PH-responsive antibacterial formaldehyde prodrug hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a pH-responsive antibacterial formaldehyde prodrug hydrogel and a preparation method and application thereof, the preparation method comprises the following steps: S1, dissolving polyethylene glycol in deionized water to obtain a polyethylene glycol aqueous solution; s2, adding formaldehyde into the polyethylene glycol aqueous solution to obtain a mixed solution; s3, stirring the mixed solution at high temperature for reaction; and S4, carrying out reduced pressure distillation on the fully reacted mixed solution to obtain the pH-responsive antibacterial formaldehyde prodrug hydrogel. The invention also provides an application of the pre-formaldehyde hydrogel medicine in pharmacy, and the pre-formaldehyde hydrogel can release low-dose gas formaldehyde when the pH value is less than or equal to 7. The pH-responsive antibacterial formaldehyde prodrug hydrogel prepared according to the invention has the advantages of slow release of formaldehyde in an acidic environment, good antibacterial property, high biocompatibility, simple synthesis, mild reaction conditions and convenient cutting, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically to a pH-responsive antibacterial formaldehyde prodrug hydrogel and a preparation method and application thereof. Background Art

[0002] The deepening of bacterial resistance and the protection of bacterial biofilms continue to weaken the antibacterial effect of traditional antibiotics. Therefore, the development of non-antibiotic drugs and treatment strategies is urgent. The use of endogenous gases such as NO, CO, H2S and H2 can achieve efficient and rapid removal of a variety of bacteria, which has attracted widespread attention from researchers. Formaldehyde molecules, which are also endogenous gases, also have broad-spectrum antibacterial properties. At present, the most famous formaldehyde as an antibacterial drug is formalin solution (37% formaldehyde aqueous solution). Its antibacterial properties come from the fact that formaldehyde can quickly destroy bacterial membranes and cross-link bacterial DNA and proteins. However, the high dose of gas molecules and non-selectivity greatly limit the application of formaldehyde gas. Formaldehyde cresol solution (PDA approved) prepared by reducing the formaldehyde concentration is widely used for disinfection of infected root canals, in which the formaldehyde concentration is only 2-8%. Another formaldehyde prodrug product-Hexamethane hippurate (FDA approved) is a broad-spectrum antibacterial drug. Hexamethane is commonly used for urinary tract infections, body odor, and various skin bacterial infections. Hexamethylenetetramine itself does not have a strong antibacterial effect, but after entering the body, it can be hydrolyzed into hippuric acid and formaldehyde under the condition of acidic urine (pH about 6.5). Formaldehyde can denature pathogen proteins and exert a nonspecific antibacterial effect. The concentration of formaldehyde in urine has bactericidal or antibacterial effects depending on the pH value, urine volume and excretion rate. Hippuric acid can maintain the acidity of urine and promote the release of formaldehyde (Nature, 2024, DOI: 10.1038 / d41586-024-01322-y).

[0003] However, these existing formaldehyde drugs, including formaldehyde cresol aqueous solution and hexamethylenetetramine and its derivatives, generally have the problem of difficulty in sustained controlled release, so it is necessary to design prodrugs for formaldehyde and achieve its precise controlled release. In addition, the biocompatibility of prodrugs and their metabolites also needs to be further improved. Summary of the invention

[0004] The purpose of the present invention is to provide a pH-responsive antibacterial formaldehyde prodrug hydrogel and a preparation method and application thereof, thereby solving the problems of complex synthesis, difficult controlled release and high toxicity of metabolites of formaldehyde antibacterial drugs in the prior art.

[0005] In order to solve the above problems, the present invention adopts the following technical solutions:

[0006] According to a first aspect of the present invention, a method for preparing a pH-responsive antibacterial formaldehyde prodrug hydrogel is provided, and the method comprises the following steps: S1, dissolving polyethylene glycol in deionized water to obtain a polyethylene glycol aqueous solution; S2, adding formaldehyde to the polyethylene glycol aqueous solution to obtain a mixed solution; S3, stirring the mixed solution at a high temperature for reaction; S4, performing reduced pressure distillation on the mixed solution after sufficient reaction to obtain a pH-responsive antibacterial formaldehyde prodrug hydrogel.

