| In recent years,malignant tumors have become worldwide public health problems that seriously threaten human health and life safety.Therefore,it is an urgent task to be solved for medical workers to realize the safe and effective treatment of tumors.At present,the main treatment modalities of tumors in clinic involve surgery,radiation therapy and chemotherapy.Among them,drug-based chemotherapy can achieve antitumor effects by inhibiting the proliferation of tumor cells or killing tumor cells.However,traditional chemotherapeutic drugs have exhibited some drawbacks in the applications:(?)instability in systemic circulation,(?)non-specific distribution in vivo,(?)toxicity and side effects and(?)multidrug resistance of tumor cells.Ideal nano drug carriers have a series of advantages,such as prolonged systemic circulation,improved pharmacokinetics and pharmacodynamics of drugs,sustained drug release behavior as well as low side effects,which have been widely used in the field of antitumor therapy.Generally speaking,nanocarriers can be divided into organic materials,inorganic materials,and hybrid materials composed of organic components and inorganic components.Conventional organic or inorganic drug nanocarriers might have some disadvantages,such as low drug loading capacity and uncontrolled drug release behavior.Metal organic frameworks(MOFs)are a class of hybrid materials crafted from metal ions,metal clusters and organic ligands via coordination.MOFs have large pore volume,high specific surface area and easily adjustable frame structure to realize high drug loadings.Zeolite imidazolate framework 8(ZIF-8)is a non-toxic and biocompatible MOF constructed from zinc ions and 2-methylimidazole,which is structurally stable under neutral physiological condition while degrade in tumor cells with low pH value.Therefore,ZIF-8 can be used as a potential carrier for antitumor drugs to achieve high drug loadings and pH-responsive drug release at tumor siteHowever,there are limited studies on MOFs-based nano drug delivery systems and related studies fail to make full use of the advantages of MOFs to solve the major problems in antitumor therapy.For example,tumor cells could show multidrug resistance toward some chemotherapeutic drugs as a result of the overexpression of efflux transporters(such as P-glycoprotein),thus to reduce the antitumor effect.In addition,chemotherapy alone has limited efficacy,and it is difficult to completely remove the tumors.The problem of poor prognosis such as tumor recurrence and tumor metastasis might arise after the discontinuation of treatment.MOFs have also exhibited some limitations in the biological application of drug delivery.Currently,two approaches have been applied to realize the MOFs-based drug loading and drug delivery.(?)Post absorbing drugs after the synthesis of MOFs:this way is suitable for drug molecules with smaller structures than that of the pores in MOFs,which can easily cause drug leakage during the cleaning process after drug loading,resulting in low drug loading contents and burst drug release.(?)One-step in situ encapsulation of drugs during the construction of MOFs:this method can achieve the loading of drugs with smaller or larger molecular structures via the generation of flaws in MOFs,avoiding drug leakage before the degradation of carriers.However,in the second case,the strong interaction between drug molecules and MOFs is essential for achieving high drug loadings and controlled drug release.Up to now,only a few drug molecules with specific functional groups could be successfully loaded by MOFs.Therefore,it is of great significance to rationally design and construct multifunctional MOFs nanoplatforms those can universally deliver multiple drugs for enhancing the antitumor treatment.In this study,three MOFs-based active targeting drug delivery nanoplatforms were constructed for multimodal antitumor therapy,focus on solving the major problems in cancer treatment and the limitations of MOFs as drug carriers.The basic characterization,in vitro antitumor evaluation and in vivo antitumor study were carried out for each formulation.The main research contents and experimental results are as follows:Relevant studies about the MOFs nanocarrier-based codelivery system of doxorubicin hydrochloride/verapamil hydrochloride for overcoming