Preparation method and application of malonic acid precursor polymer nanomedicine

Abstract

The present application relates to the field of precursor polymer drug preparation, in particular to a malonic acid precursor polymer self-assembly nanodrug preparation and application taking malonic acid as a loading object. The present application forms a drug precursor polymer through a covalent connection mode of a malonic acid precursor molecular drug, solves the metabolic kinetics obstacle of malonic acid, and increases the bioavailability of malonic acid. The present application can effectively release malonic acid as a specific inhibitor of succinate dehydrogenase in an oxidative state, has the advantages of non-toxicity, non-immunogenicity and degradability. And the polymer of the present application can self-assemble to form a nanodrug, and has stronger curative effect on the inhibition of succinate dehydrogenase in some mitochondrial metabolic activities, and has potential application value in the preparation of myocardial infarction treatment drugs. The preparation process of the present application is simple, the components are clear, and the quality is controllable.

Description

Technical Field

[0001] This invention relates to the field of precursor polymer drug preparation, and particularly to the preparation and application of a malonic acid precursor polymer self-assembled nanomedicine with malonic acid as the loading object. Background Technology

[0002] Malonic acid is mainly used as a pharmaceutical intermediate, but as a succinate dehydrogenase inhibitor, it has the potential to treat ischemia-reperfusion injury. Studies have found that malonic acid can effectively inhibit excessive intracellular reactive oxygen species, thus protecting against renal ischemia-reperfusion injury and also providing good protection against myocardial injury.

[0003] Due to limitations in dosage and side effects from large doses, there are currently no suitable prodrugs available for preclinical research. Although some small-molecule malonic acid prodrugs, such as dimethyl malonate, can exert their effects by being degraded by esterases in vivo to form malonic acid, their lack of targeting and poor pharmacokinetic issues make them unsuitable for the actual delivery of malonic acid and thus unable to treat ischemia-reperfusion myocardial injury.

[0004] Polymer drug precursors offer numerous advantages in the biopharmaceutical field. They possess excellent biocompatibility, low immunogenicity, and biodegradability. Furthermore, they allow for precise design of drug-loading units and can self-assemble through amphiphilic interactions to form suitable nanomedicines. Simultaneously, polymer drug precursors can be flexibly modified with targeting groups to achieve targeted delivery to specific tissues. Therefore, polymer drug precursors are highly ideal drug delivery carrier materials.

[0005] Polymer self-assembly refers to a technique that spontaneously forms ordered structures based on non-covalent or weakly covalent interactions. This technique allows drug molecules to spontaneously form stable nanomedicines with other drug molecules or functional molecules. The preparation process of nanomedicines through self-assembly is green and simple, avoiding cumbersome synthesis processes, and it also achieves higher drug loading capacity compared to nanomedicines formed by loading drugs onto traditional nanocarriers.

[0006] In summary, forming nanomedicines by covalently linking drug precursors to form polymers, and then self-assembling these polymers, not only increases drug loading efficiency and addresses the need for specific drug release, but also significantly increases drug bioavailability and enhances therapeutic efficacy. Compared to traditional nanomedicines, these nanomedicines also offer advantages such as high biocompatibility and high drug loading capacity, while their synthesis process is green and simple, making them promising for industrial production. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned technological status quo by providing a method for the preparation and application of self-assembled nanomedicines using malonic acid precursor polymers as the loading material. This method can solve the problems of low safety and low drug loading capacity in existing nanomedicine delivery systems.

[0008] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0009] A method for preparing a malonic acid precursor polymer nanomedicine includes the following steps:

[0010] Step 1: Dissolve the malonic acid precursor molecules and polymer molecules in tetrahydrofuran, add disodium diselenide solution, and react under nitrogen protection.

[0011] Step 2: After the reaction is complete, add chloroform, centrifuge to remove the black precipitate, and obtain the organic phase. Evaporate the organic phase to obtain the dry precipitate.