[0007] Preferably, in step S1, the polyethylene glycol is an amino-terminated polyethylene glycol with a molecular weight ranging from 200 Da to 10000 Da. More preferably, the molecular weight of the polyethylene glycol is 2000 Da.

[0008] Preferably, in step S1, the mass ratio of polyethylene glycol to deionized water is (0.5-2) : 10. More preferably, the mass ratio of polyethylene glycol to deionized water is 1:10.

[0009] Preferably, the molar ratio of formaldehyde to polyethylene glycol is (1.5-3): 1. It should be understood that the excess formaldehyde can be removed in the subsequent vacuum distillation process, and the molar ratio of formaldehyde to polyethylene glycol can be greater than 1.5: 1, but for economic considerations, the molar ratio of formaldehyde to polyethylene glycol is preferably (1.5-3): 1, more preferably 2: 1.

[0010] Preferably, in step S2, the formaldehyde is selected from: 35-40% formaldehyde aqueous solution, paraformaldehyde, and gaseous formaldehyde. In a preferred embodiment, the formaldehyde is paraformaldehyde.

[0011] Preferably, in step S3, the reaction temperature of the mixed solution is 50°C to 100°C, and the reaction time is greater than 60 seconds. In a preferred embodiment, the reaction temperature of the mixed solution is 70°C, and the reaction time is 10 minutes.

[0012] Preferably, in step S4, the water content of the formaldehyde prodrug hydrogel is reduced to less than 90% by vacuum distillation, where the water content is a mass fraction. More preferably, it is less than 80%. In a preferred embodiment, the water content of the hydrogel is 77%.

[0013] Preferably, the method further comprises step S5 of subjecting the formaldehyde hydrogel to a hot pressing treatment at a temperature of 60 to 80° C. for 10 to 20 minutes.

[0014] According to a second aspect of the present invention, a pH-responsive antibacterial formaldehyde prodrug hydrogel prepared by the above-mentioned preparation method is provided, wherein the antibacterial formaldehyde prodrug hydrogel releases a low-dose gaseous formaldehyde when the pH is less than or equal to 7.

[0015] According to the present invention, the aminal bonds in the formaldehyde prodrug hydrogel are subjected to the action of pH, resulting in structural destruction and release of gaseous formaldehyde. Thus, the formaldehyde prodrug hydrogel utilizes its unique pH response characteristics to destroy its supramolecular network and realize the release of gas molecules stored in covalent bonds.

[0016] Preferably, the pH range is less than or equal to 7.0, that is, when the pH is less than or equal to 7.0, the formaldehyde prodrug hydrogel begins to release low-dose gaseous formaldehyde. It should be understood that the lower the pH of the acutely infected wound, the better the effect of the antibacterial formaldehyde hydrogel prodrug. In a preferred embodiment, the bacteria is Staphylococcus aureus, and the pH is equal to 6.5.

[0017] According to a third aspect of the present invention, a method for preparing the pH-responsive antibacterial formaldehyde prodrug hydrogel or application of the pH-responsive antibacterial formaldehyde prodrug hydrogel in pharmaceutical manufacturing is provided.

[0018] The key invention of the present invention is mainly that a supramolecular network is formed by reacting highly efficient formaldehyde with amino-terminated polyethylene glycol, and a formaldehyde prodrug hydrogel is prepared for the first time using polyethylene glycol and formaldehyde. The formaldehyde prodrug hydrogel responds to pH, and the imine bond of the amino group and the aldehyde group condensation in the formaldehyde prodrug hydrogel is attacked by hydrogen ions, resulting in structural destruction and release of formaldehyde gas drug molecules, so that it exerts antibacterial properties; secondly, pure polyethylene glycol has high safety, and the product after degradation is polyethylene glycol with a molecular weight of 2kDa (FDA approved), so the formaldehyde prodrug hydrogel prepared according to the present invention further improves the biocompatibility of the drug on the basis of urotropine. The present invention also uses pure water as a reaction solvent, breaks through the reaction solvent limitation of the aldehyde-amine reaction, and achieves more efficient reaction activity.

