Silk fibroin-tannic acid esterified microspheres as well as preparation method and application thereof

Abstract

The invention discloses silk fibroin-tannic acid esterified microspheres as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: firstly, dissolving silk fibroin and tannic acid in a mixed solvent of N, N-dimethylformamide and water to form a precursor solution, adding a condensing agent DMTMM, stirring at normal temperature to carry out esterification reaction, and after the reaction is completed, carrying out centrifugal washing to obtain microspheres; the DMTMM is utilized to efficiently catalyze the esterification reaction in a water phase, the process is green and mild, and the use of a toxic cross-linking agent is avoided; the prepared microspheres are uniform in particle size, good in dispersity and high in stability, and due to introduction of tannic acid, the microspheres are endowed with inherent antioxidant, antibacterial and tissue adhesion properties; the prepared microspheres are loaded with the periplaneta americana extract and the doxycycline hydrochloride and have high drug loading capacity, slow release characteristic and good biocompatibility, and the microspheres with target effects such as antibacterial, antioxidant and healing promoting functions can be obtained through drug loading.

Description

TECHNICAL FIELD

[0001] The present application relates to a silk fibroin-based microsphere-tannic acid esterification microsphere and a preparation method and application thereof, in particular to a silk fibroin-tannic acid (SF-TA) microsphere prepared by esterification crosslinking and a method for loading active ingredients (such as periplaneta americana extract, doxycycline hydrochloride, etc.) thereof, and belongs to the technical field of biomedical materials. BACKGROUND

[0002] Traditional wound dressings and topical administration often have problems such as poor drug permeability, short retention time, and the need for frequent dressing changes when treating irregular, small deep wounds, and periodontitis with a complex environment. In addition, the drug function is single, and it is difficult to achieve comprehensive treatment of anti-inflammatory, antibacterial and tissue repair. Therefore, it is of great clinical significance to develop a multifunctional microsphere which is simple to prepare, easy to use and has good curative effect.

[0003] The emerging nanomicrosphere technology provides a new strategy to solve these challenges. Nanomicrospheres constructed based on biocompatible materials can serve as excellent drug carriers to achieve targeted drug delivery and controlled release due to their small size, high specific surface area and functional modification. In addition, they can effectively promote cell migration through their own physical and chemical properties.

[0004] When treating small deep wounds, nanomicrospheres loaded with antibiotics (doxycycline hydrochloride) and healing-promoting drugs (periplaneta americana extract) can be made into a suspension and injected locally by a syringe. The microspheres can quickly penetrate into the deep wound, continuously release antibiotics to prevent deep infection, and accelerate the migration of fibroblasts and keratinocytes through growth factors, achieving healing from the inside out and effectively avoiding "false healing" and cavity formation.

[0005] Silk fibroin (SF) is a natural high molecular structure protein with good biocompatibility, biodegradability and controllable mechanical properties, and has been widely used in drug delivery systems. Traditional methods for preparing silk fibroin microspheres, such as emulsion-solvent evaporation and salting-out, often have problems such as the use of organic solvents, uneven particle size distribution, and low drug encapsulation efficiency. In addition, in order to enhance the stability or function of the microspheres, chemical modification of silk fibroin is often required, which is complex and may introduce cytotoxicity.

[0006] Tannic acid (TA) is a natural polyphenolic compound with excellent antioxidant, anti-inflammatory and antibacterial properties. However, the combination of TA and SF is not stable, and it is challenging to achieve stable covalent crosslinking (such as esterification) of SF and TA in a simple aqueous environment. Conventional esterification conditions are often harsh.

[0007] Therefore, it is of great significance to develop a new method for preparing silk fibroin microspheres with mild conditions, green process, high efficiency of drug encapsulation and good biological activity of the product. SUMMARY

[0008] The present application aims to solve some problems in the prior art, such as the use of toxic reagents in the preparation process of silk fibroin microspheres, low drug encapsulation efficiency, single function, and difficulty in achieving stable chemical crosslinking in aqueous phase. The present application provides a silk fibroin-tannic acid microsphere based on esterification and a preparation method thereof. The method uses a specific condensing agent to efficiently catalyze the esterification of SF and TA in aqueous phase, forming microspheres with stable structure and uniform particle size, and simultaneously achieving high efficiency of loading of target drugs such as PAE and DCH.

[0009] To achieve the above technical purposes, the present application adopts the following technical solutions: Firstly, the present application provides a preparation method of silk fibroin-tannic acid esterification microspheres, which comprises the following steps: S1. Obtain silk fibroin after enzymatic hydrolysis or without enzymatic hydrolysis: Take natural silkworm cocoons, prepare silk fibroin fibers by alkali degumming, add lithium bromide solution after drying for a period of time, then water bath, take out and cool naturally after finishing; dialysis and freeze-drying to obtain silk fibroin sponge; preferably, the silk fibroin sponge can be used after enzymatic hydrolysis.

[0010] The method for preparing silk fibroin fibers by alkali degumming comprises: cutting the silkworm cocoons, then ultrasonic cleaning to remove impurities, and degumming with sodium carbonate solution to obtain degummed silk, i.e. silk fibroin fibers; the concentration of the sodium carbonate solution is 0.05 %(w / v).

[0011] The ratio of silk fibroin fibers to lithium bromide solution is 1g:46-60mL, the lithium bromide solution is added for 4-6h; the water bath condition is 70-85℃ for 3-6h; the concentration of the lithium bromide solution is 8-10mol / L, preferably 8.5-9.5mol / L, and further preferably 9.3mol / L.

[0012] The dialysis is to put the mixed solution after natural cooling into a 3500 Da dialysis bag, and dialysis desalination in deionized water, and the water solution is replaced every 2 hours, and dialysis for three days.

