Self-assembled micelle hydrogel integrating regulation and control of macrophage polarization, inhibition of bacterial infection and promotion of wound healing and preparation method of self-assembled micelle hydrogel

Abstract

The invention belongs to the field of pharmaceutical preparations, and discloses a self-assembled micelle hydrogel integrating regulation and control of macrophage polarization, inhibition of bacterial infection and promotion of wound healing and a preparation method of the self-assembled micelle hydrogel. The self-assembled micelle hydrogel comprises a base material and a drug entrapped by the base material, the base material comprises gelatin, carbomer 940, triethanolamine, glycerin, 1, 2-propylene glycol and a bletilla striata polysaccharide grafted stearic acid polymer (SA-BSP), and the entrapped drug comprises a bacterial infection treatment drug. The self-assembled micelle hydrogel is used for efficiently delivering bletilla striata polysaccharide and bacterial infection treatment drugs, is beneficial to enhancing antibacterial activity and related immunoregulation to promote wound healing, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a self-assembled micelle hydrogel capable of regulating macrophage polarization, inhibiting bacterial infection and promoting wound healing, and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body and bears the primary responsibility of defending against external invasion. Its main function is to prevent infections caused by microorganisms and non-microorganisms. However, after accidental injuries, surgical operations, thermal burns, bites or stings, the risk of bacterial infection increases significantly. Pathogenic bacteria will tightly adhere to the surface of damaged tissues, forming biofilms to resist clearance by the immune system and hinder the penetration of antibiotics, resulting in stubborn infections that are difficult to heal. As the infection progresses, the toxins produced by bacteria will stimulate the immune system and cause inflammatory responses in an attempt to resist further spread of the infection. Stubbornly infected wounds are notorious for their uncontrolled bacterial invasion and local persistent inflammatory responses, which may cause serious complications, especially sepsis. In order to deal with wound infections, antibiotics are often overused, which has led to increased bacterial resistance to antibiotics, resulting in multidrug-resistant strains, which has become a major threat to public health. Therefore, how to effectively kill bacteria and destroy biofilms without promoting the emergence of multidrug-resistant bacteria has become one of the main challenges that need to be solved urgently.

[0003] As an important component of the innate immune system, macrophages play a variety of key functions, mainly involving host defense mechanisms and immune responses to a variety of foreign microorganisms (including bacteria, viruses, fungi and parasites). These cells have a wide range of receptors on their surfaces and are rich in internal mediators and required secretory molecules, which enable them to effectively identify, engulf and eliminate invading pathogens, while regulating the activities of other immune cells. Depending on the environment in which they are located, macrophages can differentiate into two functionally distinct subpopulations, namely pro-inflammatory macrophages (M1 type) and anti-inflammatory macrophages (M2 type). At the site of infection wounds, macrophages are activated and transformed into M1 type. This type of macrophage will excessively release a variety of pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6), which may lead to delayed wound healing. M2 macrophages can secrete interleukin 10 (IL-10), angiogenic factor (VEGF) and transforming growth factor (TGF-β), thereby promoting skin re-epithelialization. As wound healing progresses, the phenotype of macrophages needs to be strictly regulated, and the transition from M1 to M2 will help promote the smooth progress of the repair process. However, at the infected wound, if the inflammatory response is too strong or lasts too long, it may cause damage to the body's tissues, hinder the healing process, and even induce a systemic response, resulting in an imbalance in the ratio of macrophages M1 and M2. Therefore, regulating the process of macrophage transformation to M2 has become an important strategy for treating bacterial biofilm infections.

[0004] Hydrogel is a unique material composed of water and a macromolecular cross-linked polymer network. Its structure and properties make it have broad application prospects in the fields of tissue engineering and regenerative medicine. As a dressing that meets the requirements of "wet healing", hydrogel plays an important role in promoting wound healing. It can effectively simulate soft tissue and extracellular matrix, provide necessary support for wounds, and has good filling, adhesion, hemostasis and other functions, thereby accelerating the healing process. The superior properties of hydrogel make it perform well in clinical use. Its porous structure and high water content help absorb exudate and retain moisture, which can not only effectively inhibit infection but also reduce inflammatory response. In addition, hydrogel can be used as a carrier of therapeutic factors such as cytokines. This property makes it play an important role in promoting wound repair. In recent years, more and more natural drug molecules have been introduced into the preparation of hydrogels. Compared with traditional hydrogels, the biggest advantage of these self-assembled micellar hydrogels containing natural drug molecules is that they can not only provide a scaffold for the gel, but also have inherent pharmacological activity and can achieve sustained and controlled release of drugs, which has a good effect on alleviating the production of multidrug-resistant bacteria. Bletilla striata polysaccharide (BSP) is a natural glucomannan extracted from the traditional Chinese medicine Bletilla striata. As the main active ingredient in Bletilla striata, BSP is widely used in the treatment of traumatic bleeding, sores, swelling and skin cracks. In the field of reducing swelling and promoting tissue regeneration, Bletilla striata has demonstrated significant therapeutic effects on sores, swelling and skin ulcers. Its mechanism of action includes promoting the growth of granulation tissue, collagen deposition, and blood vessel regeneration.

[0005] Therefore, it is completely feasible to introduce Bletilla striata polysaccharide into hydrogel to prepare a new type of hydrogel, which can solve the problems existing in the prior art. Summary of the invention

[0006] In view of the above-mentioned technical problems, the present invention provides a self-assembled micelle hydrogel and a preparation method thereof that integrates macrophage polarization regulation, bacterial infection inhibition, and wound healing promotion. The present invention forms a nanomaterial SA-BSP (Bletilla striata polysaccharide grafted stearic acid polymer) that self-assembles into micelles by grafting BSP (Bletilla striata polysaccharide) with SA (stearic acid); AZI (azithromycin) is encapsulated by using SA-BSP to prepare self-assembled nano-micelles; wherein BSP plays the role of "drug-adjuvant integration", not only as a micelle membrane material, but also shows a good function of regulating macrophage polarization, while encapsulating AZI, playing the purpose of continuous and controlled drug release, improving the local bioavailability of drugs in wound tissues, and inhibiting the production of multi-drug resistant bacteria; this self-assembled micelle not only significantly enhances the encapsulation capacity of drugs, but also optimizes the controlled release behavior of drugs by adjusting their hydrophilic-hydrophobic properties, thereby effectively inhibiting the development of bacterial resistance. The self-assembled micelles were embedded in gelatin-carbomer hydrogels and mixed to prepare self-assembled micelle hydrogels, which improved the mechanical properties of the hydrogels, increased the transdermal retention and bioavailability of the drugs, and provided a self-assembled micelle hydrogel that regulates macrophage polarization, inhibits bacterial infection, and promotes wound healing. It is a promising strategy for treating wound healing. The self-assembled micelle hydrogel combines nano-micelles with the hydrogel matrix, and with the dual protective effects of micelles and hydrogels, it ensures the dual sustained release of drugs and plays a role in promoting wound healing.

