Preparation method and application of a photodynamic silver nanocluster sustained-release antibacterial hydrogel

Abstract

The application discloses a preparation method of a photodynamic silver nanocluster slow-release antibacterial hydrogel, and comprises the following steps: reacting bovine serum albumin as a protective agent with a silver ion solution to obtain BSA-Ag + aqueous solution; using a wet chemical method to synthesize the BSA-Ag with a photodynamic effect at room temperature 13 NC solution; using glutaraldehyde to crosslink the BSA-Ag 13 BSA ligand in the NC, to obtain the silver nanocluster slow-release hydrogel with a photodynamic bacteriostatic property; and application of the photodynamic silver nanocluster slow-release antibacterial hydrogel in preparation of a wound dressing and antibacterial material.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicines, and in particular to a preparation method and application of a photodynamic silver nanocluster sustained-release antibacterial hydrogel. BACKGROUND

[0002] Silver nanoparticles are used as one of the antibacterial drugs. The silver nanoparticles have unique structural and functional properties, can penetrate the bacterial cell wall, destroy the bacterial membrane structure, interfere with the normal metabolic synthesis process of bacteria, and inhibit the cell division of bacteria or fungi, so that the silver nanoparticles can be used as an antibacterial drug. However, the application of the silver nanoparticle antibacterial agent still has some problems, such as that the antibacterial efficiency needs to be improved, the silver nanoparticle antibacterial agent is not easy to be stored for a long time, and the bioavailability and compatibility are low.

[0003] Since the metal nanoclusters have higher specific surface area and atom utilization rate, the antibacterial effect can be significantly improved. However, the noble metal nanoclusters still have the following problems when used as antibacterial agents alone.

[0004] 1) The antibacterial mechanism of the nanoclusters mostly relies on the internalization of bacteria, and then the ions are released to damage the bacteria. Such antibacterial preparations release a large amount of ions in a short time when antibacterial, so that the antibacterial process of the nanoclusters is relatively fast and not durable, which may affect the long-term antibacterial activity of the Au / AgNCs and limit the antibacterial application of the Au / AgNCs in actual scenes.

[0005] 2) The metal nanoclusters have the problems of easy agglomeration and photothermal instability. Under the photothermal condition, the metal nanoclusters are easily decomposed, which further leads to the low antibacterial efficiency and long-term antibacterial property of the metal nanoclusters under the photothermal condition and short service life. SUMMARY

[0006] The application aims to provide a preparation method and application of a photodynamic silver nanocluster sustained-release antibacterial hydrogel, so as to solve the technical problems of low antibacterial duration and easy photodegradation of the metal nanoclusters in the prior art.

[0007] To solve the above technical problems, the application specifically provides the following technical scheme:

[0008] The application provides a preparation method of a photodynamic silver nanocluster sustained-release antibacterial hydrogel, which comprises the following steps:

[0009] S100, reacting bovine serum albumin as a protective agent with a silver ion solution to obtain BSA-Ag with electrostatic adsorption of silver ions + aqueous solution;

[0010] S200, synthesizing BSA-Ag with a photodynamic effect by using a wet chemical method at room temperature 13 NC solution;

[0011] S300, crosslinking the BSA-Ag using glutaraldehyde 13 The BSA ligand in the NC, to obtain the silver nanocluster sustained-release hydrogel with the photodynamic antibacterial performance.

[0012] As a preferred scheme of the present application, the BSA-Ag + The preparation method of the aqueous solution comprises the following steps:

[0013] S101, preparing 1-3 ml of 50-100 g / L bovine serum albumin aqueous solution, slowly adding 1M sodium hydroxide into the bovine serum albumin aqueous solution, adjusting the pH value to 12, and uniformly stirring to obtain the bovine serum albumin aqueous solution in a space structure expansion state;

[0014] S102, mixing 1-3 ml of 0.5-3 g / L silver nitrate solution with the bovine serum albumin aqueous solution in a space structure expansion state, and magnetically stirring for 10-20 min to obtain the BSA-Ag + aqueous solution.

[0015] As a preferred scheme of the present application, the BSA-Ag 13 The preparation method of the NC crosslinking solution comprises the following steps:

[0016] S201, slowly adding a sodium borohydride aqueous solution with a concentration of 3-5 g / L into the BSA-Ag + aqueous solution, continuously stirring, and gradually changing the solution from colorless and transparent to brownish yellow to obtain the BSA-Ag 13 NC solution.

[0017] As a preferred scheme of the present application, the preparation method of the silver nanocluster sustained-release hydrogel comprises the following steps:

[0018] S301, continuously slowly adding a dilute hydrochloric acid solution into the BSA-Ag 13 NC solution under the condition of vigorous stirring until the pH of the system is stable, transferring the BSA-Ag 13 NC solution into an ultrafiltration tube, and performing filtration concentration at low temperature to obtain the BSA-Ag 13 NC crosslinking solution.

[0019] S302, slowly adding a glutaraldehyde aqueous solution into the BSA-Ag 13 NC crosslinking solution, uniformly mixing, standing for 3-12 h, and obtaining the BSA-Ag 13 NC gel solution, dialysis, and freeze-drying to obtain the silver nanocluster sustained-release hydrogel with the photodynamic antibacterial performance.

[0020] As a preferred scheme of the present application, in the S101, the volume of the sodium hydroxide aqueous solution is 50-100 muL, and the time for uniform stirring is 5-30 min.

[0021] As a preferred scheme of the present application, in the S201, the volume of the sodium borohydride aqueous solution is 40-80 muL, and the time for stirring is 30-120 min.

[0022] As a preferred scheme of the present application, in the S301, the system pH is 5-8, and the concentration multiple is 3-6.

[0023] As a preferred scheme of the present application, in the S302, the concentration of the glutaraldehyde aqueous solution is 2%-20%, and the glutaraldehyde aqueous solution and the BSA-Ag 13 The volume ratio of the NC crosslinking liquid is 1:6-1:12.

