Metal-organic Framework Immobilized Enzyme and Its Application in Screening of Enzyme Inhibitors of Traditional Chinese Medicine：A Review / 中国实验方剂学杂志

Enzymes are widely used in chemical and pharmaceutical industries because of their advantages of high efficiency and specificity. However， the shortcomings of the free enzymes， such as poor stability and difficulty in recycling， limit their application. Therefore， the immobilization and application of enzymes have become one of the research hotspots. The selection of the immobilization carriers is a critical step in the process of enzyme immobilization. Metal-organic frameworks（MOFs）， a kind of porous materials， are formed by the coordination of metal ions or metal clusters with organic ligands. As an emerging immobilization carrier， its advantages such as high porosity， strong stability， and surface modifiability make it ideal for immobilized enzyme carriers. By immobilizing the free enzyme on MOFs， the above mentioned deficiencies of the free enzymes can be effectively solved， which greatly broaden the applicable condition. Ligand fishing is a method to find receptor-specific ligands from complex components， which has the advantages of high efficiency， simple sample pretreatment and high specificity. The MOF-enzyme complex formed by enzyme immobilization can act as a "fishing rod" for ligand fishing， which can screen out the targets from the complex system of components. The complex chemical composition and various active ingredients of traditional Chinese medicine（TCM） make the ligand fishing technology to play a big role in the screening of enzyme inhibitors from TCM. And the screened enzyme inhibitors are expected to be further developed into the lead compounds with good efficacy and low adverse effects， so the immobilized enzymes of MOFs have a wide application in the screening of active ingredients from TCM. Based on this， this paper summarized the methods of immobilized enzymes of MOFs in recent years， analyzed the characteristics， advantages and disadvantages of each method， and summarized the laws of preparation conditions and mechanisms. Meanwhile， the application and future development of immobilized enzymes of MOFs in the field of enzyme inhibitor screening from TCM were also summarized and prospected， with a view to providing a reference for the development of natural ingredients and the modernization of TCM.

A chiral metal-organic framework {(HQA)(ZnCl2)(2.5H2O)}n for the enantioseparation of chiral amino acids and drugs / 药物分析学报

Chiral metal-organic frameworks(CMOFs)with enantiomeric subunits have been employed in chiral chemistry.In this study,a CMOF formed from 6-methoxyl-(8S,9R)-cinchonan-9-ol-3-carboxylic acid(HQA)and ZnCl2,{(HQA)(ZnCl2)(2.5H2O)｝n was constructed as a chiral stationary phase(CSP)via an in situ fabrication approach and used for chiral amino acid and drug analyses for the first time.The{(HQA)(ZnCl2)(2.5H2O)}n nanocrystal and the corresponding chiral stationary phase were systematically characterised using a series of analytical techniques including scanning electron microscopy,X-ray diffraction,Fourier transform infrared spectroscopy,circular dichroism,X-ray photoelectron spectros-copy,thermogravimetric analysis,and Brunauer-Emmett-Teller surface area measurements.In open-tubular capillary electrochromatography(CEC),the novel chiral column exhibited strong and broad enantioselectivity toward a variety of chiral analytes,including 19 racemic dansyl amino acids and several model chiral drugs(both acidic and basic).The chiral CEC conditions were optimised,and the enantioseparation mechanisms are discussed.This study not only introduces a new high-efficiency member of the MOF-type CSP family but also demonstrates the potential of improving the enantiose-lectivities of traditional chiral recognition reagents by fully using the inherent characteristics of porous organic frameworks.

A metabolic intervention strategy to break evolutionary adaptability of tumor for reinforced immunotherapy