[0012] Step 3: The malonic acid precursor polymer is self-assembled using an emulsification method to obtain malonic acid precursor polymer nanomedicine.

[0013] Furthermore, in step 1, the mass ratio of the malonic acid precursor molecule to the polymer molecule ranges from 1:1 to 3:1.

[0014] Further, in step 1, the preparation method of the disodium diselenide solution is as follows: one part of sodium borohydride is dissolved in water, one part of selenium powder is added, and after the solution becomes transparent under ice bath conditions, another part of selenium powder is added, and the reaction is carried out at 50°C until the solution turns wine red, thus obtaining the disodium diselenide solution.

[0015] Further, in step 1, the preparation method of the malonic acid precursor molecule is as follows: Michaelis acid and bromoethanol are dissolved in toluene solution and refluxed for 2 hours; the above reaction mixture is extracted with saturated sodium bicarbonate solution; the aqueous phase obtained by extraction is acidified with concentrated hydrochloric acid, extracted with methyl tert-butyl ether, and dried with anhydrous magnesium sulfate; the dried solvent is evaporated under vacuum to obtain an oily substance, i.e., the malonic acid precursor molecule.

[0016] Further, the bromoethanol includes one of 2-bromoethanol, 3-bromo-1-propanol, 4-bromo-1-butanol, 5-bromo-1-pentanol, and 6-bromo-1-hexanol.

[0017] Further, in step 1, the preparation method of the polymer molecule is as follows: a hydrophilic monomer and a hydrophobic bromine-containing monomer are dissolved in dioxane, a chain transfer agent and an initiator are added, and the reaction is carried out at 80°C for 24 hours under anhydrous and oxygen-free conditions; after the reaction is completed, the mixture is placed in liquid nitrogen to terminate the reaction, and the resulting mixture is precipitated with excess methyl tert-butyl ether to obtain a residue. The residue is dissolved in dichloromethane and precipitated again with methyl tert-butyl ether, and the process is repeated three times.

[0018] Furthermore, the hydrophilic monomers include: dimethylaminoethyl methacrylate, N,N-dimethylacetamide, N,N-diethylacrylamide, and methacrylamide.

[0019] Furthermore, the hydrophobic bromine-containing monomers include: 2-bromoethyl methacrylate, 3-bromopropyl methacrylate, and 4-bromobutyl methacrylate.

[0020] A malonic acid precursor polymer nanomedicine prepared by the above method is formed by connecting diselenyl bonds broken in response to reactive oxygen species to polymer matrix molecules to form a malonic acid precursor polymer, which then self-assembles to form a nanomedicine with relatively uniform particle size.

[0021] Application of a malonic acid precursor polymer nanomedicine prepared by the above method in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. This invention selects malonic acid, a succinate dehydrogenase inhibitor with a proven ability to alleviate myocardial ischemia-reperfusion injury, as the target drug, and uses a polymer as the matrix to deliver the drug. The malonic acid precursor polymer nanomedicine synthesized by the two has a grafting rate of over 80%.

[0024] 2. By specifically releasing malonic acid at the site of myocardial injury under high levels of reactive oxygen species, it can exert a stronger therapeutic effect in the treatment of specific diseases.

[0025] 3. Malonic acid precursor polymer nanomedicines are directly formed through self-assembly, with suitable particle size, controllable morphology, and good biosafety. Attached Figure Description

[0026] Figure 1 The 1H NMR spectrum of the malonic acid precursor polymer of this invention.

[0027] Figure 2 The particle size distribution of the nanomedicine carrier of this invention in ultrapure water.

[0028] Figure 3 Electron micrograph of the nanomedicine of this invention.

[0029] Figure 4 The stability of the particle size distribution of the nanomedicine carrier of the present invention in ultrapure water was observed over one week.

[0030] Figure 5 The effects of different concentrations of nanomedicine carriers on cardiomyocyte activity were investigated in this invention.

