Photosensitive gel bacteriostatic agent as well as preparation method and application thereof
By preparing a mixture of riboflavin carbon dots and aldehyde-based hyaluronic acid, a photosensitive gel antibacterial agent is formed, which solves the problem of the lack of antibacterial ability of existing antibacterial hydrogels, and achieves efficient photodynamic treatment effects, significantly inhibits bacterial growth and improves biosafety.
Patent Information
- Application Number
- CN202510151325.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-11
- Publication Date
- 2025-05-09
AI Technical Summary
The existing antibacterial hydrogels themselves lack antibacterial ability and require the introduction of additional antibacterial components. Traditional photodynamic therapy techniques are insufficient in local infection and drug resistance.
By preparing a mixture of riboflavin carbon dots (CDs) and aldehyde-based hyaluronic acid (HA), CD@ hydrogel, i.e., photosensitive gel bacteriostatic agent. The antibacterial agent generates singlet oxygen under laser irradiation to achieve the killing of bacteria.
This photosensitive gel antibacterial agent not only retains the optical properties of riboflavin, but also improves biosafety and treatment efficiency, can significantly inhibit the growth of E. coli, and does not require additional crosslinking agents, which improves bioavailability.
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Figure CN119950409A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of nanomaterials, and in particular to a photosensitive gel antibacterial agent, a preparation method and application thereof. Background Art
[0002] Bacterial infection is a serious public health problem. It not only induces inflammation of the wound surface, but also forms bacterial biofilms, which lead to a variety of chronic infections, such as chronic wounds, periodontitis, osteomyelitis, etc., and can lead to death in severe cases. Bacterial biofilms can protect bacteria from surviving in adverse environments, so traditional antibiotic treatments are usually ineffective against biofilm infections.
[0003] In recent years, hydrogels have attracted much attention due to their good biocompatibility and water absorption. They can cover the infected area and provide long-term antibacterial protection without causing excessive stimulation to the surrounding healthy tissues. They are gradually used in wound treatment, especially to prevent infection, relieve patient pain and fight bacteria. However, traditional hydrogels themselves have no antibacterial ability and need to introduce antibacterial ingredients to achieve a bactericidal effect. Therefore, it is necessary to develop new antibacterial hydrogels to deal with bacterial infections.
[0004] Photodynamic therapy (PDT) has gradually become an effective antibacterial therapy due to its good safety and precise selectivity. PDT generally requires photosensitizers, light sources and sufficient oxygen. Photosensitizers can selectively accumulate in bacteria or infected areas, and produce reactive oxygen species after light exposure to kill pathogens, such as Staphylococcus aureus, Streptococcus and Candida, without causing excessive damage to healthy tissues. As an emerging antibacterial technology, PDT is not easy to develop drug resistance, so it can be used as an effective alternative method for antibiotic-resistant bacterial infections. It has broad application prospects in treating local infections, reducing drug resistance and improving clinical treatment effects. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a photosensitive gel antibacterial agent, a preparation method and application thereof in view of the deficiencies in the above-mentioned prior art.
[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: in the first aspect of the present invention, a method for preparing a photosensitive gel antibacterial agent is provided, comprising the following steps:
[0007] S1, preparation of riboflavin carbon dots CDs;
[0008] S2, preparing aldehyde-modified hyaluronic acid;
[0009] S3, mixing riboflavin carbon dots CDs with aldehyded hyaluronic acid to prepare CD@hydrogel, i.e. the photosensitive gel antibacterial agent.
[0010] Preferably, step S1 specifically comprises:
[0011] Dissolve riboflavin in sodium hydroxide solution, stir until completely dissolved, then add sodium chloride and ethylenediamine, continue stirring, then transfer the resulting mixed solution to a reactor and heat for reaction; after the reaction is completed, centrifuge and filter, dialyze the filtrate with a dialysis bag, and finally freeze-dry the solution in the dialysis bag to obtain riboflavin carbon dots CDs.