[0013] The freeze-drying is to centrifuge the silk fibroin aqueous solution obtained by dialysis at 8000-10000 rmp / min for 8-15 min to remove impurities, and then put it into a freeze-drying machine for freeze-drying for 48-96 h to obtain a silk fibroin sponge.

[0014] The enzymatic hydrolysis is to put the silk fibroin sponge into a mixed enzyme solution of neutral protease and flavor protease for enzymatic hydrolysis.

[0015] The enzymatic hydrolysis parameters are as follows: the total enzyme amount of the neutral protease and the flavor protease is 10000 U / g, the mass ratio of the neutral protease to the flavor protease is 1:2.5-3.5, preferably 1:2.8-3.4, and further preferably 1:3; the mass ratio of the total enzyme amount of the neutral protease and the flavor protease to the silk fibroin sponge is 0.8-1.3:1, and further preferably 1:1; the enzymatic hydrolysis time is 4-5 h; the enzymatic hydrolysis temperature is 47-55℃; and the enzymatic hydrolysis pH value is 7.4; after the reaction is completed, the enzymatic hydrolysis solution is subjected to suction filtration with a nanofiltration membrane with a molecular weight cutoff of 500-1000 Da, and the filtrate is freeze-dried for 72 h to obtain the enzymatically hydrolyzed silk fibroin.

[0016] S2. Preparation of a precursor solution: the enzymatically hydrolyzed silk fibroin (SF) and tannic acid (TA) are dissolved in a mixed solvent composed of an organic solvent and water, and stirred to fully dissolve to form a uniform precursor solution.

[0017] Preferably, the organic solvent is N,N-dimethylformamide (DMF), and the volume ratio of DMF in the mixed solvent is 10%-20%, preferably 15%.

[0018] Preferably, the mass ratio of the silk fibroin to the tannic acid is 2-6:1, and preferably 4:1.

[0019] S3. Carboxyl activation treatment: 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) is dissolved in deionized water and shaken to perform the carboxyl activation treatment, to obtain a DMTMM aqueous solution.

[0020] Preferably, the concentration of DMTMM in the DMTMM aqueous solution is 10%.

[0021] S4. Catalytic esterification reaction: the DMTMM aqueous solution is added to the precursor solution, and the esterification reaction is performed under stirring at room temperature.

[0022] Preferably, the esterification reaction is stirring at room temperature for 2-24 hours, preferably 8-16 hours, and more preferably 12 hours. Preferably, the amount of the added DMTMM aqueous solution, in terms of the mass of DMTMM in the total reaction solution, is 0.5-2% of the mass of the total reaction solution, and preferably 1%.

[0023] S5. Post-treatment purification: after the reaction is completed, the product is collected by centrifugation, and washed to obtain the silk fibroin-tannic acid esterified microspheres.

[0024] Specifically, after the reaction is completed, the reaction solution is centrifuged to collect the precipitate; specifically, the precipitate is dispersed and centrifugally washed with deionized water, and this process is repeated 2-4 times to remove unreacted raw materials and by-products; finally, the purified microspheres are redispersed in deionized water, stored at 4 DEG C in the dark, or freeze-dried to obtain solid microsphere powder.

[0025] The application further provides silk fibroin-tannin esterification microspheres prepared by the method, the microspheres are uniform spherical and have good dispersibility, the microspheres are formed by ester bonds between carboxyl groups of silk fibroin and phenolic hydroxyl groups of tannin, the Zeta potential of the microspheres is not higher than -32 mV, and the particle size distribution is between 400 nm and 2000 nm.

[0026] The application further provides application of the silk fibroin-tannin esterification microspheres in loading drugs or preparing drug-loaded products.

[0027] Further, the application includes application in preparing medical dressings or cosmetics with antioxidant, antibacterial and healing functions; preferably, the drugs include Periplaneta americana extract (PAE) or doxycycline hydrochloride (DCH), and the loading amount of the drugs is 1%-10% of the mass of the silk fibroin, preferably 7.5%.

[0028] The application further provides a pharmaceutical composition, which comprises the silk fibroin-tannin esterification microspheres and drugs loaded therein.

[0029] Further, the application further provides a preparation method of the pharmaceutical composition, which comprises: according to the preparation method of the silk fibroin-tannin esterification microspheres, adding drugs to be loaded into the precursor solution prepared in step S2, and stirring and mixing uniformly, so that the drugs are encapsulated in the microspheres in the subsequent esterification reaction and microsphere formation process; and then adding DMTMM to perform subsequent reaction, to obtain the silk fibroin-tannin esterification microspheres loaded with drugs.

[0030] The application has the following advantages and beneficial effects:

[0031] Compared with the prior art, the silk fibroin-tannin esterification microspheres prepared by the application have the following overturning advantages and beneficial effects: In the application, tannin is first integrated into the network skeleton of the silk fibroin microspheres as a structural element and a functional element through covalent bonds (ester bonds) instead of simple physical mixing, which realizes the essential functionalization of the microsphere material.

[0032] The preparation method of this invention is green and mild: This invention utilizes DMTMM as a highly efficient and relatively biocompatible condensing agent to achieve esterification crosslinking of SF and TA in a mixed system of water and a small amount of organic solvent. This avoids the crosslinking reaction that occurs when silk fibroin directly contacts tannic acid, and avoids the harsh conditions such as strong acid and high temperature required for traditional esterification reactions, as well as the use of toxic crosslinking agents such as glutaraldehyde. The process is greener and safer.