[0007] To achieve the above object, the present invention provides the following solution.

[0008] A self-assembled micellar hydrogel that regulates macrophage polarization, inhibits bacterial infection, and promotes wound healing. The self-assembled micellar hydrogel comprises a base material and a drug entrapped in the base material; the base material comprises gelatin, carbomer 940, triethanolamine, glycerol, 1,2-propylene glycol, and bletilla striata polysaccharide grafted stearic acid polymer (SA-BSP); the entrapped drug comprises a drug for treating bacterial infection.

[0009] Furthermore, the mass ratio of the bletilla striata polysaccharide grafted stearic acid polymer (SA-BSP) to the encapsulated drug is 20:3.

[0010] Furthermore, the mass ratio of the gelatin, carbomer 940, triethanolamine, glycerol and 1,2-propylene glycol is 12:3:3:10:20.

[0011] Furthermore, the preparation method of the bletilla striata polysaccharide grafted stearic acid polymer (SA-BSP) comprises the following steps:

[0012] Step 1: SA (stearic acid), DMAP (4-dimethylaminopyridine) and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) in a molar ratio of 1:1:1.2 are activated at 25° C. with magnetic stirring for 1 h to obtain an activated reaction solution;

[0013] Step 2: Weigh 400 mg of BSP (Bletilla striata polysaccharide) and dissolve it in 4 mL of DMSO (dimethyl sulfoxide), then add it dropwise to the activation reaction solution, react at 38° C. for 48 hours, add 10 times the volume of ice-cold anhydrous ethanol to the reaction solution after the reaction is completed, let it stand overnight, and filter;

[0014] Step 3: Wash the precipitate with 100 mL of anhydrous ethanol and 100 mL of ether alternately for 3 times, and then dry it under reduced pressure at 50° C. to obtain SA-BSP (Bletilla striata polysaccharide grafted stearic acid polymer).

[0015] Furthermore, the bacterial infection treatment drug includes azithromycin (AZI).

[0016] A method for preparing a self-assembled micelle hydrogel that can regulate macrophage polarization, inhibit bacterial infection, and promote wound healing, comprising the following steps:

[0017] Step 1, dissolve SA-BSP in DMSO (dimethyl sulfoxide), dissolve it by ultrasound, transfer it to a dialysis bag (molecular weight cutoff 8000-12000), and dialyze it in 500 mL of distilled water with a magnetic stirring rate of 100 r / min;

[0018] Step 2: dialyze for 32 hours, replace distilled water every 2 hours, filter twice with a polycarbonate membrane (pore size 0.45 μM), and adjust the volume of the filtrate with distilled water to obtain SA-BSP micelles;

[0019] Step 3, dissolve AZI in ethanol by ultrasonication, add the solution dropwise to the SA-BSP micelles in step 2 under stirring (100 rpm / min), continue stirring for 2 h after the dropwise addition, remove the ethanol by rotary evaporation at 40°C, and distill water to make up to volume to obtain SA-BSP-AZI micelles;

[0020] Step 4: Dissolve gelatin and carbomer in SA-BSP-AZI micelles to form solutions with final concentrations of 6% and 1.5% w / v, respectively, fully swell them at 50°C, add triethanolamine, glycerol, and 1,2-propylene glycol to blend until a transparent and well-gelled hydrogel is formed, i.e., a self-assembled micelle hydrogel that regulates macrophage polarization, inhibits bacterial infection, and promotes wound healing.

[0021] Compared with the prior art, the present invention has the following beneficial effects.

[0022] The present invention has developed a self-assembled micelle hydrogel that integrates macrophage polarization regulation, bacterial infection inhibition, and wound healing promotion. The micelle first utilizes the grafting reaction of BSP and SA to self-assemble into micelles, and encapsulates AZI; then the self-assembled micelles are embedded in gelatin-carbomer hydrogels for blending to prepare self-assembled micelle hydrogels. The BSP in the self-assembled micelle hydrogel plays the role of "drug-adjuvant integration", and not only serves as a micelle membrane material, but also plays the purpose of continuous and controlled drug release, improves the local bioavailability of drugs in wound tissues, and inhibits the production of multi-drug resistant bacteria; and also serves as a good macrophage polarization regulator, inhibits the transformation of macrophages to M1 type, and promotes its transformation to M2 type, improves the wound inflammatory microenvironment, and thus accelerates the healing process of the wound surface. The composite hydrogel combines nano-micelles with the hydrogel matrix, and with the dual protective effects of micelles and hydrogels, ensures that the dual sustained release of drugs is achieved. The preparation process of this composite hydrogel is simple and reliable, with good biocompatibility, economical and safe, and it shows great application potential in the field of wound healing. It has broad application prospects in many fields such as tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 IR spectra of BSP, SA and SA-BSP;

[0025] Figure 2 is the H NMR spectra of BSP, SA and SA-BSP;

[0026] Figure 3 is the infrared spectrum of SA-BSP-AZI@MS;

[0027] Figure 4 Representative images of SA-BSP@MS particle size distribution and its appearance image;

[0028] Figure 5 Representative images of the particle size distribution of SA-BSP-AZI@MS and its appearance image;

[0029] Figure 6 Representative images of the SA-BSP@MS Zeta potential distribution and its Tyndall effect image;

[0030] Figure 7Representative images of the Zeta potential distribution of SA-BSP-AZI@MS and its Tyndall effect image;

[0031] Figure 8 are the pH and PDI values ​​of SA-BSP@MS and SA-BSP-AZI@MS;

[0032] Fig. 9 Quantitative analysis of the particle size of SA-BSP@MS and SA-BSP-AZI@MS;

