Silk fibroin composite hydrogel dressing for treating chronic wounds and preparation method thereof

Abstract

In the research, modified silk fibroin (SF-TK), methacrylamide gelatin (GelMA) and quaternary ammonium salt chitosan (QCS) are taken as matrix hydrogel (GCS), metformin (Met) and IL2-loaded PLGA particles (IL2-coated PLGA) are loaded, Met can promote polarization of macrophages from M1 to M2, interleukin 2 with a specific concentration can specifically promote proliferation of Regulatory T cells (Tregs for short), and the Met and IL2-loaded PLGA particles (IL2-coated PLGA) can be used for preparing an anti-inflammatory drug for treating the macrophages. Due to different loading modes of the metformin and the interleukin, the highest peak values accumulated by concentration of the metformin and the interleukin appear in a staggered manner, so that the effect that macrophages are polarized firstly and then Tregs are proliferated is achieved, and different immune cells can be adjusted in a time sequence manner, so that wound healing is promoted. The research starts from two immune cells in a wound microenvironment, macrophage polarization and Tregs proliferation are sequentially induced, angiogenesis is promoted through paracrine action of the immune cells, and diabetic chronic wound repair is sequentially promoted.

Description

Technical Field

[0001] The present invention relates to a silk fibroin composite hydrogel dressing integrating antibacterial and anti-inflammatory functions and a preparation method thereof, belonging to the field of biomedical technology. Background Art

[0002] Inflammatory cells and cytokines produced by them are the key causes of chronic wounds. The commonly used approach to treat wound infections is to avoid bacterial infection of the wound, regulate the cell state, thereby restoring the wound microenvironment and ending the inflammatory period, so as to promote wound healing. Traditional wound dressings mainly include gauze, bandage, and transparent dressing. Compared with traditional dressings, hydrogel dressings, as a kind of moist dressing, have unique water permeability, porosity, and moisture retention, significantly shortening the wound healing time, reducing wound exudation, and alleviating the pain caused by patients replacing traditional dressings. However, the current healing mechanism of hydrogel dressings providing antibacterial and anti-inflammatory effects for chronic wounds usually only involves a single type of inflammatory immune cell. Therefore, there is a need in the art for a silk fibroin composite hydrogel dressing integrating different immune cells and antibacterial and anti-inflammatory functions and a preparation method thereof. Summary of the Invention

[0003] The object of the present invention is to solve the problem that the healing mechanism of hydrogel dressings providing antibacterial and anti-inflammatory effects for chronic wounds in the prior art usually only involves a single type of inflammatory immune cell.

[0004] To achieve the above object, the technical solution adopted by the present invention is to provide a silk fibroin-chitosan composite hydrogel dressing integrating antibacterial and anti-inflammatory functions and a preparation method thereof.

[0005] In the first aspect of the present invention, a preparation method of a silk fibroin (SF)-chitosan composite hydrogel dressing integrating antibacterial and anti-inflammatory functions is provided, including the following steps:

[0006] Step 1: Extract silk fibroin: Weigh silkworm cocoons, measure deionized water, add Na2CO3 and stir evenly. After boiling the water, add the silkworm cocoons for degumming and drying until fluffy, and store them at room temperature in a sealed manner for later use; Take an appropriate amount of silk fibroin fibers, cut them into pieces and put them into a lithium bromide solution, heat and stir until dissolved; Put the dissolved SF solution into a dialysis bag for dialysis, and after dialysis, obtain a desalted SF solution;

[0007] Step 2. Modification of silk fibroin: Dissolve dithioketal with dicarboxyl group (TK) in deionized water, filter to obtain a clear TK solution, add EDC and NHS solutions dropwise to the TK solution in an ice bath, and activate it at low temperature for standby; add methyl ester sulfonate (MES) to the SF solution obtained in Step 1, add the activated TK solution to the SF solution to prepare an SF / TK solution, stir, and react in an ice bath. Then, add EDC and NHS to the mixed solution in sequence, remove bubbles, dialyze, and freeze-dry to obtain a TK-SF solution, that is, a modified silk fibroin solution.

