CN114377149B - Mn-based degradable MOF nano-reactor and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a Mn-based degradable MOF nano-reactor, a preparation method and application thereof, wherein the nano-reactor comprises inner core Mn-based MOF nano-particles, the surfaces of which are functionalized and grafted with pH responsive shell copolymer PEG-CDM-PEI, and the Mn-based MOF nano-particles are loaded with biological enzymes GOx and IDO immunosuppressant. The invention prepares the pH/ROS dual-responsiveness MOF nano-reactor drug controlled release system for the co-load immune checkpoint IDO inhibitor 1-MT and GOx by utilizing the advantages of high load capacity, restriction enzyme reaction space, ROS responsiveness degradation and the like of the MOF nano-reactor, and regulates and controls the tumor hunger/oxidation/IDO immune combined treatment. The drug control system can respond to weak acidic tumor microenvironment, induce size reduction/charge reversal, overcome in vivo tumor permeation and cell barrier, and improve delivery efficiency; in addition, the invention has the self-enhanced MOF degradation and drug release performance; the drug control system mediated immunotherapy can effectively inhibit immune tolerance, enhance the anti-tumor immune response of organisms and inhibit tumor growth, metastasis and recurrence.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a Mn-based degradable MOF nano-reactor and a preparation method and application thereof.
Background
Malignant proliferation of tumor seriously endangers human health, and immunotherapy can restore anti-tumor immune response of organism, and can effectively kill tumor, so that it has potential clinical application prospect. For example, the immune therapy represented by inhibiting immune check points (such as indoleamine 2, 3-dioxygenase, IDO) can obviously inhibit the immune tolerance of organisms and restore the activity of various effector T cells, and has obvious early clinical effects. However, inadequate immune response and low bioavailability have affected its clinical use. For example, glucose oxidase (GOx) mediated starvation therapy of tumors can not only competitively consume glucose necessary for their growth, but also generate Reactive Oxygen Species (ROS). Starvation/oxidation synergistically induces tumor immunogenic death, which is effective in initiating and enhancing immune responses, but this strategy is also limited by low bioavailability. Therefore, how to safely and efficiently deliver the small molecules to tumor focus, regulate and control the starvation/oxidation/IDO immune combined treatment of the tumor, realize efficient immune response and improve the tumor treatment effect, and have important scientific significance and potential application prospect.
Considering the defects of poor stability, easy inactivation and the like of GOx and 1-MT, the development of a metal organic framework polymer (MOF) nano reactor with high fidelity enzyme activity provides guarantee for maintaining the biological activity of the molecules. However, the physicochemical properties and action mechanisms of GOx and 1-MT are greatly different, so that the complex in vivo delivery barriers (such as blood barriers, tumor tissue barriers, cell uptake barriers and the like) are required to be safely and efficiently overcome, the bioavailability of the drug is improved, the loaded drug is also required to be responsively released at the tumor focus, the combined therapy of tumor starvation/oxidation/IDO immunity is activated, and the necessary functional modification is required to be carried out on the MOF nano-reactor, so that the MOF nano-reactor has the characteristics of tumor permeation, controllable release and in vivo degradability, and thus the efficient tumor killing is realized. Based on the method, the pH/ROS dual-response degradable Mn-based MOF nano-reactor with the characteristics of enhancing tumor permeation and self-amplifying drug release is constructed, GOx and 1-MT are loaded together, and the hunger/oxidation and immunotherapy strategies are combined, so that tumors are effectively killed, tumor growth and metastasis are inhibited, and scientific basis and practical reference are provided for construction of a functional MOF controlled release system and hunger/oxidation/IDO immune combined therapy.
Disclosure of Invention
The invention aims to solve the technical problem of providing a Mn-based degradable MOF nano-reactor, a preparation method and application thereof, wherein the Mn-based degradable MOF nano-reactor can load biological enzymes and immunotherapeutic drugs with high fidelity, can programmatically overcome a plurality of physiological barriers in a body, improves drug delivery efficiency and bioavailability, is beneficial to combining starvation/oxidation treatment and tumor immunotherapy by a delivery system, and can efficiently kill tumors and inhibit tumor growth and metastasis.
The technical problems to be solved by the invention are realized by the following technical scheme:
a Mn-based degradable MOF nanoreactor comprising inner core Mn-based MOF nanoparticles surface functionalized grafted pH responsive sheath copolymer PEG-CDM-PEI loaded with biological enzymes GOx and IDO immunosuppressants.
Preferably, the Mn-based MOF nanoparticle is Mn-DTA and the IDO immunosuppressant is 1-MT.
Preferably, the nano-reactor is spherical and has a particle size of 100nm.
The preparation method of the Mn-based degradable MOF nano reactor comprises the following steps:
(1) Preparation of inner core Mn-based MOF nanoparticles Mn-DTA:
(2) Synthesizing pH responsive shell PEG-CDM-PEI;
(3) Preparation of PEG-CDM-PEI-Mn-DTA particles;
(4) Preparation of PCP-Mn-DTA@GOx@1-MT controlled release system.
Preferably, step (1) comprises:
11 Synthesis of ROS-responsive organic ligand 5, 5-dimethyl-4, 6-dithioazelaic acid (DTA): dissolving 3-mercaptopropionic acid (MPA) in an acetone solution, and continuously stirring at room temperature; placing the mixed system in an ice bath for overnight crystallization; filtering and collecting crystals; repeatedly washing filtrate crystals with normal hexane and cold water, and vacuum drying to obtain DTA;
12 Synthesis of ROS-sensitive core Mn-based MOF nanoparticles Mn-DTA by hydrothermal reaction: manganese chloride (MnCl) 2 ) And DTA are dissolved in N, N-Dimethylformamide (DMF), respectively; adding the solution into a centrifuge tube, and adding polyvinylpyrrolidone K30 (PVP-K30) and triethylamine into the solution; adding the prepared DMF/ethanol solvent into a pipe, wherein the volume ratio of DMF/ethanol is 5/3, and fixing the volume to a certain volume; ultrasonically dispersing the mixture, transferring the mixture into a hydrothermal synthesis reactor, and reacting at a high temperature; after naturally cooling to room temperature, collecting the product by centrifugation, and then dispersing in ethanol for later use; the obtained MOF nanoparticle was designated Mn-DTA.
Preferably, step (2) comprises:
21 Synthesis of PEG-CDM: firstly, reacting maleic anhydride (CDM) with oxalyl chloride, and vacuum drying to obtain CDM; next, adding the product into methylene chloride (DCM) solution in which polyethylene glycol monomethyl ether (mPEG-OH) and pyridine are dissolved, and reacting at room temperature; subsequently, the reaction was terminated with a saturated ammonium chloride solution. Extracting, separating and drying the organic phase, and precipitating for 2-3 times by ice bath to obtain PEG-CDM;
22 Synthesis of PEG-CDM-PEI: PEG-CDM and branched Polyethylenimine (PEI) were dissolved in DMSO, 4-Dimethylaminopyridine (DMAP) was added dropwise, stirred at 0deg.C for 0.5h, then the system was left at room temperature and continued to react in the absence of light; the mixture was dialyzed against double distilled water and freeze-dried to give the final product PEG-CDM-PEI.
Preferably, the step (3) specifically comprises:
dissolving Mn-DTA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC.HCl (6 mM) and NHS (6 mM) obtained in the step (2) in PBS (pH 7.4), and stirring at room temperature; PEG-CDM-PEI (0.06 mmol) dispersed in PBS (pH 7.4) was then added to the above solution and the reaction continued for 36h; the product was collected by centrifugation (PEG-CDM-PEI-Mn-DTA, designated PCP-Mn-DTA).
Preferably, the step (4) specifically comprises:
Mn-DTA, GOx and 1-MT are dissolved in PBS (pH 7.4) and stirred at room temperature for reaction for 24 hours; centrifuging to remove the unloaded medicine; the mixture was then resuspended in PBS (pH 7.4) solution at pH7.4 consisting of EDC. HCl (15 mM) and NHS (15 mM); referring to the synthesis method of PCP-Mn-DTA nano-particles, centrifugally collecting a nano-reaction controlled-release system loaded with the drug, and freeze-drying to obtain a final product, which is named as PCP-Mn-DTA@GOx@1-MT.
An application of Mn-based degradable MOF nano-reactor in preparing functional drug delivery system for tumor hunger, oxidization or immune combination therapy.
The principle of the application is as follows:
the application utilizes a hydrothermal synthesis method to prepare N, N-dimethylformamide, ethanol and other reducing solvents and organic ligands of 5, 5-dimethyl-4, 6-dithioazelaic acid and MnCl 2 Mixing and adding the mixture into a polytetrafluoroethylene reaction kettle to synthesize the MOF nanoparticle inner core. Subsequently, a pH responsive sloughable shell copolymer PEG-CDM-PEI is synthesized, chemically grafted on the surface of MOF core particles and loaded with drug molecules, and the Mn-based MOF nano-reactor controlled release system is prepared. Mn prepared by the applicationThe MOF-based controlled release system integrates the advantages of high drug loading capacity, easiness in surface modification, reduced pH response size, charge reversal, ROS response degradability and the like, is easy to prepare and store, and has potential application in the aspects of biological enzyme, drug delivery and tumor treatment.
