Efficient antibacterial healing-promoting phenytoin quaternary ammonium salt as well as preparation method and effect thereof

Abstract

The invention discloses efficient antibacterial healing-promoting phenytoin quaternary ammonium salt as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials and medicine reutilization. The compound is named as 2-(4-oxo-5, 5-diphenyl-4, 5-dihydro-1h-imidazole-2-acyl) acid tetrabutylammonium salt according to a systematic naming method. The phenytoin quaternary ammonium salt is synthesized for the first time, not only has the spectral antibacterial ability, but also has the stronger ability of promoting cell proliferation and migration and promoting angiogenesis compared with phenytoin sodium, and is more suitable for wound treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and drug recycling, and relates to a phenytoin quaternary ammonium salt (Pn-NH4) having the dual functions of anti-infection and tissue repair, a preparation method thereof, and an application thereof in wound treatment. Background Art

[0002] Wound healing is a complex biological process involving inflammation control, angiogenesis, cell proliferation, and tissue remodeling. However, chronic wounds and infected wounds (such as diabetic foot ulcers and pressure ulcers) are often difficult to heal due to bacterial infection and insufficient tissue regeneration capacity. Current treatments, such as antibiotics, growth factors, and high-performance dressings, are costly and have limited efficacy, while drug resistance is becoming increasingly serious. Therefore, the development of low-cost, highly effective, and novel treatment options with both antibacterial and healing-promoting properties is of great clinical value. Phenytoin sodium (PS) is a classic antiepileptic drug. Recent studies have found that it can promote wound healing, but its low water solubility and lack of antibacterial activity limit its application in wound treatment. Quaternary ammonium salts are widely used in the modification of biomedical materials due to their good hydrophilicity, charge characteristics, and broad-spectrum antibacterial activity. To date, no quaternary ammonium salts of phenytoin have been reported. Summary of the Invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a highly effective antibacterial and healing-promoting phenytoin quaternary ammonium salt, a preparation method and application thereof.

[0004] Technical solution: The present invention provides a highly effective antibacterial and healing-promoting phenytoin quaternary ammonium salt, wherein the phenytoin quaternary ammonium salt has the following structure:

[0005]

[0006] The compound was named as 2-(4-oxo-5,5-diphenyl-4,5-dihydro-1h-imidazol-2-yl) acid tetrabutylammonium salt according to the systematic nomenclature.

[0007] The present invention also provides a preparation method of the phenytoin quaternary ammonium salt, comprising the following steps: completely dissolving phenytoin sodium in ultrapure water, adding ion exchange resin, stirring until a precipitate is completely precipitated, then dropwise adding tetrabutylammonium hydroxide solution to the supernatant solution until white insoluble matter disappears and the solution becomes clear, centrifuging the solution, taking the supernatant solution, dialyzing and freeze-drying to obtain the phenytoin quaternary ammonium salt.

[0008] Furthermore, the centrifugation condition is 4000r for 5 minutes.

[0009] The present invention also provides the use of the phenytoin quaternary ammonium salt in the preparation of antibacterial and / or wound healing promoting drugs or products.

[0010] Furthermore, the concentration of phenytoin quaternary ammonium salt in the medicine or product is 5 to 20 μg / ml.

[0011] Furthermore, the concentration of phenytoin quaternary ammonium salt in the medicine or product is 5 to 10 μg / ml.

[0012] Furthermore, the antibacterial species include Gram-negative bacteria and Gram-positive bacteria.

[0013] Furthermore, the Gram-negative bacteria include Escherichia coli.

[0014] Furthermore, the Gram-positive bacteria include Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention synthesizes a quaternary ammonium salt of phenytoin for the first time, which not only has a broad spectrum of antibacterial ability, but also has a stronger ability to promote cell proliferation and migration and promote angiogenesis than phenytoin sodium, and is more suitable for wound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a scanning electron microscope image of phenytoin quaternary ammonium salt.

[0017] Figure 2 The H NMR spectrum of phenytoin quaternary ammonium salt is shown.

[0018] Figure 3 The infrared absorption spectrum of phenytoin quaternary ammonium salt is shown.