multidrug resistance with targeting antitumor therapyThe doxorubicin hydrochloride(DOX)and verapamil hydrochloride(VER)co-loaded ZIF-8 drug codelivery nanoparticles were synthesized in water via the in situ one-pot method.Then,polyethylene glycol-folate(PEG-FA)was modified on the surface of nanoparticles by coordination to construct the tumor cells-targeted nanoparticles(PEG-FA/(DOX+VER)@ZIF-8).The PEG-FA/(DOX+VER)@ZIF-8 was characterized in vitro to investigate the crystal structure,thermal stability,morphology,as well as its particle size distribution and zeta potential in water.X-ray powder diffraction pattens showed that PEG-FA/(DOX+VER)@ZIF-8 was of high crystallinity,and thermogravimetric analysis confirmed the high thermal stability of formulations.Above experimental results could verify the successful synthesis of ZIF-8.Transmission electron microscopy(TEM)images showed that the PEG-FA/(DOX+VER)@ZIF-8 nanoparticles were of uniform spherical-like morphology and the particle size of the nanoparticles was measured by dynamic light scattering(DLS)to be approximately 185.0 nm(PDI=0.005),satisfied with the request of EPR effect.In addition,the zeta potential of PEG-FA/(DOX+VER)@ZIF-8 nanoparticles in aqueous solution was determined to be-(34.5±7.43)mV.The UV spectrophotometry was employed to evaluate the drug loading capacity of(DOX+VER)@ZIF-8,and the drug loading contents of DOX and VER were determined to be approximately 8.9%and 32.0%,respectively.Next,PBS with the pH values of 5.0 and 7.4 was chosen as the release medium for the in vitro drug release experiments.After the same period of time,(DOX+VER)@ZIF-8 immersed in the PBS with the pH value of 5.0 could release more DOX and VER compared with the sample in the PBS of pH=7.4,confirming the pH-responsive drug release behavior of(DOX+VER)@ZIF-8.MCF-7 cells and drug resistant MCF-7/ADR cells with overexpressed FA receptors were selected to evaluate the cellular uptake for formulations.Fluorescence inverted microscope images and the results of flow cytometry together demonstrated that the cellular uptake for PEG-FA/(DOX+VER)@ZIF-8 was at high levels.The contrast experiments verified the improvement of cellular uptake for chemotherapeutic drug DOX through the FA receptors-mediated endocytosis and VER-mediated reversal of multidrug resistance.The MTT method was employed to investigate the in vitro cytotoxicity of formulations toward multiple tumor cells.The experimental results showed that the inhibition rates of PEG-FA/(DOX+VER)@ZIF-8 toward tumor cells were higher than those of DOX and DOX@ZIF-8.Near-infrared fluorescence imaging was employed to monitor the biodistribution of formulations in tumor-bearing mice.The results showed that PEG-FA/ZIF-8 could be used as a tumor-targeted drug nanocarrier to prolong the circulation of drugs in body and increase the accumulation of drugs at the tumor site.During the in vivo antitumor evaluation,the antitumor efficacy and side effects of different formulations were studied.PEG-FA/(DOX+VER)@ZIF-8 exhibited the highest tumor inhibition ratio and its low systemic toxicity toward major organs was verified by the hematoxylin and eosin(H&E)staining results.The construction of cytarabine-IR820 prodrug encapsulated ZIF-8 based on a versatile prodrug strategy and its application in chemo-photothermal combined antitumor therapy6-aminocaproic acid was used as the linker to synthesize the cytarabine-new indocyanine green(Ara-IR820)small molecule prodrug and the products were purified by silica gel column chromatography.1H nuclear magnetic resonance spectrometry(1H NMR)and mass spectrometry(MS)were employed to identify the chemical structure of the intermediate products and final products,confirming the successful synthesis.The ZIF-8 drug delivery nanoparticles loaded with Ara-IR820 were synthesized via the in situ one-pot method.Finally,hyaluronic acid(HA)was modified on the surface of Ara-IR820@ZIF-8 nanoparticles to get the tumor cells-targeted nanoparticles.The synthesized HA/Ara-IR820@ZIF-8 was characterized in vitro to study its morphology,particle size distribution in aqueous solution as well as the temperature-rising ability.TEM images showed that the HA/Ara-IR820@ZIF-8 nanoparticles were of uniform spherical-like morphology and the hydrodynamic diameter of the nanoparticles was measured