The system can safely and efficiently load glucose oxidase GOx and immunological adjuvant 1-MT, and targets tumor focus by virtue of EPR effect. Then, in response to low pH stimulation of tumor microenvironment, the pH sensitive bond bridged CDM molecules are broken, the shell PEG polymer is rapidly detached, and PEI modified MOF inner core carriers are remained on the exposed surface, so that carrier size reduction and charge reversal are realized, small-size carriers (Mn-based MOF inner core size is about 50 nm) are easier to permeate into the deep part of the tumor, positive charge characteristic materials are selectively and efficiently absorbed by tumor cells, physiological barriers in vivo are further overcome, and in vivo drug delivery efficiency is improved. More importantly, after being ingested by tumor cells, the system can further respond to the high intracellular level ROS in the tumor, degrade MOF in situ and release loaded drugs. On the one hand, the released GOx can competitively consume glucose in tumors to cause tumor starvation, not only can kill tumor cells and improve tumor immunogenicity, but also can recruit a large number of immune cells to attack tumor lesions, and can synchronously generate ROS, amplify the degradation of MOF in a cascading way and promote drug release. On the other hand, released 1-MT inhibits the IDO activity of immune checkpoints, restores T cell activity, and thereby relieves immune tolerance. The MOF controlled release system combines GOx-regulated tumor starvation/oxidation treatment and 1-MT-regulated immunotherapy, so that not only is the internal immune tolerance effectively relieved, but also the anti-tumor immune response of an organism is effectively enhanced, the tumor treatment effect is synergistically improved, and the MOF controlled release system has good biological safety and clinical potential medical prospect.
The technical scheme of the invention has the following beneficial effects:
(1) The invention uses ROS sensitive bond and Mn 2+ The Mn-based degradable MOF nano-reactor is synthesized by covalent crosslinking, can respond to the ROS over-expressed in tumor cells, can rapidly degrade and release the loaded medicine in situ, and has good biological safety; (2) The surface of the nano-reactor prepared by the invention is functionalized with the PEG-CDM-PEI shell, so that nonspecific adsorption can be avoidedAnd prolong the blood circulation time and overcome the blood circulation barrier; the controlled release system also has the characteristics of pH response size reduction and charge reversal, can overcome tumor penetration and cell uptake barriers, and improves the delivery efficiency; in addition, the system also has ROS responsive degradable performance, can respond to the ROS with high intracellular concentration of the tumor, degrade and release the loaded medicine in situ, and further improve the bioavailability of the medicine; (3) The PCP-Mn-DTA@GOx@1-MT nanometer controlled release system prepared by the method can enhance in-vivo immune response through starvation/oxidation/immune combination treatment. In one aspect, GOx-mediated starvation/oxidation therapy promotes effector T cell recruitment and immune response; on the other hand, the 1-MT mediated IDO blocking immunotherapy relieves the immune tolerance, and the two are combined to cascade and amplify the anti-tumor immune response of the organism, so that the tumor is killed efficiently; (4) The PCP-Mn-DTA@GOx@1-MT nano controlled release system prepared by the method successfully activates the immune memory of the organism, inhibits the metastasis and recurrence of tumors, and has a long-acting immune memory function.
Drawings
FIG. 1 is an XRD characterization of Mn-DTA in the present invention: wherein A is the XRD pattern of Mn-DTA-MOF (MnS, JCPDS No. 89-4089); b is the unit cell structure of MnS calculated by the CCDC database (ICSD entry: 44765); c and D are crystal structures (3×3); e is a schematic representation of the combination and crystal morphology of Mn-DTA-MOF, with purple and yellow spheres representing Mn and S atoms, respectively.
FIG. 2 is a transmission electron microscope and particle size distribution plot of the product of the present invention: wherein A and B are TEM image and EDS map of Mn-DTA, respectively: yellow represents Mn element, blue represents S element, and green represents O element; c is a TEM image of PCP-Mn-DTA@GOx@1-MT; d is a DLS plot of Mn-DTA and PCP-Mn-DTA@GOx@1-MT, scale A and scale B: 50nm, scale C: 100nm.
FIG. 3 is N of Mn-DTA in the present invention 2 Absorption-desorption diagram: wherein A is an isothermal diagram and B is a corresponding BJH pore size distribution diagram.
FIG. 4 is a nuclear magnetic, gel permeation chromatography and infrared spectrum of the product of the present invention: wherein A, B, C is a nuclear magnetic spectrum of DTA, PEG-CDM-PEI, respectively; d is the gel permeation chromatogram of PEG-CDM-PEI, and A and B are the infrared chromatograms of PEG-CDM and PEG-CDM-PEI, respectively, in E.
FIG. 5 is a DLS map of the product of the present invention under different conditions &Zeta diagram&TEM image&UV diagram: wherein A is PCP-Mn-DTA@GOx@1-MT at pH 7.4, pH 6.8, pH 7.4+H 2 O 2 And pH 6.8+H 2 O 2 DLS plot after 4h treatment and Mn-DTA; b is Zeta potential value of Mn-DTA and PCP-Mn-DTA nano-particles at pH 7.4 and pH 6.8 respectively; c and D are each H 2 O 2 TEM images of Mn-DTA and PCP-Mn-DTA@GOx@1-MT were processed for 4H, E being GOx, 1-MT and PCP-Mn-DTA@GOx@1-MT containing different concentrations of H 2 O 2 Or absorption spectrum incubated in glucose for 12H, F is H with different concentrations 2 O 2 Or GOx cumulative release level of PCP-Mn-DTA@GOx@1-MT system after glucose treatment; c scale: 50nm, scale d: 100nm.
FIG. 6 is a graph of cellular activity and intracellular ROS assay: wherein A is a cell activity diagram of the PCP-Mn-DTA@GOx@1-MT controlled release system; b is CLSM plot of intracellular ROS detection; c is a fluorescent quantitative statistical graph; the scale of B is 100 μm.
FIG. 7 is an in vitro tumor penetration graph of the PCP-Mn-DTA@GOx@1-MT controlled release system of the invention: wherein, A is a CLSM graph of incubating tumor multicellular balls with the system for 4h and 12h under the conditions of pH 7.4 and pH 6.8 respectively, and B is fluorescence quantification statistics; a scale: 100 μm.
FIG. 8 is a graph of M analysis of tumor cell uptake efficiency of the MOF controlled release system of the present invention at different pH conditions: wherein A is CLSM image of B16F10 cells incubated with PCP-Mn-DTA@GOx@1-MT controlled release system under pH 7.4 and pH 6.8 for 4h and 12h respectively, and DAPI (blue) and ActinRed are used for cell nucleus and cytoskeleton respectively TM 555 (red) staining; b is quantitative statistical analysis corresponding to FCM under the processing conditions; scale of a: 50 μm.
FIG. 9 is a Western blot of induced apoptosis-related proteins of B16F10 cells treated with different nanoparticles according to the invention: wherein A is a western blot image, B and C are quantitative statistical analysis images of protein level, C and D are flow cytometry analysis images and quantitative analysis images of apoptosis by using Annexin V-FITC/PI staining after B16F10 cells are treated by different nano particles.
FIG. 10 is a graph of in vitro inhibition of IDO expression and promotion of T cell proliferation analysis of a controlled release system of MOF of the present invention: wherein a is IDO western blot of induced B16F10 cells treated with different nanoparticles; b is a quantitative analysis chart of IDO protein; c is the analysis of EdU with FCM after co-culture of different nanoparticles with B16F10 cells + Ratio of T cells graph (T cells not treated with B16F10 cells as positive control); d is EdU + Quantitative analysis of T cells, E is a schematic diagram of the mechanism of inhibition of IDO1 in tumor cells by the PCP-Mn-DTA@GOx@1-MT controlled release system.
FIG. 11 is a graph of an in vitro analysis of inhibition of tumor cell proliferation, metastasis and invasion by the MOF controlled release system of the present invention: wherein A is a microscopic image of scratch healing of B16F10 cells after incubation of different drugs; b is a quantitative analysis graph of scratch healing, C and D are quantitative analysis graphs of migration and attack tests; scale of a: 100 μm.
FIG. 12 is a graph showing in vivo therapeutic and pathological analysis of the MOF controlled release system of the present invention: wherein A is the tumor size of a tumor-bearing mouse after treatment of physiological saline, PCP-Mn-DTA, 1-MT, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT for different times; b is a plot of relative tumor volume change during the dosing treatment; c and D are mice survival curves and weight change plots; e is tumor tissue H & E, TUNEL and Ki-67 immunofluorescence staining; scale of E: 100 μm.
FIG. 13 is a graph of a flow assay for modulating T cell immune response in vivo using a controlled release system of MOF of the invention: wherein A and B are CD8 + Toxic T cells and CD4 + Auxiliary T cell flow detection and corresponding quantitative statistics; c and D are flow-through detection and quantitative statistics of tumor-infiltrating Tregs cells.
FIG. 14 is a graph showing the flow assay of the immune response of the MOF controlled release system of the present invention to regulate DC cells and B, NK cells in vivo: wherein A and B are flow detection and quantification statistical graphs of tumor infiltrating DC cells; c and D are flow detection and quantitative statistical graphs of tumor infiltrating B cells; e and F are flow-through detection and quantification statistical graphs of tumor infiltrating NK cells.
FIG. 15 is a graph showing in vivo IDO inhibition activity and expression of related pathway proteins in a MOF controlled release system according to the present invention: wherein A is Kyn/Trp ratio of C57BL/6 mice bearing B16F10 tumor 4 days after administration; b is immunofluorescence staining image of tumor sections stained with anti IDO1, anti mTOR and anti STAT3, respectively; scale of B: 100 μm.