The typical hallmark of tumor evolution is metabolic dysregulation. In addition to secreting immunoregulatory metabolites, tumor cells and various immune cells display different metabolic pathways and plasticity. Harnessing the metabolic differences to reduce the tumor and immunosuppressive cells while enhancing the activity of positive immunoregulatory cells is a promising strategy. We develop a nanoplatform (CLCeMOF) based on cerium metal-organic framework (CeMOF) by lactate oxidase (LOX) modification and glutaminase inhibitor (CB839) loading. The cascade catalytic reactions induced by CLCeMOF generate reactive oxygen species "storm" to elicit immune responses. Meanwhile, LOX-mediated metabolite lactate exhaustion relieves the immunosuppressive tumor microenvironment, preparing the ground for intracellular regulation. Most noticeably, the immunometabolic checkpoint blockade therapy, as a result of glutamine antagonism, is exploited for overall cell mobilization. It is found that CLCeMOF inhibited glutamine metabolism-dependent cells (tumor cells, immunosuppressive cells, etc.), increased infiltration of dendritic cells, and especially reprogrammed CD8+ T lymphocytes with considerable metabolic flexibility toward a highly activated, long-lived, and memory-like phenotype. Such an idea intervenes both metabolite (lactate) and cellular metabolic pathway, which essentially alters overall cell fates toward the desired situation. Collectively, the metabolic intervention strategy is bound to break the evolutionary adaptability of tumors for reinforced immunotherapy.

Preparation of Fe3O4@SW-MIL-101-NH2 for selective pre-concentration of chlorogenic acid metabolites in rat plasma,urine,and feces samples / 药物分析学报

An innovative sandwich-structural Fe-based metal-organic framework magnetic material(Fe3O4@SW-MIL-101-NH2)was fabricated using a facile solvothermal method.The characteristic properties of the material were investigated by field emission scanning electron microscopy,transmission electron mi-croscopy(TEM),energy-dispersive X-ray spectroscopy,Fourier transform infrared spectroscopy,X-ray powder diffraction,vibrating sample magnetometry,and Brunauer-Emmett-Teller measurements.Fe3O4@SW-MIL-101-NH2 is associated with advantages,such as robust magnetic properties,high specific surface area,and satisfactory storage stability,as well as good selective recognition ability for chlorogenic acid(CA)and its metabolites via chelation,hydrogen bonding,and π-interaction.The results of the static adsorption experiment indicated that Fe3O4@SW-MIL-101-NH2 possessed a high adsorption capacity toward CA and its isomers,cryptochlorogenic acid(CCA)and neochlorogenic acid(NCA),and the adsorption behaviors were fitted using the Langmuir adsorption isotherm model.Then,a strategy using magnetic solid-phase extraction(MSPE)and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF MS/MS)was developed and suc-cessfully employed for the selective pre-concentration and rapid identification of CA metabolites in rat plasma,urine,and feces samples.This work presents a prospective strategy for the synthesis of magnetic adsorbents and the high-efficiency pretreatment of CA metabolites.

Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7 / 上海预防医学

ObjectiveA rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads. MethodsUsing rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated. ResultsThe detection limit of Escherichia coli O157∶H7 was 1.18×105 CFU‧mL-1， and the detection limit of DNA concentration was 9 pg‧μL-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）. ConclusionA method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.

Research progress on Fe-based metal-organic frameworks in antitumor drug delivery / 药学学报

Fe-based metal-organic frameworks (MOFs) are a class of polymer crystals formed by the combination of Fe ions or Fe clusters with organic ligands through coordination bonds. At present, Fe-based MOFs can be mainly prepared by solvothermal synthesis, ultrasonic synthesis, microwave synthesis, and dry-gel conversion, etc. Fe-based MOFs have the characteristics of strong drug loading capacity of inorganic nano-carrier and high safety of organic nano-carrier, and have good tumor targeting and the capacity of inducting tumor's ferroptosis, which have high potential in the delivery of antitumor drugs. Recently, Fe-based MOFs have also been developed with various functions such as imaging, magnetic hyperthermia, photothermal therapy, photodynamic therapy, and intelligent response, which can facilitate diagnosis and monitor drug distribution while delivering antitumor drugs, and can produce synergistic antitumor effects combined with thermotherapy and phototherapy, and can also control the precise release of drugs. Reviewing the advances in the synthesis methods, characteristics as well as functions and types of Fe-based MOFs can provide a basis for the further applications of Fe-based MOFs in antitumor drug delivery.

Simultaneous improvement to solubility and bioavailability of active natural compound isosteviol using cyclodextrin metal-organic frameworks

Cyclodextrin metal-organic framework (CD-MOF) as a highly porous supramolecular carrier could be one of the solutions to the insolubility of isosteviol (STV). The solubility of STV was lower than 20.00 ng/mL at pH 1.0 and pH 4.5, whilst its solubility increased to 20,074.30 ng/mL at pH 6.8 and 129.58 ng/mL in water with a significant pH-dependence. The

Synthesis of (S)-4-fluorophenylglycine by using immobilized amidase based on metal-organic framework / 生物工程学报

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 μmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.

