Preparation method of antidote zeolite imidazate skeleton for central nervous agent poisoning

Abstract

The invention relates to a preparation method of an antidote zeolite imidazate skeleton for central nervous agent poisoning, which comprises the following steps: S100, weighing 2.6 mg of Mal-PEG2000-COOH, dissolving in 5ml of PBS (Phosphate Buffer Solution) with the pH value of 7.4, and carrying out ultrasonic full dissolution to form a first reaction solution; s200, 2 mg of RVG15 is weighed and dissolved in 5 ml of a PBS solution, the PH value of the PBS solution is 7.4, and a second reaction solution is formed after sufficient ultrasonic dissolution; s300, adding the first reaction solution in the step S100 and the second reaction solution in the step S200 into a reaction bottle, reacting for 20-28 hours in a dark place under the condition of nitrogen protection, dialyzing through a 3000Da dialysis bag, removing unreacted substances, and freeze-drying the obtained product for 20-28 hours to prepare RVG15-PEG2000-COOH; s400, weighing HI-6, dissolving HI-6 in a blank ZIF-8 suspension, uniformly stirring to form drug-loaded ZIF-8, centrifuging the drug-loaded ZIF-8 to remove free drugs, and alternately washing the drug-loaded ZIF-8 with methanol and water to prepare HI-6 ZIF-8; and S500, adding the RVG-PEG2000-COOH prepared in the step S300 into the HI-6 (at) ZIF-8 prepared in the step S400, so as to prepare the HI-6 loaded brain targeting ZIF-8.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and in particular to a method for preparing a zeolite imidazole ester skeleton as an antidote for central nervous system poisoning. Background Technology

[0002] Organophosphates (OP) are compounds that can disrupt the normal conduction function of the nervous system. They are used in agriculture as pesticides (such as dichlorvos (DDVP) and trichlorfon (Dipterex)) and in the military as chemical weapons (such as tabun (GA), sarin (GB), cyclosarin (GF), soman (GD), and VX). The phosphorus atoms in these nerve agents irreversibly covalently bind to acetylcholinesterase (AChEs), a key enzyme in the human cholinergic system, to form phosphorylated cholinesterase, thus rendering it unable to break down acetylcholine (ACh), causing ACh to accumulate in large quantities in the body. At the same time, they inhibit the activity of AChEs, causing over-excitation of the central nervous system (CNS) and cholinergic nerves, eventually leading to inhibition and exhaustion, manifesting a series of symptoms and signs of organophosphate poisoning. Generally, typical symptoms of central and peripheral poisoning include loss of consciousness, tremors, convulsions, coma, as well as smooth muscle contraction, increased glandular secretion, bronchospasm, decreased heart rate, decreased myocardial contractility, weakness, and difficulty breathing.

[0003] Currently, the main treatments for OP (opium poisoning) poisoning involve muscarinic antagonists (such as atropine) and acetylcholinesterase (AChE) reactivators. Acetylcholinesterase reactivators can efficiently and rapidly reactivate AChE in the poisoned enzyme. These "reactivators" can separate the poison from AChE in a relatively short time, thereby restoring AChE's ability to hydrolyze ACh. Among them, pralidoxime (HI-6) is a bispyridine quaternary ammonium salt AChE reactivator, showing good efficacy in treating GB, GF, GD, and VX poisoning, and exhibiting significant advantages compared to other reactivators such as pralidoxime and bispyridine. The blood-brain barrier (BBB) ​​generally refers to the barrier between blood plasma and cerebrospinal fluid in a living organism. This special structure is composed of BMECs, atherosclerotic ascites (AS), and the vascular basement membrane. Due to the presence of the dense structure (Tj) of the BBB, brain tissue can selectively absorb necessary nutrients and excrete unwanted or harmful substances. This characteristic also prevents most drugs and biotechnological drugs from reaching the brain parenchyma and exerting their therapeutic effects. Therefore, one of the biggest challenges in the development of central nervous system drugs is effective brain-targeting technology. Currently, nanocarrier-mediated strategies are emerging as a non-invasive and effective method for exploring the treatment of brain diseases (such as lipid nanocarriers, polymer micelles, polymer nanoparticles, nanogels, microemulsions, and metal-organic frameworks). Among these, metal-organic frameworks (such as ZIF-8 and Uio-66) are considered promising tools for delivering drugs to the brain, as they can not only improve drug brain targeting but also effectively enhance drug bioavailability and maintain the normal physiological function of the blood-brain barrier.