[0024] The application further provides a photodynamic silver nanocluster sustained-release antibacterial hydrogel, which is prepared by the preparation method and has a silver ion sustained-release gel porous structure crosslinked by silver nanoclusters of bovine serum albumin and glutaraldehyde and a crosslinked structure for enhancing photodynamic performance.

[0025] The hydrogel has photodynamic performance and silver ion activity, thereby playing a dual sterilization role.

[0026] The application provides an application of the photodynamic silver nanocluster sustained-release antibacterial hydrogel in the fields of bacteriostasis and wound dressing.

[0027] As a preferred scheme of the present application, the photodynamic silver nanocluster sustained-release antibacterial hydrogel can inhibit the proliferation of gram-positive and gram-negative bacteria.

[0028] As a preferred scheme of the present application, the photodynamic silver nanocluster sustained-release antibacterial hydrogel (Ag 13 NCs@gel) has good porosity, water vapor permeability, biocompatibility and biodegradability.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] The material prepared by loading nanometal particles on a BSA as a bearing matrix has good biological performance, the silver nanoclusters obtained by using bovine serum albumin as a protective agent have high biological availability and compatibility.

[0031] The present application directly crosslinks bovine serum albumin (BSA) protectant with a crosslinking agent to construct a porous structure hydrogel, fixes the relative position of the BSA carrier and the functional group structure, plays a supporting and stabilizing role of the gel structure on the BSA carrier and silver nanoclusters, and further improves the stability of the Ag 13 The linking and structural stability of the BSA carrier in the NCs@gel hydrogel indirectly strengthen the structural stability of the silver nanoclusters, endow the silver nanoclusters with a more long-acting functional structural stability depending on the carrier structure, and prolong the existence and service life of the silver nanoclusters; the problems of unstable photothermal degradation of the silver nanoclusters, short service life under photothermal conditions, and easy functional structure change are solved, thereby improving the stability of the silver nanoclusters and the application range and service life of the silver nanomaterials;

[0032] The present application provides a kind of photodynamic silver nanocluster sustained-release antibacterial hydrogel, with stable porous structure, support and stabilizing effect on silver nanoclusters, endow silver nanomaterials with efficient silver ion release pathway, make it have silver ion controllable release characteristics, greatly improve the Ag 13 The antibacterial durability of the NCs@gel hydrogel; while further enhancing the photodynamic performance of the silver nanoclusters, active oxygen can be generated under white light to play a rapid sterilization effect, so that the photodynamic silver nanocluster sustained-release antibacterial hydrogel has dual sterilization function, and its sterilization effect is better than that of silver nanoclusters alone;

[0033] The present application can obtain physically stable hydrogel by optimizing the formula of antibacterial hydrogel, and can adjust its antibacterial effect while ensuring the properties of the hydrogel material, so that it can be applied to various fields and scenarios.

[0034] The present application can be applied to the fields of antibacterial and wound dressing, and has significant inhibitory effect on escherichia coli and staphylococcus aureus, and can be applied in the field of preparing wound dressing and antibacterial materials. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and those skilled in the art can obtain other implementation drawings according to the provided drawings without creating any creative labor.

[0036] Figure 1 The present application provides a flowchart of the preparation method of the photodynamic silver nanocluster sustained-release antibacterial hydrogel;

[0037] Figure 2 The bovine serum albumin silver nanocluster BSA-Ag 13UV-Vis absorption spectrum of the NCs;

[0038] Figure 3 Silver nanoclusters BSA-Ag prepared for example 1 of the present application 13 Transmission electron microscope image of the NCs;

[0039] Figure 4 Silver nanoclusters BSA-Ag prepared for example 1 of the present application 13 Particle size distribution diagram of the NCs;

[0040] Figure 5 Silver nanoclusters BSA-Ag prepared for example 1 of the present application 13 Actual picture of the NCs aqueous solution;

[0041] Figure 6 Silver nanoclusters hydrogel Ag prepared for example 1 of the present application 13 Actual picture of the NCs@gel;

[0042] Figure 7 Silver nanoclusters hydrogel Ag prepared for example 1 of the present application in application example 1 13 Scanning electron microscope image of the NCs@gel;

[0043] Figure 8 Detection result picture of ROS generated by the silver nanoclusters sustained-release antibacterial hydrogel prepared for example 1 of the present application in application example 1;

[0044] Figure 9 Result picture of the change of the antibacterial performance of the silver nanoclusters solution prepared for example 1 of the present application in application example 1 on E. coli with time;

[0045] Figure 10 Result picture of the change of the antibacterial performance of the silver nanoclusters sustained-release antibacterial hydrogel prepared for example 1 of the present application in application example 1 on E. coli with time;

[0046] Figure 11 Result picture of the change of the antibacterial performance of the silver nanoclusters solution prepared for example 1 of the present application in application example 1 on drug-resistant S. aureus with time;

[0047] Figure 12 Result picture of the change of the antibacterial performance of the silver nanoclusters sustained-release antibacterial hydrogel prepared for example 1 of the present application in application example 1 on drug-resistant S. aureus with time;

[0048] Figure 13 Result picture of the antibacterial experiment of the silver nanoclusters and the silver nanoclusters sustained-release antibacterial hydrogel prepared for example 1 of the present application in application example 2 on E. coli;

[0049] Figure 14 A statistical chart of the antibacterial experiment results of the silver nanoclusters and the silver nanocluster sustained-release antibacterial hydrogel prepared in Example 1 in application example 2 on Escherichia coli;

[0050] Figure 15 A chart of the antibacterial experiment results of the silver nanoclusters and the silver nanocluster sustained-release antibacterial hydrogel prepared in Example 1 in application example 2 on drug-resistant Staphylococcus aureus;

[0051] Figure 16 A statistical chart of the antibacterial experiment results of the silver nanoclusters and the silver nanocluster sustained-release antibacterial hydrogel prepared in Example 1 in application example 2 on drug-resistant Staphylococcus aureus;

[0052] Figure 17 A chart of the biological safety evaluation results of the silver nanocluster sustained-release antibacterial hydrogel prepared in Example 1 in application example 3. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0054] The present application provides a kind of photodynamic silver nanocluster sustained-release antibacterial hydrogel (Ag 13 NCs@gel), silver nanocluster is crosslinked to form hydrogel by glutaraldehyde, can solve the short-term problem of bactericidal of silver nanocluster itself, play sustained-release long-acting antibacterial performance; It can also solve the problem of light stability while improving the photodynamic performance.