[0033] The microspheres prepared by this invention represent a fundamental breakthrough in microsphere function: the microspheres prepared by the method of this invention possess potent antibacterial and antioxidant activity. In this invention, tannic acid endows the microspheres with inherent, broad-spectrum antibacterial capabilities through multiple mechanisms, such as disrupting bacterial cell membranes and inhibiting biofilm formation, no longer entirely dependent on the loaded antibiotics. The abundant phenolic hydroxyl groups of tannic acid can efficiently scavenge excess reactive oxygen species (ROS), effectively alleviating the oxidative stress microenvironment in disease sites such as periodontitis, creating favorable conditions for tissue repair. Experiments have shown that its scavenging rate of DPPH free radicals is as high as 93% or more. Even without loading any drugs, the blank microspheres prepared by this invention can be used as an effective antibacterial and anti-inflammatory dressing. By loading other functional drug molecules, the microspheres prepared by this invention are expected to yield multifunctional microspheres with specific applications.

[0034] In this invention, the size of the microspheres prepared by the esterification reaction is closely related to the molecular weight of the silk fibroin. By using nanofiltration membranes with different molecular weight cutoffs in the filtration step after enzymatic hydrolysis, silk fibroin of different molecular weights can be obtained.

[0035] In this invention, tannic acid endows the microspheres with excellent wet tissue adhesion, enabling them to remain at the lesion site (such as periodontal pockets) for extended periods, continuously releasing drugs and exerting their own functions to achieve long-term therapeutic effects. During the preparation process, the microspheres of this invention can simultaneously encapsulate the drug, utilizing the π-π stacking and electrostatic interactions between drug molecules and microsphere components to achieve highly efficient drug loading.

[0036] The drug-loaded microspheres prepared in this invention exhibit controllable drug release and good biocompatibility. In this invention, the drug-loaded microspheres, acting as independent drug release units, demonstrate excellent sustained-release properties, which is beneficial for achieving long-acting therapy and reducing drug toxicity. Blood compatibility tests show that the microspheres prepared in this invention have good biosafety and are suitable for use in the biomedical field.

[0037] The blank microspheres and drug-loaded microspheres prepared by the method of this invention have intact structures, uniform sizes, good dispersibility, negative surface charge (Zeta potential above -32 mV), and high stability. The introduction of tannic acid endows the microspheres with inherent antibacterial and antioxidant capabilities.

[0038] In summary, this invention involves esterifying silk fibroin (SF) with tannic acid (TA) to form nanoparticles, while simultaneously loading American cockroach extract or doxycycline hydrochloride into these nanoparticles. Leveraging the unique tissue adhesion ability of tannic acid, a drug-controlled release nanocarrier with high adhesion to wound tissue can be obtained. The drug-loaded microspheres remain at the wound site for an extended period, effectively achieving local drug release. The microspheres adhere to the wound, forming a protective layer that better inhibits bacterial invasion and reduces excessive inflammatory response through their strong free radical scavenging ability. Finally, further loading of the target drug and synergistic interaction with the polyphenolic nanocarrier containing tannic acid promises to achieve comprehensive effects of antibacterial, immunomodulatory, and tissue repair in wound healing. Attached Figure Description

[0039] Figure 1 SEM images of microspheres catalyzed by DMTMM at different concentrations (0.2, 0.1, 0.05 and 0.025 g / mL) are shown. a, b, c and d correspond to microspheres catalyzed by DMTMM at concentrations of 0.2, 0.1, 0.05 and 0.025 g / mL, respectively.

[0040] Figure 2 The images show the SEM images of microspheres with SF and TA reaction ratios of 6:1 (a), 4:1 (b), and 2:1 (c). Images d, e, and f are the corresponding microsphere diameter distributions for a, b, and c.

[0041] Figure 3 SEM images of the microsphere morphology of SF / TA-NPs (a), SF / TA / DCH-NPs (b), and SF / TA / PAE-NPs (c) are shown.

[0042] Figure 4 A statistical graph of microsphere yields collected for different reaction times.

[0043] Figure 5 The images show TEM comparisons of the prepared microspheres, where a represents SF / TA-NPs and b represents SF / TA / PAE-NPs.

[0044] Figure 6 In the figure, a, b, and c are the zeta potential distributions of blank particles SF / TA-NPs, DCH-loaded microspheres SF / TA / DCH-NPs, and PAE-loaded microspheres SF / TA / PAE-NPs, respectively.

[0045] Figure 7 Fourier transform infrared spectra of the esterification reaction of SF / TA-NPs were prepared.

[0046] Figure 8Raman spectra of SF / TA-NPs, SF / TA / PAE-NPs, SF, and TA.

[0047] Figure 9 The solid-state UV absorption curves are for SF / TA-NPs, SF, and TA.

[0048] Figure 10 DPPH free radical scavenging test of SF / TA-NPs at different concentrations; 1-6 in Figure a represent 6.25, 12.5, 25, 50, 100, and 200 μL microsphere suspensions, respectively, and AF in Figures b and c correspond to 1-6 in Figure a, respectively.

[0049] Figure 11 Blood compatibility tests were performed on blank microspheres SF / TA-NPs at different concentrations; Figure a shows hemolysis images of SF / TA-NPs at different concentrations, and figure b shows the hemolysis rate of SF / TA-NPs at different concentrations.

[0050] Figure 12 The experiment tested the antibacterial activity of SF / TA-NPs. Group a was the antibacterial experiment against Escherichia coli, with a1 as the control group and a2 as the result of co-culturing with 0.1 mg / mL microspheres. Group b was the antibacterial experiment against Staphylococcus aureus, with b1 as the control group and b2 as the result of co-culturing with 0.1 mg / mL microspheres.

[0051] Figure 13 SEM images of microspheres prepared from undigested silk fibroin. The top image is taken with a 5000× lens, and the bottom image is taken with a 12000× lens. Detailed Implementation

[0052] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0053] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. Before describing the present invention with reference to the accompanying drawings, it should be particularly noted that: The technical solutions and features provided in the various parts of this invention, including the following description, can be combined with each other without conflict.