[0008] Step 3: Preparation of PLGA microspheres carrying interleukin-2: Measure an appropriate amount of IL-2 and Dextran, and prepare a clear Dextran solution with a mass fraction of 6% according to a certain ratio. Then add 10 times the volume of a 6% PEG 8000 solution, stir evenly, and freeze-dry the sample. Under the action of a vortex mixer, wash it thoroughly with dichloromethane to dissolve PEG, centrifuge at 12000 rpm for 6 min to obtain precipitated polysaccharide-protein composite particles. This process is repeated at least 3 times. Place it in a fume hood to volatilize completely and transfer it to a vacuum drying oven to dry thoroughly. Weigh PLGA and PLA, add dichloromethane to obtain a 10 - 12.5% PLGA dichloromethane solution. Then weigh 10 - 30 mg of the previously prepared IL-2 polysaccharide-protein composite particles and add them to the above solution. After dissolving into a clear solution, select PVA as a stabilizer and add an appropriate amount. After stirring for 10 min, quickly transfer it to a mixed solution of ethylene glycol and glycerol. Before transferring this solution, place the mixed solution of ethylene glycol and glycerol in a magnetic stirrer and adjust the stirring speed to 500 - 700 rpm for standby. After forming semi-cured microspheres, transfer them to a 5% NaCl solution with a volume of 500 ml or more to completely cure for 2 h or more. Collect and wash by sedimentation method, and freeze-dry for later use.

[0009] Step 4. Weigh a modified silk fibroin solution with a mass fraction of 8 - 10%, a quaternary ammonium salt chitosan with a mass fraction of 3 - 6%, a GelMA with a mass fraction of 6 - 8%, an appropriate amount of PLGA microspheres, and metformin with a concentration of 0.06 mM - 0.10 mM, and carry out a photocrosslinking process under the system of a photoinitiator LAP (lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate). Use 405 - nanometer light to crosslink and activate LAP to promote the crosslinking reaction of molecules in the hydrogel, and obtain a silk fibroin composite hydrogel dressing integrating antibacterial and anti-inflammatory functions.

[0010] Preferably, in the above Step 1, the ratio of raw silk to deionized water is 1:50 (w:v); the mass fraction of Na2CO3 is 0.06%, the concentration of lithium bromide solution is 9.3 M, the volume is 100 mL, and the heating temperature is 65 ± 2 °C.

[0011] Preferably, in step 2, the added MES accounts for 20% of the mass of SF, and the supplemented EDC and NHS account for 20% and 10% of the mass of SF respectively.

[0012] Preferably, in step 3, the mass ratio of IL-2 freeze-dried powder to Dextran is 1:5, the volume ratio of 6% Dextran solution to 6% PEG 8000 solution is 1:10, the mass ratio of PLGA to PLA is 70:30, the PLGA dichloromethane solution is 12.5%, the mass of IL-2 polysaccharide vitreous particles is 20 mg, the PVA solution is formulated at 1%, and a mixed solution is prepared from 4 g of ethylene glycol and 1 g of glycerol, and the stirring speed of the ethylene glycol-glycerol mixed solution is 500 rpm.

[0013] Preferably, in step 4, the mass fraction of the modified silk fibroin solution is 8%, the mass fraction of quaternary ammonium salt chitosan is 4%, the mass fraction of GelMA is 7%, the concentration of metformin is 0.08 mM, and the mass ratio of the photoinitiator is 0.2 wt%.

[0014] In the second aspect of the present invention, a silk fibroin composite hydrogel dressing integrating antibacterial and anti-inflammatory functions prepared by the above method is provided.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] In the preparation method of the present invention, ketone thiol TK modified with carboxyl groups at both ends is crosslinked with the remaining amino groups on silk fibroin to obtain modified silk fibroin. This modification can endow the hydrogel made of silk fibroin with the ability to scavenge free radicals ROS in the microenvironment of chronic wounds, bringing an anti-inflammatory effect. Quaternary ammonium salt chitosan provides antibacterial ability. In addition, GelMA is incorporated into the mixed solution to endow crosslinking ability, and the microparticles are mixed into the un-gelled liquid before crosslinking and mixed evenly.