FIG. 16 is biosafety and pharmacokinetics of the MOF controlled release system of the invention: wherein A is the change in blood glucose levels of the mice before and after 1 hour of treatment; b and C are AST and ALT concentrations that assess liver function after 10d of treatment; d is H & E staining of each organ of the tumor-bearing mice; e and F are drug generation curves of tumor-bearing mice after intravenous injection of 1-MT and PCP-Mn-DTA@GOx@1-MT nano particles for 24 hours and the distribution of 1-MT in various organs and tumors.
FIG. 17 is an in vivo anti-tumor metastasis profile of a controlled release system of MOFs of the present invention: wherein, A and B are lung tumor metastasis nodules and quantitative statistical graphs of B16F10 malignant tumor lung metastasis model mice of each administration treatment group.
FIG. 18 is a graph of an in vivo anti-tumor recurrence and long-term immunomemory analysis of a controlled-release system of MOF according to the present invention: wherein, A is a schematic diagram for evaluating the recurrence degree of tumor by the administration route of the above treatment group, B is tumor recurrence rate statistics of mice after various administration, C, D and E are in vivo effector memory T cells and central memory T cells horizontal flow detection and quantification statistics after secondary injection of tumor cells into mice.
FIG. 19 is a schematic representation of the preparation of the PCP-Mn-DTA@GOx@1-MT controlled release system of the invention in combination with starvation/oxidation/IDO immunotherapy in vivo.
Detailed Description
Specific embodiments of the invention are described in detail below to facilitate a further understanding of the invention.
All experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples were commercially available unless otherwise specified.
Example 1 reactor and method of making the same
A controlled release system PCP-Mn-DTA@GOx@1-MT of a Mn-based degradable MOF nano reactor is prepared by taking Mn-DTA of Mn-based ROS responsive degradable MOF nano particles as an inner core, functionally grafting a pH responsive PEG-CDM-PEI shell on the surface of the inner core, and loading GOx and 1-MT together.
The preparation process of the Mn-based degradable MOF nano-reactor controlled release system PCP-Mn-DTA@GOx@1-MT comprises the following steps:
(1) Preparation of inner core Mn-based MOF nanoparticles Mn-DTA:
11 Synthesis of ROS-responsive organic ligand 5, 5-dimethyl-4, 6-dithioazelaic acid (DTA):
4.9mmol of 3-mercaptopropionic acid (MPA) was weighed out and dissolved in 9.82mmol of acetone solution and stirred continuously at room temperature for 8 hours. The above mixture was then placed in an ice bath (ice water mixture) overnight to crystallize. Filtering and collecting crystals. Subsequently, the filtrate crystals were repeatedly washed with n-hexane and cold water, and after vacuum drying, the resulting product, DTA, was collected as follows:
12 Preparation of Mn-DTA:
firstly, 50mg of manganese chloride (MnCl) 2 ) And 15mg of DTA were dissolved in 1mL of N, N-Dimethylformamide (DMF), respectively; next, 107. Mu.L of MnCl2 solution and 347. Mu.L of DTA solution were added to a 15mL centrifuge tube; then, 300mg of polyvinylpyrrolidone K30 (PVP-K30) and 200. Mu.L of triethylamine were further added thereto; thereafter, the prepared DMF/ethanol (v/v=5/3) solvent was added to the tube until the volume was 13mL. Next, the mixture was ultrasonically dispersed, transferred to a hydrothermal synthesis reactor, and then reacted at a high temperature of 150 ℃ for 24 hours. After naturally cooling to room temperature, the product was collected by centrifugation and then dispersed in ethanol for use. The obtained MOF nanoparticle was designated Mn-DTA.
(2) Carrying out surface functionalized grafting on the inner core:
21 Synthesis of PEG-CDM:
firstly, uniformly dispersing 6mmol of cis-aconitic anhydride (CDM) in 5mL of anhydrous Dichloromethane (DCM), and stirring for 15min; placing the system in an ice-water mixture at 0 ℃, adding 12mmol of oxalyl chloride, and uniformly stirring; then 160 mu L DMF is added into the mixed solution drop by drop, and the reaction is continued for 15min at 0 ℃; the reaction was then allowed to warm to room temperature and stirred for 2h. And (3) after the mixed solution is dried in vacuum, collecting the acyl chloride CDM. Next, the product was added to 10mL of DCM solution having dissolved therein 0.8mmol of polyethylene glycol monomethyl ether (mPEG-OH) and 120. Mu.L of pyridine, and the reaction was continued with stirring at room temperature for 2 hours; subsequently, the reaction was terminated by adding 10mL of a saturated ammonium chloride solution. Finally, extracting, separating and drying the organic phase, and precipitating for 2-3 times by ice bath to obtain a final product, namely PEG-CDM, wherein the structure is as follows:
22 Synthesis of PEG-CDM-PEI:
PEG-CDM (0.6 mmol) and branched Polyethylenimine (PEI) (0.9 mmol) were dissolved in 10mL DMSO at 0deg.C, then 1.2mmol 4-Dimethylaminopyridine (DMAP) was added dropwise and stirred at 0deg.C for 0.5h, after which the reaction was left at room temperature and was reacted in the dark for 2h. The mixture was dialyzed against double distilled water (MWCO 3.5 kDa) for 4 days, and the water change operation was performed at the corresponding time, and the product was freeze-dried and collected to give the final product PEG-CDM-PEI having the structure:
(3) Preparation of PEG-CDM-PEI-Mn-DTA particles
Mn-DTA (10 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC.HCl (6 mM) and NHS (6 mM) were dissolved in PBS (pH 7.4, 10 mL) and stirred at room temperature for 1.5h. Subsequently, PEG-CDM-PEI (0.06 mmol) dispersed in PBS (pH 7.4,5 mL) was added to the above solution and the reaction was continued for 36h. The product was collected by centrifugation (PEG-CDM-PEI-Mn-DTA, designated PCP-Mn-DTA) and stored under vacuum.
(4) Preparation of PCP-Mn-DTA@GOx@1-MT controlled release system
Mn-DTA (10 mg), GOx (1.33 mg) and 1-MT (2 mg) were dissolved in PBS (pH 7.4, 10 mL), and the reaction was stirred at room temperature for 24 hours; the unloaded drug was removed by centrifugation. The mixture was then resuspended in PBS (pH 7.4, 10 mL) consisting of EDC. HCl (15 mM) and NHS (15 mM). With reference to the synthesis method of the PCP-Mn-DTA nano-particles, a nano-reaction controlled release system loaded with the drug is collected centrifugally, and a final product is obtained through freeze drying, which is abbreviated as PCP-Mn-DTA@GOx@1-MT.
Experimental example 1
The product obtained in each step of example 1 was subjected to XRD, TEM, BET, 1 Modern nano-test analysis technologies such as H NMR, DLS and Zeta potential analyzers, GPC, FTIR and the like systematically study the morphology, the components and the chemical bonds, and the results are as follows:
the present invention analyzes the crystal structure of Mn-DTA obtained in the examples by X-ray diffraction, and the result is shown in FIG. 1A, in which the XRD pattern of Mn-DTA is 100 DEG, 002 DEG, 101 DEG, 102 DEG, 110 DEG, 103 DEG and 112 DEG, showing strong diffraction peaks, which are consistent with the crystal structure of MnS compound. Among them, mn-DTA also showed relatively sharp diffraction peaks, indicating that Mn-DTA single crystals have good crystallinity. In addition, CCDC database calculations indicate Mn 2+ In fact, coordinates with 3 DTA ligands to form a regular hexagonal prism structure (shown in the B-D diagram of FIG. 1), three peaks of the bottom hexagon are composed of 3 Mn 2+ Occupying two apexes of a quadrilateral of each prism side by 2 Mn 2+ Occupied by them with Mn from DTA 2+ and-COOH, which form the sides of the hexagonal prism. At the same time, each Mn 2+ Is shared by two adjacent quadrilaterals and a hexagon (as shown in figure 1, E). Together, the above results indicate that Mn-DTA is a MOF particle having a three-dimensional porous network structure.
The Mn-DTA and the PCP-Mn-DTA obtained in the examples are characterized by a transmission electron microscope and dynamic light scattering. As shown in FIG. 2A, mn-DTA has a spherical structure with a size of about 50nm. The EDS elemental maps of Mn, S and O further confirm the structure of Mn-DTA and its elemental composition (as shown in FIG. 2, panel B). The above results confirm that Mn-DTA nanoparticles of suitable size have been successfully synthesized. After PEG-CDM-PEI functionalization modification, the PCP-Mn-DTA nanoparticles showed a spherical morphology similar to that of the Mn-DTA core (as shown in figure 2, panel C) and a distinct outer layer was observed around the PCP-Mn-DTA. Furthermore, the overall structure of PCP-Mn-DTA became obscured, and the particle diameter increased directly from 49.5±7.3 to 97.0±4.5nm (n=300) due to successful grafting of the PEG-CDM-PEI shell. This result is consistent with DLS statistical analysis (as shown in the D plot of fig. 2). The above results confirm successful preparation of PCP-Mn-DTA.
The invention is realized by N 2 The specific surface area and pore diameter of Mn-DTA obtained by the measurement and implementation of the absorption-desorption technology are shown as a graph A of FIG. 3, the Mn-DTA shows a typical IV type isothermal adsorption curve, which shows that the Mn-DTA has an obvious mesoporous structure, and meanwhile, the Mn-DTA has high specific surface area and larger pore diameter, which are respectively 107.8m 2 g -1 And 8.61nm (as shown in panel B of FIG. 3), further confirming successful synthesis of Mn-DTA.