[0055] Specifically, silver ions are electrostatically adsorbed on bovine serum albumin (BSA), and are converted into silver nanoclusters in the loading structure by reduction of NaBH4. Silver nanoclusters are a special size of nano-silver form, and silver nanoclusters have smaller cluster size in physical form, usually less than 2nm, with bovine serum albumin as ligand, sodium borohydride (NaBH4) as reducing agent, glutaraldehyde crosslinking silver nanoclusters in the hydrogel have the photodynamic performance of producing active oxygen under white light irradiation.

[0056] The above-mentioned nano-silver can be nano-gold, nano-copper.

[0057] This antibacterial silver nanocluster sustained-release hydrogel has the dual effects of silver ion bacteriostasis and photodynamic bacteriostasis, thereby improving its antibacterial effect.

[0058] In order to solve the problems of short antibacterial time and decreased antibacterial effect of noble metal nanoclusters, the present application first uses bovine serum albumin (BSA) as a protective agent and sodium borohydride (NaBH4) as a reducing agent to quickly synthesize a bovine serum albumin silver nanocluster (BSA-Ag 13 NC) solution with a photodynamic effect at room temperature by using a wet chemical method; and then crosslinks the BSA ligand in the BSA-Ag 13 NC by using glutaraldehyde to synthesize a BSA silver nanocluster slow-release hydrogel (Ag 13 NCs@gel) with a photodynamic antibacterial performance.

[0059] When silver ions are loaded on bovine serum albumin (BSA), under the influence of the reducing agent NaBH4, due to the special silver ion electrostatic adsorption sites in BSA and suitable synthesis pH conditions, the nanosilver becomes silver nanoclusters with smaller sizes. Compared with ordinary nanosilver, the silver nanoclusters in the present application have more efficient membrane penetration and silver ion release performance. In terms of antibacterial performance, small-sized silver nanoclusters are more likely to penetrate the bacterial wall and enter the bacterial interior than ordinary nanosilver, thereby interfering with the normal physiological activities of bacteria. However, compared with nanosilver, the silver nanoclusters are more difficult to be stored and utilized for a long time, and the functional structure thereof is more easily destroyed by complex physical and chemical factors from the outside.

[0060] Further, the silver nanoclusters in the hydrogel of the present application are proved to have excellent photodynamic performance. This photodynamic performance can be excited to produce reactive oxygen under white light, and the reactive oxygen has been verified to have the effect of inhibiting and killing cancer cells and bacteria. The photodynamic properties of the specific silver nanoclusters can produce a rapid bactericidal effect of reactive oxygen, combined with the antibacterial effect of nanosilver itself, which meets the comprehensive needs of rapid antibacterial and healing period sustained antibacterial and promotion of wound repair in the early debridement of wounds. By the method of loading silver nanoclusters on a bovine serum albumin carrier and crosslinking the ligand with a crosslinking agent, the stability and antibacterial performance of the silver nanoclusters can be significantly improved, the biological safety can be improved, and better application effects can be achieved.

[0061] The silver nanoclusters in the present application use bovine serum albumin (BSA) as a carrier of the silver nanoclusters. Bovine serum albumin (BSA) can protect and cover enzymes and other biological macromolecules, reduce the denaturation of functional molecules, and reduce the adverse effects caused by adverse environmental factors such as heating, surface tension and chemical factors. Bovine serum albumin (BSA) is a natural animal protein that can be obtained and utilized in large quantities, has low acquisition cost, and also has excellent biocompatibility.

[0062] The material prepared by loading nanometal particles on BSA as a carrier substrate has good biological performance. The silver nanoclusters obtained by using bovine serum albumin as a protective agent have high bioavailability and compatibility, improve the stability of silver nanoclusters, and improve the application range and service life of silver nanomaterials.

[0063] Meanwhile, the process of directly crosslinking the bovine serum albumin (BSA) protective agent to construct the porous structure hydrogel is a relative position and functional group structure fixation of the BSA carrier, which plays a supporting and stabilizing role of the gel structure on the BSA carrier and silver nanoclusters, and further improves the Ag 13 The linkage and structural stability of the BSA carrier in the NCs@gel hydrogel indirectly strengthen the structural stability of the silver nanoclusters, endow the silver nanoclusters with more long-acting functional structural stability depending on the carrier structure, prolong the existence and service life of the silver nanoclusters, and solve the problems of unstable photothermal degradation of the silver nanoclusters, short service life under photothermal conditions, and easy functional structure change.

[0064] In particular, due to the Ag 13 The loose porous structure of the NCs@gel hydrogel endows the silver nanocluster material with an efficient silver ion release pathway. At the same time, the crosslinked structure of the silver nanoclusters can stabilize silver ions and limit the massive release of silver ions in a short time, forming an effective silver ion sustained-release structure. The bactericidal silver ions can be released slowly, improving the service life of the gel, and the antibacterial effect of the gel does not change dramatically during the service life.

[0065] As an excellent carrier of silver nanocluster materials, bovine serum albumin is electrostatically adsorbed with silver ions, sodium borohydride (NaBH4) is used as a reducing agent, and a specific morphology of silver nanoclusters with photodynamic properties is synthesized and constructed by a wet chemical method. The bovine serum albumin is crosslinked and fixed by glutaraldehyde to directly construct a protein hydrogel crosslinking framework, that is, the excellent carrier and application function of the protein hydrogel are combined to prepare a silver nanocluster sustained-release antibacterial hydrogel material (Ag 13 NCs@gel) with photodynamic properties. The structure of this hydrogel is stable. This photodynamic performance can be excited by white light to produce reactive oxygen species, which has been verified to have the effect of inhibiting and killing cancer cells and bacteria.