[0012] Preferably, step S1 specifically comprises:
[0013] 150-600 mg of riboflavin is dissolved in 15-60 mL of a sodium hydroxide solution with a concentration of 0.05-0.2 mol / L, and stirred until completely dissolved. Then, 0.05-0.2 g of sodium chloride and 1-4 mL of ethylenediamine are added, and stirring is continued for 5-20 minutes. Then, the resulting mixed solution is transferred to a reactor, and heated at 160-200° C. for reaction for 3-12 hours. After the reaction is completed, centrifugation is performed, and then filtration is performed with a filter. The filtrate is dialyzed with a dialysis bag with a molecular cutoff of 500-2000D for 6-24 hours. Finally, the solution in the dialysis bag is freeze-dried to obtain riboflavin carbon dots CDs.
[0014] Preferably, step S2 specifically comprises:
[0015] The hyaluronic acid is dissolved in deionized water, and then a sodium periodate solution is added, and stirring is continued. Finally, ethylene glycol is added to terminate the reaction. The product is dialyzed with a dialysis bag, and the solution in the dialysis bag is freeze-dried to obtain aldehyde-modified hyaluronic acid.
[0016] Preferably, step S2 specifically comprises:
[0017] Dissolve 250-1000 mg of hyaluronic acid in 25-100 mL of deionized water, stir until completely dissolved, then add 2.5-10 mL of 0.15-0.6 mol / L sodium periodate solution, continue stirring at 20-40° C. for 2.5-10 hours, and finally add 0.5-2 g of ethylene glycol to terminate the reaction; the product is dialyzed with a dialysis bag with a molecular cutoff of 3000-6000D for 24-96 hours, and the solution in the dialysis bag is freeze-dried to obtain aldehyde-modified hyaluronic acid.
[0018] Preferably, step S3 specifically comprises:
[0019] Dissolve riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid in PBS solution, control the mass concentrations of riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid to be 10-40% and 7.5-30% respectively, mix and shake well to obtain CD@hydrogel, i.e. the photosensitive gel antibacterial agent.
[0020] Preferably, the preparation method of the photosensitive gel antibacterial agent comprises the following steps:
[0021] S1. Preparation of riboflavin carbon dots CDs:
[0022] 300 mg of riboflavin was dissolved in 30 mL of 0.1 mol / L sodium hydroxide solution, stirred for 10 minutes until completely dissolved, then 0.1 g of sodium chloride and 1 mL of ethylenediamine were added, and stirring was continued for 10 minutes. Then, the resulting mixed solution was transferred to a reactor and heated at 180°C for 6 hours. After the reaction was completed, centrifugation was performed, and then filtered with a 0.22 μm filter. The filtrate was dialyzed with a dialysis bag with a molecular cutoff of 1000 D for 12 hours. Finally, the solution in the dialysis bag was freeze-dried to obtain riboflavin carbon dots CDs.
[0023] S2, preparing aldehyde-modified hyaluronic acid;
[0024] 500 mg of hyaluronic acid was dissolved in 50 mL of deionized water, and stirred until completely dissolved. Then, 5 mL of a sodium periodate solution with a concentration of 0.3 mol / L was added, and the mixture was stirred continuously at 30° C. for 5 hours. Finally, 1 g of ethylene glycol was added to terminate the reaction. The product was dialyzed for 48 hours using a dialysis bag with a molecular cutoff of 5000 D, and the solution in the dialysis bag was freeze-dried to obtain aldehyde-modified hyaluronic acid.
[0025] S3. Dissolve riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid in PBS solution, control the mass concentrations of riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid to be 20% and 15% respectively, mix and shake well at room temperature to obtain CD@hydrogel, i.e., the photosensitive gel antibacterial agent.
[0026] The second aspect of the present invention provides a photosensitive gel antibacterial agent, which is prepared by the method as described above.