[0019] Figure 4 The activity of HaCaTs (human immortalized keratinocytes) was measured by CCK-8 method after treatment with different concentrations of Pn-NH4 for 24 h and 72 h (*, p < 0.05, **, p < 0.01; ***, p < 0.001).

[0020] Figure 5 Shown are live-dead staining images of HaCaTs cells when Pn-NH4 and PS acted on them for 24h and 72h at the same concentration.

[0021] Figure 6 The relative expression levels of proliferation-related genes (A) Efemp-1 and (B) Tgf-β in HaCaTs under different treatments are shown (*, p<0.05, **, p<0.01; ***, p<0.001).

[0022] Figure 7The viability of HaCaTs at day 1 (A) and day 3 (B) under different treatments is shown (*, p<0.05, **, p<0.01; ***, p<0.001).

[0023] Figure 8 The migration images and migration quantitative analysis of HaCaTs under different treatments are shown (*, p<0.05, **, p<0.01; ***, p<0.001).

[0024] Figure 9 The images and quantitative analysis of HUVECs tube formation under different treatments are shown (*, p<0.05, **, p<0.01; ***, p<0.001).

[0025] Figure 10 The results of the antibacterial experiment under different treatments are shown (*, p < 0.05, **, p < 0.01; ***, p < 0.001). DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0027] In the following examples, the experimental materials and their sources include: phenytoin sodium and Dowex 66 ion exchange resin were purchased from Maclean, and tetrabutylammonium hydroxide was purchased from Anhui Zesheng Technology Co., Ltd. The beef extract and peptone used in LB medium were purchased from OXOID.

[0028] Example 1: Synthesis and Identification of Phenytoin Quaternary Ammonium Salt

[0029] 1.1 Synthesis of phenytoin quaternary ammonium salt

[0030] First, dissolve 0.4 mg of phenytoin sodium in 20 ml of ultrapure water and stir at a constant temperature of 50°C until completely dissolved. Add 0.2 mg of cation exchange resin and continue stirring. During the stirring process, white insoluble matter will continue to precipitate and stir for 3 hours. After the reaction stops, titrate the supernatant solution with 3M tetrabutylammonium hydroxide solution until the white insoluble matter disappears and the solution becomes clear. The pH of the solution is measured with a pH meter to be approximately 11. Centrifuge the clear solution at 4000 r for 5 minutes and take the supernatant solution. Use a 400 kDa dialysis membrane to dialyze for two days to remove byproducts such as sodium hydroxide. Freeze-dry for 3 days to obtain a white granular powder, which is the quaternary ammonium salt of phenytoin Pn-NH4, which is dried and weighed.

[0031] 1.2 Characterization of phenytoin quaternary ammonium salt

[0032] 1. The microstructure of the particles was observed using a scanning electron microscope (SEM, VEGA3, Tescan). The surface of Pn-NH4 after sputtering and gold plating was observed using SEM.

[0033] The results showed (see Figure 1 ), Pn-NH4 is blocky particles.

[0034] 2. The chemical properties of the compounds were characterized by Fourier transform infrared spectroscopy (FTIR, JASCO, Japan) in the wavelength range of 4000-400 cm-1, with a focus on the analysis of functional groups and chemical bonds.

[0035] The results showed (see Figure 2 ), according to the infrared spectrum comparison between PS and Pn-NH4, at 2960cm -1 A new peak appeared at , indicating that tetrabutylammonium successfully replaced hydrogen, confirming the addition of a quaternary ammonium group to the phenytoin structure.

[0036] 3. Adoption 1 H NMR system ( 1 The chemical structure of the Pn-NH4 particles was characterized by H NMR (Bruker-400 MHz, USA).