[0054] Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0055] In this invention, DMF and water are used as a mixed solvent. The reason for preferentially preparing an aqueous DMF solution is that the silk fibroin molecular chain contains a large number of hydrophobic regions (such as amino acid residues like tyrosine, alanine, and valine). Tannic acid, as a polyphenol compound, has a highly hydrophobic benzene ring structure. Therefore, in aqueous solution, these hydrophobic parts tend to approach and aggregate to escape the aqueous environment, thus aggregating silk fibroin and tannic acid together to form a complex. Adding a highly polar aprotic solvent, DMF (N,N-dimethylformamide), fundamentally alters the solvent environment. DMF and water, when miscible in a suitable ratio, can effectively solvate (dissolve) both hydrophobic and hydrophilic groups. In the DMF-water mixed solvent, the solvent environment becomes more favorable to hydrophobic groups. Therefore, preferentially preparing an aqueous DMF solution is particularly important when performing the esterification reaction of silk fibroin and tannic acid into microspheres.

[0056] An optimal process parameter is as follows: the precursor solution consists of 0.4 g SF, 0.1 g TA, 34 mL water and 6 mL DMF, and the condensing agent solution consists of 0.4 g DMTMM and 4 mL water. Based on this, the industrial-scale preparation of this microsphere can be achieved by scaling up the ratio.

[0057] Unless otherwise specified, all raw materials or reagents used in this invention are commercially available products well known to those skilled in the art.

[0058] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0059] Example 1: Preparation of silk fibroin by enzymatic hydrolysis Cut the dried, shaved cocoon into 1cm pieces. 2 The silkworm cocoons, which are lumpy and cut into pieces, are ultrasonically cleaned to remove impurities. The lumpy cocoons are then added to a 0.05% (w / v) sodium carbonate solution at a ratio of 1g:50mL and boiled for 1 hour. The silk is then removed, and the lumpy cocoons are boiled again at a ratio of 1g:50mL with the added 0.05% (w / v) sodium carbonate solution for another 1 hour. The silk is then removed again, and the lumpy cocoons are boiled again at a ratio of 1g:50mL with the added 0.05% (w / v) sodium carbonate solution for another 30 minutes. The mixture is then repeatedly rinsed with deionized water to remove residual sericin, yielding silk fibroin fibers. These fibers are then dried at 60℃.

[0060] Following a material-to-liquid ratio of 1g:50mL for silk fibroin fiber, 9.3mol / L lithium bromide solution was added to the dried silk fibroin fiber and dissolved for 5h. Then, the fiber was placed in an oil bath at 80℃ for 4h. After the fiber was fully dissolved, it was removed and allowed to cool naturally before being placed in a 3500Da dialysis bag for desalting.

[0061] The desalted silk fibroin solution was frozen at -20°C, and then placed in a freeze dryer at -80°C for freeze drying to obtain silk fibroin sponge.

[0062] Enzymatic hydrolysis: A compound enzyme was prepared by mixing a total enzyme amount of 10,000 U / g with a mass ratio of neutral protease to flavor protease of 1:3. The compound enzyme was added to the aqueous solution of the dissolved silk sponge at a mass ratio of 1:1. Enzymatic hydrolysis was carried out at 50°C and pH 7.4 for 5 hours, followed by enzyme inactivation by heating to 98°C for 10 minutes.

[0063] The enzymatically hydrolyzed silk fibroin solution was filtered using a nanofiltration membrane with a molecular weight cutoff of 1000 Da. The filtered solution was then freeze-dried for 72 hours to obtain enzymatically hydrolyzed silk fibroin (SF).

[0064] The enzymatically hydrolyzed silk fibroin was used in subsequent experiments in Examples 2-9.

[0065] Example 2: Preparation of a direct mixture of silk fibroin (SF) and tannic acid (TA) (SF / TA) Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh and add 0.4 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) to the DMF aqueous solution (a mixture of DMF and water), and stir magnetically for 30 minutes until completely dissolved. Freeze the resulting solution at -20°C, then freeze-dry it in a freeze dryer at -80°C to obtain the unesterified reaction product SF / TA.

[0066] Example 3: Preparation of esterified microspheres (SF / TA-NPs) with a silk fibroin (SF) to tannic acid (TA) mass ratio of 6:1. Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.6 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the above DMF aqueous solution (a mixed solvent of DMF and water). Stir magnetically for 30 minutes to completely dissolve the DMF to obtain the precursor solution.

[0067] Weigh 0.4 g of DMTMM and dissolve it completely in 4 mL of deionized water to obtain an aqueous solution of DMTMM.

[0068] Under continuous stirring, the DMTMM aqueous solution was slowly added dropwise to the precursor solution. After the addition was complete, the reaction was continued to be stirred at room temperature for 12 hours.

[0069] After the reaction was complete, the reaction solution was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded. The precipitate was re-dispersed by sonication with 40 mL of deionized water and centrifuged again. This washing process was repeated 3 times.

[0070] Finally, the purified microsphere precipitate was dispersed in 5 mL of deionized water to obtain an SF-TA-NPs dispersion, which was stored at 4°C in the dark. Alternatively, a portion of the dispersion could be freeze-dried to obtain a white solid powder, which is the esterified microsphere SF / TA-NPs.

[0071] Example 4: Preparation of esterified microspheres (SF / TA-NPs) with a silk fibroin (SF) to tannic acid (TA) mass ratio of 4:1. Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.4 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the above DMF aqueous solution (a mixed solvent of DMF and water). Stir magnetically for 30 minutes to completely dissolve the DMF to obtain the precursor solution.