[0017] The mechanism of action of the present invention is that the two drugs carried by the gel start from two types of immune cells in the wound microenvironment, and induce macrophage polarization and Tregs proliferation through the difference in release sequence in a time-dependent manner. Through the paracrine effect of immune cells, it promotes angiogenesis and chronologically promotes the repair of diabetic chronic wounds. Description of the Drawings

[0018] Figure 1 It is a macroscopic photograph of a silk fibroin-chitosan composite hydrogel integrating antibacterial and anti-inflammatory functions prepared in Example 1;

[0019] Figure 2 Among them, A: Microscopic image of a silk fibroin-chitosan composite hydrogel integrating antibacterial and anti-inflammatory functions; B: Microscopic image after the morphology of the hydrogel is destroyed by ROS in the solution due to the presence of TK bonds;

[0020] Figure 3 Micrograph of hydrogel - carried microspheres;

[0021] Figure 4 Micrograph of microspheres;

[0022] Figure 5 FITR of SF - TK;

[0023] Figure 6 FITR diagrams of silk fibroin with different degrees of TK modification. In A, SF:TK = 100:6; in B, SF:TK = 100:4; in C, SF:TK = 100:2; in D, SF:TK = 100:0;

[0024] Figure 7 Strain tests when the mass percentages of the modified silk fibroin solution are set to 8%, 12%, and 16% respectively;

[0025] Figure 8 In it, A: PBS negative control group; B: mass percentage of modified silk fibroin component is 8%, and mass percentage of quaternary ammonium salt chitosan is 1%; C: antibacterial effect diagrams when the mass percentage of modified silk fibroin component is 8% and the mass percentage of quaternary ammonium salt chitosan is 4%;

[0026] Figure 9 The five groups in it are respectively the Control group, the GCS hydrogel extract, the GCS gel - loaded Met extract, the GCS hydrogel - loaded IL2 extract, and the GCS hydrogel simultaneously loaded with metformin and IL2 extract. Macrophages polarized by LPS for 8 hours are cultured for 24 hours, and the anti - inflammatory transformation effect of the five groups on macrophages is detected by flow cytometry;

[0027] Figure 10 The five groups of it are respectively the Control group (LPS - treated), the GCS hydrogel extract group, and the GCS / Met hydrogel extract group. After culturing macrophages for 24 hours, the supernatant of RAW is collected, and then the supernatant of macrophages is divided into five groups to culture Tregs cells for 72 hours respectively. Flow cytometry is used to perform Treg gating analysis on spleen cells cultured in different environments, and the GCS / Met / IL2 group has the best proliferation effect;

[0028] Figure 11 The five groups of it are the supernatant obtained after culturing Tregs cells for 72 hours with the Control group, the GCS group, the GCS / Met group, the GCS / IL2 group, and the GCS / Met / IL2 group, and then continue to culture HUVEC for angiogenesis testing - tube formation experiment. The GCS / Met / IL2 group has the best culturing effect. Specific implementation manners

[0029] To make the present invention more obvious and understandable, the following is a detailed description with preferred embodiments and accompanied by drawings:

[0030] The present invention provides a silk fibroin-chitosan composite hydrogel dressing with integrated antibacterial and anti-inflammatory properties and a preparation method thereof.

[0031] Preparation Example 1

[0032] Step 1: Extraction of silk fibroin: Weigh 40 g of silkworm cocoons, measure 1 L of deionized water, add Na2CO3 with a mass fraction of 0.06% and stir evenly. After boiling, add the silkworm cocoons. After 1 h, take them out and wash them with deionized water until there is no foam, repeating at least 3 times. Dry the degummed silk fibroin in a constant temperature drying oven at 60 °C (about 24 h) until it is fluffy, and then store it sealed for later use. Measure 100 mL of 9.3 M lithium bromide solution, weigh 20 g of degummed silk fibroin fibers, cut them into pieces and put them into the lithium bromide solution, and stir and heat in a water bath at 65 ± °C with a magnetic stirrer until dissolved. After filtering the impurities, put the solution into a dialysis bag and dialyze at room temperature for 4 days, changing the solution more than three times a day. After freeze-drying, seal it and put it in a -20 °C refrigerator for later use;