[0037] The results showed (see Figure 3 ),pass 1 H NMR (400 MHz, DMSO-d6) confirmed the structural elucidation of Pn-NH4. Characteristic signals for aromatic protons were observed at δ = 7.42-7.35 (m, 4H), 7.27-7.18 (m, 4H), and 7.18-7.12 (m, 2H). Methylene protons associated with the quaternary ammonium group were detected at δ = 3.20-3.12 (m, 6H), while alkyl chain methylene and methyl protons were detected at δ = 1.56 (td, J = 11.8, 10.0, 6.1 Hz, 6H), 1.31 (h, J = 7.4 Hz, 6H), and 0.93 (t, J = 7.3 Hz, 9H), respectively. These data, along with complementary spectroscopic and analytical results, unequivocally confirmed the compound's structure.

[0038] 1.3 Healing-promoting function of phenytoin quaternary ammonium salt

[0039] HaCaTs cells are immortalized human epidermal cells, derived from normal human skin, and are a non-tumor-derived immortalized keratinocyte cell line. They have differentiation characteristics similar to normal human keratinocytes and lack tumor-like properties. In vitro experiments were conducted using HaCaTs cells as a model cell line.

[0040] 1. Assessing sample cytocompatibility using the CCK-8 assay. Cells were seeded at a density of 3,000 cells per well in 96-well plates and allowed to adhere for 24 hours. Pn-NH4 was initially dissolved in DMSO to a final concentration of 10 mg / ml and then serially diluted in 1640 medium (Sevier Biotech, China) to achieve final concentrations of 5, 10, 20, and 40 μg / ml for HaCaTs. Cells were incubated under standard culture conditions (37°C, 5% CO2). After 24 and 72 hours of co-culture, 100 μL of culture medium containing 10% CCK-8 reagent (Beyotime, China) was added to each well. After a 1-hour incubation, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA), and a standard curve of OD values was generated to quantify cell viability. HaCaTs cells without Pn-NH4 were also maintained as a control.

[0041] CCK-8 results showed (see Figure 4 ), after stimulation with Pn-NH4 at concentrations of 5 and 10 μg / mL for 24 h ( Figure 4 A), cell viability was significantly enhanced, and reached its peak value when stimulated with 10 μg / mL Pn-NH4 for 72 h ( Figure 4 B). Therefore, subsequent experiments used 10 μg / mL Pn-NH4 and PS as experimental groups.

[0042] 2. The morphology of HaCaTs in direct contact with the sample was examined using a live / dead staining method. Cells were seeded into 6-well plates at a density of 4×104 cells per well and cultured under standard conditions for 24 hours. Subsequently, 10 μg / mL of Pn-NH4 or PS was added to the wells and incubated at 37°C in a 5% CO2 atmosphere for 24 and 72 hours. At the same time, new complete medium was replaced in the control group without drug addition for cell culture. Cells were stained using a live / dead cell double staining reagent (Solarbio, China).

[0043] Live / dead staining results (see Figure 5 ), under the same conditions, the proportion of living cells in the Pn-NH4 group was significantly higher than that in other groups, with the best cell morphology and vitality.

[0044] 3. Cell viability was assessed using the CCK-8 assay. The method and steps were the same as above, except that the experimental groups were treated with 10 μg / mL of Pn-NH4 and PS.

[0045] The results showed (see Figure 6 ), incubate for 24 h ( Figure 6 A) and 72h( Figure 6 B) After treatment, the cell viability of the Pn-NH4 group was the highest.

[0046] 4. qRT-PCR was used to detect the expression of HaCaTs proliferation genes under the stimulation of 10 μg / mL Pn-NH4 or PS. 4 Cells were seeded into 6-well plates at a density of 100 μg / mL and cultured under standard conditions for 24 h to allow cell adhesion. Subsequently, Pn-NH4 or PS was added to each well and incubated at 37°C in a 5% CO2 atmosphere for 72 h.

[0047] RNA extraction: After cells have grown confluently, remove the culture medium and gently rinse once with pre-chilled PBS. Aspirate any remaining PBS. Add 1 mL of Trizol Reagent to each well and incubate at room temperature for 5 minutes. Use a pipette to repeatedly pipette until cells are completely detached from the bottom of the dish. Collect the lysate into a 1.5 mL EP tube and place on ice for 10 minutes to fully lyse the cells.