[0031] Figure 6Cell imaging (CLSM) of cells treated with fluorescein Rhodamine B for 4 hours during the assembly of the nanomedicine carrier of this invention. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0033] Example 1

[0034] The preparation of a malonic acid precursor polymer nanomedicine includes the following steps:

[0035] (1) Synthesis of malonic acid precursor (3-(2-bromoethoxy)-3-oxomalonic acid) (EMal):

[0036] A. Reflux the solution of Michaelis-Müller's acid (14.1 mmol) and 2-bromoethanol (14.1 mmol) in 10 mL of dry toluene for two hours;

[0037] B. The reaction mixture was washed with saturated NaHCO3 solution (20 ml);

[0038] C. The aqueous phase was acidified with concentrated sulfuric acid and extracted twice with methyl tert-butyl ether (20 mL);

[0039] D. After drying on MgSO4, the solvent is evaporated and dried under high vacuum to obtain a light yellow oily substance.

[0040] (2) Preparation of polymer P (DMAEMA-co-BrMA)

[0041] A. The chain transfer agent (CTABD) was dissolved in anhydrous dioxane at a molar ratio of 1:80:20 with purified hydrophilic monomer N,N-dimethylacetamide (DMAEMA) and hydrophobic bromine-containing monomer 2-bromoethyl methacrylate (BrMA). The molar ratio of the initiator (AIBN) to the chain transfer agent was 0.2:1. The total amount of all monomers and the weight ratio of the chain transfer agent to dioxane were 0.5.

[0042] B. Place in liquid nitrogen, reduce pressure, and then dissolve. Repeat this process three times.

[0043] C. Then seal the reaction system and polymerize at 80°C for 24 hours;

[0044] D. After the reaction is complete, the mixture is placed in liquid nitrogen to terminate the reaction. The resulting mixture is precipitated with excess methyl tert-butyl ether to obtain the residue. The residue is dissolved in dichloromethane and precipitated again with methyl tert-butyl ether. This process is repeated three times.

[0045] (3) Preparation of Na2Se2 solution

[0046] A. Dissolve 50 mg of sodium borohydride in 10 mL of water, add 50 mg of selenium powder, and stir slowly in an ice bath until the solution becomes colorless and transparent;

[0047] B. Add 50 mg of selenium powder to the above colorless and transparent solution, and react at 50 °C under nitrogen protection. The solution turns wine red, yielding a sodium diselenide (Na2Se2) solution.

[0048] (4) Polymer molecules grafted with malonic acid precursor P (DMAEMA-co-SeEMal)

[0049] A. Dissolve polymer P (DMAEMA-co-BrMA) in anhydrous tetrahydrofuran (1 mg / mL) and stir slowly until fully dissolved;

[0050] B. Then, under nitrogen protection, add (3-(2-bromoethoxy)-3-oxomalonic acid)(EMal) and stir rapidly;

[0051] C. Add freshly prepared Na2Se2 solution to the above solution and continue stirring for 5 hours;

[0052] D. After the reaction is complete, chloroform is added, the black precipitate is removed by centrifugation, and the organic phase is obtained. The organic phase is removed by evaporation to obtain dry P(DMAEMA-co-SeEMal).

[0053] (5) Preparation of nanomedicines

[0054] P(DMAEMA-co-SeEMal) was dissolved in CCl3 (1 mg / mL). After complete dissolution, it was added to rapidly stirred deionized water, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain the nanomedicine.

[0055] The degree of polymerization of the polymer was calculated to be P[(DMAEMA0.43-co-BrMA0.57)]35, and the grafting rate of the malonic acid precursor was 90%. The P(DMAEMA-co-SeEMal) nanomedicine prepared in Example 1 has a spherical structure with a diameter of 60 nm and good dispersibility. The nanomedicine carrier continuously releases the drug, exhibiting good sustained-release performance and a good protective effect against oxidative stress on cardiomyocytes.