[0027] The third aspect of the present invention provides an application of the above-mentioned photosensitive gel antibacterial agent as a photodynamic therapy agent, wherein the application method is: applying light while applying the photosensitive gel antibacterial agent to sterilize.
[0028] A fourth aspect of the present invention provides a use of the photosensitive gel antibacterial agent as described above in the preparation of a photodynamic therapy agent.
[0029] The beneficial effects of the present invention are:
[0030] The present invention provides a novel injectable hydrogel based on riboflavin carbon dots (CDs) and hyaluronic acid (HA): first, yellow fluorescent CDs based on riboflavin are prepared, and then reacted with aldehyded hyaluronic acid to prepare CD@hydrogel, i.e., a photosensitive gel antibacterial agent, which can be used in antibacterial treatment.
[0031] The CDs in the present invention not only retain the optical properties of riboflavin, but also greatly improve the water solubility and fluorescence quantum yield, thus having higher biological safety and therapeutic efficiency; after laser irradiation, the CDs can excite oxygen to generate singlet oxygen ( 1 O2), thereby achieving a bactericidal effect; in addition, the abundant -NH2 on the surface of the CDs can make the carbon dots act as a cross-linking agent, and cross-link with the aldehyde-modified hyaluronic acid (HA-CHO) through the Schiff base reaction to prepare CD@hydrogel, that is, the photosensitive gel antibacterial agent; at this time, the carbon dots are not only highly efficient photosensitizers, but also can act as cross-linking agents.
[0032] The preparation process of the invention is simple and efficient, and does not require the introduction of an additional cross-linking agent, thereby improving bioavailability; biological experiments show that the CD@ hydrogel has good biosafety, and bactericidal experiments show that the hydrogel system has a significant inhibitory effect on the growth of Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 TEM image of CDs prepared in Example 1;
[0034] Figure 2 is the UV-absorption spectrum of the CDs prepared in Example 1 in aqueous solution;
[0035] Figure 3 Excitation spectrum and emission spectrum of CDs prepared in Example 1: (A) excitation spectrum (blue line) and emission spectrum (red line) of CDs, (B) emission spectrum of CDs at different excitation wavelengths (400nm-520nm);
[0036] Figure 4 is the Fourier transform infrared spectrum of the CDs prepared in Example 1;
[0037] Figure 5 This is the SEM image of the CD@ hydrogel prepared in Example 1;
[0038] Figure 6 Test results of the injectability of the CD@ hydrogel prepared in Example 1: (A) Rheological property test of CD@ hydrogel: (A) Frequency sweep test of CD@ hydrogel at 1% strain, (B) Strain amplitude measurement of CD@ hydrogel at a fixed frequency of 1 Hz in the strain range of 1% to 1000%;
[0039] Figure 7 The rheological test results of the CD@ hydrogel prepared in Example 1;
[0040] Figure 8 The singlet oxygen generation performance test results of CDs and CD@ hydrogel prepared in Example 1;
[0041] Fig. 9 The cytotoxicity test results of the CD@ hydrogel prepared in Example 1;
[0042] Fig.10 The test results of the killing ability of CD@hydrogel prepared in Example 1 on Escherichia coli: (A) the antibacterial activity of CD@hydrogel against Escherichia coli (+ indicates illumination, - indicates no illumination), (B) the effect of laser irradiation of different powers on the activity of Escherichia coli (without adding CD@hydrogel). DETAILED DESCRIPTION
[0043] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.
[0044] It should be understood that terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.
[0045] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.
