Fabrication Of Mitochondria-targeting/Fluorescent Multi-functional Nanoplatform And Micelles For Biomedical Applications

Malignant tumor(cancer)has become one of the major diseases that endanger human life and health in the 21st century.Up to date,there is no great breakthrough in cancer therapy owing to three big obstacles such as highly toxic side effect of chemotherapy drugs,tumor metastasis and multidrug resistance of cancers.The current strategies for cancer therapy include surgical treatment,chemotherapy,radiation therapy and so on.Among them,chemotherapy is one of the most commonly used strategies in comprehensive treatment of cancer,but the potency and efficacy of chemotherapy is not ideal.When the traditional chemotherapy drugs enter the human body through intravenous injection,they must undergo a series of processes of serum protein interactions,metabolism and excretion before they finally arrive at the tumor tissue.These processes make the blood drug concentration decrease very fast,so that the quantity of drug that can reach the specific site of tumor is limited.In order to increase the drug concentration in tumor,the dose of injected chemotherapeutic drug must be added,however,which may cause a worse side effect on normal cells or tissues.Even worse,most of the chemotherapeutic drugs lack of specific pharmacological activities,which will lead to multidrug resistance(MDR)easily and make cancer treatment more difficult.The concept of targeted therapy is proposed to solve these problems.The purpose of targeting is to increase drug concentration in lesion locations,reduce the side effects,and enhance the safety and efficiency of drugs.Mitochondrion is one of the most important organelles inside a cell.Mitochondria take charge of the energy supply for metabolism of eukaryotic cells,and also have genetic material(MitoDNA)like nucleus.In addition,cell apoptosis can be triggered and mediated by mitochondria pathway oxidative stress and the induced mitochondria dysfunction.Therefore,mitochondrion is recognized as one of the most effective targets in cancer therapy and treatment of other mitochondria-related diseases.With the development of nanoscience and nanotechnology,new nanomaterials provide new materials and new methods for cancer targeting treatment.In this dissertation,we have synthesized triphenylphospine(TPP)-modified Fe3O4@mSiO2 nanoparticles and then conjugated with biocompatible and fluorescent carbon quantum dots(CDs)to construct a novel magnetic "all-in-one" nanoplatform(Fe3O4@mSiO2-TPP/CDs)with multifunctional ities,including mitochondria targeting,long time cell imaging,drug loading and controlled-release property etc.Further,a model anticancer drug(doxorubicin,DOX)has been loaded onto CQDs-TPGS-TPP nanomicelles by means of nanomaterials self-assembly and bioconjugation technique,which shows mitochondria targeting,controlled drug release behavior as well as a possibility of overcoming the multidrug resistance(MDR)of cancer cells.Our main research works and the obtained results can be summarized as follows:1.The nitrogen and sulfur co-doped carbon quantum dots(g-CQDs)were prepared by pyrolysis of garlic under mild conditions followed with a simple extraction by ethanol and water repeatedly.We find that(1)the doping contents of N and S elements in g-CQDs are 6.46%and 0.34%,respectively.The g-CQDs are quasi-spherical in an average size of 3.83 nm with a graphitic inner core;(2)the QY of the g-CQDs in distilled water can reach 13.76%while quinoline sulfate used as a standard;(3)the g-CQDs show a excitation-dependent photoluminescence(PL)both in distilled water and ethanol;(4)the g-CQDs possess robust pH stability and preserve stable fluorescence under wide ranging pH values(2-12);(5)the g-CQDs also exhibit a solvent-dependent PL,and show stronger emissions in the solvents of higher dielectric constants;(6)the g-CQDs have a highly selectivity towards Fe3+ ions,fluorescence quenching will happen to when 0.5 mM Fe3+ ions are added into the aqueous solution containing g-CQDs.Further,we select human breast cancer cell line(MCF-7)and human neuroblastoma cell line(SH-SY5Y)as the research models to test the cytotoxicity of g-CQDs employing a MTT assay,the results indicate that the cell viability can keep as high as 80%even with the concentration of g-CQDs increasing up to 400?g/mL.Subsequently,2-dimensional(2D)monolayer-cultured cells and 3-dimensional(3D)multicellular spheroids(MCs)of MCF-7 and SH-SY5Y are taken as models to perform 3D in vitro cell signaling analyses.The confocal laser scanning microscopy(CLSM)observations reveal that both 2D cells and 3D MCs labeled with g-CQDs can exhibit bright and blue fluorescence under 405 nm laser