[0033] Fig.10 Quantitative analysis of the Zeta potential of SA-BSP@MS and SA-BSP-AZI@MS;

[0034] Fig.11 is the change of encapsulation efficiency of SA-BSP-AZI@MS within 21 days;

[0035] Fig.12 The changes of particle size and PDI value of SA-BSP-AZI@MS within 21 days;

[0036] Fig.13 Transmission electron microscopy image of SA-BSP-AZI@MS;

[0037] Fig.14 The formation process of hydrogel;

[0038] Fig.15 For gelation time analysis;

[0039] Fig.16 is the SEM image of the hydrogel;

[0040] Fig.17 EDS element mapping of hydrogel;

[0041] Fig.18 The compressive stress-strain curve and test photos of the hydrogel;

[0042] Fig.19 is the water evaporation rate of the hydrogel;

[0043] Fig. 20 The CLSM image shows that the drug is evenly distributed in the hydrogel;

[0044] Fig.21 is the swelling rate of the hydrogel;

[0045] Fig. 22 is the pH value of the hydrogel;

[0046] Fig.23 is the rheological curve of the hydrogel;

[0047] Fig.24 for the adhesion of the hydrogel to the finger at different bending angles;

[0048] Fig.25 Representative images of hydrogel compression and stretching;

[0049] Fig.26 for the adhesion of hydrogels to different substrates;

[0050] Fig. 27 to provide representative images of wounds during treatment;

[0051] Fig.28 Schematic diagram of wound closure marks;

[0052] Fig.29 For quantitative analysis of wound area;

[0053] Fig.30 Representative images and quantitative analysis of bacterial colonies in wound fluid;

[0054] Fig.31 Representative images for H&E staining;

[0055] Fig.32 Representative images of Masson's trichrome staining;

[0056] Fig.33 Representative images stained for Sirius red;

[0057] Fig.34 Representative images of immunofluorescence staining for COL-1 and COL-3;

[0058] Fig.35 Representative images of KI67 immunofluorescence staining;

[0059] Fig.36 Representative images of immunofluorescence staining for CD31 and α-SMA;

[0060] Fig.37 Representative images of immunofluorescence staining for MMP-2 and MMP-9;

[0061] Fig.38 Representative images of NF-κB immunofluorescence staining;

[0062] Fig.39 Representative images of immunofluorescence staining for CD86, CD206, and F4 / 80;

[0063] Fig.40 Representative images of subcutaneous blood vessels at the wound site after different drug treatments;

[0064] Fig.41 For quantitative analysis of granulation tissue width;

[0065] Fig.42 For quantitative analysis of collagen deposition;

[0066] Fig.43 For quantitative analysis of COL-1 and COL-3 immunofluorescence staining;

[0067] Fig.44 For quantitative analysis of KI67 immunofluorescence staining;

[0068] Fig.45 Quantitative analysis of CD31 and α-SMA immunofluorescence staining;

[0069] Fig.46 For quantitative analysis of immunofluorescence staining of MMP-2 and MMP-9;

[0070] Fig.47 For quantitative analysis of NF-κB immunofluorescence staining;

[0071] Fig.48 Quantitative analysis of immunofluorescence staining for CD86, CD206, and F4 / 80;

[0072] Fig.49 For quantitative analysis of related factor expression and related blood cells. DETAILED DESCRIPTION

[0073] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0074] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0075] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0076] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0077] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0078] The main experimental instruments and materials used in the following examples are:

[0079] Experimental instruments: DZKW-S-4 electric constant temperature water bath (Shanghai Medical Equipment Company Medical Equipment Factory No. 5); SG3300H ultrasonic cleaner (Shanghai Guante Ultrasonic Instrument Company); RE52CS rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); XS105 one-hundred-thousandth balance (Mettler Toledo); Ti-S fluorescent inverted microscope (Nikon Company, Japan); HBS-1096A microplate reader (Nanjing Detie Experimental Equipment Co., Ltd.); 1100 liquid chromatograph (UV detector, Dalian Yilite); C18 chromatographic column (250mm×4.6mm, 5μm, Shanghai Yuexu). Sephadex G-50 column (Shanghai Hualan Chemical Technology Co., Ltd.); MD44 dialysis bag (Beijing Solebold Technology Co., Ltd., molecular weight cutoff 12000-14000); polycarbonate membrane (Milipore Co., Ltd., USA); 500A nanoparticle size and Zeta potential analyzer (Anton Paar, Austria); JEM-2000EX transmission electron microscope (Japan Electronics Co., Ltd.); SW-CJ-1D single-person clean bench (Shanghai Sujing Industrial Co., Ltd.); gas-jacketed carbon dioxide cell culture incubator (Thermo Flesher Scientific (Asheville) LLC, USA); FACSCalibur flow cytometer (BD Company, USA); multispectral in vivo imaging system (Carestream Health, Inc., USA); RM2235 paraffin sectioner (Leica, Germany); confocal microscope (Leica, Germany).

[0080] Experimental materials: Bletilla striata polysaccharide (purity ≥ 90%) was purchased from Xi'an Huilin Biotechnology Co., Ltd. (Xi'an, China). Azithromycin (purity > 96%), lipopolysaccharide (LPS), and Carbopol 940 were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Gelatin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai). ELISA kits, HE staining kits, Masson trichrome staining kits (fast green method), and Sirius red staining kits were purchased from Beijing Solebow Technology Co., Ltd. (Beijing). Dimethyl sulfoxide and stearic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. Triethanolamine, glycerol, 1,2-propylene glycol, and butylene glycol were purchased from Tianjin Damao Chemical Reagent Factory. KI67, CD31, α-SMA, MMP-2, MMP-9, and NF-κB antibodies were purchased from Boster Biotechnology Co., Ltd. (Wuhan). CD86, CD206, F4 / 80, COL-1, and COL-3 antibodies were purchased from Abcam (Shanghai).

[0081] The technical concept of the present invention is as follows: severe bacterial infection will significantly hinder the wound healing process, thereby causing tissue necrosis. In order to better promote wound healing, nano-micelles containing azithromycin are self-packed by bletilla polysaccharide as a precursor solution, and carbomer 940 and gelatin are physically blended to improve the gel properties. As a hydrogel matrix, the precursor solution is combined with the gel matrix to obtain a preparation, which is intended to be used for treating wound infection and promoting wound healing. Relevant experiments show that it has excellent biocompatibility. The hydrogel can not only promote cell migration, proliferation, angiogenesis and collagen deposition related to skin healing, but also regulate the polarization of macrophages from M1 to M2 phenotype and the expression of related factors, and relevant experiments also show its good antibacterial activity. The following is a detailed description with specific embodiments.