[0019] Current formaldehyde drugs include aqueous formaldehyde cresol solutions and urotropine and its derivatives. Compared with the prior art, the pH-responsive antibacterial formaldehyde prodrug hydrogel provided by the present invention and its preparation method and application have the following significant advantages:

[0020] 1) The supermolecular network formed by the reaction of efficient formaldehyde and amino-terminated polyethylene glycol can respond to the breakage of covalent bonds in an acidic environment and release low-dose gas formaldehyde, which has good antibacterial properties, especially the killing effect on Staphylococcus aureus is higher than that on normal cells, and has the ability to selectively kill bacteria;

[0021] 2) According to the method for preparing the pH-responsive antibacterial formaldehyde prodrug hydrogel of the present invention, the gaseous formaldehyde molecules are fixed to the hydrogel supramolecular network by the aldehyde-amine cross-linking method, the preparation process is simple, a one-pot hydrothermal method is adopted, the reaction conditions are mild, the repeatability is good, and it is convenient for large-scale production;

[0022] 3) Pure polyethylene glycol is highly safe, and the product after degradation is polyethylene glycol with a molecular weight of 2 kDa (FDA approved), so the formaldehyde prodrug hydrogel prepared according to the present invention has higher biocompatibility;

[0023] 4) Hydrogel has the characteristics of sustained release of drugs, its water content is close to the water content in the body, and the foreign body sensation is low;

[0024] 5) The formaldehyde prodrug hydrogel prepared by the present invention has unique temperature sensitivity and can be transformed from a solid into a fluid for secondary molding by heating;

[0025] 6) The formaldehyde prodrug hydrogel prepared by the present invention has excellent mechanical properties and can be prepared into a flexible patch form through secondary molding. It can be designed and cut according to the wound size, so it is easy to use and easy to promote.

[0026] In summary, a pH-responsive antibacterial formaldehyde prodrug hydrogel provided by the present invention has an acidic pH response function, ensuring that it responds to release in the acidic environment of acute wound tissue, has good antibacterial effect, promotes wound repair, and will not penetrate deep into the tissue, with less harm, that is, killing bacteria promotes wound healing, and will not affect normal tissue. The pH-responsive antibacterial formaldehyde prodrug hydrogel prepared according to the present invention has the advantages of sustained release of formaldehyde in an acidic environment, good antibacterial properties, high biocompatibility, simple synthesis, mild reaction conditions, and easy cutting, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of a method for preparing a formaldehyde prodrug hydrogel provided by the present invention;

[0028] Figure 2 It is a hydrogen nuclear magnetic resonance spectrum of the amino-terminated polyethylene glycol and the formaldehyde prodrug hydrogel supramolecular network before and after reduced pressure concentration according to Example 1 of the present invention;

[0029] Figure 3 The water contact angles of the formaldehyde prodrug hydrogels in Examples 1 to 5 are shown;

[0030] Figure 4 is a scanning electron micrograph of the freeze-dried formaldehyde prodrug hydrogel obtained according to Example 1 of the present invention;

[0031] Figure 5 is a scanning electron micrograph of the freeze-dried formaldehyde prodrug hydrogel obtained according to Example 6 of the present invention;

[0032] Figure 6 is a scanning electron micrograph of the freeze-dried formaldehyde prodrug hydrogel obtained according to Example 7 of the present invention;

[0033] Figure 7 This is a film image obtained by hot pressing the formaldehyde prodrug hydrogel obtained in Example 7 of the present invention;

[0034] Figure 8 is a formaldehyde release graph of the formaldehyde prodrug hydrogel obtained according to the preparation method of the present invention at a concentration of pH 6.5;

[0035] Fig. 9 This is a statistical graph of the toxicity of the formaldehyde prodrug hydrogel obtained according to the preparation method of the present invention to normal cells (mouse embryonic fibroblasts);

[0036] Fig.10 The figure is a diagram showing the inhibitory effect of the formaldehyde prodrug hydrogel and raw materials obtained according to the preparation method of the present invention on Staphylococcus aureus under different pH conditions. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The raw materials and instruments used in the following examples are all commercially available; the equipment and preparation processes used are conventional equipment and conventional processes unless otherwise specified. Example 1