[0046] Example 1
[0047] A photosensitive gel antibacterial agent, the preparation method of which comprises the following steps:
[0048] S1. Preparation of riboflavin carbon dots CDs by one-step hydrothermal method:
[0049] 300 mg of riboflavin was dissolved in 30 mL of 0.1 mol / L sodium hydroxide solution, and magnetic stirring was performed for 10 minutes until it was completely dissolved. Then, 0.1 g of sodium chloride and 1 mL of ethylenediamine were added, and stirring was continued for 10 minutes. Then, the resulting mixed solution was transferred to a high-pressure reactor and heated at 180°C for 6 hours. After the reaction was completed, centrifugation was performed, and then filtrated with a filter with a membrane of 0.22 μm. The filtrate was dialyzed with a dialysis bag with a molecular cutoff of 1000D for 12 hours. Finally, the solution in the dialysis bag was freeze-dried to obtain riboflavin carbon dots CDs.
[0050] S2, preparing aldehyde-modified hyaluronic acid (HA-CHO);
[0051] 500 mg of hyaluronic acid (HA) was dissolved in 50 mL of deionized water and stirred until completely dissolved. Then, 5 mL of 0.3 mol / L sodium periodate (NaIO4) solution was added and stirred continuously at 30°C for 5 hours. Finally, 1 g of ethylene glycol was added to terminate the reaction. The product was dialyzed for 48 hours using a dialysis bag with a molecular cutoff of 5000D to remove the remaining reactants. The solution in the dialysis bag was freeze-dried to obtain aldehyde-modified hyaluronic acid.
[0052] S3. Dissolve riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid in PBS solution, control the mass concentrations of riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid to be 20% (w / v) and 15% (w / v), respectively, mix and shake at room temperature to obtain CD@hydrogel, i.e., a photosensitive gel antibacterial agent.
[0053] Performance Test:
[0054] Morphological and optical tests of CDs and CD@hydrogels
[0055] The morphology and structure of CDs and CD@ hydrogels were characterized by transmission electron microscopy (JEOL Ltd, Japan, 200 kV accelerating voltage) and scanning electron microscopy (SEM, FEIQuanta 200F). The average size of CDs was measured using a Zeta sizer Nano ZS (MalvernInstruments, USA). The Fourier transform infrared spectra (FTIR) of CDs were recorded using a Cary 660FT-IR spectrometer (Agilent, USA). The UV-visible absorption spectra of CDs were measured using a U-3310 spectrophotometer (Hitachi). The fluorescence spectra were analyzed using a Hitachi 135F-4600 fluorescence spectrophotometer. The surface properties of CD@HA hydrogels were tested using a Kratos AXIS Ultra DLD X-ray photoelectron spectrometer (Shimadzu, Japan).
[0056] Reference Figure 1 , which is the transmission electron microscope image of CDs. It can be seen from the figure that CDs are uniform spherical structures with a size of 3-5nm and are evenly dispersed.
[0057] Reference Figure 2 , is the UV-absorption spectrum of CDs in aqueous solution, Figure 2 It can be seen that the absorption range of CDs is 350nm-500nm, with an obvious absorption peak at 460nm.
[0058] Reference Figure 3 , where (A) is the excitation spectrum (blue line) and emission spectrum (red line) of CDs, (B) is the emission spectrum of CDs at different excitation wavelengths (400nm-520nm); Figure 3A It can be seen that when the excitation wavelength is 465nm, the emission wavelength of CDs is 560nm. Figure 3 B shows the effect of different excitation wavelengths on the fluorescence emission of CDs. The emission wavelength of CDs is obviously dependent on the excitation. Within the excitation wavelength range of 400nm-520nm, the emission intensity is maximum when the excitation light is 470nm.
[0059] Reference Figure 4 , is the Fourier transform infrared spectrum (FT-IR) of CDs, CDs at 3470cm -1 and 1640cm -1 The peaks of -NH2 and -C=O appeared at the positions respectively, indicating the existence of -NH2 and unsaturated bonds (such as -C=O) on the surface of CDs.
[0060] Reference Figure 5 , which is the SEM image of CD@hydrogel. The SEM image shows that the hydrogel as a whole presents a three-dimensional porous structure similar to that of loofah, with obvious pores, so it helps to absorb tissue exudate and reduce inflammatory response.