The chemical structures of DTA, PEG-CDM and PEG-CDM-PEI obtained in each step in the examples were analyzed by nuclear magnetic spectroscopy, GPC and FTIR. As shown in a graph A of fig. 4, in the nuclear magnetic spectrum of the DTA, the position and the integral area of the characteristic signal peak of each group are accurate, which indicates the successful synthesis of the DTA; as shown in B of fig. 4, the nuclear magnetic spectrum of PEG-CDM showed distinct signal peaks at 3.45ppm and 3.71ppm, respectively, due to-OCH 3 methoxy peak and-OCH 2CH 2O-methylene proton peak in PEG, whereas signal peaks at 2.93ppm and 7.95ppm were respectively assigned to characteristic peaks of-OOCH 2C-methylene and-CHCOO-methine in CDM, and the integrated areas of the respective characteristic peaks were accurate, indicating that PEG-CDM was successfully synthesized; after coupling PEI and PEG-CDM, the characteristic peak of PEI (c+d, as shown in the graph C of FIG. 4) appears in the PEG-CDM-PEI nuclear magnetic spectrum, and the integral area is accurate, indicating that PEG-CDM-PEI has been successfully synthesized. As shown in FIG. 4D, there is only a single elution peak in the GPC chart of PEG-CDM-PEI, and the calculated average molecular weight is 7200g/mol, consistent with the calculated results of the nuclear magnetism and the theoretical molecular weight in the following table, again indicating successful synthesis of PEG-CDM-PEI.
The chemical structures of PEG-CDM and PEG-CDM-PEI obtained in each step of example 1 were analyzed by FTIR. As shown in FIG. 4, the infrared spectra of PEG-CDM and PEG-CDM-PEI were found at 1115cm -1 And 2885cm -1 There is a strong absorption peak due to the stretching vibration of C-O and-CH 2 in PEG, respectively. In addition, at 1633cm -1 And 1569cm -1 The absorbance peaks at (a) are ascribed to the stretching vibrations of amide I and amide ii (as shown in figure 4, panel E, panel a), which indicates successful synthesis of PEG-CDM; after grafting PEI, the PEG-CDM-PEI has an infrared spectrum (shown as B in FIG. 4, E) of 1739cm -1 The characteristic peak of carbonyl group (c=o) appears at this point because amide bond is formed during grafting of PEI to PEG-CDM, and 1645cm -1 And 1562cm -1 The characteristic peaks of typical amide I and amide II appear further confirm the successful synthesis of PEG-CDM-PEI.
Experimental example 2
Mn-based degradable MOF nano-reactor controlled release system pH/ROS responsive size reduction/charge reversal and drug release characteristics.
The invention utilizes H 2 O 2 To simulate tumor microenvironment ROS, and detecting the pH response size reduction of the system&Charge reversal, ROS-responsive drug release properties. The specific results are as follows:
1) pH responsive size reduction & charge reversal characteristics of PCP-Mn-DTA@GOx@1-MT controlled release system
The size reduction & charge reversal characteristics of the PCP-Mn-DTA@GOx@1-MT controlled release system pH response were examined using DLS and Zeta. As shown in FIG. 5A, the size of PCP-Mn-DTA@GOx@1-MT is 97+ -4.5 nm under the condition of pH 7.4 (simulating normal physiological conditions), and after incubation for 4 hours under the condition of pH 6.8 (simulating tumor microenvironment low acid stimulation), the particle size is reduced to 55nm, and is similar to the size of Mn-DTA, and the result is consistent with DLS, so that the system has the size reduction characteristic of pH response; in addition, zeta potential results showed that the surface charge of PCP-Mn-DTA@GOx@1-MT was reversed from-13.7 mV to +34.2mV (as shown in FIG. 5, panel B) under the stimulus of pH 6.8, confirming that the system has the charge reversal characteristic of pH response.
2) ROS responsive self-enhanced degradation and drug controlled release characteristics of PCP-Mn-DTA@GOx@1-MT controlled release system
First, the degradable properties of the PCP-Mn-DTA@GOx@1-MT controlled release system under ROS response conditions were examined by TEM and DLS, respectively. As shown in the A and C of FIG. 5, in the absence of H 2 O 2 (control group) in the presence of Mn-DTA and PCP-Mn-DTA@GOx@1-MT exhibit a typical spherical structure; and in the presence of H 2 O 2 Under the stimulated conditions of (a) the spherical structure of the nanoparticle collapses and complete disintegration occurs (see fig. 5 panels C and D). The DLS (shown in FIG. 5, panel A) results also exhibited similar changes, indicating that the PCP-Mn-DTA@GOx@1-MT controlled release system had the degradable property of ROS response.
Next, UV-Vis was used to examine the drug release profile of the ROS responsiveness of the PCP-Mn-DTA@GOx@1-MT controlled release system. As shown in the E chart of FIG. 5, the PCP-Mn-DTA@GOx@1-MT controlled release system is combined with different concentrations of H 2 O 2 After incubation, the characteristic peak intensities of GOx and 1-MT showed a significant enhancement, and their respective characteristic peak intensities and H 2 O 2 The concentrations are positively correlated (e.g. FIG. 5, E), i.e. H 2 O 2 The higher the concentration, the faster the degradation of the controlled release system and the release rates of GOx and 1-MT. Notably, when the PCP-Mn-DTA@GOx@1-MT controlled release system is treated with glucose, the peak intensities of the absorption peaks of GOx and 1-MT in the PCP-Mn-DTA@GOx@1-MT system are also drastically improved, because GOx catalyzes the production of a large amount of ROS by glucose, thereby accelerating the degradation of particles and the release of drugs. The results indicate that the PCP-Mn-DTA@GOx@1-MT controlled release system has the characteristics of ROS-responsive self-enhanced degradation and drug release.
Finally, cumulative drug release levels under different ROS-stimulated conditions were quantified. As shown in the F graph of FIG. 5, after 24h incubation under physiological conditions (pH 7.4), the GOx release amount of the control group is negligible (less than 16%), which indicates that the PCP-Mn-DTA@GOx@1-MT controlled release system has good stability. When compared with the control group, the nano particles are respectively compared with H at 0.1mM and 1mM 2 O 2 After incubation, 58% and 79% of GOx was released from the PCP-Mn-DTA@GOx@1-MT controlled release system, again confirming that the PCP-Mn-DTA@GOx@1-MT system has ROS-sensitive drug release characteristics. In addition, PCP-Mn-DTA@GOx@1-MT exhibited nearly complete GOx release (about 81% and 90%) after the additional glucose stimulus, which results further confirm that the PCP-Mn-DTA@GOx@1-MT controlled release system had ROS-responsive self-enhancing drug release properties.
Experimental example 3
In vitro biological evaluation of Mn-based degradable MOF nanoreactor controlled release System PCP-Mn-DTA@GOx@1-MT.
(1) Cell Activity and intracellular ROS detection
Cell culture: the invention selects the high metastasis cell (B16F 10) of the mouse melanoma provided by the cell research institute of China academy of sciences. B16F10 cells were cultured in DMEM high-sugar medium containing 10% FBS and 1% diabodies (100. Mu.g/mL penicillin and 100. Mu.g/mL streptomycin) at 37℃under 5% CO 2 Is included in the incubator.
In order to evaluate the in vitro tumor cytotoxicity of the PCP-Mn-DTA@GOx@1-MT controlled release system, the invention adopts a CCK-8 method to respectively detect the effects of PCP-Mn-DTA, 1-MT, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT on the activity of B16F10 cells by being assisted by glucose/glucose-free treatment. B16F10 cells were grown at 2X 10 4 cells/cm 2 When the cell fusion reached about 60-70%, B16F10 cells were co-cultured with the above drugs for 24h and 48h, respectively, and the cells were washed with PBS, followed by addition of 200 μl of a mixed solution of fresh medium and 20 μl of CCK 8. The reaction was continued at 37℃for 1.5-2 hours, and then the reaction solution in the well plate was transferred to a 96-well plate, and the absorbance of the solution at a wavelength of 450nm was measured with a microplate reader, thereby calculating the cell activity. As shown in panel A of FIG. 6, both the control group and the PCP-Mn-DTA group showed higher cell activity regardless of the length of incubation time; free 1-MT produced slight cellular damage; PCP-Mn-DTA@GOx@1-MT causes more serious cytotoxicity than PCP-Mn-DTA@1-MT group; while the PCP-Mn-DTA@GOx@1-MT plus glucose group treatment exhibited the lowest level of cellular activity in all treatment groups.The result further proves that the PCP-Mn-DTA@GOx@1-MT controlled release system has good in-vitro tumor killing effect.
In order to examine the ROS production characteristics of the PCP-Mn-DTA@GOx@1-MT controlled release system, ROS production levels of induced tumor cells of each experimental group are examined by utilizing ROS detection probes DCF-DA and CLSM. The method mainly comprises the following steps: B16F10 cells were grown at 1X 10 5 cells/cm 2 After cell fusion reached 60-70%, co-cultured with GOx, PCP-Mn-DTA, PCP-Mn-DTA@GOx, PCP-Mn-DTA@GOx@1-MT and PCP-Mn-DTA@GOx@1-MT+ low sugar medium, respectively, for 12h, followed by addition of a serum-free medium solution (1 mL) containing 10. Mu.M of DCFH-DA, co-incubation at 37℃for 20min, washing of the cells with PBS, observation of ROS fluorescence generation by CLSM and quantification of statistical fluorescence intensity. As a result, as shown in Panel B of FIG. 6, neither the PCP-Mn-DTA group nor the free 1-MT group produced visible green fluorescence as compared with the control group; while the PCP-Mn-DTA@GOx group and the PCP-Mn-DTA@GOx@1-MT group both excite bright green fluorescence in cells. In addition, in the absence of glucose, intracellular ROS production in the PCP-Mn-DTA@GOx@1-MT group incubated with low-sugar medium was significantly reduced. The quantitative statistical analysis results further confirm the above phenomenon (as shown in the graph C of FIG. 6). The results demonstrate that the PCP-Mn-DTA@GOx@1-MT group can effectively catalyze and metabolize glucose, self-generate ROS, and have ROS-responsive self-enhanced drug release characteristics.