[0004] Zeo-lite imidazole framework (ZIF) is a structure composed of zinc ions (Zn). 2+ Metal-organic frameworks (ZIFs) composed of zinc and imidazole or their derivatives are the most widely used drug carriers among zinc-based metal-organic frameworks. The most representative ZIF is ZIF-8, composed of Zn... 2+ ZIF-8, formed by coordination with the N atom on the 2-methylimidazolium ring, possesses significant advantages such as large specific surface area, high porosity, good stability, simple preparation, and acid sensitivity. Furthermore, it exhibits excellent biocompatibility; compared to heavy metals like Zr, Cr, and Cu, Zn poses less harm and lower toxicity to organisms. ZIF-8's acid sensitivity and pH-responsive release mechanism make it well-suited for controlled drug release. Studies have reported that ZIF-8's pH sensitivity is due to acidic conditions causing Zn to... 2+The ZIF-8 framework breaks down by cleaving the coordination bonds with nitrogen atoms, releasing the drug. Under normal pH conditions (pH 7.4), ZIF-8 can retain the drug within its pores, protecting the environment from the drug's toxic side effects. Organophosphate poisoning causes inflammation in the central nervous system, leading to a decrease in pH, which further facilitates drug release into the brain. Furthermore, numerous studies have documented the use of ZIF-8 as a drug carrier for brain delivery, providing reliable evidence for this experiment. Therefore, this study utilizes the unique stable porous framework of ZIF-8 to efficiently encapsulate and transfer the drug into the brain, aiming to improve the central nervous system detoxification effect of pralidoxime.

[0005] A glycoprotein (RVG) in the rabies virus capsid specifically recognizes acetylcholine receptors expressed on the surface of nerve cells. Kumar et al. discovered a 29-amino acid polypeptide on the rabies virus glycoprotein that can bind to the specific acetylcholine receptor on the blood-brain barrier, ultimately allowing the rabies virus to cross the blood-brain barrier and infect nerve cells. However, RVG 29 is characterized by its long structure and high molecular weight, which negatively impacts the particle size of the modified nanoparticles, thus limiting its application. Therefore, those skilled in the art are dedicated to developing a method for preparing a zeolite imidazole ester skeleton as an antidote for central nervous system poisoning, which can effectively improve the reactivation ability of the reactivator HI-6 on central nervous system poisoning enzymes after nerve agent poisoning. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing a zeolite imidazole ester skeleton as an antidote for central nervous system poisoning, which can effectively improve the reactivation ability of the reactivator HI-6 on central nervous system poisoning enzymes after nerve agent poisoning. The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for preparing a zeolite imidazole ester skeleton as an antidote for central nervous system poisoning, characterized by comprising the following steps: S100. Weigh 2.6 mg of Mal-PEG2000-COOH and dissolve it in 5 ml of PBS solution. The pH of the PBS solution is 7.4. After sonication to fully dissolve the solution, the first reaction solution is formed. S200. Weigh 2 mg of RVG15 and dissolve it in 5 ml of PBS solution with a pH of 7.4. After sonication to fully dissolve the PBS solution, a second reaction solution is formed. S300. Add the first reaction solution from step S100 and the second reaction solution from step S200 into a reaction flask, and react under nitrogen protection and in the dark for 20 to 28 hours. After dialysis through a 3000 Da dialysis bag to remove unreacted substances, freeze-dry the obtained product for 20 to 28 hours to prepare RVG15-PEG2000-COOH. S400. Weigh HI-6 and dissolve it in blank ZIF-8 suspension, stir evenly to form drug-loaded ZIF-8, centrifuge the drug-loaded ZIF-8 to remove free drug, and wash it with methanol and water alternately to prepare HI-6@ZIF-8. S500. The RVG-PEG2000-COOH prepared in step S300 is added to the HI-6@ZIF-8 prepared in step S400 to prepare HI-6-loaded brain-targeting ZIF-8.