by DLS to be approximately 132.0 nm(PDI=0.086),satisfied with the request of EPR effect.The UV spectrophotometry was employed to evaluate the drug loading capacity of HA/Ara-IR820@ZIF-8,and the drug loading content of Ara-IR820 was determined to be approximately 39.8%.The in vitro temperature-rising experiment was carried out to investigate the photothermal performance of formulations.Under the same concentration,the temperature-rising capacity of HA/Ara-IR820@ZIF-8 was higher than that of IR820 aqueous solution.After the irradiation of 5 min(1 W/cm2),the temperature of HA/Ara-IR820@ZIF-8(ciRS20=118 ?M)increased by 26.7?.The drug release behavior of HA/Ara-IR820@ZIF-8 was investigated in PBS with the pH values of 5.0 and 7.4,and the results verified the pH-responsive drug release behavior of the formulation.The hemolysis test was performed to preliminarily evaluate the biocompatibility and safety of HA/Ara-IR820@ZIF-8 for intravenous injection.The results showed that the hemolysis ratio of this formulation was less than 5%,which could be acceptable for intravenous administration.4T1 cells with overexpressed HA receptors(CD44)were selected to evaluate the cellular uptake for formulations.Fluorescence inverted microscope images and the results of flow cytometry together demonstrated that the cellular uptake for HA/Ara-IR820@ZIF-8 was at high levels.The competitive inhibition experiments verified that the CD44-mediated endocytosis could promote the actively targeting uptake of tumor cells for formulations.The MTT method was employed to investigate the therapeutic efficacy of HA/Ara-IR820@ZIF-8 toward tumor cells via chemotherapy and photothermal therapy(PTT).The results showed that the formulation exhibited strong cytotoxicity through the combination effect of chemotherapy and PTT.Near-infrared fluorescence imaging was employed to monitor the biodistribution of formulations in tumor-bearing mice.The results confirmed the excellent tumor-targeted accumulation of HA/Ara-IR820@ZIF-8 and provided the optimal time to give laser for the fluorescence imaging guided PTT.Subcutaneously tumor-bearing mice were built to study the in vivo temperature-rising capacity,antitumor efficacy and side effects of formulations.HA/Ara-IR820@ZIF-8 exhibited great temperature-rising ability and could be employed for antitumor PTT.During the in vivo antitumor evaluation,HA/Ara-IR820@ZIF-8 could inhibit tumor growth to a great extent and showed low side effects on the treated mice.Task-specific MOFs-based hybrid nanoplatforms for synergistic tumor cells-targeted treatment and dendritic cells-targeted immunomodulation toward enhanced anti-tumor immunotherapyThe IR820 loaded ZIF-8 and imiquimod(R837)and 1-methyl-D-tryptophan(1MT)co-loaded ZIF-8 nanoparticles were synthesized via the one-pot method.HA was modified on the surface of IR820@ZIF-8 to get the tumor cells-targeted nanoparticles(HA/IR820@ZIF-8).Mannan(MAN)was modified on the surface of R837 and 1MT co-loaded ZIF-8 nanoparticles to obtain the dendritic cells(DCs)-targeted nanoparticles(MAN/(R837+1MT)@ZIF-8).The prepared HA/IR820@ZIF-8 and MAN/(R837+1MT)@ZIF-8 were characterized in vitro to investigate the crystal structure,morphology,as well as their particle size distributions and zeta potentials in water.TEM images showed that HA/IR820@ZIF-8 and MAN/(R837+1MT)@ZIF-8 had spherical-like structures with relatively uniform particle size distributions.The hydrodynamic sizes of HA/IR820@ZIF-8 and MAN/(R837+1MT)@ZIF-8 were measured by DLS to be approximately 120.0 nm(PDI=0.119)and 221.0 nm(PDI=0.185),respectively.The UV spectrophotometry was employed to evaluate the drug loading capacity of HA/IR820@ZIF-8,and the drug loading content of IR820 was determined to be approximately 34.4%.The drug loading contents of R837 and 1MT in MAN/(R837+1MT)@ZIF-8 were determined to be 8.9%and 10.7%,respectively.The in vitro temperature-rising experiment verified the excellent photothermal performance of HA/IR820@ZIF-8,suggesting its potential for PTT.The hemolytic toxicity of the mixed formulation of HA/IR820@ZIF-8 and MAN/(R837+1MT)@ZIF-8 was measured,preliminarily suggesting the feasibility of this formulation for intravenous injection.The results of cell uptake experiments showed that the cellular uptake of HA/IR820@ZIF-8 by B16F10 cells was at high levels and