(2) In vitro tumor penetration and cellular uptake
First, a 3D multicellular spheroid (MCS), i.e., a tumor sphere model, based on B16F10 cells was constructed. 80. Mu.L of a 1.5% hot agarose solution was added to a 96-well plate and after it had cooled to solidify into a gel, it was sterilized by ultraviolet irradiation overnight. Then B16F10 cells were grown at 2X 10 3 Initial density of cells/well cells were inoculated into the above well plate, and after co-culture for 6d, tumor balls were formed. Subsequently, the FITC-labeled PCP-Mn-DTA@GOx@1-MT controlled release system is incubated with the tumor balls, and the CLSM monitors the MCSs of the controlled release system to permeate under the conditions of pH 7.4 (physiological conditions) and pH6.8 (simulated weak acid tumor microenvironment) to examine the pH-enhanced tumor permeation behavior of the controlled release system. As shown in panel A of FIG. 7, after incubation for 4 hours at pH 7.4. The green fluorescent-labeled PCP-Mn-DTA@GOx@1-MT is mostly dispersed around the outer boundary of the MCSs; this is the case even if the incubation time is extended to 12 hours, and only a small amount of green fluorescence enters the interior of the MCS. After 4h incubation at pH6.8, the green fluorescent-labeled PCP-Mn-DTA@GOx@1-MT is widely distributed in the MCSs core region; when incubated for 12 hours, the whole MCS spheres disperse stronger green fluorescence. Quantitative analysis further confirmed that the PCP-Mn-DTA@GOx@1-MT controlled release system had pH-enhanced tumor permeability characteristics (as shown in a B panel of FIG. 7).
To further examine the cellular uptake efficiency, the endocytosis level of B16F10 cells to the PCP-Mn-DTA@GOx@1-MT controlled release system was monitored in real time using CLSM and FCM. Will be 1X 10 5 cells/cm 2 B16F10 cells of (A) were seeded in confocal dishes, and when the degree of cell fusion reached 60-70%, the cells were treated with PBS, FITC-labeled GOx (abbreviated as GOx-FITC,10 mU/mL), PCP-Mn-DTA@GOx-FITC@1-MT, and incubated for 4h and 12h at pH 7.4 and 6.8, respectively. The nuclei and the skeletons of the cells were then separately stained for analysis and quantitative endocytosis efficiency was measured using a flow cytometer. As shown in panels a and B of fig. 8, the PCP-Mn-dta@gox@1-MT concentration and intensity of B16F10 endocytosis were significantly higher than that of free GOx at pH 7.4 or pH6.8 compared to the control; at pH6.8, the endocytosis amount of B16F10 cells to PCP-Mn-DTA@GOx@1-MT is significantly higher than that of pH 7.4, which is consistent with the result of FCM quantitative analysis.
(3) In vitro tumor cell apoptosis
In order to study the in vitro tumor killing effect of the PCP-Mn-DTA@GOx@1-MT controlled release system, the apoptosis level of B16F10 cells induced by PCP-Mn-DTA@GOx@1-MT is analyzed by a Western blotting method and an Annexin V-FITC/PI apoptosis kit flow assay method respectively. The method comprises the following steps:
1)Western blotting
expression of Bcl-2 family related genes plays an important role in tumor cell apoptosis signaling pathways, where expression of the apoptosis inhibiting proteins Bcl-2 and the pro-apoptotic protein Bax are used to characterize the apoptotic state of tumor cells. In addition, cytochrome C (Cyt-C) is also an important protein indicator indispensable to the apoptotic pathway. B16F10 cells seeded on 6-well plates were treated with PBS, 1-MT, PCP-Mn-DTA, PCP-Mn-DTA@1-MT, PCP-Mn-DTA@GOx@1-MT and PCP-Mn-DTA@GOx@1-MT+glucose, respectively, for 24 hours, and the cells were lysed and protein samples were collected by centrifugation. Western blotting and ImageJ software was used to detect and analyze Bax, bcl-2 and Cyt-C expression. The results are shown in FIG. 9A: bax/Bcl-2 protein ratio and Cyt-C in the free 1-MT group showed weak up-regulation compared to the control group; compared with the 1-MT group, the PCP-Mn-DTA@1-MT group loaded with 1-MT induces more obvious apoptosis protein expression; while the PCP-Mn-DTA@GOx@1-MT group, which provided additional glucose, induced the highest proportion of up-regulated Bax/Bcl-2 and Cyt-C expression, exhibiting the highest level of apoptosis. Panels B and C of fig. 9 are quantitative statistical analyses of apoptosis-related proteins from different treatment groups, and the conclusion is the same as panel a of fig. 9.
2) Annexin V-FITC/PI flow cytometer detection
To further investigate the level of apoptosis in tumor cells, the proportion of apoptosis induced in B16F10 cells in different experimental groups was quantitatively determined using an Annexin V-FITC/PI staining kit and FCM. The specific experimental process is as follows: incubating B16F10 cells seeded on 6-well plates with PBS, 1-MT, PCP-Mn-DTA, PCP-Mn-DTA@1-MT, PCP-Mn-DTA@GOx@1-MT and PCP-Mn-DTA@GOx@1-MT+ glucose for 24 hours, washing with PBS, adding EDTA-free pancreatin to digest the cells, collecting the cells by centrifugation, and referring to an Annexin V-FITC/PI kit, staining with FITC in the absence of light for 10 minutes; washing once with binding buffer, staining with PI, detecting on-machine, and analyzing apoptosis level with flow cytometry FCM. Results as shown in panels D and E of fig. 9, the blank vector PCP-Mn-DTA induced a negligible rate of apoptosis compared to the control group; free 1-MT induced weak apoptosis and was significantly lower than the PCP-Mn-DTA@1-MT group (12.91% vs 21.7%); furthermore, PCP-Mn-DTA@GOx@1-MT group induced the most severe apoptosis (47.4%), which was due to oxidative damage and cell starvation by GOx. Notably, the PCP-Mn-DTA@GOx@1-MT plus glucose group caused the most severe apoptosis of tumor cells (54.1%), which again demonstrated that the PCP-Mn-DTA@GOx@1-MT controlled release system induced efficient tumor killing was caused by the combined effects of GOx-induced starvation/oxidative damage and 1-MT-induced cytotoxicity.
(4) Inhibition of IDO in vitro and effects of T cell proliferation
In order to study the in vitro inhibition effect of the PCP-Mn-DTA controlled release system on IDO1 activity, the expression level of the B16F10 cell IDO1 was induced by using a western blotting examination PCP-Mn-DTA@GOx@1-MT controlled release system. After B16F10 cells seeded on 6-well plates were treated with PBS, 1-MT, PCP-Mn-DTA, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT, respectively, for 24 hours, the cells were lysed, and protein samples were collected by centrifugation. Western blotting and ImageJ software was used to detect and analyze IDO1 expression. The results are shown in FIG. 10A, in which free 1-MT group has a significant inhibitory effect on IDO1 expression compared with the control group and the PCP-Mn-DTA group; both the PCP-Mn-DTA@1-MT group and the PCP-Mn-DTA@GOx@1-MT group exhibited a higher level of down-regulation of IDO1 expression compared to the free 1-MT group. FIG. 10B is a quantitative statistical analysis of apoptosis-related proteins from different treatment groups, and the conclusion is the same as FIG. 10A. This indicates that the PCP-Mn-DTA@GOx@1-MT controlled release system can inhibit IDO activity more efficiently because of the excellent drug delivery efficiency of the PCP-Mn-DTA@GOx@1-MT controlled release system.
A co-culture system comprising B16F10 cells and lymphocytes was established to determine IDO1 mediated T cell proliferation. B16F10 cells were stimulated overnight with IFN-. Gamma.50 ng/mL to induce IDO1 expression. After collection of spleen cell suspensions from C57 mice, lymphocytes were obtained by density gradient centrifugation and purified by nylon fiber column. Purified T cells were then activated with anti-CD 3/CD28 magnetic beads and IL-2 (20 ng/mL). Next, IFN-gamma stimulated B16F10 cells (1X 10 5 cells/cm 2 ) Is identical to the T cells (5X 10) 5 cells/cm 2 ) After mixing well in a 24-well plate, the cells were co-cultured with PCP-Mn-DTA, 1-MT, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT for 12 hours, and lymphocytes in the supernatant were collected and incubated with EdU (10. Mu.M) for 2 hours. Cells were then collected by centrifugation, fixed with 4% paraformaldehyde, followed by permeabilization of the cell membrane with 0.5% Triton X-100, followed by488 staining 10min (Cell-Light) TM EdU/>488 kit), and finally quantitatively analyzed using a flow cytometer. As a result, as shown in FIG. 10C, 1-MT and 1-MT-loaded drug (PCP-Mn-DTA@1-MT)&PCP-Mn-DTA@GOx@1-MT) treated intra-group EdU + The proportion of T cells increases significantly; the number of T-cell proliferation in the PCP-Mn-DTA@GOx@1-MT group was significantly higher than that in the free 1-MT group, without significant difference from the PCP-Mn-DTA@1-MT group (as shown in the D panel of FIG. 10). The explanation is as shown in the E diagram of fig. 10: first, the absorption efficiency and subsequent bioavailability of free 1-MT is limited by its poor solubility; secondly, the PCP-Mn-DTA carrier with high drug loading rate can effectively solve the problem of weak solubility of 1-MT and improve the bioavailability of the drug; thirdly, PCP-Mn-DTA@GOx@1-MT and PCP-Mn-DTA@1-MT can be endocytosed by B16F10 cells, and in response to intracellular ROS, GOx and 1-MT are released, and high-dose 1-MT accumulated in the cells can effectively inhibit the activity of IDO1 and restore the activity of T cells, so that T cell proliferation is promoted. These results demonstrate the great potential of the PCP-Mn-DTA controlled release system to enhance anti-tumor immunity in vivo.