excitation,and shows a good cell imaging effect.2.The magnetite(Fe3O4)nanocrystals were synthesized by a hydrothermal method and then encapsulated with mesoporous silica shells by stober method.And then,the as-synthesized nitrogen doped carbon dots(N-CDs)from konjac flour were conjugated with triphenylphospine(TPP)-modified Fe3O4@mSiO2 to construct a novel biocompatible nanoplatform(Fe3O4@mSiO2-TPP/CDs)with multifunctionalities,such as mitochondria targeting,long time cell imaging,and magnetic field-response cellular uptake.The morphology,structure,and magnetic properties of the samples were characterized by using transmission electron microscopy(TEM),physical property measurement system(PPMS),ultraviolet-visible(UV-Vis)spectroscopy and PL spectra etc.The results have shown that the nanoplatform have uniform core-shell structure in an average size of 43 nm,with superparamagnetism at room temperature,and exhibit similar fluorescence to the CDs.According to the TGA(thermal gravimetric analysis)curves of nanoplatform,the contents of TPP and CDs on the surface of Fe3O4@mSiO2 nanoparticles are calculated to be about 12.18%and 1.76%,respectively.In addition,the ?-potential of the Fe3O4@mSiO2-TPP nanoparticles decreases from +33.47 mV to+16.33 mV after conjugation with CDs.However,dynamic light scattering(DLS)results show that the hydrodynamic sizes of Fe3O4@mSiO2-TPP/CDs become larger.All the results have demonstrated that multifunctional mitochondria targeting/fluorescent nanoplatform of Fe3O4@mSiO2-TPP/CDs has been successfully constructed.3.We select human pulmonary adenocarcinoma(A549),Chinese hamster ovary(CHO),human cervical cancer(HeLa),SH-SY5Y,human foreskin fibroblast(HFF)and human microvascular endothelial(HMEC-1)as cell line models to test the cytotoxicity of Fe3O4@MSiO2-TPP/CDs.The results indicate that this nanoplatform has a very low cytotoxicity and will not induce cell apoptosis.After the nanoplatform has co-incubated with A549,CHO,HeLa,SH-SY5 Y,HFF,and HMEC-1 cell lines for 24 h,respectively,in CLSM observation,stable and bright blue,green and red fluorescence can be observed under different channels,which means Fe3O4@mSiO2-TPP/CDs nanoplatform is indeed suitable for long time cell imaging.Moreover,CLSM,flow cytometry and Bio-TEM results demonstrate that Fe3O4@mSiO2-TPP could more co-localized with Mitotracker Red in mitochondria compared with non-targeting Fe3O4@mSiO2.The z-stacked scanning observations eliminate the possibility of fluorescence coming from mitochondria targeting nanoparticles that adhered to the cell surface.In addition,the influence of an applied static magnetic field(?0.3 T)on cellular uptake of the nanoplatform has also been studied.We find a magnetically enhanced cellular uptake within a short co-incubation time of 1 h.4.D-a-tocopheryl polyethylene glycol succinate(TPGS)is one of the nonionic surfactants.We modified TPGS with a mitochondria-targeting group,TPP,through a DCC/DMAP coupling method.Subsequently,the TPGS-TPP conjugate and hexadecylamine-capped carbon quantum dots(CQDs)self-assembled into spherical nanomicelles(CQDs-TPGS-TPP).TEM and DLS results show that the CQDs-TPGS-TPP can form nanomicelles with hydrodynamic sizes of?100 nm,and part of these nanomicelles will further assemble into larger-sized nested vesicles.MCF-7 and multidrug resistant MCF-7/ADR cancer cells were co-incubated with CQDs-TPGS-TPP nanomicelles for 24 h,MTT assay results indicate that the nanomicelles has little cytotoxicity.More experiments show that this nanomicelles not only has mitochondria targeting ability,but also be suitable for directly cell imaging because of incorporation of fluorescent CQDs.In addition,TPGS,one major composition of the nanomicelles,could inhibit the expression of P-glycoprotein(P-gp)to reduce the pump-out of drugs in cytoplasm and used in reversal of the MDR.Thus,the in vitro drug loading experiments of DOX show CQDs-TPGS-TPP had a drug loading content of 3.4%.Furthermore,the release of DOX from CQDs-TPGS-TPP/DOX can be accelerated at lower pH value.CQDs-TPGS-TPP/DOX could induce more cytotoxicity in MCF-7/ADR cells compared with equivalent free DOX when they co-incubated with MCF-7/ADR cells.Further,the tumor penetration of CQDs-TPGS-TPP/DOX were evaluated by using MCF-7 and MCF-7/ADR MCs as the in vitro 3D model.The results show that mitochondria targeting nanomicelles could improve the efficiency of DOX penetration on MCs compared with free DOX.As for MCF-7/ADR MCs,DOX-loaded CQDs-TPGS-TPP/DOX results in more efficient cell killing to inhibit the growth of MCs compared with equivalent free DOX,which means drug-loaded CQDs-TPGS-TPP nanomicelled could achieve MDR reversal both in cancer cells and cancer tissues.