[0066] It can be seen that the application discloses a brand-new silver nanocluster hydrogel, has a double sterilization effect, and the sterilization effect is remarkable. The silver nanoclusters obtained by taking bovine serum albumin as a protective agent have a photodynamic property due to the special structure and functional group distribution of the BSA carrier. After the silver nanoclusters are crosslinked, the photodynamic property is further improved, and the active oxygen can be generated by the silver nanoclusters under white light, so that the effect of killing bacteria is further improved. Meanwhile, due to the support and stability of the porous structure of the gel to the silver nanoclusters, the silver nanomaterial has an efficient silver ion release path, the crosslinked structure of the silver nanoclusters can stabilize the silver ions, and the release of the silver ions in a short time is limited, so that the silver nanoclusters have a slow-release function, have a controllable silver ion release characteristic, greatly improve the antibacterial effect of the silver nanoclusters, and prolong the antibacterial duration of the silver nanoclusters. 13 The Ag 13 NCs@gel hydrogel has a double long-acting antibacterial advantage.

[0067] As shown in the accompanying drawings, Figure 1 the application further provides a preparation method of the above-mentioned photodynamic silver nanocluster slow-release antibacterial hydrogel (Ag 13 NCs@gel), and steps are as follows:

[0068] The bovine serum albumin-silver ion (BSA-Ag + ) aqueous solution obtained by reacting bovine serum albumin as a protective agent with a silver ion solution is electrostatically adsorbed with silver ions;

[0069] The bovine serum albumin silver nanocluster (BSA-Ag 13 NC) solution with a photodynamic effect is quickly synthesized by using sodium borohydride (NaBH4) as a reducing agent by means of a wet chemical method at room temperature;

[0070] The BSA ligand in the BSA-Ag 13 NC is crosslinked by using glutaraldehyde, so that the short chains are combined together to form a reticular structure, so that the photodynamic silver nanocluster slow-release antibacterial hydrogel (Ag 13 NCs@gel) with a photodynamic antibacterial property is obtained.

[0071] The silver nanocluster material prepared by the method has good dispersibility and stability, the Ag 13 NCs have a photodynamic antibacterial property due to the fact that bovine serum albumin (BSA) is a protective agent and sodium borohydride (NaBH4) is a reducing agent. After the ligand protection and gel crosslinking fixation, the prepared hydrogel has a silver ion slow-release property and enhanced antibacterial property, is an excellent wound dressing material, has a wide application prospect, and the above-mentioned preparation process is simple and easy to popularize.

[0072] Further, the preparation method of the antibacterial silver nanocluster slow-release hydrogel specifically comprises the following steps:

[0073] S101, 1-3 ml of 50-100 g / L aqueous solution of bovine serum albumin is prepared, 1M sodium hydroxide is slowly added into the BSA aqueous solution until the pH value is adjusted to 12, and uniform stirring is performed to obtain the aqueous solution of bovine serum albumin in a space structure expansion state (BSA aqueous solution);

[0074] S102, 1-3 ml of 0.5-3 g / L silver nitrate solution is mixed with the BSA aqueous solution, and magnetic stirring is performed for 10-20 min to obtain the bovine serum albumin-silver ion (BSA-Ag + ) aqueous solution in which silver ions are electrostatically adsorbed;

[0075] S201, the aqueous solution of sodium borohydride with a concentration of 3-5 g / L is slowly added into the BSA-Ag + aqueous solution, and continuous stirring is performed until the solution gradually changes from colorless and transparent to brown yellow, and the BSA-Ag 13 NC solution is obtained;

[0076] S301, under the condition of vigorous stirring, the dilute hydrochloric acid solution is continuously and slowly added into the BSA-Ag 13 NC solution until the pH of the system is stable, the BSA-Ag 13 NC solution is transferred into an ultrafiltration tube, and filtration and concentration are performed at low temperature to obtain the BSA-Ag 13 NC crosslinking solution;

[0077] S302, the aqueous solution of glutaraldehyde is slowly added into the BSA-Ag 13 NC crosslinking solution, and uniform mixing is performed, and the BSA-Ag 13 NC gel solution is obtained after standing for 3-12 h, dialysis, and freeze-drying, and the silver nanocluster slow-release hydrogel with photodynamic antibacterial performance is obtained.

[0078] In step S301, the pH is adjusted to be constant within an accurate value after a period of time of vigorous stirring, and according to different concentration multiples, the physical state of the hydrogel is different, and the hydrogel can be applied to different scenarios of sterilization process.

[0079] The concentration of the aqueous solution of sodium hydroxide can be selected in any range, preferably, in the S201, the volume of the aqueous solution of sodium borohydride is 40-80 μL, and the stirring time is 30-120 min.

[0080] The volume of the aqueous solution of sodium borohydride can be selected in any range, preferably, in the S101, the volume of the aqueous solution of sodium hydroxide is 50-100 μL, and the uniform stirring time is 5-30 min.

[0081] The pH of the system and the concentration multiple can be selected in any range, preferably, in order to keep the sterilization effect unchanged and have higher safety performance, in the S301, the pH of the system is 5-8, and the concentration multiple is 3-6.

[0082] The concentration of the aqueous glutaraldehyde solution can be selected in any range, preferably, in the S302, the concentration of the aqueous glutaraldehyde solution is 2%-20%.

[0083] The aqueous glutaraldehyde solution and the BSA-Ag 13 The volume ratio of the NC crosslinking liquid can be selected in any range, preferably, the volume ratio of the aqueous glutaraldehyde solution and the BSA-Ag 13 The volume ratio of the NC crosslinking liquid is 1:6-1:12.