[0082] Example 1.

[0083] 1. Preparation of micelles

[0084] 1.1 Preparation of polymer membranes

[0085] n(SA):n(DMAP):n(EDC)=1:1:1.2, activate with magnetic stirring at 25℃ for 1h to obtain activation reaction solution; weigh 400mg BSP and dissolve it in 4mL DMSO, then add it dropwise to the activation reaction solution, and react at 38℃ for 48h; after the reaction, add 10 times the volume of ice anhydrous ethanol to the reaction solution, let it stand overnight, and filter; wash the precipitate alternately with 100mL anhydrous ethanol and 100mL ether for 3 times, and then dry it under reduced pressure at 50℃ to obtain SA-BSP (Bletilla striata polysaccharide grafted stearic acid polymer).

[0086] 1.2 Preparation of blank micelles

[0087] SA-BSP was dissolved in DMSO and dissolved by ultrasound. The product was transferred to a dialysis bag (molecular weight cutoff 8000-12000) and dialyzed in 500 mL of distilled water at a magnetic stirring rate of 100 r / min. The product was dialyzed for 32 hours, with the distilled water replaced every 2 hours. The product was then filtered twice with a polycarbonate membrane (pore size 0.45 μM), and the filtrate was fixed to volume with distilled water to obtain SA-BSP@MS.

[0088] 1.3 Preparation of drug-loaded micelles

[0089] AZI was dissolved in ethanol by ultrasonic treatment, and the solution was added dropwise into SA-BSP@MS under stirring (100 rpm / min). After the addition was completed, stirring was continued for 2 h. The ethanol was removed by rotary evaporation at 40 °C, and the volume was fixed with distilled water to obtain SA-BSP-AZI@MS.

[0090] 1.3 Preparation of coumarin micelles

[0091] The same method was used to prepare coumarin-loaded micelles, essentially replacing the drug with a fluorescent probe.

[0092] 2. Characterization of micelles

[0093] 2.1 Transmission electron microscopy observation of micelle morphology

[0094] Take an appropriate amount of micelles and dilute them to a suitable concentration, drop one drop on a copper grid for electron microscopy, stain with 2% phosphotungstic acid, and observe their morphology using a transmission electron microscope after drying. The acceleration voltage of the transmission electron microscope is set to 120 kV.

[0095] 2.2 Particle size, PDI and zeta potential

[0096] The particle size, PDI and zeta potential of the samples were determined by dynamic light scattering using a 500A nanoparticle size and zeta potential analyzer.

[0097] 2.3 Evaluation of micelle stability

[0098] The drug content was determined by HPLC, and the calculation formula of EE was: EE% = (W1 / W2) × 100%, where W1 and W2 represent the drug content before and after elution, respectively. In order to analyze the storage stability of micelles, the micelles were stored at 4 °C for 21 days, and the EE, particle size and PDI of the micelles were measured at specific time points.

[0099] 3. Preparation and characterization of hydrogel

[0100] 3.1 Preparation of hydrogel

[0101] Gelatin and carbomer were dissolved in each precursor solution to make them fully swell, and triethanolamine, glycerol, and 1,2-propylene glycol were added to blend until a transparent and well-gelled hydrogel was formed. The mass ratio of gelatin, carbomer 940, triethanolamine, glycerol, and 1,2-propylene glycol was 12:3:3:10:20.

[0102] 3.2 Characterization of hydrogels

[0103] 3.2.1 Gel time analysis

[0104] Place each solution in a flat-bottomed test tube without touching the tube wall. Use magnetic stirring. Then add a magnetic stirrer to the flat-bottomed test tube at a speed of 100 rpm. When the magnetic stirrer stops rotating, the total time spent is the gel time.

[0105] 3.2.2 Morphology observation

[0106] The hydrogel was cut open and freeze-dried for 48 hours, then gold-sprayed. The morphology of the freeze-dried hydrogel samples was observed using a Japanese Regulus 8100 SEM and energy dispersive spectrometer.

[0107] 3.2.3 Compression performance analysis

[0108] The hydrogel was made into a cylindrical shape with a height of 2 cm and a diameter of 1.5 cm. The sample was compressed at a speed of 5 mm min-1 using an INSTRON 5982 electronic universal testing machine to measure the compression properties.

[0109] 3.2.4 Drug distribution detection in hydrogel

[0110] Different formulations were prepared using fluorescent probes instead of drugs and then observed using a Leica confocal laser scanning microscope.

[0111] 3.2.5 Swelling analysis

[0112] The hydrogel sample was freeze-dried and its dry weight (W0) was recorded. Then, the hydrogel was placed in PBS at 37°C. When the predetermined time was reached, the sample was taken out of PBS, the surface water was removed with filter paper, and then the hydrogel was weighed (Wt). The swelling rate was calculated as follows: swelling rate (%) = (Wt-W0) / W0×100%.

[0113] 3.2.6 Analysis of water evaporation rate

[0114] Accurately weigh the initial weight of the hydrogel (W0), then place it in an incubator at 50°C for 24 hours, and accurately measure the weight of the sample (Wt) every 4 hours. The water evaporation rate of the hydrogel is calculated as follows: Water evaporation rate (%) = (Wt-W0) / W0×100%.

[0115] 3.2.7 Rheological properties test

[0116] The rheological properties of the hydrogels were measured using Anton Paar MCR302e by frequency sweep and strain sweep.

[0117] 3.2.8 Adhesion performance test

[0118] Test the hydrogel's adhesion to different material surfaces, its compatibility with finger joints, and its tensile and compressive capabilities.

[0119] 4. Pharmacodynamic evaluation of different hydrogels in promoting wound healing

[0120] 4.1 Mouse grouping

[0121] The mice were divided into the following groups: ① normal saline (Blank); ② blank gel (GC); ③ free Bletilla striata polysaccharide gel (BSP-GC); ④ free azithromycin gel (AZI-GC); ⑤ Bletilla striata polysaccharide self-assembled micelle gel (SA-BSP@MS-GC); ⑥ Bletilla striata polysaccharide self-assembled micelle gel loaded with azithromycin (SA-BSP-AZI@MS-GC).