[0048] Add chloroform to the Eppendorf tubes in a ratio of 1:5 chloroform:Trizol. Add 200 μL of chloroform to each tube, vortex for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge in a pre-cooled centrifuge at 12,000 rpm at 4°C for 10 minutes. Gently remove the tubes. Separate the liquids into separate layers, with the upper layer being the aqueous phase containing the RNA. After centrifugation, aspirate 450 μL of the upper aqueous phase and transfer it to a fresh enzyme-free Eppendorf tube (be gentle during this operation to avoid aspirating the lower and middle organic phases).

[0049] Add an equal volume of isopropanol to a new EP tube, gently invert to mix, place at room temperature for 15 minutes, centrifuge at 12000 rpm and 4℃ for 15 minutes, discard the supernatant, and the white precipitate visible at the bottom of the tube is RNA.

[0050] Add 1 mL of pre-cooled 75% alcohol (anhydrous ethanol: DEPC water = 3:1) to the EP tube to wash the precipitate. Centrifuge at 12,000 rpm at 4°C for 5 minutes. Repeat the washing operation and discard the supernatant as much as possible to obtain high-purity RNA.

[0051] Dry at room temperature for 5-10 minutes and add 20-40 μL RNase-free ddH2O to fully dissolve the RNA.

[0052] Load 1 μL of RNA solution and measure RNA concentration and OD260 / 280 using an ultra-micro spectrophotometer. Using the Takara reverse transcription kit, add the components listed in Table 1 to a PCR tube. Gently mix with a pipette tip, then centrifuge briefly. Place the tube in a PCR instrument for RT reaction to reverse transcribe the RNA into cDNA. The reverse transcription reaction process is shown in Table 2.

[0053] Table 1 Addition volume of each component of RT reaction system

[0054]

[0055] Table 2 PCR reverse transcription reaction procedure

[0056]

[0057] After diluting the resulting cDNA sample 10-fold, subsequent experiments can be performed directly. This study used a Yeasean kit with a 20 μL system for RT-qPCR. The RT-qPCR reaction system was prepared according to Table 3. The components are listed below.

[0058] Table 3 RT-qPCR reaction system components

[0059]

[0060]

[0061] The specific information of primers is shown in Table 4.

[0062] Table 4 Primer information for HaCaTs used in this experiment

[0063]

[0064] The relative expression data were obtained using the 2-ΔΔCT method.

[0065] The results showed that (see Figure 7 To further validate its proliferative effects, we quantitatively analyzed the expression of proliferation-related genes, focusing on Efemp-1 (a fibrin-like extracellular matrix protein gene containing an epidermal growth factor-like domain) and Tgf-β (transforming growth factor β). Compared with the control group, the expression levels of proliferation-related genes were significantly increased in the Pn-NH4 group. However, there was no significant difference in Efemp-1 expression in the PS group. This suggests that, compared with PS, Pn-NH4 significantly promotes the expression of proliferation genes, thereby promoting cell proliferation.

[0066] 5. Epidermal cell migration plays a key role in skin wound healing, directly affecting the speed and quality of wound healing and tissue regeneration. In order to evaluate the effects of Pn-NH4 and PS on the migration ability of HaCaTs cells, a scratch healing assay was used. Cells were plated at 5×10 5Cells were seeded at a density of 100 cells / well in a 6-well plate and cultured to approximately 90% confluence. A scratch was then made on the cell monolayer using a sterile 200 μL pipette tip and washed three times with PBS to remove detached cells and debris. Subsequently, the regular culture medium was replaced with medium containing 10 μg / mL of Pn-NH4 or PS. Cell migration was observed and photographed using an inverted microscope at 0, 6, and 12 hours. A control group received regular culture medium replacement. Finally, the percentage of the scratch area closed was calculated using ImageJ software and analyzed relative to the initial scratch area to assess cell migration ability.