(5) In vitro anti-tumor migration
Considering that GOx and 1-MT regulated energy metabolism is closely related to tumor migration invasion, scratch healing, migration and invasion experiments are adopted to evaluate the anti-migration effect of the PCP-Mn-DTA@GOx@1-MT controlled release system on B16F10 cells.
B16f10 cells seeded in 6-well plates were treated with PBS, PCP-Mn-DTA, 1-MT, PCP-Mn-dta@1-MT, PCP-Mn-dta@gox@1-MT for 24 hours, gently scratched between the cultured cells of the monolayer with a yellow gun head (gun head was not inclined vertically to the bottom of six-well plates so that the scratches were in the same direction), then the cells were washed to remove the nanoparticles and the exfoliated cells, washed with PBS after 24 hours, fixed with paraformaldehyde, stained with 1% crystal violet, and finally the image of scratch healing was photographed with a microscope. As shown in panel A of FIG. 11, the scratches of the B16F10 cells of the control group and the PCP-Mn-DTA treated group disappeared, showing an inherently stronger healing ability of the tumor cells. Whereas the cell healing rates of the 1-MT group and the PCP-Mn-DTA@1-MT group were 75% and 58%, respectively (as shown in the B chart of FIG. 11), showing a certain level of anti-tumor migration effect. In addition, the PCP-Mn-DTA@GOx@1-MT group had the lowest cell healing rate of 39%, which indicates that PCP-Mn-DTA@GOx@1-MT can achieve the most effective cell migration inhibition effect.
In agreement with the scratch healing experiments, B16F10 cells were incubated with the above drugs for 24h, the cells were digested and collected by centrifugation. For cell migration experiments: the cells were resuspended in serum-free medium followed by 1X 10 cells 5 cells/cm 2 Cells were transferred to the Transwell upper chamber. For invasive experiments: will be 2X 10 5 cells/cm 2 The B16F10 cells of (C) were seeded on a Transwell pre-coated matrigel. In this test, a medium containing 10% serum was added as a chemoattractant to the lower chamber. After incubation of the cells for 24h, cells on the upper surface of the Transwell were removed with a cotton swab and cells on the lower surface were stained with crystal violet. After staining, the cells were counted and then imaged under a microscope. We specified that the untreated control group had a cell migration/invasion rate of 100%. The results are shown in FIG. 11A&11C&11D shows that migration and invasion experiments also showed a similar trend to the scratch healing experiments described above, wherein the PCP-Mn-DTA@GOx@1-MT group showed the strongest inhibition of migration and invasion of B16F10 cells, and the corresponding rates of tumor cell migration and invasion were 30.1% and 24.9%, respectively. The result shows that the PCP-Mn-DTA@GOx@1-MT controlled release system can effectively inhibit in-vitro tumor metastasis.
Experimental example 4
In vivo antitumor evaluation of Mn-based degradable MOF nano-reactor controlled release system PCP-Mn-DTA@GOx@1-MT.
(1) Construction of tumor-bearing mouse model:
all animal experiments of the present invention were performed according to the rules related to the laboratory animal administration and use committee working manual. About 20g female C57BL/6 mice of 5-6 weeks old were purchased from Beijing pharmaceutical laboratory, china. The invention adopts B16F10 cells in exponential growth phase to prepare 100 mu L of cell suspension, the suspension is inoculated subcutaneously in the right inguinal of each mouse, a tumor-bearing mouse model is established to observe the health condition and behavior of the mouse every day, and the load is recorded every 2 daysBody weight and tumor volume of tumor mice tumor volume (V) was calculated according to the following formula: v=l×s 2 2 (L, major diameter of tumor; S, minor diameter of tumor).
(2) Drug intervention and histopathological analysis:
tumor-bearing mice of the successfully constructed tumor-bearing mice are divided into five groups according to the principle of random weight, and are intravenously injected with physiological saline, 1-MT, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT, and are administrated 2 times per week for 20 days. After the end of the administration, the mice were euthanized. Mice were collected for heart, liver, spleen, lung, kidney and tumors and stained for H & E, TUNEL and Ki-67 for histopathological analysis.
After mice are sacrificed, main organs and tumor tissues are collected, and the results are shown as a graph A of FIG. 12, wherein the sizes of tumors in the PCP-Mn-DTA group and the physiological saline group are similar, and the tumors show rapid growth trend during the administration period, so that the blank carrier has no obvious anti-tumor effect and has good biological safety; the free 1-MT group and PCP-Mn-DTA@1-MT group showed slight and moderate inhibition of tumor growth, respectively, and PCP-Mn-DTA@GOx@1-MT group induced the most significant tumor growth inhibition in all the treatment groups, and B16F10 malignant tumor was almost disappeared after 18 days of administration treatment, indicating that PCP-Mn-DTA@GOx@1-MT mediated tumor starvation/oxidation/IDO immune combination treatment was able to kill tumors with high efficiency. The tumor volume statistics of fig. 12B confirm the same tumor growth inhibition trend. In addition, the PCP-Mn-dta@gox@1-MT group not only significantly prolonged the survival time of tumor-bearing mice (30 days survival rate was 67%, figure 12, panel C, significantly higher than other treatment groups), but also did not lose the weight of tumor-bearing mice during administration (as shown in figure 12, panel D), indicating that the PCP-Mn-dta@gox@1-MT controlled release system had good biosafety and excellent antitumor effect.
The results of the histopathological analysis are shown in the E graph of FIG. 12, and compared with the control group and the PCP-Mn-DTA group, the 1-MT group and the PCP-Mn-DTA@1-MT group induce a certain degree of tumor tissue apoptosis; the PCP-Mn-DTA@GOx@1-MT group induced the most severe apoptosis of tumor tissue, manifested by significant cell collapse, deformation and chromatin concentration in the tumor section H & E map and a large number of magenta spots in the TUNEL map marking DNA lesions. In addition, the Ki-67 immunofluorescence staining results show that the PCP-Mn-DTA@1-MT group and the PCP-Mn-DTA@GOx@1-MT group effectively down regulate the expression of the tumor cells Ki-67, wherein the PCP-Mn-DTA@GOx@1-MT group presents the weakest expression of the Ki-67, which again proves that the PCP-Mn-DTA@GOx@1-MT controlled release system has good anti-tumor effect.
(3) In vivo immune response detection:
the invention adopts FCM to quantitatively analyze various immune cell proportions of tumor site infiltration to verify the in vivo anti-tumor immune response effect of PCP-Mn-DTA@GOx@1-MT controlled release system-mediated hunger/oxidation/immunotherapy, and the specific experimental process is as follows: after the administration treatment, mice were euthanized, tumor tissues were collected, and lysed using a lysate consisting of 0.2% collagenase IV, 0.1% hyaluronidase V, 0.002% dnase I and DMEM medium at 37 ℃ on a shaker for 1h. The lysed solution was then passed through a 75 μm filter to separate monodisperse lymphocytes, collected by centrifugation and washed with wash solution, and the cells resuspended in PBS solution. Next, the cells were stained with Live/read dye for 20min.
1) For tumor infiltration CD4 + CD8 + Detection of T cells: combining the collected cells with anti-CD3-APC/Cy7,
anti-CD4-FITC and anti-CD8-PerCP-Cy5.5 were incubated at 4deg.C for 30min followed by detection of tumor infiltrating CD4 with FCM + CD8 + T cell level;
2) Detection of Tregs cells: after the above-mentioned collected cells were co-incubated with anti-CD3-APC/Cy7 and anti-CD4-FITC for 30min, they were fixed and permeabilized by referring to the procedure on Foxp3 kit, followed by incubating the cells with anti-Foxp3-Alexa 647 at 4℃for 30min and detecting tumor-infiltrating Treg cells with FCM (CD 3 + CD4 + Foxp3 + ) Is a level of (2);
3) Detection of tumor infiltrating mature DC cells: the collected cells were co-stained with anti-CD11b-FITC, anti-CD80-APC and anti-CD86-PE for 30min and analyzed for DC cells (CD 11 b) with FCM (FCM + CD80 + CD86 + ) Is water of (2)Leveling;
4) Detection of tumor infiltrating NK cells: the above-mentioned collected cells were co-stained with anti-CD3-APC/Cy7 and anti-CD49b-PE/Cy7 for 30min, and NK cells (CD 3) were analyzed by FCM - CD49b + ) Is a level of (2);
5) Detection of tumor infiltrating B cells: the collected cells were co-stained with anti-CD3-APC/Cy7 and anti-CD45R/B220-PE for 30min and B cells were analyzed by FCM (CD 3 - CD45R/B220 + ) Is a level of (2);
the results were as follows:
1) Immune response of T cells
Direct inhibition of tumor infiltration of CD8 due to IDO-mediated immune escape + Toxic T cells and CD4 + The activity of helper T cells, so that tumor infiltration of cd8+ toxic T cells and cd4+ helper T cells is the most directly reflected in anti-tumor immune responses. Thus, the present invention first quantitatively characterizes CD8 in tumor lesions of different treatment groups with FCM + Toxic T cells, CD4 + Helper T cells and levels of Tregs. As shown in FIGS. 13A-D, tumor-infiltrating CD8 in the control group + Low proportion of cytotoxic T Cells (CTLs) and high Tregs cell level; the number of CTLs in the free 1-MT group increased moderately, and Tregs showed some decrease; the trend of PCP-Mn-DTA@1-MT group loaded with 1-MT is obviously higher than that of free 1-MT, and the PCP-Mn-DTA@1-MT is beneficial to excellent drug delivery efficiency, and anti-tumor immune response is activated through IDO blocking mediated by 1-MT, so that a large amount of tumor infiltrating CD8 is recruited + Helps to restore effector T cell activity and inhibits Tregs proliferation. In addition, PCP-Mn-DTA@GOx@1-MT presents the highest CTL infiltration proportion and the lowest Tregs proportion in all treatment groups, which shows that the PCP-Mn-DTA@GOx@1-MT controlled release system can effectively activate immune response, strengthen anti-tumor immune response and inhibit immune tolerance.