[0056] Figure 1 The present invention uses 2-bromoethanol as a bromoethanol to prepare malonic acid precursor molecules, and uses N,N-dimethylacetamide as a hydrophilic monomer and 2-bromoethyl methacrylate as a hydrophobic monomer to prepare polymer molecules. The NMR spectrum of the polymer prepared from the two is 87%, showing that the grafting rate of malonic acid precursor is 87%.

[0057] Figure 2 , Figure 3 These are the particle size distribution and electron micrographs of the nanomedicine carrier of this invention in ultrapure water, with a particle size of approximately 60 nm.

[0058] Figure 4 The stability of the particle size distribution of the nanomedicine carrier of this invention in ultrapure water was observed over a week, and it maintained good particle size stability in water.

[0059] Figure 5 This invention relates to the effect of different concentrations of nanomedicine carriers on cardiomyocyte activity, demonstrating that P(DMAEMA-co-SeEMal) nanomedicine did not affect cell activity at a concentration of 30 μg / mL. In cardiomyocytes induced by 100 μM hydrogen peroxide oxidation damage, P(DMAEMA-co-SeEMal) nanomedicine exhibited concentration-dependent cardiomyocyte protection.

[0060] Figure 6 The cell imaging (CLSM) of cells treated with Rhodamine B for 4 hours during the assembly of the nanomedicine carrier of this invention shows that P(DMAEMA-co-SeEMal) can be well taken up by cardiomyocytes.

[0061] Example 2

[0062] The preparation of a malonic acid precursor polymer nanomedicine includes the following steps:

[0063] (1) Synthesis of malonic acid precursor (3-(3-bromopropoxy)-3-oxopropionic acid) (PMal):

[0064] A. Reflux the solution of Michaelis-Müller's acid (14.1 mmol) and 2-bromopropanol (14.1 mmol) in 10 mL of dry toluene for two hours;

[0065] B. The reaction mixture was washed with saturated NaHCO3 solution (20 ml);

[0066] C. The aqueous phase was acidified with concentrated sulfuric acid and extracted twice with methyl tert-butyl ether (20 mL);

[0067] D. After drying on MgSO4, the solvent is evaporated and dried under vacuum to obtain a light yellow oily substance.

[0068] (2) Preparation of polymer P (DMAEMA-co-BrMA)

[0069] A. The chain transfer agent (CTABD) was dissolved in anhydrous dioxane at a molar ratio of 1:80:20 with purified hydrophilic monomer N,N-dimethylacetamide (DMAEMA) and hydrophobic bromine-containing monomer 2-bromoethyl methacrylate (BrMA). The molar ratio of the initiator (AIBN) to the chain transfer agent was 0.2:1. The total amount of all monomers and the weight ratio of the chain transfer agent to dioxane were 0.5.

[0070] B. Place in liquid nitrogen, reduce pressure, and then dissolve. Repeat this process three times.

[0071] C. Then seal the reaction system and polymerize at 80°C for 24 hours;

[0072] D. After the reaction is complete, the mixture is placed in liquid nitrogen to terminate the reaction. The resulting mixture is precipitated with excess methyl tert-butyl ether to obtain the residue. The residue is dissolved in dichloromethane and precipitated again with methyl tert-butyl ether. This process is repeated three times.

[0073] (3) Preparation of Na2Se2 solution

[0074] A. Dissolve 50 mg of sodium borohydride in 10 mL of water, add 50 mg of selenium powder, and stir slowly in an ice bath until the solution becomes colorless and transparent;

[0075] B. Add 50 mg of selenium powder to the above colorless and transparent solution, and react at 50 °C under nitrogen protection. The solution turns wine red, yielding a sodium diselenide (Na2Se2) solution.

[0076] (4) Polymer molecules grafted with malonic acid precursor P (DMAEMA-co-SePMal)

[0077] A. Dissolve polymer P (DMAEMA-co-BrMA) in anhydrous tetrahydrofuran (1 mg / mL) and stir slowly until fully dissolved;

[0078] B. Then, under nitrogen protection, add (3-(3-bromopropoxy)-3-oxopropionic acid)(PMal) and stir rapidly;

[0079] C. Add freshly prepared Na2Se2 solution to the above solution and continue stirring for 5 hours;

[0080] D. After the reaction is complete, chloroform is added, the black precipitate is removed by centrifugation, and the organic phase is obtained. The organic phase is removed by evaporation to obtain dry P(DMAEMA-co-SePMal).