[0033] Step 2: Modification of silk fibroin: Prepare a clear and impurity-free solution of TK with a concentration of 8 mg / ml. Slowly drop the EDC and NHS solutions into the TK solution in an ice bath (the molar mass ratio of TK to EDC is 1:4, and the molar mass ratio of TK to NHS is 1:2), and then activate it in the ice bath for 30 min; add MES accounting for 20% of the mass of SF to the SF solution (mass percentage 4%), and slowly stir it in the ice bath for 20 min. According to the mass ratio of SF:TK = 100:4, slowly drop TK into the SF solution to prepare an SF / TK solution, stir it slowly on a magnetic stirrer, and react in an ice bath for 30 min. Then, sequentially add 20% and 10% of EDC and NHS accounting for the mass of SF in the solution to the mixed solution, put it into a vacuum drying oven to remove air bubbles, take it out after 10 min, dialyze it with water, and freeze-dry to obtain SF-TK. The modified silk fibroin is used as component A, represented by FITR, which is the FITR curve of the SF-TK composite porous membrane. The displacement and peak shape changes of the SF-TK composite porous membrane and the pure SF porous membrane with all ratios are not significant. 3294.7 CM -1 The N-H stretching vibration of primary amine -NH2 is at this position. After adding TK, this peak is enhanced. 3072.7 CM -1 The -OH vibration of -COOH is at this position. After adding TK, the peak becomes wider, indicating that adding TK has an impact on the chemical structure of the material. 1643.4 CM -1 is the -C=O- stretching vibration of amide. 1529.1 CM -1 is the -N-H- bending vibration of amide. 1221.9 CM -1It is the C-N stretching vibration of the amide. After adding TK, the characteristic absorption peaks of these three amide groups become stronger, indicating the formation of a secondary structure. For the porous material mainly composed of β-sheet structure, with the increase of TK, the characteristic peak of the β-sheet structure slightly increases, as shown in Figure 6 shown in Figure 6 Example A of silk fibroin in

[0034] Step 3: Preparation of PLGA microspheres carrying interleukin-2: Measure appropriate amounts of IL-2 and Dextran, and prepare a 6% Dextran clear solution according to the mass ratio of IL-2:Dextran of 1:5. Then add 10 times the volume of 6% PEG 8000 solution to obtain a clear and transparent solution. Place the sample in a -80°C refrigerator for 12 h and then freeze-dry. Under the action of a vortex mixer, wash it thoroughly with dichloromethane to dissolve PEG completely, and centrifuge at 12000 rpm for 6 min to obtain precipitated polysaccharide-protein particles. This process is repeated 3 times. Place it in a fume hood to volatilize and transfer it to a vacuum drying oven to dry thoroughly. Weigh 70 mg of PLGA and 30 mg of PLA, add dichloromethane to obtain a 10% PLGA dichloromethane solution. Then weigh 30 mg of the previously prepared IL-2 polysaccharide vitreous particles and add them to the above solution. After stirring evenly, select 1% PVA as a stabilizer and add it. After stirring for 10 min, quickly transfer it to a mixed solution of 4 g of ethylene glycol and 1 g of glycerol. Before transfer, place a magnetic stirrer in the ethylene glycol-glycerol mixed solution and adjust the stirring speed to 700 rpm for standby. After forming semi-cured microspheres, transfer them to a 500 ml 5% NaCl solution to complete curing, and the curing time is 2 h. Collect, wash, and freeze-dry for later use; the microscopic image of the microspheres is as shown in Figure 4 shown in

[0035] Step 4: Weigh 8% (by mass) of the modified silk fibroin solution, 4% of quaternary ammonium salt chitosan, 7% of GelMA, 30 mg of PLGA microspheres, and 0.08 mM Met. Carry out a photocrosslinking process under the system of 0.2 wt% photoinitiator LAP (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate). Use 405-nm light to activate LAP and irradiate with visible light (450 nm) for 2 min to promote the crosslinking reaction of the molecules in the hydrogel, and obtain a silk fibroin-chitosan composite hydrogel dressing with antibacterial and anti-inflammatory integration. The macroscopic image is as shown in Figure 1 shown, and the microscopic image is as shown in Figure 3 shown.