[0084] After the carrier protection and the gel crosslinking fixation, the light-driven silver nanocluster slow-release antibacterial hydrogel (Ag 13 NCs@gel) has the silver nanocluster stabilized by the bovine serum albumin ligand and the bovine serum albumin glutaraldehyde crosslinking gel structure, that is, has the long-acting bacteriostatic effect of silver ions and the light-driven reinforced bacteriostatic dual effect.

[0085] The silver nanocluster with a specific structure is synthesized by taking the bovine serum albumin as a protection carrier and has the light-driven property, that is, generates the ability of active oxygen, and the active oxygen has excellent bacteriostatic capacity, and cooperates with the silver nanocluster to release silver ions to kill bacteria, realizes the double bacteriostatic effect, and strengthens the bacteriostatic effect of the hydrogel.

[0086] The bovine serum albumin ligand protection and the gel crosslinking fixation together improve the biological safety and the structural stability of the silver cluster, the Ag 13 The gel porous structure of the NCs@gel regulates the silver nanocluster silver ion slow-release performance and prolongs the bacteriostatic effect of the silver nanocluster. The material can be used in the bacteriostatic and wound dressing fields and has significant inhibition effect on bacteria such as escherichia coli and staphylococcus aureus and has excellent application potential.

[0087] The Ag 13 NCs@gel hydrogel material has excellent water vapor permeability, biocompatibility and biodegradability. In addition, by adjusting the synthesis conditions of the hydrogel material, the softness and hardness, and the swelling performance of the hydrogel can be adjusted to cope with diversified application scenarios. The material can be used in the bacteriostatic, wound and biological engineering fields and is a good choice for loading functional materials and wound dressing materials and has wide application prospects.

[0088] The silver nanocluster of the application can be practically applied to the biological medicine field in the form of a hydrogel.

[0089] The function and structure of the hydrogel are discussed in detail below through examples.

[0090] Example 1

[0091] (Example 1 is different from Examples 2 and 3 in that the pH is adjusted differently and the physical properties such as the texture and swelling performance of the obtained hydrogel are different from the application scenarios)

[0092] Step 1: Preparation of BSA-Ag 13 NC solution:

[0093] 0.8 ml of 74 g / L aqueous solution of bovine serum albumin was prepared, 1M sodium hydroxide was slowly added to the BSA aqueous solution until the pH value was adjusted to 12, and uniform stirring was performed to obtain a spatially expanded bovine serum albumin aqueous solution. 1.6 ml of 1.274 g / L silver nitrate solution was mixed with the BSA aqueous solution, and magnetic stirring was performed at a speed of 1500 r / min for 20 min to obtain a bovine serum albumin-silver ion (BSA-Ag + ) aqueous solution in which silver ions were electrostatically adsorbed; a 4.24 g / L aqueous solution of sodium borohydride was slowly added to the BSA-Ag + aqueous solution, and stirring was continued until the solution gradually changed from colorless and transparent to brownish yellow, obtaining a BSA-Ag 13 NC solution.

[0094] As shown in Figure 2 , the results of the ultraviolet-visible absorption spectrum show that a new substance different from the silver ion and BSA solution is generated, which has the characteristic of a shoulder peak at 460 nm. As shown in Figure 3 , the results of the transmission electron microscope show that the size distribution of the synthesized BSA-Ag 13 NC particles is uniform, and there is no obvious aggregation and silver nanoparticle deposition. As shown in Figure 4 , the results of the particle size distribution show that the synthesized BSA-Ag 13 NC is about 2 nm. The actual picture of the BSA-Ag 13 NC aqueous solution is shown in Figure 5 .

[0095] Step 2: Preparation of a photodynamic silver nanocluster slow-release antibacterial hydrogel:

[0096] Under the condition of vigorous stirring, 1M dilute hydrochloric acid solution was slowly added to the BSA-Ag 13 NC solution obtained in step 1 above until the pH of the system was stable at 7, and the BSA-Ag 13 NC solution was transferred into an ultrafiltration tube and concentrated by filtration at low temperature to obtain a BSA-Ag 13 NC crosslinking solution.

[0097] A 10% aqueous solution of glutaraldehyde was slowly added dropwise to BSA-Ag at a volume ratio of 1:8. 13 Mix thoroughly in NC crosslinking solution, let stand for 12 hours, and obtain BSA-Ag. 13 NC gel solution, dialysis, lyophilization, to obtain silver nanocluster sustained-release hydrogel (Ag) with photodynamic antibacterial properties. 13 NCs@gel), store in a brown sample vial at 4°C protected from light, such as Figure 6 As shown.

[0098] After swelling and absorbing water, the hydrogel obtained in Example 1 has a relatively soft texture, with certain water absorption and swelling properties. It can swell to a degree of 150% and has excellent surface adsorption properties.

[0099] Scanning electron microscope image of hydrogel as shown Figure 7 As shown, the hydrogel has large gel pores and a dense and stable structure.

[0100] Example 1: The hydrogel can be used as a wound dressing. The hydrogel in Example 1 has a moist and soft texture, excellent surface adsorption and breathability. Combined with its antibacterial properties, it possesses excellent performance for wound dressings, meeting the comprehensive needs of rapid antibacterial debridement in the early stages of wound healing and continuous antibacterial action to promote wound repair during the healing process.

[0101] Example 2

[0102] (Example 2 differs from Examples 1 and 3 in that the pH is adjusted differently, and the resulting hydrogel has different physical properties such as texture and swelling properties, as well as different application scenarios.)