[0007] The beneficial effects of the above scheme are as follows: ZIF-8 is selected as the drug carrier to encapsulate the hydrophilic oxime reactivator HI-6, and its surface is modified by linking PEG with peptide RVG15 to prepare a ZIF-8 formulation with high encapsulation efficiency and drug loading, and central targeting. It is expected to effectively improve the reactivation ability of the reactivator HI-6 on central nervous system poisoning enzymes after nerve agent poisoning, with the characteristics of low cytotoxicity and active central targeting, thereby improving the treatment level of central nervous system poisoning enzymes in organophosphate poisoning.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, in step S400, the preparation of the blank ZIF-8 suspension includes the following steps: S410. Weigh out 2-methylimidazole and dissolve it in ultrapure water by sonication to form a solvent; S420. Weigh out zinc acetate dihydrate and dissolve it in ultrapure water by ultrasonication to fully dissolve it and form a dropping agent; S430. Slowly add the dropper prepared in step S420 to the solvent prepared in step S410, and stir for 0.5 h to 1.5 h to prepare a blank ZIF-8 suspension.

[0010] The beneficial effect of adopting the above-mentioned further scheme is that it facilitates the rapid preparation of blank ZIF-8 suspension. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the HI-6 structure in a specific embodiment of the present invention; Figure 2 This is a schematic diagram of the main components of ZIF-8 in a specific embodiment of the present invention; Figure 3This is a schematic diagram of the ZIF-8 connection method in a specific embodiment of the present invention; Figure 4 This is a schematic diagram of the RVG15 structure in a specific embodiment of the present invention; Figure 5 This is a synthetic route diagram of RVG-PEG2000-COOH in a specific embodiment of the present invention; Figure 6 The above is the 1H NMR spectrum of Mal-PEG2000-COOH in a specific embodiment of the present invention; Figure 7 The above is the proton NMR spectrum of RVG15-PEG2000-COOH in a specific embodiment of the present invention; Figure 8 This is a physical characterization diagram of RVG15-HI-6@ZIF-8 in a specific embodiment of the present invention; Figure 9 The images show the TEER monitoring data of the BMECs monolayer culture model and the BMECs co-culture model with AS in a specific embodiment of the present invention. Figure 10 This is a standard FLU curve diagram from a specific embodiment of the present invention; Figure 11 The above is a graph showing the FLU permeability coefficients of two in vitro BBB models in a specific embodiment of the present invention. Figure 12 This is a graph showing the cytotoxicity of different concentrations of free HI-6 and different ZIF-8 to BMECs in a specific embodiment of the present invention. Figure 13 This is a permeability coefficient diagram of free HI-6 and different ZIF-8 in an in vitro BBB model in a specific embodiment of the present invention. Detailed Implementation

[0012] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0013] In the description of this invention, it should be understood that the terms "center," "length," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "inner," "outer," "circumferential," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the system or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0014] In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0015] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0016] A method for preparing a zeolite imidazole ester skeleton as an antidote for central nervous system poisoning, characterized by comprising the following steps: S100. Weigh 2.6 mg of Mal-PEG2000-COOH (maleimide-polyethylene glycol-carboxyl) and dissolve it in 5 ml of PBS (phosphate buffered saline) solution. The pH of the PBS solution is 7.4. After sonication to fully dissolve the solution, the first reaction solution is formed. S200. Weigh 2 mg of RVG15 (a short peptide consisting of 15 amino acids, a 15-peptide fragment derived from rabies virus glycoprotein (RVG)) and dissolve it in 5 ml of PBS solution with a pH of 7.4. After sonication to fully dissolve, a second reaction solution is formed. S300. Add the first reaction solution from step S100 and the second reaction solution from step S200 into a reaction flask, and react under nitrogen protection and in the dark for 20 to 28 hours. After dialysis through a 3000 Da dialysis bag to remove unreacted substances, freeze-dry the obtained product for 20 to 28 hours to prepare RVG15-PEG2000-COOH. S400. Weigh HI-6 (phosphatidylcholine) and dissolve it in blank ZIF-8 (zeolite imidazole skeleton) suspension. Stir evenly to form drug-loaded ZIF-8. After centrifuging the drug-loaded ZIF-8 to remove free drug, wash it with methanol and water alternately to prepare HI-6@ZIF-8. The preparation of the blank ZIF-8 suspension includes the following steps: S410. Weigh 2.8g of 2-methylimidazole and dissolve it in 5ml of ultrapure water by sonication to form a dispensing agent; S420. Weigh 150 mg of zinc acetate dihydrate and dissolve it in 5 ml of ultrapure water by sonication to form a solvent. S430. Slowly add the dropper prepared in step S420 to the solvent prepared in step S410, and stir for 0.5 h to 1.5 h to prepare a blank ZIF-8 suspension.