[0122] 4.2 Determination of wound area

[0123] All wounds were photographed and recorded on days 0, 1, 3, 7, 10, 14, and 18 after injury, and the images were analyzed using Image J software.

[0124] 4.3 HE staining

[0125] Mice were anesthetized with 7% chloral hydrate, and skin tissue was quickly removed. The skin tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and the coronal sections were 5 μm thick. The skin tissue was dewaxed twice in xylene, 5 minutes each time. The skin tissue was rehydrated with 100%, 95%, 85%, and 75% ethanol, and then washed with tap water for 2 minutes. The skin tissue was stained with hematoxylin solution for 15 minutes and then washed with tap water. The skin tissue was differentiated with differentiation solution for 30 seconds and then washed with tap water for 20 minutes. The skin tissue was stained with eosin solution for 1 minute and then washed with tap water for 2 minutes. The skin tissue was dehydrated with 75%, 85%, 95%, and 100% ethanol, and then transparentized with xylene twice. The skin tissue was sealed with neutral gum, and the healing condition was observed under an inverted microscope.

[0126] 4.4Masson trichrome staining

[0127] Mice were anesthetized with 7% chloral hydrate, and skin tissue was quickly removed. The skin tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and the coronal sections were 5 μm thick. The skin tissue was dewaxed twice in xylene, 5 minutes each time. The skin tissue was rehydrated with 100%, 95%, 85%, and 75% ethanol, and washed with tap water for 2 minutes. The skin tissue was stained with iron hematoxylin staining solution for 10 minutes, differentiated with differentiation solution for 30 seconds, blued with bluing solution for 3 minutes, stained with Ponceau fuchsin staining solution for 5 minutes, washed with weak acid working solution for 1 minute, stained with fast green staining solution for 1 minute, washed with weak acid working solution for 1 minute, dehydrated with 75%, 85%, 95%, and 100% ethanol, transparentized with xylene twice, sealed with neutral gum, and collagen deposition was observed under an inverted microscope.

[0128] 4.5 Sirius red staining

[0129] Mice were anesthetized with 7% chloral hydrate, and skin tissue was quickly removed. The skin tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and the coronal sections were 5 μm thick. The skin tissue was dewaxed in xylene twice, 5 min / time. The skin tissue was hydrated with 100%, 95%, 85%, and 75% ethanol, respectively, and washed with tap water for 2 minutes. The skin tissue was stained with iron hematoxylin solution for 10 minutes, washed with tap water for 5 minutes, stained with Sirius red solution for 15 minutes, rinsed with running water, dehydrated with 5%, 85%, 95%, and 100% ethanol, transparentized with xylene twice, sealed with neutral gum, and the distribution of collagen was observed under an inverted microscope.

[0130] 4.6 Immunofluorescence staining of COL-1 and COL-3

[0131] Mice were anesthetized with 7% chloral hydrate, and skin tissue was quickly removed. The skin tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and the coronal sections were 5 μm thick. The skin tissue was dewaxed twice in xylene, 5 minutes each time. The skin tissue was hydrated with 100%, 95%, 85%, and 75% ethanol, respectively, and washed with tap water for 2 minutes. The skin tissue was placed in citric acid buffer for antigen repair for 10 minutes, blocked with 5% BSA for 2 hours, and then incubated overnight at 4°C with appropriately diluted primary antibodies. After the primary antibody incubation, the skin tissue was rinsed with PBS for 3-5 times, and appropriately diluted fluorescent labeled secondary antibodies were added. The skin tissue was incubated at room temperature in the dark for 1 hour, stained with DAPI for 15 minutes, and then sealed. The expression of COL-1 and COL-3 was observed under a fluorescence microscope.

[0132] 4.7 Immunofluorescence staining of KI67

[0133] Mice were anesthetized with 7% chloral hydrate, skin tissue was quickly removed, fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and coronal sections were cut into 5μm thickness. Dewaxed in xylene twice, 5min / time. Rehydrated with 100%, 95%, 85%, and 75% ethanol, respectively, and washed with tap water for 2min. Antigen repair was performed in citric acid buffer for 10min, blocked with 5% BSA for 2h, and incubated overnight at 4℃ with appropriately diluted primary antibody. After the primary antibody incubation, the sections were rinsed with PBS 3-5 times, and appropriately diluted fluorescent-labeled secondary antibody was added. Incubated at room temperature in the dark for 1h, stained with DAPI for 15min, and sealed. The expression of KI67 was observed under a fluorescence microscope.

[0134] 4.8 Immunofluorescence staining of CD31 and α-SMA

[0135] Mice were anesthetized with 7% chloral hydrate, and skin tissue was quickly removed. The skin tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and the coronal sections were 5 μm thick. Dewaxed in xylene twice, 5 minutes per time. Rehydrated with 100%, 95%, 85%, and 75% ethanol, and washed with tap water for 2 minutes. Antigen retrieval was performed in citric acid buffer for 10 minutes, blocked with 5% BSA for 2 hours, and then the primary antibody was added with appropriate dilution and incubated overnight at 4°C. After the primary antibody incubation, the sections were rinsed with PBS for 3-5 times, and the fluorescent labeled secondary antibody was added with appropriate dilution. The sections were incubated in the dark for 1 hour at room temperature, stained with DAPI for 15 minutes, and sealed. The expression of CD31 and α-SMA was observed under a fluorescence microscope.

[0136] 4.9 Immunofluorescence staining of MMP-2 and MMP-9

[0137] Mice were anesthetized with 7% chloral hydrate, and skin tissue was quickly removed. The skin tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and the coronal sections were 5 μm thick. The skin tissue was dewaxed in xylene twice, 5 minutes each time. The skin tissue was hydrated with 100%, 95%, 85%, and 75% ethanol, respectively, and washed with tap water for 2 minutes. The skin tissue was placed in citric acid buffer for antigen repair for 10 minutes, blocked with 5% BSA for 2 hours, and then incubated overnight at 4°C with appropriately diluted primary antibodies. After the primary antibody incubation, the skin tissue was rinsed with PBS for 3-5 times, and appropriately diluted fluorescent labeled secondary antibodies were added. The skin tissue was incubated at room temperature in the dark for 1 hour, stained with DAPI for 15 minutes, and then sealed. The expression of MMP-2 and MMP-9 was observed under a fluorescence microscope.