Preparation and evaluation of methotrexate sustained-release particles using crosslinked cyclodextrin metal-organic frameworks / 药学学报

Methotrexate (MTX) injection has a short half-life and significant toxic side effects. In order to overcome the demerits of MTX injection, MTX@COF was prepared for subcutaneous injection by loading MTX in crosslinked cyclodextrin metal-organic framework (COF) in this study. The cationic lipid material (2, 3-dioleoyl-propyl)-trimethylamine (DOTAP) was then coated on the MTX@COF surface by solvent evaporation. Different surface charge characteristics were observed in the coated MTX@COF@DOTAP with no significant change in particle morphology. The in vitro release behaviors of sustained-release particles were investigated in water and phosphate buffer (pH 7.4), and the in vivo release characteristics were evaluated for pharmacokinetics in rats. The in vitro release results showed that the cumulative release of MTX, MTX@COF and MTX@COF@DOTAP within 6 h was 92.70%, 36.31% and 18.19% in water, respectively; the cumulative release of MTX, MTX@COF and MTX@COF@DOTAP within 4 h was 90.82%, 79.37% and 58.30% in phosphate buffer, respectively; the results showed that MTX@COF can significantly delay the release of MTX, the modification to MTX@COF by DOTAP can further delay the release of MTX. Pharmacokinetic studies in rats showed that the mean retention time [MRT(0-t)] and the time to peak (Tmax) of the subcutaneous injection of MTX@COF@DOTAP group were significantly prolonged compared with the MTX@COF group and the MTX group. The area under the concentration-time curve [AUC(0-t)] of the MTX@COF@DOTAP subcutaneous injection group was 1.8 times high as that of the MTX group. In this study, MTX@COF@DOTAP particles had a certain sustained-release effect, and could prolong the bioavailability of MTX by subcutaneous injection, which provided a new idea for the development of new MTX dosage forms.

Recent advances of metal-organic frameworks applied in biomedicine / 药学学报

Metal-organic frameworks (MOFs) are porous crystalline polymers constructed from the coordination reaction between organic ligands and metal ions. Due to their advantages: adjustable periodic pore structure, large specific surface area and easy functional modification, etc., MOFs have been widely used in the fields of gas storage/separation, catalysis, sensing, biological imaging and drug delivery. In recent years, MOFs have shown great potential in disease diagnosis and treatment. This review summarizes the application of MOFs in the fields of bio-sensing, cell imaging, in vivo imaging, drug delivery, etc., discusses the problems and corresponding solutions in the application of MOFs for biomedicine. We hope this review can provide reference for the designing new methods for disease diagnosis and treatment.

Magnetic metal organic framework for pre-concentration of ampicillin from cow milk samples / 药物分析学报

The aim of this study is a present of a simple solvothermal synthesis approach to preparation of Cu-based magnetic metal organic framework (MMOF) and subsequently its application as sorbent for ultrasound assisted magnetic solid phase extraction (UAMSPE) of ampicillin (AMP) from cow milk samples prior to high performance liquid chromatography-Ultraviolet (HPLC-UV) determination. Characteristics of pre-pared MMOF were fully investigated by different techniques which showed the exclusive properties of proposed sorbent in terms of proper functionality, desirable magnetic property and also high specific surface area. Different influential factors on extraction recovery including sorbent dosage, ultrasonic time, washing solvent volume and eluent solvent volume were assessed using central composite design (CCD) based response surface methodology (RSM) as an operative and powerful optimization tool. This is the first report for determination of AMP using MMOF. The proposed method addressed some drawbacks of other methods and sorbents for determination of AMP. The presented method decreases the extraction time (4 min) and also enhances adsorption capacity (250 mg/g). Moreover, the magnetic property of presented sorbent (15 emu/g) accelerates the extraction process which does not need filtration, centrifuge and precipitation procedures. Under the optimized conditions, the proposed method is applicable for linear range of 1.0-5000.0μg/L with detection limit of 0.29μg/L, satisfactory recoveries (≥95.0％) and acceptable repeatability (RSD less than 4.0％). The present study indicates highly promising perspectives of MMOF for highly effective analysis of AMP in complicated matrices.