[0072] The subsequent steps are the same as in Example 3: prepare DMTMM aqueous solution (0.4g DMTMM dissolved in 4 mL deionized water), add it dropwise to the precursor solution, react at 4°C for 12 hours, and then centrifuge, wash and collect the microspheres.

[0073] The preparation method of SF / TA-NPs (SF to TA mass ratio of 4:1) provided in this embodiment is the same as that in Example 3, except for the different ratio of silk fibroin to tannic acid. The resulting microspheres have intact morphology and a smaller average diameter distribution compared to Example 3.

[0074] Example 5: Preparation of esterified microspheres (SF / TA-NPs) with a silk fibroin (SF) to tannic acid (TA) mass ratio of 2:1 Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.2 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the above DMF aqueous solution (a mixed solvent of DMF and water). Stir magnetically for 30 minutes until completely dissolved to obtain the precursor solution. Subsequent preparation steps are the same as described in Example 3.

[0075] The subsequent steps are the same as in Example 3: prepare DMTMM aqueous solution (0.4g DMTMM dissolved in 4 mL deionized water), add it dropwise to the precursor solution, react at 4°C for 12 hours, and then centrifuge, wash and collect the microspheres.

[0076] The preparation method of SF / TA-NPs (SF to TA mass ratio of 2:1) microspheres provided in this embodiment is the same as that in Example 3, except for the different ratio of silk fibroin to tannic acid. The resulting microspheres have intact morphology and a smaller average diameter distribution compared to Example 4.

[0077] Example 6: Microspheres prepared from silk fibroin (SF) and tannic acid (TA) under the catalysis of different concentrations of DMTMM solution.

[0078] Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.4 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the above DMF aqueous solution (a mixed solvent of DMF and water). Stir magnetically for 30 minutes to completely dissolve the DMF to obtain the precursor solution.

[0079] Different concentrations of DMTMM aqueous solutions were prepared (0.8, 0.4, 0.2 and 0.1 g of DMTMM were weighed and dissolved in 4 mL of deionized water to form reaction solutions with concentrations of 0.2, 0.1, 0.05 and 0.025 g / mL, respectively), and slowly added dropwise to the precursor solution. The reaction was carried out at 4 °C for 12 hours, and then centrifuged, washed and collected.

[0080] The morphology of microspheres prepared under the catalysis of DMTMM solutions of different concentrations was observed by SEM. The micromorphology of the nanospheres prepared in Examples 1-7 was examined using a field emission scanning electron microscope (SU8600, Hitachi). The loaded samples were prepared using single-sided polished silicon wafers (5 mm × 5 mm). The prepared microsphere solution was sonicated for 10 minutes, then diluted 100 times with deionized water, and sonicated again for 5 minutes. 10 μL of the solution was added dropwise onto the silicon wafer, allowed to dry naturally in a desiccator, sputter-coated with gold, and observed using SEM. Figure 1 SEM images of the prepared microspheres, in which Figure 1 Figures a, b, c, and d show the morphology of microspheres produced by DMTMM catalysis at different concentrations (0.2, 0.1, 0.05, and 0.025 g / mL), respectively. It can be seen that the red area in figure a shows microsphere polymerization; the microspheres in figures b and c have uniform morphology and good dispersibility; and the yellow area in figure d shows uneven microsphere size and structural breakage. The morphology and structure of the microspheres are shown in the table below: Table 1. Preparation of microspheres under different concentrations of DMTMM catalysis

[0081] Example 7: Preparation of microspheres from silk fibroin (SF) and tannic acid (TA) using DMTMM (0.1 g / mL) solution at different catalytic times. Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.4 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the above DMF aqueous solution (a mixed solvent of DMF and water). Stir magnetically for 30 minutes to completely dissolve the DMF to obtain the precursor solution.

[0082] A 0.1 g / mL DMTMM solution was prepared. After adding the DMTMM aqueous solution to the precursor solution, the esterification reaction was carried out by stirring at room temperature. Different reaction times were set, while other conditions remained the same. For the experimental groups with different reaction times (2, 4, 8, 12, and 16 h), the reaction solution was centrifuged, washed, and the product was collected at the corresponding time points. The collected microspheres were then lyophilized and weighed. The ratio of the product weight to the total mass of added silk fibroin and tannic acid was defined as the microsphere yield. Results are as follows: Figure 4 As shown, the esterification product generation efficiency over time is plotted. The results show that within 12 hours, the mass of the product increases with the extension of reaction time. After 12 hours, the microsphere yield only increases slightly. At 16 hours and after 16 hours, the mass of the product collected is similar to that collected at 12 hours. Therefore, choosing 12 hours as the product collection point can save preparation time and obtain the optimal product collection amount.

[0083] Example 8: Preparation of silk fibroin-tannin-esterified microspheres (SF / TA / PAE-NPs) loaded with American cockroach extract (PAE)

[0084] Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.4 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the above DMF aqueous solution. Stir magnetically for 30 minutes until completely dissolved to obtain the precursor solution.

[0085] Add 0.015 g of American cockroach extract (PAE) to the above solution and continue stirring for 1 hour to ensure thorough mixing of the drug.

[0086] The subsequent steps are the same as in Example 3: prepare an aqueous solution of DMTMM (0.4 g DMTMM dissolved in 4 mL of deionized water), add it dropwise to the precursor solution, react at 4°C for 12 hours, then centrifuge, wash, and collect the microspheres to obtain SF / TA / PAE-NPs dispersion or lyophilized powder.

[0087] The method for preparing SF / TA / PAE-NPs microspheres provided in this embodiment is the same as that in Example 3, except that PAE is added before the esterification reaction occurs due to the addition of a condensing agent. The resulting microspheres have intact morphology and a reduced average diameter distribution.