[0017] S500. The RVG-PEG2000-COOH prepared in step S300 is added to the HI-6@ZIF-8 prepared in step S400 to prepare HI-6-loaded brain-targeting ZIF-8.

[0018] Example 1 like Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 As shown, The synthesis of the brain-targeting peptide RVG15-PEG2000-COOH mainly involves the following steps: S100. Accurately weigh Mal-PEG2000-COOH (compound A, 2.6 mg) and dissolve it completely in PBS (pH=7.4); S200. Accurately weigh RVG15-Cys (compound B, 2 mg), dissolve it thoroughly in PBS (pH=7.4), and mix the two thoroughly in a 20 mL round-bottom flask. Under S300 nitrogen (N2) protection, the above mixture was reacted in the dark for 24 h, dialyzed through a 3000 Da dialysis bag to remove unreacted substances, and the resulting product was freeze-dried for 24 h.

[0019] Preparation of RVG15-modified centrally targeting ZIF-8 loaded with HI-6: A well-morphologically optimized ZIF-8 organometallic framework loaded with the acetylcholinesterase reactivator HI-6 was designed and prepared using the impregnation method. Impregnation is a traditional method for preparing ZIF-8, and it allows for easier control of the ZIF-8 size. Therefore, this study used the impregnation method to encapsulate HI-6. Based on previous studies, the optimal formulation of the HI-6-loaded ZIF-8 was determined.

[0020] S400. Accurately weigh the prescribed amount of 2-methylimidazole and dissolve it in 15 ml of water; dissolve 150 mg of zinc acetate dihydrate in 5 ml of water; sonicate to ensure complete dissolution. Slowly add zinc acetate dihydrate dropwise to the 2-methylimidazole solution, stir for 1 hour, let stand for 1 hour, centrifuge, wash three times alternately with methanol and water, freeze-dry, and store at -20℃. This yields blank ZIF-8. Next, accurately weigh 20 mg of ZIF-8 and dissolve it in 10 ml of deionized water to obtain a blank ZIF-8 suspension. Accurately weigh the prescribed amount of HI-6 and dissolve it in the blank ZIF-8 suspension. After stirring and loading the drug, centrifuge the drug-loaded ZIF-8 to remove free drug, finally obtaining HI-6@ZIF-8. After the preparation of HI-6@ZIF-8, a synthesized brain-targeting peptide-polyethylene glycol-carboxylic acid conjugate (RVG15-PEG2000-COOH) was introduced to prepare three batches of HI-6-loaded brain-targeting ZIF-8. The final average encapsulation efficiency was (62.51±0.36)%, and the drug loading was (8.57±0.25)%. The morphology of ZIF-8 was observed by transmission electron microscopy. Figure 8 As can be seen from -C), RVG15-PEG2000-HI-6@ZIF-8 particles are spherical and dispersed. The average particle size is 188.9 nm (polydispersity index is 0.247), and the zeta potential is -4.13 mV. Figure 8 -A、 Figure 8 -B).

[0021] Figure 8 In the diagram, A: Zeta potential diagrams for different ZIF-8 molecules; B: Particle size distribution of different ZIF-8 particles; C: Transmission electron microscope image of ZIF-8; Transmission electron microscope image of D:RVG15-HI-6@ZIF-8; Mapping analysis of E:RVG-HI-6@ZIF-8.

[0022] The establishment of the BBB model: A non-contact co-culture model of the blood-brain barrier (BBB) ​​was established by seeding BMECs (brain capillary endothelial cells) in the inner chamber and AS (astrocytes) in the lower chamber of a Transwell 24-well plate. A single-culture BBB model was also established by seeding BMECs in the inner chamber of a Transwell 24-well plate. Transendothelial cell resistance (TEER) and sodium fluorescein permeability coefficient were used as indicators to evaluate the success of the in vitro BBB model establishment.

[0023] Measurement of TEER (transendothelial resistance) The TEER value was monitored daily starting from day 3 of culture for both the BMECs monoculture BBB model and the BMECs+AS co-culture BBB model. The TEER value reached its maximum on day 7, with the co-culture BBB model reaching 66 and the monoculture BBB model reaching 58. The lower TEER value of the monoculture BBB model suggests that the BMECs + AS co-culture BBB model has better barrier function.