[0138] 4.10 Immunofluorescence staining of NF-κB

[0139] Mice were anesthetized with 7% chloral hydrate, skin tissue was quickly removed, fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin wax, and coronal sections were cut into 5μm thickness. Dewaxed in xylene twice, 5min / time. Rehydrated with 100%, 95%, 85%, and 75% ethanol, respectively, and washed with tap water for 2min. Antigen retrieval was performed in citric acid buffer for 10min, blocked with 5% BSA for 2h, and incubated overnight at 4℃ with appropriately diluted primary antibody. After the primary antibody incubation, the sections were rinsed with PBS 3-5 times, and appropriately diluted fluorescent labeled secondary antibody was added. Incubated at room temperature in the dark for 1h, stained with DAPI for 15min, and sealed. The expression of NF-κB was observed under a fluorescence microscope.

[0140] 4.11 Immunofluorescence staining of CD86, CD206 and F4 / 80

[0141] Mice were anesthetized with 7% chloral hydrate, skin tissue was quickly removed, fixed with 4% paraformaldehyde for 24h, embedded in paraffin, and coronal sections were cut into 5μm thickness. Dewaxed in xylene twice, 5min / time. Rehydrated with 100%, 95%, 85%, and 75% ethanol, respectively, and washed with tap water for 2min. Antigen retrieval was performed in citric acid buffer for 10min, blocked with 5% BSA for 2h, and incubated overnight at 4℃ with appropriately diluted primary antibody. After the primary antibody incubation, the sections were rinsed with PBS 3-5 times, and appropriately diluted fluorescent labeled secondary antibody was added. Incubated at room temperature in the dark for 1h, stained with DAPI for 15min, and sealed. The expression of CD86, CD206, and F4 / 80 was observed under a fluorescence microscope.

[0142] 4.12 Subcutaneous blood vessels at wound sites after different treatments

[0143] The mice were anesthetized with 7% chloral hydrate, the wound skin tissue was quickly removed, and the blood vessel conditions were recorded with a camera.

[0144] 4.13 Quantitative analysis of related factor expression and related blood cells

[0145] Blood was collected 1, 3, and 7 days after injury, and the levels of blood indicators were analyzed using a Bowringman BM830 fully automatic blood cell analyzer. Serum samples were prepared 3 and 7 days after injury, and the levels of TNF-α, IL-1β, TGF-β, and IL-10 in serum were detected according to the instructions of the ELISA kit.

[0146] 5. Statistical analysis

[0147] Graphpad Prism 9.0 software was used for statistical analysis. All experimental data were expressed as Mean ± SD. One-way analysis of variance was used for comparison among multiple groups, and LSD-t test was used for further pairwise comparison. P < 0.05 indicated that the difference was statistically significant.

[0148] 6. Results and Analysis

[0149] 6.1 Characterization of micelles

[0150] Figure 1 The infrared spectra of BSP, SA and SA-BSP are 914.1 and 847.4 cm -1 The absorption peaks of β-glucose residue and mannose residue are 1072.4 and 1155.8 cm -1 It is the characteristic absorption peak of the pyranoside configuration in the polysaccharide structure of Bletilla striata. SA-BSP polymer has an absorption peak of 1743.2 cm -1 The characteristic stretching vibration peak of C=O in the ester carbonyl group appears at 2917.8cm -1 The absorption peak of methyl group (-CH3) appeared at 40°, indicating that stearic acid had been successfully grafted onto the hydroxyl group of BSP. Figure 2 The H NMR spectra of BSP, SA and SA-BSP show that the peak at 4.55 ppm is the absorption peak of hydrogen connected to 1,4 positions of BSP. The peak at 1.24 ppm is the characteristic peak of methylene (-CH2) of stearic acid. The peak at 0.8 ppm is the characteristic peak of methyl (-CH3) of stearic acid. This shows that stearic acid has been successfully grafted onto the hydroxyl group of BSP.

[0151] Fourier transform infrared spectroscopy was used to analyze the drug encapsulation effect. Figure 3 As shown in the infrared spectrum of AZI, the characteristic peak at 1453.1cm-1 is the bending vibration of CH2 plane, and the peaks at 1164.5 and 1050.5cm -1 The characteristic peak at 1741.8 cm -1 The characteristic peak at 2924.9cm-1 is the characteristic stretching vibration peak of C=O in the ester carbonyl group, and the absorption peak at 2924.9cm-1 is the absorption peak of methyl (-CH3). In the SA-BSP-AZI@MS spectrum, multiple absorption peaks of AZI become smaller or even disappear, and these changes indicate that AZI is embedded in the micelles.

[0152] The average particle size of SA-BSP@MS is 76.97±2.32nm, the average zeta potential is -10.8±1.28mV, the average pH is 6.83±0.06, and the average PDI is 11.2%±1.63%. The average particle size of SA-BSP-AZI@MS is 115.06±3.38nm, the average zeta potential is -11.4±1.83mV, the average pH is 7.25±0.21, and the average PDI is 26.46%±1.63%. Both are clear and transparent in appearance and have Tyndall effect. The results are shown in Figure 4-Figure 10 .

[0153] 6.2 Stability of micelles

[0154] The micelles were placed at 4°C for 21 days to analyze their stability. After 21 days, the EE of the micelles decreased slightly, and the particle size and PDI increased slightly. This was because the micelles had slight leakage, but it was within a reasonable range, indicating that the micelles had good stability. The results are shown in Figure 11-Figure 12 . Fig.13 This is the transmission electron microscopy image of SA-BSP-AZI@MS. It can be seen that the prepared micelles are round and spherical and are evenly distributed.

[0155] 6.3 Characterization of hydrogels

[0156] Gelatin and carbomer are common clinical materials. They are physically blended to obtain a composite gel. Fig.14 The formation process of hydrogel. Fig.15 For gelation time analysis, it was found that the addition of amphiphilic micelles would slightly increase the gelation time. Fig.16 The SEM image of the hydrogel shows that the introduction of the drug did not significantly change the structure of the hydrogel. Fig.17 This is the EDS element mapping of the hydrogel. The presence of elements such as C, O and S in SA-BSP-AZI@MS-GC was detected, which further proved the structural formation of the hydrogel. Fig.18 Figure 2 shows the compressive stress-strain curve and test photos of the hydrogel. The compressive stress-strain results of the hydrogel show that the SA-BSP-AZI@MS-GC group exhibits slightly higher stress under the same strain.