[0061] Injectability and rheological testing of hydrogels:
[0062] The injectability of CD@ hydrogel was tested with a disposable sterile syringe (22-gauge needle). The rheological properties of the hydrogel were tested at 37°C using a rheometer (MCR302, Anton Paar). The storage modulus (G') and loss modulus (G") of the hydrogel were tested by time sweep. The hydrogel sample was placed between the 25 mm parallel plates of the rheometer, the frequency was set to 10 rad / s, and the strain was 1%. Figure 6 For test results.
[0063] Figure 6 The storage modulus (G') and loss modulus (G") vary with angular frequency. Within the angular frequency test range (1-100 rad / s), the G' value of CD@hydrogel is higher than the G" value, proving that the hydrogel has good mechanical stability ( Figure 6 A). The dynamic strain sweep of the hydrogel was then tested. At low strain (1%-100%), G' and G" remained almost unchanged, showing good linear viscoelasticity ( Figure 6 B).
[0064] Reference Figure 7 , is the swelling test result of CD@ hydrogel. The swelling rate of the hydrogel increased rapidly within the first 20 hours, indicating that it has good water absorption capacity and is beneficial to wound healing.
[0065] The singlet oxygen green fluorescence probe (SOSG) was used to test the generation of singlet oxygen (ROS) in aqueous solution by CDs and CD@hydrogel. Figure 8 As shown in the figure, the fluorescence intensity of CDs and CD@Hydrogel increased significantly compared with water after laser irradiation, which proved that CDs and CD@Hydrogel could effectively generate ROS and have good potential for photodynamic therapy.
[0066] Reference Fig. 9 , are the results of CD@ hydrogel cytotoxicity test, Fig. 9 The results showed that different concentrations of CD@hydrogel (0-200ug / ml) did not produce obvious cytotoxicity when incubated with 4T1 cells for 24h or 48h, proving that the hydrogel has high biosafety.
[0067] The standard plate colony counting method was used to study the killing ability of CD@ hydrogel on Escherichia coli. The test results are as follows Fig.10 The experimental results show that laser irradiation (wavelength 470nm, power 60mW / cm 2 After 60 minutes, the colony activity gradually decreased with the increase of hydrogel concentration. When the hydrogel concentration increased to 200 μM, the colony survival rate was less than 10%. At the same time, when only CD@hydrogel was added but no light was applied, there was no significant change in bacterial activity. Fig.10 B shows that when the laser power is less than 100mW / cm 2 This experiment proves that CD@hydrogel-mediated photodynamic therapy has a significant killing effect on Escherichia coli and is expected to become a promising bactericidal material.
[0068] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.
Claims
1. A method for preparing a photosensitive gel antibacterial agent, characterized in that: The following steps are involved: S1, preparation of riboflavin carbon dots CDs; S2, preparing aldehyde-modified hyaluronic acid; S3, mixing riboflavin carbon dots CDs with aldehyded hyaluronic acid to prepare CD@hydrogel, i.e. the photosensitive gel antibacterial agent.
2. The method for preparing the photosensitive gel antibacterial agent according to claim 1, characterized in that: Step S1 is specifically as follows: Dissolve riboflavin in sodium hydroxide solution, stir until completely dissolved, then add sodium chloride and ethylenediamine, continue stirring, then transfer the resulting mixed solution to a reactor and heat for reaction; after the reaction is completed, centrifuge and filter, dialyze the filtrate with a dialysis bag, and finally freeze-dry the solution in the dialysis bag to obtain riboflavin carbon dots CDs.
3. The method for preparing the photosensitive gel antibacterial agent according to claim 2, characterized in that: Step S1 is specifically as follows: 150-600 mg of riboflavin is dissolved in 15-60 mL of a sodium hydroxide solution with a concentration of 0.05-0.2 mol / L, and stirred until completely dissolved. Then, 0.05-0.2 g of sodium chloride and 1-4 mL of ethylenediamine are added, and stirring is continued for 5-20 minutes. Then, the resulting mixed solution is transferred to a reactor, and heated at 160-200° C. for reaction for 3-12 hours. After the reaction is completed, centrifugation is performed, and then filtration is performed with a filter. The filtrate is dialyzed with a dialysis bag with a molecular cutoff of 500-2000D for 6-24 hours. Finally, the solution in the dialysis bag is freeze-dried to obtain riboflavin carbon dots CDs.