[0036] Application Example 1

[0037] The modified silk fibroin composite hydrogel is placed in a ROS solution simulating the diabetic microenvironment for 24 h, and the microscopic morphology changes. Due to the presence of TK bonds in the hydrogel, the morphology will be destroyed by ROS in the solution, as shown in Figure 2 B shown

[0038] Application Example 2

[0039] With other component mass fractions unchanged, the mass percentages of A were set to 8%, 12%, and 16% respectively. After the stock solution was injected into a 10 ml syringe and crosslinked by the above method, the hydrogel was cut out according to the modulus of every 1 ml for rheological testing. The test results are as Figure 6 shown

[0040] Application Example 3

[0041] Keep the mass percentage of the modified silk fibroin component at 8%, and adjust the mass percentages of quaternary ammonium chitosan to 1% and 4% respectively. Three different substrates, a PBS negative control group and two hydrogels with different mass fractions of chitosan, were designed for the antibacterial experiment with Escherichia coli. The increase in the mass percentage of quaternary ammonium chitosan can improve the antibacterial performance of the hydrogel. When component B is 4%, the hydrogel has the best antibacterial effect on Escherichia coli. The results are as Figure 8 shown

[0042] Application Example 4

[0043] Five groups including the Control group, the GCS hydrogel extract, the GCS gel loaded with Met extract, the GCS hydrogel loaded with IL2 extract, and the GCS hydrogel simultaneously loaded with metformin and IL2 extract were used to culture macrophages polarized by LPS for 8 hours for 24 hours. The anti-inflammatory transformation effect of the five groups on macrophages was detected by flow cytometry. In the flow cytometry experiment, there was no obvious change in the gating ratio of macrophages in the positive control group, the GCS group, and the GCS / IL2 group. Although there was no obvious change between the GCS / Met group and the GCS / Met / IL2 group, the macrophage cell ratio in these two groups was significantly increased compared with the above three groups. Excluding the obvious pro-inflammatory effect of IL2 on macrophages, it was proved that metformin has a promoting effect on the M2 polarization of macrophages. The results are as Figure 9 shown

[0044] Application Example 5

[0045] After culturing macrophages for 24 hours with the Control group (treated with LPS), the GCS hydrogel extract group, and the GCS / Met hydrogel extract group, the supernatant of RAW was collected. Then, the supernatant of macrophages was divided into five groups to culture Tregs cells for 72 hours respectively. Flow cytometry was used to perform Treg gating analysis on spleen cells cultured in different environments. At the same time, in the supernatant obtained after drug Met treatment of macrophage M2 polarization, interleukin stimulated the most obvious proliferation of Tregs. The results are as Figure 10 shown

[0046] Application Example 6

[0047] Preserve the supernatant obtained after culturing Tregs cells for 72 h in the Control group, GCS group, GCS / Met group, GCS / IL2 group, and GCS / Met / IL2 group, and continue to culture HUVEC for angiogenesis testing - tube formation assay. The results are as Figure 11 shown.