[0103] Step 1: Use the BSA-Ag obtained in Example 1 13 NC solution;

[0104] Step 2: Preparation of photodynamic silver nanoclusters sustained-release antibacterial hydrogel:

[0105] Under vigorous stirring conditions, the BSA-Ag obtained in Example 1 was continuously stirred. 13 Slowly add 1M dilute hydrochloric acid solution dropwise to the NC solution until the pH of the system stabilizes at 6, then add BSA-Ag. 13 The NC solution was transferred into an ultrafiltration tube and concentrated by filtration at low temperature to obtain BSA-Ag. 13 NC crosslinking solution;

[0106] A 10% aqueous solution of glutaraldehyde was slowly added dropwise to BSA-Ag at a volume ratio of 1:8. 13 Mix thoroughly in NC crosslinking solution, let stand for 12 hours, and obtain BSA-Ag. 13 NC gel solution, dialysis, lyophilization, to obtain silver nanocluster sustained-release hydrogel (Ag) with photodynamic antibacterial properties.13 BSA-Ag NCs@gel) was obtained and stored in a brown sample bottle at 4°C in the dark.

[0107] The hydrogel of Example 2 was relatively hard in texture, had toughness and elasticity, poor water absorption and swelling performance, and hardly swelled.

[0108] The application scenario of the hydrogel of Example 2 can be a medical hard filling gel, which can be used for filling of cartilage and supporting soft tissue material. The hydrogel of Example 2 has low swelling degree and sustained antibacterial property required for such gel applications, to resist the swelling deformation and infection risk that may occur to exogenous implants, and the BSA silver cluster and silver ion material has also been proved to have anti-inflammatory effect to resist the possible inflammatory reaction, and has good biological safety.

[0109] Example 3

[0110] (Different from Examples 1 and 2 in that the pH is adjusted, and the physical properties such as texture and swelling performance of the obtained hydrogel and the application scenario are different)

[0111] Step 1: BSA-Ag NC solution obtained in Example 1 was used 13 NC solution;

[0112] Step 2: Preparation of a photodynamic silver nanocluster slow-release antibacterial hydrogel:

[0113] Under the condition of vigorous stirring, 1M dilute hydrochloric acid solution was slowly added to the BSA-Ag NC solution obtained in Example 1 until the pH of the system was stable at 8, and the BSA-Ag NC solution was transferred into an ultrafiltration tube and concentrated by filtration at low temperature to obtain a BSA-Ag NC cross-linking solution. 13 13 NC solution; 13 NC cross-linking solution;

[0114] A 10% concentration glutaraldehyde aqueous solution was slowly added to the BSA-Ag NC cross-linking solution at a volume ratio of 1:8, mixed uniformly, and placed for 12h to obtain a BSA-Ag NC gel solution. 13 13 NC gel solution, dialyzed, freeze-dried, and obtained a silver nanocluster slow-release hydrogel (Ag NCs@gel) with photodynamic antibacterial performance. 13 BSA-Ag NCs@gel) was obtained and stored in a brown sample bottle at 4°C in the dark.

[0115] The hydrogel obtained in Example 3 was softer in texture, had certain water absorption and swelling properties, could swell to a degree of 300%, and had surface adsorption properties.

[0116] ​​The hydrogel of the embodiment 3 can be used as a substitute for the paste wound dressing. The hydrogel has a soft texture and a certain deformation capacity, and can be closely attached to the wound. The gel powder also has a strong water absorption capacity, and can absorb the exudate on the wound surface to meet the application requirement of keeping the wound dry.

[0117] In the above embodiments, the hydrogels with different textures have similar silver cluster density and antibacterial performance. The preparation process of the hydrogel does not affect the antibacterial function of the silver cluster. Therefore, although the hydrogels with different textures have different textures, they also have the dual performance of silver ion antibacterial and photodynamic antibacterial.

[0118] Therefore, by adjusting the synthesis conditions of the hydrogel material, the softness and swelling performance of the hydrogel can be adjusted to meet the diversified application scenarios. The material can be used in the fields of antibacterial, wound and biological engineering, and is a good choice for loading functional materials and wound dressing materials, and has a wide application prospect. Further, the embodiments show that under the limited preparation conditions, stable and uniform hydrogels can be obtained.

[0119] The following provides multiple embodiments for further illustration:

[0120] Embodiment 4

[0121] Embodiment 4 is different from example 1 in that the volume ratio of glutaraldehyde aqueous solution and BSA-Ag 13 NC crosslinking solution is different

[0122] Step 1: using the BSA-Ag 13 NC solution obtained in embodiment 1;

[0123] Step 2: preparation of photodynamic silver nanocluster slow-release antibacterial hydrogel:

[0124] Under the condition of vigorous stirring, 1M dilute hydrochloric acid solution was slowly added to the BSA-Ag 13 NC solution obtained in embodiment 1 until the pH of the system was stable at 7. The BSA-Ag 13 NC solution was transferred into an ultrafiltration tube and concentrated by filtration at low temperature to obtain a BSA-Ag 13 NC crosslinking solution;

[0125] 10% concentration of glutaraldehyde aqueous solution was slowly added to the BSA-Ag 13 NC crosslinking solution at a volume ratio of 1:6, mixed uniformly, and placed for 12h to obtain a BSA-Ag 13 NC gel solution. After dialysis and freeze-drying, a silver nanocluster slow-release hydrogel with photodynamic antibacterial performance (Ag 13 NCs@gel) was obtained and stored in a brown sample bottle at 4°C in the dark.

[0126] The hydrogel obtained in Example 4 has the same physical morphology as that in Example 3, but its bactericidal effect is slightly weaker than that in Example 1.

[0127] Example 5

[0128] (Example 5 differs from Example 3 in that it contains glutaraldehyde aqueous solution and BSA-Ag) 13 (Different volume ratios of NC crosslinking solutions)

[0129] Step 1: Use the BSA-Ag obtained in Example 1 13 NC solution;

[0130] Step 2: Preparation of photodynamic silver nanoclusters sustained-release antibacterial hydrogel:

[0131] Under vigorous stirring conditions, the BSA-Ag obtained in Example 1 was continuously stirred. 13 Slowly add 1M dilute hydrochloric acid solution dropwise to the NC solution until the pH of the system stabilizes at 7, then add BSA-Ag. 13 The NC solution was transferred into an ultrafiltration tube and concentrated by filtration at low temperature to obtain BSA-Ag. 13 NC crosslinking solution;

[0132] A 10% aqueous solution of glutaraldehyde was slowly added dropwise to BSA-Ag at a volume ratio of 1:12. 13 Mix thoroughly in NC crosslinking solution, let stand for 12 hours, and obtain BSA-Ag. 13 NC gel solution, dialysis, lyophilization, to obtain silver nanocluster sustained-release hydrogel (Ag) with photodynamic antibacterial properties. 13 Store NCs@gel in a brown sample vial at 4°C protected from light.

