutathione（GSH）is abundant in the tumor tissue and will prevent Pt-based chemical drugs binding with nucleic acid of cancer cells,inducing decreased therapeutic effect.This study aims to investigate the responsive release of HMOS@ZC during the drug delivery and the GSH consumption by the doping copper within the framework of ZC-MOF,as well as the successive hydroxyl radical production through catalyzing the decomposition of intracellular hydrogen peroxide（H2O2）.Methods: Conventional chemical drug cis-Diammineplatinum（II）dichloride（CDDP）was used in this study.CDDP was loaded within the hollow cavity of HMOS,followed by ZC-MOF coating to obtain the organic-inorganic hybrid nanomaterials,Pt@HMOS@ZC.Inductively Coupled Plasma-Mass Spectrometry（ICP-MS）was used to detect CDDP release of Pt@HMOS and Pt@HMOS@ZC under different p H（7.4,6.5,5.5）,respectively.The degradation of MOF component of HMOS@ZC and copper release were also investigated.5,5′-Dithiobis（2-nitrobenzoic acid）（DTNB）and 3,3′,5,5′-Tetramethylbenzidine（TMB）were used to measure GSH consumption and ·OH generation,respectively.Results: Drug leakage could be significantly reduced after coating ZC-MOF on HMOS.The ICP results showed that no significant difference of CDDP release could be detected of Pt@HMOS at various p H（7.4,6.5,5.5）.Oppositely,CDDP release displayed a p H-dependent manner after ZC-MOF coating on HMOS.Similar trend could be observed for copper release.Moreover,the released copper could remarkably decrease the intracellular GSH level and catalyze H2O2 into ·OH generation.Conclusion: HMOS@ZC nanoparticles could improve the drug delivery efficiency through preventing premature leakage by ZC-MOF coating.The loading drugs of HMOS@ZC could be released in a p H-dependent manner.Additionally,ZC-MOF could be degraded in acid environment and the doping copper also could be released.The continuously released copper was able to consume intracellular GSH and successively catalyze the abundant H2O2 into highly toxic ·OH,which provided great support for chemo-chemodynamic synergistic therapy.Part III In vitro study on the enhanced effect of chemotherapy/chemodynamic therapy for bi-metallic hybrid nanoparticlesObjectives: This study aims to investigate the inhibitory effect of Pt@HMOS@ZC on human derived non-small cell lung cancer cells（A549）and the ability of ZC-MOF component to consume GSH and produce ·OH in vitro.Additionally,in order to evaluate the synergistic effect of Pt@HMOS@ZC especially in comparison with CDDP,the formation of Pt-DNA adducts and DNA damage of cancer cells are detected after treating with Pt@HMOS@ZC or CDDP,respectively.Methods: Confocal microscope imaging and flow cytometry were used to detect the cellular uptake of HMOS@ZC at different time points（1,3,and 6 h）.Methylthiazolyldiphenyltetrazolium bromide（MTT）test,Calcein-AM/propidium iodide（AM/PI）staining,and apoptosis test were used to evaluate the inhibitory effect of Pt@HMOS@ZC on A549 cancer cells.GSH detection kit and GSH probe(Thiol Trace^TM Violet)were used to detect the intracellular GSH level after treatment of Pt@HMOS@ZC.Confocal microscope imaging and flow cytometry was performed using 2′,7′-Dichlorofluorescin diacetate（DCF-DA）to measure the ·OH level.ICP-MS and γ-H2 AX staining were used to assess the formation of Pt-DNA adducts and DNA damage,respectively.Results: Confocal laser scanning microscopy（CLSM）images and flow cytometric analyses demonstrated the elevated A549 tumor cellular uptake of HMOS@ZC with increasing incubation time.Results of cytotoxic tests showed Pt@HMOS@ZC had a much higher toxicity than those of single administration of ZC-MOF or CDDP.Copper in Pt@HMOS@ZC could remarkably reduce the intracellular GSH level,further produce ·OH.Compared with CDDP,the decreased level of GSH promoted the loading CDDP to bind with DNA and form more PtDNA adducts,as well as more intense DNA damage of cancer cells.Conclusion: Pt@HMOS@ZC could achieve the synergistic effect of chemo-chemodynamic therapy through GSH consumption and further produced ·OH which resulting from the doping copper.Part IV In vivo study on anti-tumor effect and biosafety/stability of bimetallic hybrid nanoparticlesObjectives: This study aims to study the biodistribution in A549 tumor bearing mice after administration of HMOS@ZC,as well as the anti-tumor effect of Pt@HMOS@ZC.Besides,the biosafety and bio-stability tests of Pt@HMOS@ZC are also investigated.Methods: Indocyanine green（ICG）was loaded into HMOS@ZC（denoted as ICG@HMOS@ZC）and used for biodistribution.ICG@HMOS@ZC（ICG equiv 1 mg/kg body weight）was intravenously injected into A549 tumor-bearing mice.Then the mice were imaged to observe the biodistribution at designed time points（1,2,4,8,12,24,and 48 h）using In Vivo Imaging System（IVIS）.For the anti-tumor effect,the mice were randomly divided into six groups: 1)PBS;2)HMOS;3)Pt@HMOS;4)HMOS@ZC;5)Pt@HMOS@ZC;6)Pt@HMOS@ZC plus N-Acetyl-L-cysteine（NAC）.The dose for CDDP was 1.5 mg/kg body weight.The dose for copper is 0.4 mg/kg body weight.NAC was injected intraperitoneally one hour before intravenous administration,and the dose was 10 mg/kg body weight.The nanoparticles were administrated every other day for 3 doses.After that,the body weight and tumor size were measured every two days,and the organs of mice and tumor tissues were harvested at day 22 for further histopathological tests.The blood of the mice was also collected for hematological analysis.DLS was used to monitor the changes of the sizes after incubating in PBS,DMEM,DMEM plus 10% fetal bovine serum at different time points in order to investigate the stability of HMOS@ZC.Hemolysis test was performed by incubating different concentrations of HMOS@ZC with collecting red blood cells at 37℃ for two hours.The pharmacokinetics of HMOS@ZC was also detected.Urine and feces at different time points（2 h,1,2,3,4,7,and 10 days）post injection of Pt@HMOS@ZC were collected for excretion test.Results: In vivo imaging showed that ICG@HMOS@ZC could accumulate in the tumor tissue quickly and reached its peak level at 4 h post injection.The tumor inhibition tests and further pathological tests showed the Pt@HMOS@ZC possessed superior synergistic anti-tumor effect,higher apoptosis rate,and more intense damage of DNA,especially compared with the single use of CDDP.Additionally,hematological indexes demonstrated that Pt@HMOS@ZC could prevent hepatotoxicity and myelosuppression.DLS results showed that HMOS@ZC could stay stable in different buffers.Results of excretion and hemolysis tests indicated the HMOS@ZC possess great biosafety.Conclusion: Pt@HMOS@ZC had the synergistic effect of chemo-chemodynamic therapy and was able to improve the efficacy of CDDP.Besides,it exhibited good biological safety and stability.