[0048] As mentioned above, it is only the preferred embodiment of the present invention, and there is no limitation in any form and substance to the present invention. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any equivalent changes, such as slight modifications, evolutions, and changes made by those who are familiar with the technology in this field without departing from the spirit and scope of the present invention, using the technical content disclosed above, are equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the substantial technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a silk fibroin composite hydrogel dressing with integrated antibacterial and anti-inflammatory properties, characterized in that: The following steps are involved: Step 1: Extract silk fibroin: weigh raw silk, measure deionized water, add Na2CO3 and stir evenly, add raw silk to degumming after the water boils, dry until fluffy, and seal and store at room temperature for later use; weigh degummed silk fibroin fibers, cut into pieces and put into lithium bromide solution, heat and stir until dissolved; put the dissolved SF solution into a dialysis bag for dialysis, and obtain a desalted SF solution after dialysis; Step 2: Modification of silk fibroin: dissolve dicarboxyl ketone thioacetal in deionized water to obtain a clear TK solution without impurities, add EDC and NHS solution dropwise to the TK solution in an ice bath, and activate in an ice bath for later use; add MES to the SF solution obtained in step 1, add the activated TK solution to the SF solution to prepare an SF / TK solution, stir, and react in an ice bath, then, add EDC and NHS to the mixed solution in sequence, degas, dialyze with water, and freeze-dry to obtain a TK-SF solution, i.e., a modified silk fibroin solution; Step 3: Preparation of PLGA microspheres carrying interleukin-2: Measure IL-2 and Dextran to prepare a 6% Dextran clear solution, then add 10 times the volume of 6% PEG 8000 solution, stir evenly and freeze-dry the sample; wash with dichloromethane under the action of a vortex mixer to dissolve PEG, centrifuge at 12000rpm for 6min to obtain precipitated IL-2 polysaccharide protein composite particles, and repeat this process at least 3 times; put it in a fume hood to evaporate and transfer it to a vacuum drying oven to dry it thoroughly; weigh PLGA and PLA and add dichloromethane to obtain a 10-12.5% ​​PLGA dichloromethane solution, then weigh 10-30mg of the previously reserved IL-2 polysaccharide protein composite particles and add them to the above solution, dissolve it into a clear solution, select PVA as a stabilizer and add an appropriate amount, stir for 10min and then transfer it to a mixed solution of ethylene glycol and glycerol, before transferring the solution, put the ethylene glycol and glycerol mixed solution into a magnetic son to adjust the stirring speed to 500-700rpm for standby, after forming semi-solidified microspheres, transfer them to a 5% NaCl solution of 500ml or more to completely solidify, the time is 2h or more, collect and wash by sedimentation method, freeze-dry and set aside; Step 4: Weigh 8-10% modified silk fibroin solution, 3-6% quaternary ammonium chitosan, 6-8% GelMA, PLGA microspheres and 0.06mM-0.10mM metformin, and carry out photocrosslinking in the system of photoinitiator LAP (phenyl (2,4,6-trimethylbenzoyl) lithium phosphate). Use 405 nm photocrosslinking to activate LAP and promote the crosslinking reaction of molecules in the hydrogel to obtain a silk fibroin chitosan composite hydrogel with integrated antibacterial and anti-inflammatory properties.

2. The method for preparing the antibacterial and anti-inflammatory silk fibroin chitosan composite hydrogel dressing according to claim 1, characterized in that: In the step 1, the ratio of raw silk to deionized water is 1:50 (w:v); the mass fraction of Na2CO3 is 0.06%, the concentration of lithium bromide solution is 9.3M, the volume is 100mL, and the heating temperature is 65±2°C.

3. The method for preparing the antibacterial and anti-inflammatory silk fibroin chitosan composite hydrogel dressing according to claim 1, characterized in that: In the step 2, the added MES accounts for 20% of the mass of SF, and the supplemented EDC and NHS account for 20% and 10% of the mass of SF respectively.

4. The method for preparing the antibacterial and anti-inflammatory silk fibroin chitosan composite hydrogel dressing according to claim 1, characterized in that: In the step 3, the mass ratio of IL-2:Dextran is 1:5, a 6% Dextran clear solution is prepared, the mass fraction of PLGA dichloromethane solution is 10-12.5%, 10-30 mg of IL-2 polysaccharide vitreous particles are weighed, the ethylene glycol glycerol mixed solution is placed in a magnetic stirring speed of 500-700 rpm, and the volume of the NaCl solution transferred to after the semi-solidified microspheres are formed is 500 ml-1 L.

5. The method for preparing the antibacterial and anti-inflammatory silk fibroin chitosan composite hydrogel dressing according to claim 1, characterized in that: In step 4, the mass fraction of the modified silk fibroin solution is 8%, the mass fraction of the quaternary ammonium salt chitosan is 4%, the mass fraction of GelMA is 6-8%, the concentration of metformin is 0.06-0.1 mM, and the mass proportion of the photoinitiator is 0.2wt%.

6. A silk fibroin chitosan composite hydrogel dressing with integrated antibacterial and anti-inflammatory properties prepared by the method according to any one of claims 1 to 5.

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