[0157] Fig.19 is the water evaporation rate of the hydrogel, which is used to test the water retention capacity of the hydrogel. The growth rate is very fast in the first 12 hours, then slows down, and becomes stable after about 24 hours. This may be due to the hydrophilicity of Bletilla striata polysaccharide and micelles, resulting in a slightly lower water evaporation rate of the corresponding gel than that of the blank gel. Fig. 20 The CLSM images show that the drug is evenly distributed in the hydrogel, and the results indicate that both free drugs and micelles are evenly distributed in the hydrogel. Fig.21 The swelling rate of the hydrogel was measured by immersing the hydrogel samples in PBS solution and taking them out after 24 hours. The results showed that each group of hydrogels had good swelling properties, which could ensure the effective absorption of exudate by the wound dressing. Fig. 22 is the pH value of the hydrogel. The pH of each hydrogel was tested and the results showed that the pH was neutral. Fig.23This is the rheological curve of the hydrogel. In the frequency scan, it can be seen that the hydrogel is in a gel state when the scanning frequency ranges from 0.1 to 100 rad / s and G' is greater than G". In the strain scan, the critical value of hydrogel collapse is explored. When the strain is ≈100%, each gel will show that G" is greater than G' and exhibits liquid properties. The rheological properties of all hydrogels show similar results, which indicates that the loading of drugs has little effect on the rheological properties of the hydrogel. Fig.24 To investigate the adhesion of hydrogels to fingers with different bending angles, the adhesion ability of hydrogels to finger joints at different angles was tested. Fig.25 Representative images of hydrogel compression and stretching. Simple compression-stretching between fingers was tested. Fig.26 To investigate the adhesion of hydrogels to different substrates, the adhesion ability of hydrogels to common materials was tested.

[0158] 6.4 Pharmacodynamic evaluation of hydrogels in promoting healing of infected wounds

[0159] 6.4.1 Effect of hydrogel on healing of infected wounds

[0160] The effects of each hydrogel on the healing of bacterial infection wounds were evaluated. The wounds were treated with GC, BSP-GC, AZI-GC, SA-BSP@MS-GC, SA-BSP-AZI@MS-GC and saline (blank), and all wounds were photographed on days 0, 1, 3, 7, 10, 14 and 18, and statistically analyzed using image J software. The results are shown in Figure 27-Figure 29 .

[0161] 6.4.2 Evaluation of wound bacterial count

[0162] Fig.30 The representative images and quantitative analysis of bacterial colonies in wound fluid show that on the first day, due to the small number of treatments, the number of bacteria in the wounds of each group was high, and the number of bacteria in the AZI-GC group and the SA-BSP-AZI@MS-GC group showed a downward trend. On the third day, the number of bacteria in the AZI-GC group and the SA-BSP-AZI@MS-GC group decreased significantly. On the seventh day, the number of bacteria in the BSP-GC group and the SA-BSP@MS-GC group also decreased significantly, and the bacteria in the AZI-GC group and the SA-BSP-AZI@MS-GC group were almost eliminated.

[0163] 6.4.3 HE staining

[0164] HE staining results showed that the skin defects of the blank group and GC group were larger, and the BSP-GC group, AZI-GC group, SA-BSP@MS-GC group and SA-BSP-AZI@MS-GC group formed a continuous epidermis, the granulation tissue became narrower, and new hair follicles were formed. Among them, the SA-BSP-AZI@MS-GC group was closest to healthy skin. Fig.31 and Fig.41 .

[0165] 6.4.4 Masson trichrome staining

[0166] In order to analyze the collagen deposition, Masson trichrome staining was used, and the results showed that the collagen area of ​​the BSP-GC group, AZI-GC group, SA-BSP@MS-GC group and SA-BSP-AZI@MS-GC group increased, among which the collagen area of ​​the SA-BSP-AZI@MS-GC group was the largest and the arrangement was the most orderly. Fig.32 and Fig.42 .

[0167] 6.4.5 Sirius red staining

[0168] Fig.33 Representative images of Sirius red staining show that the SA-BSP-AZI@MS-GC group achieved better skin tissue remodeling.

[0169] 6.4.6 Immunofluorescence staining of COL-1 and COL-3

[0170] Immunofluorescence staining of COL-1 and COL-3 showed that the BSP-GC group, SA-BSP@MS-GC group and SA-BSP-AZI@MS-GC group showed strong expression, which was similar to the results of the above collagen test. Fig.34 and Fig.43 .

[0171] 6.4.7 KI67 immunofluorescence staining

[0172] Cell proliferation is an important part of wound healing. Long-term bacterial environment and inflammation can lead to inhibition of cell proliferation. We performed immunofluorescence staining of the proliferation marker KI67. The results showed that the BSP-GC group, SA-BSP@MS-GC group and SA-BSP-AZI@MS-GC group had strong expression, while the other groups had weak expression, among which the SA-BSP-AZI@MS-GC group had the strongest expression. Fig.35 and Fig.44 .

[0173] 6.4.8 CD31 and α-SMA immunofluorescence staining and angiogenesis assessment

[0174] In order to detect angiogenesis, we performed CD31 and α-SMA immunofluorescence staining. The BSP-GC group, SA-BSP@MS-GC group and SA-BSP-AZI@MS-GC group expressed strongly, while the other groups expressed weakly. Among them, the SA-BSP-AZI@MS-GC group expressed the strongest, showing a dense vascular network. At the same time, we also took pictures of the subcutaneous blood vessels in the wound, and the results were similar to those of immunofluorescence staining. The results are shown in Fig.36 , Fig.40 and Fig.45 .

[0175] 6.4.9 Immunofluorescence staining of MMP-2 and MMP-9

[0176] Overexpression of MMP-2 and MMP-9 in chronic wounds disrupts the balance between ECM synthesis and degradation, thereby hindering the formation of granular tissue and slowing down wound healing. Immunofluorescence staining results of MMP-2 and MMP-9 showed that the expression of BSP-GC group, AZI-GC group, SA-BSP@MS-GC group and SA-BSP-AZI@MS-GC group was lower, which is consistent with the better skin regeneration trend observed above. Fig.37 and Fig.46 .