2) Immune response of DC cells and B, NK cells
The invention further discloses an enhanced anti-tumor immune response mechanism of the PCP-Mn-DTA@GOx@1-MT controlled release system by examining the maturity of DC cells. As shown in FIG. 14A&14B, 1-MT and Experimental group (PCP-Mn-DTA@1 @) loaded with 1-MTMT and PCP-Mn-DTA@GOx@1-MT) promote the maturation of DC cells to different extents in the order of maturity: 1-MT<PCP-Mn-DTA@1-MT<The result shows that the PCP-Mn-DTA@GOx@1-MT controlled release system successfully promotes the maturation of DC cells. In addition, the PCP-Mn-DTA@GOx@1-MT controlled release system also successfully recruited tumor-infiltrating B cells and NK cells (as shown in the C-F panel of FIG. 14). This is because PCP-Mn-DTA@GOx@1-MT can effectively induce tumor damage by GOx-mediated oxidation/starvation therapy, enhance tumor immunogenicity, promote exposure of tumor-associated antigen TAA and enhance cytotoxic CD8 + T cells and helper CD4 + Intratumoral recruitment of T cells; in addition, the PCP-Mn-DTA@GOx@1-MT controlled release system further enhances the anti-tumor immune response of the organism by inhibiting IDO activity and relieving IDO-mediated immune tolerance.
3) Immunofluorescence analysis of related proteins
IDO1 is an important therapeutic target for restoring anti-tumor immunity as a key mechanism for inducing immune escape, and can catalyze Trp to be metabolized into Kyn, directly inhibit mTOR and activate STAT3 through an AHR-IL-6-STAT3 pathway and regulate immune tolerance. Based on the above considerations, the ratio of Kyn to Trp, and the changes in intratumoral IDO1, mTOR and STAT3 expression can be used as indicators to reveal IDO 1-mediated suppression of immune escape. The invention respectively measures the proportions of Kyn and Trp in tumor, and performs immunofluorescence analysis on the expressions of IDO1, mTOR and STAT3 in tumor to clarify the molecular mechanism of the PCP-Mn-DTA@GOx@1-MT controlled release system for regulating and controlling the immune response of organisms. The results are shown in FIGS. 15A & B, where the Kyn/Trp ratio and IDO1 protein expression were minimal in the immunotherapeutic groups (PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT), indicating that the system effectively inhibited IDO1 activity. In addition, IFC results also show that the immune treatment group can obviously induce the up-regulation of mTOR and the down-regulation of STAT3, and the results further prove that the PCP-Mn-DTA@GOx@1-MT controlled release system can effectively block IDO1 mediated immune tolerance and enhance persistent anti-tumor immune response.
(4) Biosafety and pharmacokinetics:
first, blood glucose levels of mice before and after intravenous injection of PCP-Mn-DTA@GOx@1-MT controlled release system were measured. As shown in fig. 16A, all treatment groups did not induce a sustained decrease in peripheral blood glucose compared to the control group, indicating that the controlled release system had good blood safety. Secondly, the liver function or liver injury is estimated by detecting the levels of glutamic oxaloacetic transaminase AST and glutamic pyruvic transaminase ALT in blood after different medicaments are injected according to the kit, and the result shows that the concentrations of the two enzymes are not obviously changed and are not obviously different from those of a control group, so that the PCP-Mn-DTA@GOx@1-MT controlled release system has good biological safety on liver tissues. In addition, the analysis results of the H & E of the main organs show that all treatment groups taking PCP-Mn-DTA as the carrier do not cause damage to the organs of the organism (as shown in the D diagram of FIG. 16), which again proves that the PCP-Mn-DTA@GOx@1-MT controlled release system has good biological safety in vivo.
The pharmacokinetics of PCP-Mn-DTA@GOx@1-MT in vivo was then monitored by intravenous injection of 1-MT and PCP-Mn-DTA@GOx@1-MT into the mouse tail bearing the tumor. For pharmacokinetic analysis, the eyebox of the mice is subjected to blood sampling in a specific time period after administration, plasma is collected by centrifugation after heparinization, and acetonitrile is added to settle proteins; subsequently centrifuging to collect the supernatant; finally, the collected samples were dried, redissolved and the concentration of 1-MT in the blood was measured by HPLC and the pharmacokinetic profile was plotted. For in vivo drug distribution studies in mice, mice were euthanized 24h after the above-described dosing treatment, heart, liver, lung, spleen, kidney and tumor tissues were collected, homogenized in 0.5mL DMSO, centrifuged at 15000rpm for 15min, and finally the distribution of 1-MT in each tissue was examined by HPLC, calculated as: mg (mass of controlled release system)/g (weight of tissue). Results as shown in fig. 16, panels E and F, PCP-Mn-dta@gox@1-MT group not only significantly prolonged the drug-loaded blood circulation time, but also delivered to the tumor site at higher doses and accumulated efficiently in tumor lesions again (the content was nearly 5 times that of free 1-MT) compared to free 1-MT group; in addition, the drug content accumulated in the tumor is also significantly greater than that of other tissues due to the shielding effect of the PEG outer layer, the size reduction and charge reversal characteristics of the pH responsiveness of the PCP-Mn-DTA@GOx@1-MT controlled release system, and the EPR tumor targeting characteristics.
(5) Antitumor metastasis, recurrence efficiency and persistence of immune effect:
the invention constructs a lung metastasis model to further examine the anti-tumor metastasis effect of the PCP-Mn-DTA@GOx@1-MT controlled release system. 100 μl of the mixture containing 1×10 is injected into tail vein of mice 6 After 7d, the PBS solution of B16F10 tumor cells was used to randomly divide the mice into 5 groups, and the mice were treated with different administrations, and after 10d, the administration was terminated and euthanized. Lung tissue from mice was collected, washed with PBS and photographed with a camera, and the number of lung metastasis nodules was counted to evaluate the anti-tumor metastasis efficiency of the controlled release system. The lung tissue of the mice was then preserved in 10% formalin solution for H&E staining analysis. As a result, as shown in fig. 17A, it was observed that there were only a very small number of tumor metastasis nodules in lung tissue of PCP-Mn-dta@gox@1-MT group in all the treatment groups, and that the quantitative statistics were statistically different (as shown in fig. 17B). This indicates that the PCP-Mn-DTA@GOx@1-MT controlled release system significantly and efficiently inhibits tumor metastasis. In contrast, in the other dosing groups, there were varying degrees of tumor metastatic nodules in the lung tissue of the mice. The result shows that the PCP-Mn-DTA@GOx@1-MT system has remarkable anti-tumor metastasis effect.
In order to evaluate the anti-tumor recurrence efficiency and the persistence of the induction of the immune effect of the organism by the PCP-Mn-DTA@GOx@1-MT controlled release system, firstly, tumor of the tumor-bearing mice after the administration treatment is resected, then, tumor cells are injected subcutaneously for the second time, and the secondary immune effect and the anti-tumor recurrence level of the organism are revealed by detecting the number of the secondary tumor-bearing mice and the immune memory T cell level of the organism, and the schematic diagram is shown in a graph of 18. The experimental results are shown in a panel B of FIG. 18, and the control group, the PCP-Mn-DTA group and the 1-MT group treated mice all show higher tumor recurrence rate (100%); the tumor recurrence rate of the PCP-Mn-DTA@1-MT group is reduced to 83.3%; while the PCP-Mn-DTA@GOx@1-MT treatment group obviously reduces the tumor recurrence rate of the organism to 33.3%, which is far lower than that of other treatment groups, thus indicating that the group effectively inhibits tumor recurrence. In addition, PCP-Mn-DTA@GOx@1-MT group mouse in vivo effector memory T cells (i.e., T capable of providing protection immediately) EM ，CD3 + CD8 + CD62L - CD44 + ) Level displayIs significantly increased, while central memory T cells (T CM ，CD3 + CD8 + CD62L + CD44 + Only during antigen stimulated clonal expansion, differentiation and transport provided protection) levels were correspondingly reduced (as shown in panel C of fig. 18) and statistically different from the other treatment groups (as shown in panels D and E of fig. 18). The result shows that the PCP-Mn-DTA@GOx@1-MT controlled release system successfully activates the immune memory reaction and has long-term effective action on tumor recurrence.