4. The method for preparing the photosensitive gel antibacterial agent according to claim 1, characterized in that: Step S2 is specifically as follows: The hyaluronic acid is dissolved in deionized water, and then a sodium periodate solution is added, and stirring is continued. Finally, ethylene glycol is added to terminate the reaction. The product is dialyzed with a dialysis bag, and the solution in the dialysis bag is freeze-dried to obtain aldehyde-modified hyaluronic acid.
5. The method for preparing the photosensitive gel antibacterial agent according to claim 4, characterized in that: Step S2 is specifically as follows: Dissolve 250-1000 mg of hyaluronic acid in 25-100 mL of deionized water, stir until completely dissolved, then add 2.5-10 mL of 0.15-0.6 mol / L sodium periodate solution, continue stirring at 20-40° C. for 2.5-10 hours, and finally add 0.5-2 g of ethylene glycol to terminate the reaction; the product is dialyzed with a dialysis bag with a molecular cutoff of 3000-6000D for 24-96 hours, and the solution in the dialysis bag is freeze-dried to obtain aldehyde-modified hyaluronic acid.
6. The method for preparing the photosensitive gel antibacterial agent according to claim 1, characterized in that: Step S3 is specifically as follows: Dissolve riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid in PBS solution, control the mass concentrations of riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid to be 10-40% and 7.5-30% respectively, mix and shake well to obtain CD@hydrogel, i.e. the photosensitive gel antibacterial agent.
7. The method for preparing the photosensitive gel antibacterial agent according to claim 1, characterized in that: The following steps are involved: S1. Preparation of riboflavin carbon dots CDs: 300 mg of riboflavin was dissolved in 30 mL of 0.1 mol / L sodium hydroxide solution, stirred for 10 minutes until completely dissolved, then 0.1 g of sodium chloride and 1 mL of ethylenediamine were added, and stirring was continued for 10 minutes. Then, the resulting mixed solution was transferred to a reactor and heated at 180°C for 6 hours. After the reaction was completed, centrifugation was performed, and then filtered with a 0.22 μm filter. The filtrate was dialyzed with a dialysis bag with a molecular cutoff of 1000 D for 12 hours. Finally, the solution in the dialysis bag was freeze-dried to obtain riboflavin carbon dots CDs. S2, preparing aldehyde-modified hyaluronic acid; Dissolve 500 mg of hyaluronic acid in 50 mL of deionized water, stir until completely dissolved, then add 5 mL of 0.3 mol / L sodium periodate solution, continue stirring at 30 °C for 5 hours, and finally add 1 g of ethylene glycol to terminate the reaction; The product was dialyzed for 48 hours using a dialysis bag with a molecular cutoff of 5000D, and the solution in the dialysis bag was freeze-dried to obtain aldehyde-modified hyaluronic acid; S3. Dissolve riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid in PBS solution, control the mass concentrations of riboflavin carbon dots CDs and aldehyde-modified hyaluronic acid to be 20% and 15% respectively, mix and shake well at room temperature to obtain CD@hydrogel, i.e., the photosensitive gel antibacterial agent.
8. A photosensitive gel antibacterial agent, characterized in that: It is prepared by the method according to any one of claims 1 to 7.
9. Use of the photosensitive gel antibacterial agent as claimed in claim 8 as a photodynamic therapy agent, wherein the application method is: applying light while applying the photosensitive gel antibacterial agent to sterilize.
10. Use of the photosensitive gel antibacterial agent according to claim 8 in the preparation of photodynamic therapy agents.