[0024] Determination of the permeability coefficient of sodium fluorescein like Figure 11 As shown, 200 μL of 200 μg·mL⁻¹ FLU solution was added to the upper chamber of the Transwell apparatus, and 800 μL of D-Hanks solution was added to the lower chamber. 200 μL of liquid was collected from the lower chamber at time points of 15, 30, and 60 min, with 200 μL of D-Hanks solution added simultaneously. The OD value of the collected liquid was measured. Compared to the monoculture in vitro BBB model, the co-cultured BBB model exhibited lower permeability, indicating a higher membrane tightness in the co-cultured BBB model. Therefore, the non-contact BMECs+AS co-cultured BBB model was chosen for subsequent experiments. Cytotoxicity studies: like Figure 12 As shown, different concentrations of ZIF-8 formulations were diluted to predetermined concentrations (based on HI-6 concentration). Six replicates were set for each concentration, and a control group and a zero-adjustment group were included. An appropriate amount of BMECs passaged to the third generation was seeded into 96-well plates and cultured for 24 hours. Then, different concentrations of ZIF-8 solution were added, and the cells were cultured for another 24 hours. 200 μl of 10% CCK 8 reagent was added to each well. After incubation for another 2 hours, the absorbance was measured at 450 nm to calculate cell viability.

[0025] The blank metal-organic framework ZIF 8 maintained BMEC cell viability above 85% within a concentration range of 5-100 μg·mL⁻¹, indicating that the material itself has low cytotoxicity. This demonstrates the safety advantage of the metal-organic framework ZIF 8 provided in this study. Penetration experiments of ZIF-8 solutions with different HI-6 loadings in an in vitro BBB model Different HI-6 formulations were dispersed in D-Hanks solution, and 200 μL of the solution was added to the upper chamber at a concentration of 1 mg·mL⁻¹. The liquid in the lower chamber was collected at 15, 30, and 60 minutes to detect the HI-6 content and calculate the permeability coefficient.

[0026] like Figure 13As shown, the in vitro BBB penetration of HI-6@ZIF-8 modified with peptide RVG was higher than that of free HI-6 and HI-6@ZIF-8. Furthermore, the maximum penetration coefficient was reached at 30 minutes. This indicates that the peptide-modified ZIF-8 formulation can rapidly and effectively increase the intracerebral transport rate of HI-6. In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a zeolite imidazole ester skeleton as an antidote for central nervous system poisoning, characterized in that, Includes the following steps: S100. Weigh 2.6 mg of Mal-PEG2000-COOH and dissolve it in 5 ml of PBS solution. The pH of the PBS solution is 7.

4. After sonication to fully dissolve the solution, the first reaction solution is formed. S200. Weigh 2 mg of RVG15 and dissolve it in 5 ml of PBS solution with a pH of 7.

4. After sonication to fully dissolve the PBS solution, a second reaction solution is formed. S300. Add the first reaction solution from step S100 and the second reaction solution from step S200 into a reaction flask, and react under nitrogen protection and in the dark for 20 to 28 hours. After dialysis through a 3000 Da dialysis bag to remove unreacted substances, freeze-dry the obtained product for 20 to 28 hours to prepare RVG15-PEG2000-COOH. S400. Weigh HI-6 and dissolve it in blank ZIF-8 suspension, stir evenly to form drug-loaded ZIF-8, centrifuge the drug-loaded ZIF-8 to remove free drug, and wash it with methanol and water alternately to prepare HI-6@ZIF-8. S500. The RVG-PEG2000-COOH prepared in step S300 is added to the HI-6@ZIF-8 prepared in step S400 to prepare HI-6-loaded brain-targeting ZIF-8.

2. The method for preparing the zeolite imidazole ester skeleton as an antidote for central nervous system poisoning according to claim 1, characterized in that, In step S400, the preparation of the blank ZIF-8 suspension includes the following steps: S410. Weigh out 2-methylimidazole and dissolve it in ultrapure water by sonication to form a solvent; S420. Weigh out zinc acetate dihydrate and dissolve it in ultrapure water by ultrasonication to fully dissolve it and form a dropping agent; S430. Slowly add the dropper prepared in step S420 to the solvent prepared in step S410, and stir for 0.5 h to 1.5 h to prepare a blank ZIF-8 suspension.

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