[0088] Example 9: Preparation of silk fibroin-tannin-esterified microspheres (SF / TA / DCH-NPs) loaded with doxycycline hydrochloride (DCH).

[0089] Prepare 40 mL of a 15% (v / v) DMF aqueous solution. Weigh 0.4 g of silk fibroin (SF) and 0.1 g of tannic acid (TA) and add them to the DMF aqueous solution. Stir magnetically for 30 minutes until completely dissolved.

[0090] Add 0.015 g of doxycycline hydrochloride (DCH) to the above solution and continue stirring for 1 hour to ensure thorough mixing of the drug, thus obtaining the precursor solution.

[0091] The subsequent steps are the same as in Example 3: prepare an aqueous solution of DMTMM (0.4 g DMTMM dissolved in 4 mL of deionized water), add it dropwise to the precursor solution, react at 4°C for 12 hours, then centrifuge, wash, and collect the microspheres to obtain SF / TA / DCH-NPs dispersion or lyophilized powder.

[0092] The method for preparing SF / TA / DCH-NPs microspheres provided in this embodiment is the same as that in Example 3, except that DCH is added before the esterification reaction occurs due to the addition of a condensing agent. The resulting microspheres have intact morphology and a reduced average diameter distribution.

[0093] Characterization and analysis of the microspheres prepared in the above embodiments: SEM morphological observation: The method was the same as described in Example 6. The micromorphology of the nanospheres prepared in Examples 1-7 was examined using a field emission scanning electron microscope (SU8600, Hitachi). Loaded samples were prepared using single-sided polished silicon wafers (5 mm × 5 mm). The prepared microsphere solution was sonicated for 10 minutes, then diluted 100-fold with deionized water, and sonicated again for 5 minutes. 10 μL of the solution was added dropwise to the silicon wafer, allowed to air dry in a desiccator, sputter-coated with gold, and observed using SEM. Figure 2 and Figure 3 All images are SEM images of the prepared microspheres. Figure 2 a, b, and c are the microsphere morphology diagrams obtained when the reaction ratio of SF to TA is 6:1, 4:1, and 2:1, respectively. d, e, and f are the microsphere diameter distribution diagrams corresponding to a, b, and c. The results show that the higher the ratio of SF to TA, the larger the microsphere diameter. Figure 3 In the diagram, a represents SF / TA-NPs (microspheres prepared in Example 4, with an SF to TA mass ratio of 4:1), b represents SF / TA / DCH-NPs, and c represents SF / TA / PAE-NPs microspheres. Figure 3It can be seen that both the blank microspheres SF / TA-NPs and the drug-loaded microspheres exhibit uniform spherical shape and good dispersibility, with particle diameters ranging from 400 to 2000 nm. Among them, the microsphere diameters of SF / TA / DCH-NPs and SF / TA / PAE-NPs are smaller than those of SF / TA-NPs. This is because the added drug molecules form a "cross-linking" effect between the esterification matrix of the microspheres, pulling the polymer network and thus obtaining esterified drug-loaded microspheres with smaller diameters.

[0094] TEM morphology test: SF / TA-NPs (microspheres prepared in Example 4, with a mass ratio of SF to TA of 4:1) and SF / TA / PAE-NPs were deposited onto copper mesh with aqueous solutions for TEM imaging. Figure 5 The images are TEM morphology images, where a represents SF / TA-NPs and b represents SF / TA / PAE-NPs. Compared to a, image b shows that drug molecules are encapsulated inside the microspheres, and during the deposition process with aqueous solution, drug molecules are continuously released from inside the microspheres, confirming that the drug-loaded microspheres can adhere to wound tissue and achieve effective release of the loaded drug.

[0095] ZETA Potential Test: First, the solutions of 15 μg / mL SF / TA-NPs (microspheres prepared in Example 4, with a mass ratio of SF to TA of 4:1), SF / TA / DCH-NPs, and SF / TA / PAE-NPs were sonicated for 10 minutes. Then, an appropriate amount of dispersion was placed in a test tube, and the ZETA potential was tested using a Zeta potential analyzer (Malvin, NANOZS90, UK). Figure 6 The graph shows the potential test data from Zeta testing, where a represents SF / TA-NPs, b represents SF / TA / DCH-NPs, and c represents SF / TA / PAE-NPs. The Zeta data results show that the potentials of SF / TA-NPs, SF / TA / DCH-NPs, and SF / TA / PAE-NPs are all below -32 mV, indicating good stability of the prepared microspheres, consistent with the SEM results.

[0096] Fourier Transform Infrared Spectroscopy (FTIR) Analysis: To investigate the crosslinking mechanism of microsphere nanoparticles, SF / TA (unesterified product) and esterified product SF / TA-NPs (microspheres prepared in Example 4, with a mass ratio of SF to TA of 4:1) were analyzed using Fourier transform infrared spectroscopy (FTIR). Figure 7 This is a Fourier transform infrared (FTIR) spectral image. From... Figure 7As can be seen, compared with the SF / TA (unesterified product) prepared in Example 2, the esterification reaction product of the SF-TA-NPs prepared in Example 4 has a spectrum at 1726 cm⁻¹. -1 A sharp new peak appears at 1126 cm⁻¹, which is attributed to the stretching vibration of the ester bond (C=O). The appearance of this peak is the most direct indicator distinguishing it from a physical mixture, indicating that the carboxyl group of silk fibroin has undergone a dehydration reaction with the phenolic hydroxyl group of tannic acid. As confirmation, a peak appears at 1126 cm⁻¹. -1 The newly appearing absorption peak at the same location is clearly attributed to the (COC) asymmetric stretching vibration of the ester bond. The co-occurrence of the characteristic peaks of the (C=O) stretching vibration of the ester bond and the (COC) asymmetric stretching vibration, with the newly appearing peak at 1634 cm⁻¹... -1 The peak is the result of the superposition of the amide I band in the β-sheet conformation and the C=C skeletal vibrational absorption of the tannic acid benzene ring. At 1355 cm⁻¹ -1 The newly appearing absorption peak is attributed to the in-plane bending vibration of the (OH) group of the phenolic hydroxyl group or the stretching vibration of the (CO) group of the aromatic ring in the tannic acid molecule. The clear appearance of this peak indicates that a large number of tannic acid molecules have been successfully fixed in the cross-linked network, constituting decisive evidence of covalent cross-linking. The changes in these four characteristic peaks prove that a covalent esterification reaction has occurred between the carboxyl group of silk fibroin and the phenolic hydroxyl group of tannic acid, rather than a simple physical mixing or hydrogen bonding.