[0133] After swelling and absorbing water, the physical morphology of the hydrogel obtained in Example 5 was the same as that in Example 3, but its bactericidal effect was lower than that in Example 1.

[0134] Analysis of the results of Examples 1, 4, and 5 shows that in BSA-Ag 13 Without altering the NC solution, glutaraldehyde aqueous solution reacts with BSA-Ag 13 When the volume ratio of the NC crosslinking liquid is changed, the gelation rate changes, but due to the selection of the characteristics of the protectant and crosslinking agent, the physical morphology and shape of the hydrogel remain almost unchanged.

[0135] The change in its antibacterial properties is mainly due to BSA-Ag 13 The lower the concentration of the NC crosslinking solution, the faster its gelation rate and the more stable its structure, but the slower its silver ion release rate. BSA-Ag... 13The higher the concentration of the NC crosslinking solution, the slower its gelation rate. After standing for the same amount of time, the obtained gel has weaker photodynamic properties, which further verifies that the hydrogel in Example 1 has a higher ability to generate active oxygen through photodynamics and silver ion controlled release characteristics. This invention not only improves the bactericidal effect of the hydrogel without changing its morphology, but also endows the hydrogel with dual bactericidal properties.

[0136] The following section will further verify the function of the hydrogel.

[0137] Application Example 1

[0138] Antibacterial performance evaluation

[0139] Using drug-resistant Staphylococcus aureus and Escherichia coli as research subjects, PBS buffer, silver nanocluster solution, and equal amounts of Ag were used. 13 The NCs@gel hydrogel (Example 1) solutions were used in different experimental groups to study the antibacterial effects at different times.

[0140] Samples from different experimental groups were added to 12-well plates, and 2 ml of bacteria with a concentration of 1.00 × 10⁻⁶ were added to each well. 5 CFU mL -1 The bacterial culture in LB medium was used to culture 12-well plates in a shaker at 37.0℃ and 2000 rpm. Meanwhile, the plates from different experimental groups were placed in a dark environment and then irradiated with a 150W LED lamp for 30 minutes before being transferred back to a dark environment for further culture.

[0141] During the culture process, the optical density of the bacterial culture in each group's 12-well plate was measured and statistically analyzed every hour using an ELISA reader to detect the real-time proliferation rate of bacteria in each group.

[0142] like Figures 9-12 The results shown indicate that Ag 13 NCs@gel hydrogels exhibited more significant antibacterial activity than silver nanoclusters solutions under both light and dark conditions. Furthermore, the hydrogels showed superior antibacterial performance under light conditions compared to those under dark conditions. This is attributed to the hydrogels' higher photodynamic ability to generate reactive oxygen species (see...). Figure 8 , Figure 8 (The detection results of ROS generated by photodynamic therapy of hydrogels) further inhibited bacterial proliferation.

[0143] As the experimental time increased, Ag 13 The optical density of the bacterial culture in the NCs@gel hydrogel group did not increase significantly under light irradiation, indicating that compared to liquid Ag... 13 NCs solution, Ag 13NCs@gel hydrogel has better antibacterial persistence and stability.

[0144] Therefore, this application example demonstrates that the photodynamic silver nanocluster sustained-release antibacterial hydrogel disclosed in this application, by endowing the hydrogel with photodynamic properties of silver nanoclusters, has a dual antibacterial effect. Without changing the loading amount and the loaded drug, the antibacterial ability of the hydrogel is improved by improving the use of its protective agent, reducing agent and crosslinking agent.

[0145] Application Example 2

[0146] Antibacterial performance evaluation

[0147] Further research was conducted using drug-resistant Staphylococcus aureus and Escherichia coli as subjects, with PBS buffer, silver nanocluster solution, and equal amounts of Ag-containing... 13 The NCs@gel hydrogel (Example 1) solutions were used in different experimental groups, and the antibacterial efficacy of different groups was compared using the dilution plating method.

[0148] Samples from different experimental groups were added to 12-well plates, and 2 ml of bacteria with a concentration of 1.00 × 10⁻⁶ were added to each well. 5 CFU mL -1 The bacterial culture in LB medium was placed in a 12-well plate and co-cultured in a shaker at 37.0℃ and 2000r / min.

[0149] Meanwhile, the well plates of different experimental groups were placed in a dark, light-protected environment and then irradiated with a 150W LED lamp for 30 minutes before being transferred to a dark, light-protected environment for further culture.

[0150] After 6 hours of incubation, 10 μL of the culture solution was dropped onto an LB plate in a clean bench, spread on the LB plate with a spreader, and then incubated at 37.0℃ for 24 hours before colony counting.

[0151] The antibacterial properties of the blank control, silver nanoclusters, and silver nanocluster sustained-release antibacterial hydrogel were statistically analyzed, and the results were obtained. Figures 13 to 16 .Depend on Figures 13 to 16 It can be seen that it exhibits significant antibacterial ability under both light and dark conditions, and the presence of silver nanoclusters provides Ag... 13 The NCs@gel hydrogel provides antibacterial properties, and the use of pentylene glycol to crosslink the silver nanoclusters enhances its photodynamic properties. Therefore, after light exposure, the Ag... 13 The antibacterial effect of the NCs@gel hydrogel group was the most significantly improved. Application Example 2 further demonstrates that, without changing the loading amount or the loaded drug, this invention improves the antibacterial ability of the hydrogel by improving the use of its protective agent, reducing agent, and crosslinking agent.