[0177] 6.4.10 NF-κB immunofluorescence staining

[0178] Nuclear factor κB (NF-κB) plays a key role in regulating various physiological processes such as immune response, inflammatory response, cell growth and death. Therefore, NF-κB plays an important role in regulating the inflammatory response of macrophages. Immunofluorescence staining was used to evaluate the expression of NF-κB in tissues. The results showed that the expression of AZI-GC group and SA-BSP-AZI@MS-GC group was low, indicating that it plays an anti-inflammatory and healing role through NF-κB. Results are shown in Fig.38 and Fig.47 .

[0179] 6.4.11 Immunofluorescence staining of CD86, CD206 and F4 / 80

[0180] F4 / 80 was set as a marker for macrophages, CD86 and CD206 were established as markers for M1 and M2 macrophages, respectively, and double immunofluorescence staining was used to analyze the polarization of macrophages. The proportion of M1 macrophages (CD86) in SA-BSP-AZI@MS-GC was lower than that in other treatment groups, and the proportion of M2 macrophages (CD206) was higher than that in other treatment groups. This was consistent with the results of the above in vitro and in vivo experiments. SA-BSP-AZI@MS-GC had the strongest ability to affect macrophage polarization. The results are shown in 39 and Fig.48 .

[0181] 6.4.12 Quantitative analysis of related factor expression and related blood cells

[0182] The relevant inflammatory factors and inflammatory cell indicators were analyzed by ELISA and blood cell analyzer. The results showed that the AZI-GC group and SA-BSP-AZI@MS-GC group significantly reduced the expression of TNF-α and IL-1β, while increasing the levels of TGF-β and IL-10. The results of various inflammatory cell indicators were consistent with them. Fig.49 .

[0183] In summary, through the above implementation examples, it can be seen that SA-BSP-AZI@MS-GC can significantly promote the healing of infected wounds, and promote tissue repair through multiple pathways such as regulating polarization and inhibiting bacterial infection, thereby exerting a synergistic sustained-release effect.

[0184] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A self-assembled micellar hydrogel that regulates macrophage polarization, inhibits bacterial infection, and promotes wound healing, characterized in that: The self-assembled micelle hydrogel comprises a base material and a drug encapsulated by the base material; The basic material comprises gelatin, carbomer 940, triethanolamine, glycerol, 1,2-propylene glycol and bletilla striata polysaccharide grafted stearic acid polymer, and the encapsulated drug comprises a drug for treating bacterial infection.

2. The self-assembled micellar hydrogel according to claim 1 that combines the functions of regulating macrophage polarization, inhibiting bacterial infection, and promoting wound healing, characterized in that: The mass ratio of the gelatin, carbomer 940, triethanolamine, glycerol and 1,2-propylene glycol is 12:3:3:10:

20.

3. The self-assembled micellar hydrogel capable of regulating polarization, inhibiting bacterial infection and promoting wound healing according to claim 1, characterized in that: The mass ratio of the bletilla striata polysaccharide grafted stearic acid polymer to the encapsulated drug is 20:

3.

4. The self-assembled micellar hydrogel according to claim 1, characterized in that The bacterial infection treatment drugs include azithromycin.

5. The self-assembled micellar hydrogel according to claim 1, characterized in that The preparation method of the bletilla striata polysaccharide grafted stearic acid polymer comprises the following steps: Step 1: SA, DMAP and EDC were activated at a molar ratio of 1:1:1.2 at 25° C. with magnetic stirring for 1 h to obtain an activated reaction solution; Step 2: Weigh 400 mg of BSP and dissolve it in 4 mL of DMSO, then add it dropwise to the activation reaction solution, react at 38°C for 48 hours, add 10 times the volume of ice-cold anhydrous ethanol to the reaction solution after the reaction is completed, let it stand overnight, and filter; Step 3: Wash the precipitate with 100 mL of anhydrous ethanol and 100 mL of ether alternately for 3 times, and then dry it under reduced pressure at 50°C to obtain a Bletilla striata polysaccharide grafted stearic acid polymer.

6. The method for preparing the self-assembled micellar hydrogel according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1, dissolving the Bletilla striata polysaccharide grafted stearic acid polymer in DMSO, dissolving it by ultrasound, transferring it to a dialysis bag, placing it in 500 mL of distilled water for dialysis, and the magnetic stirring rate was 100 r / min; Step 2, after dialysis, filtering the mixture twice with a polycarbonate membrane, and fixing the filtrate with distilled water to obtain Bletilla striata polysaccharide grafted stearic acid polymer micelles; Step 3, dissolving azithromycin in ethanol by ultrasonication, adding the solution dropwise to the Bletilla striata polysaccharide grafted stearic acid polymer micelles in step 2 under stirring conditions, continuing stirring for 2 h after the dropwise addition, removing the ethanol under rotary evaporation, and fixing the volume with distilled water to obtain SA-BSP-AZI micelles; Step 4: Dissolve gelatin and carbomer in SA-BSP-AZI micelles to form solutions with final concentrations of 6% and 1.5% w / v, respectively. Fully swell the solutions at 50°C, and add triethanolamine, glycerol, and 1,2-propylene glycol to blend the mixture until a transparent and well-gelled hydrogel is formed, i.e., a self-assembled micelle hydrogel that regulates macrophage polarization, inhibits bacterial infection, and promotes wound healing.

7. The preparation method according to claim 6, characterized in that: In the step 2, the dialysis time is 32 h, and the distilled water is replaced every 2 h; the polycarbonate membrane is a 0.45 μm microporous filter membrane.

8. The preparation method according to claim 6, characterized in that: In the step 3, the stirring condition is 100 rpm / min; the rotary evaporation condition is reduced pressure rotary evaporation in a 40° C. water bath.

9. Use of the self-assembled micelle hydrogel according to any one of claims 1 to 5 in the preparation of a drug for promoting wound healing.

10. Use of the self-assembled micellar hydrogel according to any one of claims 1 to 5 in the following (1) to (3): (1) Application in regulating macrophage polarization; (2) Application in inhibiting acute and chronic bacterial infections; (3) Application in promoting the healing of acute and chronic infected wounds.

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