verified the HA-mediated tumor cells-targeted drug delivery.In addition,the uptake kinetics of B16F10 cells for HA/IR820@ZIF-8 were investigated.The uptake of nanoparticles by tumor cells reached a high level at 4 h after the administration,at that time,laser irradiation could be carried out for PTT.MTT method and apoptosis experiments were used to study the dark toxicity and phototoxicity of HA/IR820@ZIF-8.The dark toxicity of formulations toward B16F10 cells was low in the absence of laser,while after the irradiation,HA/IR820@ZIF-8 showed high cell inhibition rates and induced severe cell apoptosis.The immunogenic cell death(ICD)of tumor cells induced by HA/IR820@ZIF-8 was then studied.Immunofluorescence staining was employed to examine the expression of calreticulin(CRT).Experimental results showed that the PTT based on HA/IR820@ZIF-8 could effectively induce the ICD of tumor cells and trigger the antitumor immune response.The cell uptake experiment was carried out to study the cellular uptake behavior of bone marrow-derived dendritic cells(BMDCs)for MAN/(R837+1 MT)@ZIF-8 and verified the MAN-mediated DCs-targeted drug delivery.The contents of kynurenine(kyn)in the culture medium of DCs were examined after the treatment with MAN/(R837+1MT)@ZIF-8 or 1MT.The results suggested that 1MT and MAN/(R837+1MT)@ZIF-8 both performed high inhibition ratios of kyn and could effectively inhibit the immunosuppressive indoleamine 2,3-dioxygenase(IDO)pathway.The expression of CD80 and CD86 on the surface of DCs as well as the concentrations of representative cytokines(tumor necrosis factor-a and interleukin-6)in the cell culture medium were detected to verify the stimulation effect of MAN/(R837+1MT)@ZIF-8 or tumor cell residues toward DCs maturation.These results demonstrated that the mixture of HA/IR820@ZIF-8 and MAN/(R837+1 MT)@ZIF-8 could effectively activate the antitumor immune response.Unilateral melanoma model was developed on mice to examine the in vivo biodistribution,temperature-rising effect antitumor ability and the systemic toxicity of formulations.It was determined based on the in vivo imaging that the laser irradiation could be performed at 6 h after the administration.HA/IR820@ZIF-8 exhibited efficient temperature-rising capacity.During the antitumor experiment,either HA/IR820@ZIF-8 or MAN/(R837+1MT)@ZIF-8 could inhibit tumor growth to some extent,while the mixture of HA/IR820@ZIF-8 and MAN/(R837+1MT)@ZIF-8 achieved the highest tumor inhibition ratio.To clarify the effect and mechanism of the mixed formulation in enhancing the antitumor immunotherapy,tumors were dissected from mice after the pharmacodynamic evaluation.Immunofluorescence staining and flow cytometry were employed to analysis the proportions of CD4+T cells,CD8+T cells and immunoregulatory T cells(Tregs)in lymphocytes.The results confirmed that the mixed formulation was able to synergistically increase the ratio of effector T cells(CD8+/CD4+T cells)to Tregs,enhancing the activity of antitumor immunotherapy.In addition,the proportions of CD4+T cells,CD8+T cells,Tregs and memory T cells in spleen were analyzed to verify the enhancement of systemic immunity and the generation of immune memory effect.Bilateral melanoma model was developed on mice to evaluate the abscopal antitumor effects of formulations.The results showed that the mixture of HA/IR820@ZIF-8 and MAN/(R837+1MT)@ZIF-8 could not only suppress the growth of primary tumors exposed to laser irradiation,but also inhibite the untreated abscopal tumors.The antitumor immune mechanism of formulations toward abscopal tumors was studied based on the analysis of the proportions of CD4+T cells,CD8+T cells and Tregs in the abscopal tumors.In summary,this study designed and constructed three MOFs-based active targeting nano drug delivery platforms for multifunctional antitumor therapy,focusing on solving the multidrug resistance during chemotherapy,the limitations of MOFs in drug loading,as well as the problems(multiple targets and low efficacy)in antitumor immunotherapy.On the one hand,this study offered novel,efficient and multifunctional antitumor nanoplatforms,exhibiting broad application prospects.On the other hand,this work provided new ideas for the applications of MOFs in the field of biomedicine. |
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