[0038] See also Figure 1 10 g of amino-terminated polyethylene glycol (molecular weight 2000 Da) was dissolved in 100 mL of deionized water to obtain a polyethylene glycol aqueous solution. 315 mg of paraformaldehyde was added to the above mixture and stirred at 70°C for 10 minutes to allow the paraformaldehyde and amino-terminated polyethylene glycol to fully react. The above mixture was concentrated to 42.8 g by reduced pressure distillation (0.1 atmosphere, 50°C) to obtain a formaldehyde prodrug hydrogel.

[0039] Example 2

[0040] The implementation method and basic formula were the same as those in Example 1, except that amino-terminated polyethylene glycol with a molecular weight of 1000 Da was used to obtain the formaldehyde prodrug hydrogel.

[0041] Example 3

[0042] The implementation method and basic formula were the same as those in Example 1, except that amino-terminated polyethylene glycol with a molecular weight of 5000 Da was used to obtain the formaldehyde prodrug hydrogel.

[0043] Example 4

[0044] The implementation method and basic formula were the same as those in Example 1, except that amino-terminated polyethylene glycol with a molecular weight of 10,000 Da was used to obtain the formaldehyde prodrug hydrogel.

[0045] Example 5

[0046] The implementation method and basic formula were the same as those in Example 1, except that amino-terminated polyethylene glycol with a molecular weight of 20,000 Da was used to obtain the formaldehyde prodrug hydrogel.

[0047] Example 6

[0048] The implementation method and basic formula are the same as those in Example 1, except that formaldehyde aqueous solution is used instead of paraformaldehyde to obtain the formaldehyde prodrug hydrogel in the same manner, wherein the concentration of the formaldehyde aqueous solution is 35% and the amount used is 0.85 mL.

[0049] Example 7

[0050] The implementation method and basic formula are the same as those of Example 1, except that the formaldehyde hydrogel is subjected to heat pressing (the heat pressing temperature is 70° C. and the time is 15 minutes) to obtain a formaldehyde prodrug hydrogel film with a thickness of 0.5 mm.

[0051] The H NMR spectra of the prodrug hydrogel of amino-terminated polyethylene glycol in Example 1 before and after the free formaldehyde was removed under reduced pressure are as follows: Figure 2 As shown in the figure, it can be seen that two obvious characteristic peaks appear at 2.7ppm, indicating the formation of a hydrogel network, but there is still free formaldehyde. After decompression treatment, the peak of free formaldehyde disappears.

[0052] Figure 3 The water contact angles of the formaldehyde prodrug hydrogels in Examples 1 to 5 are shown. It can be seen that the larger the molecular weight of the amino-terminated polyethylene glycol, the better the wettability of the prepared formaldehyde prodrug hydrogel. When the molecular weight of the amino-terminated polyethylene glycol is 10000Da, the formaldehyde prodrug hydrogel exhibits superhydrophilic characteristics. Considering the rapid degradation of the hydrogel, the present invention preferably uses an amino-terminated polyethylene glycol with a molecular weight of 2000Da.

[0053] Figure 4 This is a scanning electron microscope image of the formaldehyde prodrug hydrogel in Example 1. According to the scanning electron microscope image, it can be seen that the surface of the formaldehyde prodrug hydrogel prepared using paraformaldehyde is relatively rough and has uniform small pores. These pores can effectively fix water molecules and enhance the stability of the hydrogel.

[0054] Figure 5 This is a scanning electron microscope image of the formaldehyde prodrug hydrogel in Example 6. According to the scanning electron microscope image, the surface of the formaldehyde prodrug hydrogel prepared from the formaldehyde solution is relatively rough, and the pores on the surface are not uniform, indicating that the formaldehyde solution has a high reaction activity with the amino-terminated polyethylene glycol and is prone to implosion.

[0055] Figure 6This is a scanning electron microscope image of the formaldehyde prodrug hydrogel in Example 7. This shows that the surface of the hydrogel after heat pressing is flat and its surface pores are small. Compared with Examples 1 and 6, its water solubility is further reduced and its stability is further improved.