[0097] Figure 8 Raman spectra of SF, TA, PAE, SF / TA-NPs (microspheres prepared in Example 4, with an SF to TA mass ratio of 4:1), and SF / TA / PAE-NPs were compared. The results show that SF is the main component in the microspheres, and the SF / TA-NPs spectrum is concentrated in the 1514-1600 cm⁻¹ range. -1 The presence of characteristic peaks for the aromatic ring skeletal vibration (C=O stretching) of tannic acid indicates successful integration of TA into the microspheres. As a typical chemical composition of insects, the American cockroach extract, as shown in the Raman spectrum, exhibited a higher peak at 1486 cm⁻¹ in SF / TA / PAE-NPs compared to the blank SF / TA-NPs. -1 The presence of CH2 / CH3 bending vibrations and characteristic peaks of the amide II band at 1845 cm⁻¹ is an important spectral fingerprint of the protein and lipid components of the American cockroach extract; -1 The peak is attributed to the stretching vibration (C=O) of a specific bond and environment in the chitin-protein complex; 1028 cm⁻¹ -1 This is attributed to the stretching vibrations of COC and COH in chitin and carbohydrates. Therefore, from Figure 8 It can be seen that the silk fibroin and tannin esterified microspheres were successfully prepared, and that the drug molecules could be successfully loaded after the drug molecules were added before the esterification reaction.

[0098] Figure 9 Solid-state UV absorption curves for SF, TA, and SF / TA-NPs (microspheres prepared in Example 4, with an SF to TA mass ratio of 4:1). Figure 9 It can be seen that SF has a significant absorption peak at 280 nm, and TA has the strongest absorption peak at 325 nm. After the two raw materials are synthesized into nanospheres, since SF is the main component of the nanoparticles, the strongest absorption peak of the nanoparticles is between the absorption peaks of the two raw materials. This indicates that the microspheres contain silk fibroin and tannic acid, further proving that silk fibroin and tannic acid have been successfully chemically combined.

[0099] Antioxidant test: The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) solution purchased from Phygene was stored in a dark place for later use. A 0.1 mg / mL microsphere suspension (microspheres prepared in Example 4, SF to TA mass ratio 4:1) was prepared. 6.25, 12.5, 25, 50, 100, and 200 μL of the microsphere suspension were added to 2 mL of DPPH solution, and the reaction was carried out at room temperature in the dark for 1 hour. The color change of the solution was observed and detected. The absorbance of the solution at 517 nm after the reaction was measured using a UV-Vis spectrophotometer. The results were calculated using the formula, where A0 and A... S The absorbance values ​​are for the group without microspheres and the group with microspheres, respectively. Figure 10 In Figure a, 1-6 are photographs of solutions after 1 hour of reaction at room temperature in the dark, with 2 mL of DPPH added to 6.25, 12.5, 25, 50, 100, and 200 μL of microsphere suspension, respectively. The blank in the figure is the blank control, without the addition of DPPH to the microsphere suspension. Figure 10 In b and c, AF corresponds to the free radical scavenging efficiency of 6.25, 12.5, 25, 50, 100, and 200 μL microsphere suspensions, respectively. From... Figure 10 Calculations from UV spectrophotometer data in sections b and c show that the free radical scavenging efficiency increases significantly with the increase of SF / TA-NPs concentration. UV spectrophotometer results indicate that a 200 μL suspension (corresponding to a concentration of 200 μg / mL microspheres) incubated with DPPH detection reagent for 1 hour in the dark can achieve a free radical scavenging rate of over 93%, demonstrating that SF / TA-NPs possess excellent free radical scavenging capabilities and can effectively reduce the generation of traumatic inflammation.

[0100] Sample hemolytic test: Hemolysis refers to the ability of a substance (such as a chemical, drug, or extract from a medical device) to cause red blood cells to rupture. A low hemolysis rate indicates that the sample is less likely to damage red blood cell membranes upon contact with blood, thus reducing the risk of toxic side effects. According to ASTM F756-2000, the hemolysis rate of medical devices must be less than 5%. Figure 11 Hemolysis was tested using a 2% rabbit red blood cell suspension purchased from Guangzhou Hongquan Biotechnology. Microspheres (prepared in Example 4, with a SF to TA mass ratio of 4:1) were added in a gradient of 50 μg / mL, 100 μg / mL, and 300 μg / mL. Deionized water and PBS were used as positive and negative control groups, respectively. Each sample was centrifuged at 4000 rpm for 2 min, and 100 μL of the supernatant was added to a 96-well plate. The absorbance was measured at 540 nm using a microplate reader. The hemolysis rate (%) was calculated using the formula: % = (A0 / A1) × 100%, where A, A0, and A1 correspond to the absorbance at 540 nm for the experimental group, negative control group, and positive control group, respectively. Red blood cells in both the negative control group and the experimental group were completely precipitated by centrifugation, and the supernatant was clear and not red. The positive control group showed hemolysis after releasing hemoglobin, and the supernatant was distinctly red. Experiments show that even at a high concentration of 300 μg / mL, the hemolysis rate of SF / TA-NPs is far below 5%, demonstrating that the microspheres have good blood compatibility, good biocompatibility, and high safety, making them suitable for applications involving blood contact. For bleeding wounds, materials with good blood compatibility do not interfere with the body's normal coagulation process, have high acceptance by the body, and result in mild inflammatory reactions. As drug carriers, the drugs loaded onto the microspheres do not need to counteract adverse reactions caused by the material itself, thus maximizing the therapeutic effect.