FIG. 19 is a schematic representation of the preparation of the PCP-Mn-DTA@GOx@1-MT controlled release system of the invention in combination with starvation/oxidation/IDO immunotherapy in vivo.
In conclusion, the pH/ROS dual-responsiveness MOF nano-reaction controlled-release system for the co-load immune checkpoint IDO inhibitor 1-MT and GOx is prepared by utilizing the advantages of high load capacity, restriction enzyme reaction space, ROS responsiveness and degradability and the like of the MOF nano-reactor, and the tumor starvation/oxidation/IDO immune combined treatment is regulated and controlled. The nanosystems can respond to weakly acidic tumor microenvironments, induce size reduction/charge reversal, overcome tumor penetration and cell barriers in vivo and improve delivery efficiency. In addition, the invention has self-enhancing MOF degradation and drug release properties: in one aspect, GOx released by the system produces H while consuming glucose 2 O 2 By Mn of 2+ The mediated Fenton-like reaction is converted into hydroxyl radicals (.OH) with higher toxicity, and starvation/oxidation therapy is mediated to enhance immune response; on the other hand, the 1-MT released by the system can effectively enhance the anti-tumor immune response of organisms by blocking IDO activity and relieving immune tolerance, and can inhibit tumor growth, metastasis and recurrence, thereby achieving better treatment effect.
The application solves the following problems:
(1) Solves the problem of low bioavailability such as easy inactivation of in vivo delivery of biological enzyme: based on the advantages of the MOF and the nano-reactor, the MOF nano-reactor is fitted, the bio-enzyme is loaded efficiently, the enzyme-restricted reaction space is provided, the activity of the enzyme is high, and meanwhile, the bioavailability of the enzyme is improved.
(2) The problems of low in-vivo delivery efficiency, incomplete drug release and the like of therapeutic drugs are solved in a broad spectrum: based on the advantages that the size of the surface negative charge carrier is about 100nm, the surface negative charge carrier has the long circulation characteristic of blood, the small size of 50nm and the surface positive charge particle have the advantages of enhancing tumor penetration and cell uptake, an ROS-responsive degradable Mn-based MOF nano-reactor is to be constructed, and a shell polymer PEG-CDM-PEI with reduced pH-responsive size and reversed charge is introduced on the surface of the MOF in a functionalized manner, so that GOx and other therapeutic drugs are co-loaded, and a functional MOF nano-reactor delivery system is constructed; on one hand, the system responds to the stimulation of a low pH signal in the tumor microenvironment, breaks the bridging molecule CDM in the shell copolymer, further drops off the shell copolymer PEG, exposes PEI (strong positive charge characteristic) functionalized grafted MOF carrier, induces the carrier size to be reduced and reverses the surface charge, thereby improving the tumor penetration depth and the cell uptake efficiency. On the other hand, the ingested vector is able to degrade MOF vector in situ and release the loaded drug in response to intracellular ROS stimulation of the tumor. Finally, the released GOx starves the tumor and produces ROS by competing consumption of glucose, cascade amplified MOF degradation and drug release. In summary, the system overcomes many physiological barriers and improves drug delivery efficiency in a broad spectrum.
(3) Solves the problem of unsatisfactory tumor immunotherapy effect caused by organism immune tolerance: the immune checkpoint indoleamine 2, 3-dioxygenase (IDO) is highly expressed in tumors, can inhibit proliferation of effector T cells and induce expansion of regulatory T cells (Treg) by catalyzing tryptophan to be kynurenine, and is a potential tumor immunotherapy target. It has been demonstrated that the IDO competitive inhibitor, 1-methyltryptophan (1-MT), can effectively inhibit IDO-mediated immune escape by inhibiting IDO activity. However, IDO blocking therapy is not clinically effective due to poor tumor immunogenicity and inadequate immune response.
GOx-mediated starvation therapy of tumors can not only competitively consume glucose necessary for tumor growth, but also generate ROS, starve/oxidize and kill tumors, thereby causing immunogenic death of the tumors, improving the immunogenicity of the tumors and enhancing the immune response of organisms. Therefore, the research plan constructs a MOF delivery system, safely and efficiently co-delivers GOx and 1-MT to tumor lesions, and combines 1-MT-mediated IDO blocking immunotherapy and GOx-activated hunger/oxidation therapy to inhibit organism immune tolerance and enhance organism anti-tumor immune response, thereby improving tumor immunotherapy effect.
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims (8)
1. The Mn-based degradable MOF nano-reactor is characterized by comprising inner core Mn-based MOF nano-particles, wherein the surface of the Mn-based MOF nano-particles is functionalized and grafted with pH responsive shell copolymer PEG-CDM (cis aconitic anhydride) -PEI, and the Mn-based MOF nano-particles are loaded with biological enzymes GOx and IDO immunosuppressant; wherein 5, 5-dimethyl-4, 6-dithioazelaic acid (DTA) is reacted with Mn by ROS responsive organic ligand 2+ Covalent cross-linking to synthesize the Mn-based degradable MOF nanoparticle.
2. The Mn-based degradable MOF bioreactor of claim 1, wherein the Mn-based MOF nanoparticle is Mn-DTA and the IDO immunosuppressant is 1-MT.
3. The Mn-based degradable MOF bioreactor of claim 1, wherein the bioreactor is spherical and has a particle size of 100nm.
4. The preparation method of the Mn-based degradable MOF nano reactor is characterized by comprising the following steps of:
(1) Preparing a core Mn-based MOF nanoparticle Mn-DTA, which comprises the following steps:
11 Synthesis of ROS-responsive organic ligand 5, 5-dimethyl-4, 6-dithioazelaic acid (DTA): dissolving 3-mercaptopropionic acid (MPA) in an acetone solution, and continuously stirring at room temperature; placing the mixed system in an ice bath for overnight crystallization; filtering and collecting crystals; repeatedly washing filtrate crystals with normal hexane and cold water, and vacuum drying to obtain DTA;
12 Synthesis of ROS-sensitive core Mn-based MOF nanoparticles Mn-DTA by hydrothermal reaction: mnCl is added to 2 And DTA are dissolved in N, N-Dimethylformamide (DMF), respectively; adding the solution into a centrifuge tube, and adding polyvinylpyrrolidone K30 (PVP-K30) and triethylamine into the solution; adding the prepared DMF/ethanol solvent into a tube, wherein the volume ratio of DMF/ethanol is 5:3, constant volume; ultrasonically dispersing the mixture, transferring the mixture into a hydrothermal synthesis reactor, and reacting at a high temperature; after naturally cooling to room temperature, collecting the product by centrifugation, and then dispersing in ethanol for later use; the obtained MOF nanoparticle is named Mn-DTA;
(2) Synthesizing pH responsive shell PEG-CDM-PEI;
(3) Preparation of PEG-CDM-PEI-Mn-DTA particles;
(4) Preparation of PCP-Mn-DTA@GOx@1-MT controlled release system.
5. The method for preparing a Mn-based degradable MOF nanoreactor according to claim 4, wherein step (2) specifically comprises:
21 Synthesis of PEG-CDM: firstly, reacting maleic anhydride (CDM) with oxalyl chloride, and vacuum drying to obtain CDM; next, adding the product into methylene chloride (DCM) solution in which polyethylene glycol monomethyl ether (mPEG-OH) and pyridine are dissolved, and reacting at room temperature; subsequently, the reaction was terminated with a saturated ammonium chloride solution; extracting, separating and drying the organic phase, and precipitating for 2-3 times by ice bath to obtain PEG-CDM;
22 Synthesis of PEG-CDM-PEI: PEG-CDM and branched Polyethylenimine (PEI) were dissolved in DMSO, 4-Dimethylaminopyridine (DMAP) was added dropwise, stirred at 0deg.C for 0.5h, then the system was left at room temperature and continued to react in the absence of light; the mixture was dialyzed against double distilled water and freeze-dried to give the product PEG-CDM-PEI.
6. The method for preparing a Mn-based degradable MOF nanoreactor according to claim 4, wherein step (3) specifically comprises:
dissolving Mn-DTA obtained in the step (2), 6mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC.HCl and 6mM NHS in PBS with pH of 7.4, and stirring at room temperature; subsequently, 0.06mmol of PEG-CDM-PEI dispersed in PBS at pH 7.4 was added to the above solution, and the reaction was continued for 36 hours; the product PEG-CDM-PEI-Mn-DTA, designated PCP-Mn-DTA, was collected by centrifugation.
7. The method for preparing a Mn-based degradable MOF nanoreactor according to claim 4, wherein step (4) is specifically:
Mn-DTA, GOx and 1-MT are dissolved in PBS with pH of 7.4, and stirred at room temperature for reaction for 24 hours; centrifuging to remove the unloaded medicine; the mixture was then resuspended in PBS solution at pH 7.4 consisting of 15mM EDC. HCl and 15mM NHS; referring to the synthesis method of PCP-Mn-DTA nano-particles, centrifugally collecting a nano-reaction controlled-release system loaded with the drug, and freeze-drying to obtain a final product, which is named as PCP-Mn-DTA@GOx@1-MT.
8. Use of a Mn-based degradable MOF nanoreactor according to any one of claims 1 to 3 or prepared by a method of preparation according to any one of claims 4 to 7 for the preparation of a functional delivery system for tumour starvation, oxidation or immune combination therapy.
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