[0152] Example 3

[0153] Cell safety evaluation

[0154] The cells were selected from normal human epidermal keratinocytes (NHEK) and were cultured in DMEM medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin) in a cell incubator at 37°C. The cell safety and the ability to affect the metabolic state of the cells were studied by detecting the OD (optical density) value of the cells in the presence of the gel. In the experiment, NHEK cell suspension with a density of 4 x 10 5 / mL was inoculated in a 96-well culture plate, 100 μL per well.

[0155] The experimental group was Ag 13 NCs@gel hydrogel cell culture solution with different concentrations (0.1, 0.25, 0.5, 0.75, 1, 2.5 mg / mL), and the negative control group was cell culture solution, 6 replicates per group. After the cell culture plate was incubated in the incubator (37°C, 5% CO2) for 24 h, 10 μl of CCK8 solution was added to each well, and attention was paid not to generate bubbles in the well to prevent affecting the OD value reading. The culture plate was continued to be incubated in the incubator for 1-4 hours. The absorbance at 450 nm of each group of wells was measured by an enzyme marker, and the OD value was compared by recording and analyzing the data, so as to judge the biological safety of Ag 13 NCs@gel hydrogel material and its effect on cell growth.

[0156] As Figure 17 ( Figure 17 The cell viability test showed that the hydrogel at each concentration had no obvious cytotoxicity in NHEK cells, and the NHEK cells coexisting with the hydrogel also showed good cell viability.

[0157] The photodynamic silver nanocluster slow-release antibacterial hydrogel (Ag 13 NCs@gel) of the embodiment can realize slow release of silver ions and stable antibacterial function, prolong the antibacterial and service life, and has a dual antibacterial effect and improved antibacterial effect. At the same time, the preparation method of the photodynamic silver nanocluster slow-release antibacterial hydrogel of the embodiment realizes the simple preparation of the photodynamic silver nanocluster slow-release antibacterial hydrogel.

[0158] The above embodiments are only exemplary embodiments of the present application and are not used to limit the present application, and the protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements shall also be considered to fall within the protection scope of the present application.

Claims

1. A method for preparing a photodynamic silver nanocluster sustained-release antibacterial hydrogel, characterized in that, Includes the following steps: S100. Bovine serum albumin was reacted with a silver ion solution as a protective agent to obtain BSA-Ag, which electrostatically adsorbs silver ions. + Aqueous solution; S200, BSA-Ag exhibiting photodynamic effects was synthesized at room temperature using a wet chemical method. 13 NC solution; S300, BSA-Ag crosslinked using glutaraldehyde. 13 BSA ligands in NC were used to obtain silver nanocluster sustained-release hydrogels with photodynamic antibacterial properties; Step S300 includes the following steps: S301. Under vigorous stirring conditions, continuously add BSA-Ag... 13 Slowly add dilute hydrochloric acid solution dropwise to the NC solution until the pH of the system stabilizes, then add the BSA-Ag solution. 13 The NC solution was transferred into an ultrafiltration tube and concentrated by filtration at low temperature to obtain BSA-Ag. 13 NC crosslinking liquid; S302, Slowly add glutaraldehyde aqueous solution dropwise to the BSA-Ag... 13 Mix thoroughly in NC crosslinking solution and let stand for 3-12 hours to obtain BSA-Ag. 13 NC gel solution, dialyzed, lyophilized to obtain the silver nanocluster sustained-release hydrogel with photodynamic antibacterial properties.

2. The preparation method according to claim 1, characterized in that, The BSA-Ag + The method for preparing an aqueous solution includes the following steps: S101. Prepare 1-3 ml of 50-100 g / L bovine serum albumin aqueous solution, slowly add 1 M sodium hydroxide to the bovine serum albumin aqueous solution, adjust the pH value to 12, stir at a uniform speed to obtain a bovine serum albumin aqueous solution in a spatially expanded state. S102. Mix 1-3 ml of 0.5-3 g / L silver nitrate solution with the bovine serum albumin aqueous solution in the expanded state of the spatial structure, and stir magnetically for 10-20 min to obtain the BSA-Ag electrostatically adsorbed silver ions. + Aqueous solution.

3. The preparation method according to claim 1, characterized in that, The BSA-Ag 13 The preparation method of NC solution includes the following steps: S201. Slowly add a sodium borohydride aqueous solution with a concentration of 3~5 g / L to the BSA-Ag... + In an aqueous solution, with continuous stirring, the solution gradually changes from colorless and transparent to brownish-yellow, yielding BSA-Ag. 13 NC solution.

4. The preparation method according to claim 2, characterized in that, In step S101, the volume of the sodium hydroxide aqueous solution is 50-100 μL, and the stirring time is 5-30 min.

5. The preparation method according to claim 3, characterized in that, In step S201, the volume of the sodium borohydride aqueous solution is 40-80 μL, and the stirring time is 30-120 min.

6. The preparation method according to claim 1, characterized in that, In the S301, the system pH is 5-8; the concentration factor is 3-6 times.

7. The preparation method according to claim 1, characterized in that, In step S302, the concentration of the glutaraldehyde aqueous solution is 2%~20%; the glutaraldehyde aqueous solution and the BSA-Ag 13 The volume ratio of the NC crosslinking solution is 1:6 to 1:

12.

8. A photodynamic silver nanocluster sustained-release antibacterial hydrogel, characterized in that... ， The photodynamic silver nanocluster sustained-release antibacterial hydrogel, prepared according to any one of claims 1-7, has a porous structure of silver ion sustained-release gel crosslinked with bovine serum albumin silver nanoclusters and glutaraldehyde, as well as a crosslinked structure that enhances photodynamic properties.

9. The application of the photodynamic silver nanocluster sustained-release antibacterial hydrogel as described in claim 8 in the preparation of antibacterial materials.

Images