[0101] Antibacterial performance testing: Gram-negative Escherichia coli (ATCC25922) and Gram-positive Staphylococcus aureus (CMCC(B)2603) (purchased from Shanghai Luwei Technology Co., Ltd.) were used to evaluate the antibacterial properties of nanoparticles by plate counting method. Figure 12 The antibacterial properties of the microspheres (prepared in Example 4, with a SF to TA mass ratio of 4:1) were tested. Group a was an antibacterial experiment against *Escherichia coli*, where a1 was the blank control group (i.e., no microspheres were added) and a2 was the result of co-culturing with 0.1 mg / mL microspheres. Group b was an antibacterial experiment against *Staphylococcus aureus*, where b1 was the blank control group (i.e., no microspheres were added) and b2 was the result of co-culturing with 0.1 mg / mL microspheres. Figure 12The results of the plate coating method showed that, in Figures a2 and b2, the addition of 0.1 mg / mL SF / TA-NPs for bacterial co-culture significantly inhibited Escherichia coli and Staphylococcus aureus compared to the blank control group.

[0102] Furthermore, the present invention uses the silk fibroin sponge prepared in Example 1 to prepare microspheres according to the method in Example 4, and the resulting microspheres are as follows: Figure 13 As shown, with Figures 1-3 and Figure 5 Compared to microspheres prepared from enzymatically hydrolyzed silk fibroin, microspheres prepared from unhydrolyzed silk fibroin have larger diameters and more uniform morphology, but can still be loaded with drugs. Enzymatically hydrolyzed silk fibroin has a lower molecular weight and narrower distribution, resulting in more stable rheological properties in the solution. Furthermore, the molecular weight distribution of the final product can be precisely controlled by adjusting the type, concentration, reaction time, and temperature of the enzyme. During microsphere formation, this process helps to create microspheres with more uniform size and better sphericity. The consistent molecular chain length after hydrolysis reduces particle size differences and morphological defects caused by uneven long-chain entanglement. In addition, specific low-molecular-weight peptides produced by enzymatic hydrolysis may possess antioxidant and cell proliferation-promoting activities, further enhancing cell attachment, growth, and tissue regeneration.

Claims

1. A method for preparing silk fibroin-tannin esterified microspheres, characterized in that, Includes the following steps: S1. Obtain silk fibroin, whether enzymatically hydrolyzed or not; S2. Dissolve silk fibroin and tannic acid in a mixed solvent consisting of organic solvent and deionized water, and stir until fully dissolved to form a precursor solution; S3. Dissolve 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) in deionized water and perform carboxyl activation treatment to obtain an aqueous solution of DMTMM; S4. Add DMTMM aqueous solution to the precursor solution and stir at room temperature to carry out esterification reaction; S5. After the reaction is complete, the product is collected by centrifugation, washed, and the silk fibroin-tannin esterified microspheres are obtained.

2. The preparation method according to claim 1, characterized in that, The organic solvent in step S2 is N,N-dimethylformamide, and the volume percentage of N,N-dimethylformamide in the mixed solvent is 10%-20%. The mass ratio of silk fibroin to tannic acid is 2-6:

1. The concentration of DMTMM in the aqueous solution in step S3 is 10%. The esterification reaction in step S4 is a stirring reaction at room temperature for 2-24 hours. The amount of DMTMM aqueous solution added is 0.5-2% of the total mass of the reaction solution, based on the mass of DMTMM in the total reaction solution.

3. The preparation method according to claim 2, characterized in that, In step S2, the N,N-dimethylformamide has a volume percentage of 15% in the mixed solvent, and the mass ratio of silk fibroin to tannic acid is 4:

1.

4. The preparation method according to claim 2, characterized in that, The esterification reaction in step S4 is carried out by stirring at room temperature for 8-16 hours, preferably 12 hours; the amount of DMTMM aqueous solution added is 1% of the total mass of the reaction solution, based on the mass of DMTMM in the total reaction solution.

5. Silk fibroin-tannin esterified microspheres obtained by the preparation method according to any one of claims 1-4.

6. The use of the silk fibroin-tannin esterified microspheres obtained by the preparation method according to any one of claims 1-4 or the silk fibroin-tannin esterified microspheres according to claim 5 in loading drugs or preparing drug-loaded products.

7. The application according to claim 6, characterized in that, The applications include use in the preparation of products with antioxidant, antibacterial and healing-promoting functions; the drugs include American cockroach extract or doxycycline hydrochloride.

8. The application according to claim 7, characterized in that, The loading of the drug, by weight, is 1% to 10% of the silk fibroin protein, preferably 7.5%.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises silk fibroin-tannin-esterified microspheres obtained by the preparation method according to any one of claims 1-4 or silk fibroin-tannin-esterified microspheres according to claim 5, and a drug loaded thereon.

10. The method for preparing the pharmaceutical composition according to claim 9, characterized in that, The method includes: according to the preparation method of claim 1, adding the drug to be loaded into the precursor solution prepared in step S2, stirring and mixing evenly, and then adding DMTMM aqueous solution for subsequent reaction.

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