[0056] Figure 7 This is a photo of the dumbbell-shaped formaldehyde prodrug hydrogel film in Example 7.

[0057] Figure 8 This is the formaldehyde release diagram of the formaldehyde prodrug hydrogel sample in Example 1 in a pH 6.5 environment. This shows that the formaldehyde prodrug hydrogel can destroy its chemical structure in a slightly acidic environment and release a low dose of formaldehyde gas, and 74% of the gas formaldehyde can be released after 48 hours. The release of formaldehyde shows that the hydrogel has obvious sustained release and pH response.

[0058] Fig. 9 The figure is a statistical graph of the cytotoxicity of the formaldehyde prodrug hydrogel sample to mouse embryonic fibroblasts in Example 1. Obviously, the obtained formaldehyde prodrug hydrogel has no significant toxicity to normal cells at a higher concentration (mg / mL) (cell survival rate is greater than 90%).

[0059] Fig.10 The figure shows the inhibitory effect of amino-terminated polyethylene glycol (raw material group) and formaldehyde prodrug hydrogel (hydrogel group) on Staphylococcus aureus under different pH conditions in Example 1. The formaldehyde prodrug hydrogel has obvious inhibitory ability against Staphylococcus aureus at both pH 6.5 and pH 7.2, which is due to the fact that Staphylococcus aureus can metabolize organic acid. The above results prove that the formaldehyde prodrug hydrogel has pH responsiveness and can effectively inhibit the growth of bacteria.

[0060] In summary, according to the preparation method of the present invention, formaldehyde and amino-terminated polyethylene glycol are polycondensed in one pot and then concentrated under reduced pressure to obtain a formaldehyde hydrogel prodrug. This method can not only simplify the preparation of formaldehyde prodrug, but also realize the responsive release of low-dose formaldehyde by the prodrug hydrogel in a weakly acidic environment.

[0061] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a pH-responsive antibacterial formaldehyde prodrug hydrogel, characterized in that: The preparation method comprises the following steps: S1, dissolving polyethylene glycol in deionized water to obtain a polyethylene glycol aqueous solution; S2, adding formaldehyde to the polyethylene glycol aqueous solution to obtain a mixed solution; S3, stirring the mixed solution at high temperature to react; S4, subjecting the mixed solution after sufficient reaction to reduced pressure distillation to obtain a pH-responsive antibacterial formaldehyde prodrug hydrogel.

2. The preparation method according to claim 1, characterized in that: In step S1, the polyethylene glycol is amino-terminated polyethylene glycol with a molecular weight ranging from 200 Da to 10000 Da.

3. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of polyethylene glycol to deionized water is (0.5-2):

10.

4. The preparation method according to claim 1, characterized in that: The molar ratio of formaldehyde to polyethylene glycol is (1.5-3):

1.

5. The preparation method according to claim 1, characterized in that: In step S2, the formaldehyde is selected from: 35-40% formaldehyde aqueous solution, paraformaldehyde, and gaseous formaldehyde.

6. The preparation method according to claim 1, characterized in that: In step S3, the reaction temperature of the mixed solution is 50° C. to 100° C., and the reaction time is greater than 60 seconds.

7. The preparation method according to claim 1, characterized in that: Step S4, reducing the water content of the formaldehyde prodrug hydrogel to less than 90% by distillation under reduced pressure.

8. The preparation method according to claim 1, characterized in that: The method further comprises step S5 of performing a hot pressing treatment on the formaldehyde hydrogel at a temperature of 60 to 80° C. for 10 to 20 minutes.

9. A pH-responsive antibacterial formaldehyde prodrug hydrogel prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The antibacterial formaldehyde prodrug hydrogel releases low-dose gaseous formaldehyde when the pH is less than or equal to 7.

10. A method for preparing the pH-responsive antibacterial formaldehyde prodrug hydrogel according to any one of claims 1 to 8 or use of the pH-responsive antibacterial formaldehyde prodrug hydrogel according to claim 9 in pharmaceutical manufacturing.

Images