A homogenous nanoporous pulmonary drug delivery system based on metal-organic frameworks with fine aerosolization performance and good compatibility

Pulmonary drug delivery has attracted increasing attention in biomedicine, and porous particles can effectively enhance the aerosolization performance and bioavailability of drugs. However, the existing methods for preparing porous particles using porogens have several drawbacks, such as the inhomogeneous and uncontrollable pores, drug leakage, and high risk of fragmentation. In this study, a series of cyclodextrin-based metal-organic framework (CD-MOF) particles containing homogenous nanopores were delicately engineered without porogens. Compared with commercial inhalation carrier, CD-MOF showed excellent aerosolization performance because of the homogenous nanoporous structure. The great biocompatibility of CD-MOF in pulmonary delivery was also confirmed by a series of experiments, including cytotoxicity assay, hemolysis ratio test, lung function evaluation,

Drug nanoclusters formed in confined nano-cages of CD-MOF: dramatic enhancement of solubility and bioavailability of azilsartan

Tremendous efforts have been devoted to the enhancement of drug solubility using nanotechnologies, but few of them are capable to produce drug particles with sizes less than a few nanometers. This challenge has been addressed here by using biocompatible versatile -cyclodextrin (-CD) metal-organic framework (CD-MOF) large molecular cages in which azilsartan (AZL) was successfully confined producing clusters in the nanometer range. This strategy allowed to improve the bioavailability of AZL in Sprague-Dawley rats by 9.7-fold after loading into CD-MOF. The apparent solubility of AZL/CD-MOF was enhanced by 340-fold when compared to the pure drug. Based on molecular modeling, a dual molecular mechanism of nanoclusterization and complexation of AZL inside the CD-MOF cages was proposed, which was confirmed by small angle X-ray scattering (SAXS) and synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR) techniques. In a typical cage-like unit of CD-MOF, three molecules of AZL were included by the -CD pairs, whilst other three AZL molecules formed a nanocluster inside the 1.7 nm sized cavity surrounded by six -CDs. This research demonstrates a dual molecular mechanism of complexation and nanoclusterization in CD-MOF leading to significant improvement in the bioavailability of insoluble drugs.

Improving the dissolution rate of water-insoluble diflunisal by γ-cyclodextrin metal-organic framework / 药学学报

The aim of this study is to prepare porous γ-cyclodextrin metal-organic framework (CD-MOF) with good biocompatibility to improve the in vitro release properties of water-insoluble drugs. Different sizes of CD-MOF were obtained by controlling the self-assembly of γ-cyclodextrin and potassium ion and the rate of crystal growth. The poorly water-soluble diflunisal (DIF) was selected as the model drug and loaded into the interior of porous CD-MOF by the impregnation method. The DIF loaded CD-MOF (DIF-MOF) was characterized by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), nitrogen adsorption and desorption, Fourier infrared spectrometer and thermogravimetric analysis. In addition, in vitro cytotoxicity and solubilizing capability of CD-MOF were investigated. It revealed that the obtained CD-MOF was cubic-like with a narrow size distribution and high porosity. Negligible cytotoxicity was found after incubation with RAW264.7 cells. Compared with the pure CD-MOF carrier, the morphology and crystal form of DIF-MOF was not damaged during the drug loading process. Moreover, the solubility and release rate of water-insoluble DIF from the DIF-MOF were significantly increased.

Novel nano-scaled Mn-metal-organic framework for enhancing photodynamic therapy through overcoming tumor hypoxia / 中国药科大学学报

@#A Mn-clusters-porphyrin metal-organic framework nanosheet(nMn-MOF)was synthesized by coordination chelation to enhance photodynamic therapy. The nanosheet was characterized by dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy. Oxygen sensor and ICG were used to investigate the production of oxygen and the singlet oxygen(1O2)generation. The cytotoxicity of the nanosheet against tumor cells were detected by CCK-8 assay, and the anti-hypoxia and oxygen-generation ability of nanosheets were investigated by fluorescence staining assay. The results indicated that this nanosheet could catalyze the intracellular H2O2 into O2, which overcame the tumor hypoxia. Furthermore, the generated oxygen was converted to cytotoxic 1O2 under the near infrared light irradiation, thereby enhancing photodynamic therapy.