[0081] (5) Preparation of nanomedicines

[0082] P(DMAEMA-co-SePMal) was dissolved in CCl3 (1 mg / mL). After complete dissolution, it was added to rapidly stirred deionized water, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain the nanomedicine.

[0083] The P(DMAEMA-co-SePMal) nanomedicine prepared in Example 2 has a spherical structure with a diameter of 80 nm and good dispersibility. The nanomedicine carrier continuously releases the drug, exhibiting good sustained-release performance and a good protective effect against oxidative stress on cardiomyocytes.

[0084] Example 3

[0085] The preparation of a malonic acid precursor polymer nanomedicine includes the following steps:

[0086] (1) Synthesis of malonic acid precursor (3-(4-bromobutoxy)-3-oxopropionic acid) (OMal):

[0087] A. Reflux the solution of Mischel acid (14.1 mmol) and 2-bromobutanol (14.1 mmol) in 10 mL of dry toluene for two hours;

[0088] B. The reaction mixture was washed with saturated NaHCO3 solution (20 ml);

[0089] C. The aqueous phase was acidified with concentrated sulfuric acid and extracted twice with methyl tert-butyl ether (20 mL);

[0090] D. After drying on MgSO4, the solvent is evaporated and dried under vacuum to obtain a light yellow oily substance.

[0091] (2) Preparation of polymer P (DMAEMA-co-BrMA)

[0092] A. The chain transfer agent (CTABD) was dissolved in anhydrous dioxane at a molar ratio of 1:80:20 with purified hydrophilic monomer N,N-dimethylacetamide (DMAEMA) and hydrophobic bromine-containing monomer 2-bromoethyl methacrylate (BrMA). The molar ratio of the initiator (AIBN) to the chain transfer agent was 0.2:1. The total amount of all monomers and the weight ratio of the chain transfer agent to dioxane were 0.5.

[0093] B. Place in liquid nitrogen, reduce pressure, and then dissolve. Repeat this process three times.

[0094] C. Then seal the reaction system and polymerize at 80°C for 24 hours;

[0095] D. After the reaction is complete, the mixture is placed in liquid nitrogen to terminate the reaction. The resulting mixture is precipitated with excess methyl tert-butyl ether to obtain the residue. The residue is dissolved in dichloromethane and precipitated again with methyl tert-butyl ether. This process is repeated three times.

[0096] (3) Preparation of Na2Se2 solution

[0097] A. Dissolve 50 mg of sodium borohydride in 10 mL of water, add 50 mg of selenium powder, and stir slowly in an ice bath until the solution becomes colorless and transparent;

[0098] B. Add 50 mg of selenium powder to the above colorless and transparent solution, and react at 50 °C under nitrogen protection. The solution turns wine red, yielding a sodium diselenide (Na2Se2) solution.

[0099] (4) Polymer molecules grafted with malonic acid precursor P (DMAEMA-co-SeOMal)

[0100] A. Dissolve polymer P (DMAEMA-co-BrMA) in anhydrous tetrahydrofuran (1 mg / mL) and stir slowly until fully dissolved;

[0101] B. Then, under nitrogen protection, add (3-(4-bromobutoxy)-3-oxopropionic acid)(OMal) and stir rapidly;

[0102] C. Add freshly prepared Na2Se2 solution to the above solution and then continue stirring for 5 hours;

[0103] D. After the reaction is complete, chloroform is added, the black precipitate is removed by centrifugation, and the organic phase is obtained. The organic phase is removed by evaporation to obtain dry P(DMAEMA-co-SeOMal).

[0104] (5) Preparation of nanomedicines

[0105] P(DMAEMA-co-SeOMal) was dissolved in CCl3 (1 mg / mL). After complete dissolution, it was added to rapidly stirred deionized water, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain the nanomedicine.