[0067] The results showed (see Figure 8 ), creating consistent and uniform scratches in HaCaTs ( Figure 8 A). After incubation for 6 hours, the migration rate of the Pn-NH4 group reached about 28% ( Figure 8 B), which is almost twice that of the control group. At 12h, the migration rate of the Pn-NH4 group increased to about 57%, which was significantly higher than that of the other two groups ( Figure 8 C). There was no statistically significant difference between the PS and control groups. These results indicate that Pn-NH4 significantly promoted the migration of HaCaTs. In summary, Pn-NH4 can promote wound healing by promoting cell proliferation and migration.

[0068] 6. To evaluate the effects of different treatments on angiogenesis, a tube formation assay was performed on Matrigel (BD Biosciences, US) to assess the effects of each group on HUVEC morphogenesis and tube formation. The Matrigel solution was thawed overnight at 4°C and then placed in a 24-well plate and placed in a cell culture incubator for 1 hour to solidify. Subsequently, after culturing in medium containing 10 μg / mL of Pn-NH4 or PS for 3 days, HUVECs were digested and 6,000 HUVECs were seeded in a 24-well plate pre-coated with Matrigel. After culturing in endothelial cell growth medium for 3 hours and 6 hours, HUVECs were observed under an optical microscope to form tubes.

[0069] The results showed (see Figure 9 ), we evaluated the effects of different groups on the tube-forming ability of HUVECs through a tube-forming experiment. After 6 hours of intervention, microscopic observation revealed that the number of nodes, main connections, and total branch length in the Pn-NH4-treated group were significantly higher than those in the PS and control groups, and the differences were statistically significant. Quantitative analysis showed that the Pn-NH4-treated group performed almost twice as well as the PS and control groups in these indicators ( Figure 9 BC). This result indicates that Pn-NH4 can significantly enhance the tube-forming ability of HUVECs, and this effect may be closely related to its promotion of cell migration and proliferation.

[0070] 1.4 Antibacterial function of phenytoin quaternary ammonium salt

[0071] Experimental method: 20 μl of 10 μg / ml Pn-NH4 or PS was mixed with 50 μl of Gram-negative Escherichia coli (E. coli, ATCC 25922), Gram-positive Staphylococcus aureus (S. aureus, ATCC 25923) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) at a bacterial concentration of 1×10 8 Mix the CFU / ml in EP tubes and incubate in a 37°C incubator for 2 hours. After incubation, add 930 μL of normal saline to each tube, take 20 μL of the mixed solution, and serially dilute it with PBS. The serially diluted solution is dropped onto LB agar plates and incubated at 37°C for 24 hours. CFU are then counted.

[0072] The results showed (see Figure 10 ), compared with the control, the Pn-NH4 prepared by the present invention can effectively inhibit bacteria, and fewer bacteria that come into contact with Pn-NH4 survive. Its antibacterial effect is much higher than that of the PS group.

[0073] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A highly effective antibacterial and healing-promoting phenytoin quaternary ammonium salt, characterized in that: The phenytoin quaternary ammonium salt has the following structure:

2. The method for preparing the phenytoin quaternary ammonium salt according to claim 1, wherein The following steps are involved: Completely dissolve phenytoin sodium in ultrapure water, add ion exchange resin, and stir until the precipitate is completely precipitated. Then add tetrabutylammonium hydroxide solution dropwise to the supernatant solution until the white insoluble matter disappears and the solution becomes clear. Centrifuge the solution, take the supernatant solution, dialyze and freeze-dry to obtain phenytoin quaternary ammonium salt.

3. The preparation method according to claim 2, characterized in that The centrifugation condition is 4000r for 5 minutes.

4. Use of the phenytoin quaternary ammonium salt according to claim 1 in the preparation of antibacterial and / or wound healing promoting drugs or products.

5. The use according to claim 3, characterized in that The concentration of phenytoin quaternary ammonium salt in the medicine or product is 5 to 20 μg / ml.

6. The use according to claim 3, characterized in that The concentration of phenytoin quaternary ammonium salt in the medicine or product is 5-10 μg / ml.

7. The use according to claim 3, characterized in that The antibacterial species include Gram-negative bacteria and Gram-positive bacteria.

8. The use according to claim 6, characterized in that The Gram-negative bacteria include Escherichia coli.

9. The use according to claim 6, characterized in that The Gram-positive bacteria include Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

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