Synthesis and anti-myocarditis activity in a multifunctional lanthanide microporous metal-organic framework with 1D helical chain building units

A new microporous lanthanide metal-organic framework, {[Yb(BTB)(H2O) (DEF)2}n (1, DEF=N,N-Diethylformamide), with 1D nano-sized channels has been constructed by bridging helical chain secondary building units with 1,3,5-benzenetrisbenzoic acid (H3BTB) ligand. Structural characterization suggests that this complex crystallizes in the hexagonal space group P6122 and possesses 1D triangular channels with coordinated water molecules pointing to the channel center. In addition, anti-myocarditis properties of compound 1 were evaluated in vivo. The results showed that compound 1 can improve hemodynamic parameters of, and it may be a good therapeutic option for heart failure in the future.

Facile Synthesis of the Magnetic Metal Organic Framework Fe3O4@UiO-66-NH2 for Separation of Strontium / 生物医学与环境科学（英文）

A magnetic metal organic framework (MMOF) was synthesized and used to separate Sr2+ in aqueous solution. The shape and structure of prepared Fe3O4@UiO-66-NH2 were characterized, and the absorbed concentration of strontium was determined through inductively coupled plasma mass spectrometry. The results indicated that Fe3O4 and UiO-66-NH2 combined through chemical bonding. The experimental adsorption results for separation of Sr2+ in aqueous solution indicated that the adsorption of Sr2+ to Fe3O4@UiO-66-NH2 increased drastically from pH 11 to pH 13. The adsorption isotherm model indicated that the adsorption of Sr2+ conformed to the Freundlich isotherm model (R2 = 0.9919). The MMOF thus inherited the superior qualities of magnetic composites and metal organic frameworks, and can easily be separated under an external magnetic field. This MMOF thus has potential applications as a magnetic adsorbent for low level radionuclide 90Sr.

Optimization of the Preparation Technology of Ferulic Acid/K/β-CD/Metal Organic Framework Inclusion / 中国药房

OBJECTIVE:To prepare Ferulic acid/K/β-CD/metal organic framework (FA/K/β-CD/MOF) inclusion,and to opti-mize its preparation technology. METHODS:K/β-CD/MOF was synthesized by solvothermal method as inclusion material. Using FA as main component,FA/K/β-CD/MOF was prepared by grinding method. The preparation technology was optimized by orthogo-nal test using mole ratio of main component-inclusion material,grinding time,dropping time and inclusion temperature as factors, inclusion rate as index. Prepared FA/K/β-CD/MOF was indentified by IR spectrum and DSC,and inclusion rate and dissolution rate were determined. RESULTS:Optimized preparation technology was as follows as mole ratio of main main component to inclusion material 3∶1,dropping time 60 min,inclusion temperature 40 ℃,inclusion time 60 min. Prepared FA/K/β-CD-MOF had already formed a new kind of phase,and its average inclusion rate was(18.0±1.6)%(RSD=0.9%,n=6);its solubility was 15 times as much as FA(9.582 mg/ml vs. 0.647 mg/ml). CONCLUSIONS:FA/K/β-CD/MOF is prepared successfully;and the preparation tech-nology is reasonable and feasible.

Investigation of anticancer drug 5-Fu loaded porous metal-organic frameworks of MIL-53 / 中国药学杂志

OBJECTIVE: To study the drug release and cytotoxicity in vitro of 5-fluorouracil (5-Fu)-loaded porous metal-organic frameworks of MIL-53. METHODS: MIL-53 was hydrothermally synthesized, and the synthetic MIL-53 was characterized by TEM, XRD, IR and TG. 5-Fu was loaded into MIL-53, and the release pattern and cytotoxicity were investigated. RESULTS: The structure of MIL-53 was confirmed. The diameter of MIL-53 was at nanometer level and MIL-53 displayed better thermo-stabilization. The highest drug loading rate was 0.431 g · g-1 MIL-53 for 5-Fu. The drug release profile of 5-Fu-loaded MIL-53 displayed a clear biphasic release pattern. Cytotoxicity test showed that MIL-53 had good biocompatility at lower concentrations. CONCLUSION: MIL-53 is a promising platform for drug delivery due to its large pore sizes for drug encapsulation and good biocompatility at lower concentrations.