[0106] The P(DMAEMA-co-SeOMal) nanomedicine prepared in Example 3 has a spherical structure with a diameter of 100 nm and good dispersibility. The nanomedicine carrier continuously releases the drug, exhibiting good sustained-release performance and a good protective effect against oxidative stress on cardiomyocytes.

[0107] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a malonic acid precursor polymer nanomedicine, characterized in that, Includes the following steps: Step 1: Dissolve malonic acid precursor molecules and polymer molecules in tetrahydrofuran, add disodium diselenide solution, and react under nitrogen protection. The specific preparation method of the malonic acid precursor molecules is as follows: dissolve Michaelis acid and bromoethanol in toluene solution and reflux for 2 hours; extract the refluxed reaction mixture with saturated sodium bicarbonate solution; acidify the aqueous phase obtained from the extraction with concentrated hydrochloric acid, extract with methyl tert-butyl ether, and dry with anhydrous magnesium sulfate; evaporate the dried solvent under vacuum to obtain an oily substance, i.e., malonic acid precursor molecules; the mass ratio of the malonic acid precursor molecules to polymer molecules ranges from 1:1 to 3:

1. Step 2: After the reaction is complete, add chloroform, centrifuge to remove the black precipitate, and obtain the organic phase. Evaporate the organic phase to obtain the dry precipitate, which is the malonic acid precursor polymer. Step 3: The malonic acid precursor polymer is self-assembled using an emulsification method to obtain malonic acid precursor polymer nanomedicine.

2. The method for preparing a malonic acid precursor polymer nanomedicine as described in claim 1, characterized in that, In step 1, the preparation method of the disodium diselenide solution is as follows: one part of sodium borohydride is dissolved in water, one part of selenium powder is added, and after the solution becomes transparent under ice bath conditions, another part of selenium powder is added. The reaction is carried out at 50°C until the solution turns wine red, thus obtaining the disodium diselenide solution.

3. The method for preparing a malonic acid precursor polymer nanomedicine as described in claim 1, characterized in that, The bromoethanol includes one of 2-bromoethanol, 3-bromo-1-propanol, 4-bromo-1-butanol, 5-bromo-1-pentanol, and 6-bromo-1-hexanol.

4. The method for preparing a malonic acid precursor polymer nanomedicine as described in claim 1, characterized in that, In step 1, the preparation method of the polymer molecule is as follows: hydrophilic monomers and hydrophobic bromine-containing monomers are dissolved in dioxane, chain transfer agent and initiator are added, and the reaction is carried out at 80°C for 24 hours under anhydrous and oxygen-free conditions; after the reaction is completed, the mixture is placed in liquid nitrogen to terminate the reaction, and the resulting mixture is precipitated with excess methyl tert-butyl ether to obtain the residue. The residue is dissolved in dichloromethane and precipitated again with methyl tert-butyl ether, and the process is repeated three times.

5. The method for preparing a malonic acid precursor polymer nanomedicine as described in claim 4, characterized in that, The hydrophilic monomers include: dimethylaminoethyl methacrylate, N,N-dimethylacetamide, N,N-diethylacrylamide, and methacrylamide.

6. The method for preparing a malonic acid precursor polymer nanomedicine as described in claim 4, characterized in that, The hydrophobic bromine-containing monomers include: 2-bromoethyl methacrylate, 3-bromopropyl methacrylate, and 4-bromobutyl methacrylate.

7. A malonic acid precursor polymer nanomedicine prepared by the method according to any one of claims 1 to 6, characterized in that, This nanomedicine is formed by connecting diselenide bonds broken in response to reactive oxygen species to polymer matrix molecules to form a malonic acid precursor polymer, which then self-assembles to form a nanomedicine with relatively uniform particle size.

8. The use of a malonic acid precursor polymer nanomedicine prepared by the method according to any one of claims 1 to 6 in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

Images