Self-adjustable skin bionic hydrogel as well as preparation method and application thereof

Abstract

The invention provides self-adjustable skin bionic hydrogel, a preparation method and application, and belongs to the technical field of biomedical materials, methyl acrylate (MA), 3-acrylamido phenylboronic acid (AAPBA) and the like are used as raw materials to prepare organogel, polyphenol drugs and antibiotics are encapsulated, double-drug-loading organogel is prepared, and the hydrogel is used for preparing the skin bionic hydrogel. The preparation method comprises the following steps: firstly, preparing a hydrogel (QL-MAB) by replacing an organic solvent with water as a medium, carrying out rapid phase separation on an MA hydrophobic fragment to form a compact outer layer similar to'epidermis', combining an AAPBA fragment with quercetin through a borate bond to construct a glucose-responsive'pore '-similar active site, and finally constructing a skin bionic hydrogel system which simulates a skin structure and can realize intelligent drug delivery. The skin bionic hydrogel provided by the invention can realize on-demand release of drugs, and can effectively promote the repair of wound surfaces infected by diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a self-regulating skin biomimetic hydrogel, a preparation method and an application thereof, wherein the self-regulating skin biomimetic hydrogel has the properties of low preparation cost, high water retention, sustained drug release, and simulation of skin structure and function, and provides a new clinical solution for the treatment of diabetic infected wounds. Background Art

[0002] Diabetic wounds are one of the most common complications of diabetes, placing a huge burden on patients and society. Compared with traditional wounds, diabetic wounds have a more complex pathological microenvironment, which seriously delays wound healing. First, the hyperglycemic state hinders the reconstruction of the vascular network, resulting in limited delivery of nutrients and oxygen required for tissue regeneration. Secondly, persistent inflammatory responses and oxidative stress lead to upregulation of matrix metalloproteinases. In addition, diabetic wounds are more susceptible to bacterial infection due to immune system defects and high blood sugar, further hindering wound healing. Therefore, in the absence of effective intervention measures, diabetic wounds can lead to a large amount of tissue necrosis, amputation, and even death.

[0003] The main methods for clinical treatment of diabetic wounds include surgical debridement, medical treatment and wound dressings. Recently, the development of dressings such as sponges, films and hydrogels has witnessed the rapid development of diabetic wound management. In particular, hydrogels have been widely studied due to their extracellular matrix-like structure, good biocompatibility, effective absorption of exudate and maintenance of a moist pro-healing microenvironment. From the perspective of clinical translation, compared with other biological agents, the construction of functional hydrogels should be easy to obtain, highly stable and low-cost. However, clinically available hydrogels still have bottlenecks such as low drug loading efficiency, uncontrollable drug release and poor water retention capacity, resulting in frequent dressing changes and increasing the risk of secondary wound injury. To overcome these defects, people have invested a lot of effort in designing new hydrogels. For example, incorporating nano / microspheres into hydrogel matrices has been shown to be a viable option for enhancing drug loading and maintaining drug release. In addition, stimuli-responsive hydrogels can also release drugs on demand under biochemical or physical stimuli such as pH, glucose, reactive oxygen species (ROS) and light. In terms of enhancing moisturizing effects, hydrophobic membranes are usually wrapped on hydrogels as secondary modifications or laminated with hydrogels to form composites. However, it is still very challenging to overcome the many shortcomings of clinical hydrogels in a single hydrogel system.

[0004] Considering that wound dressings will serve as temporary replacements for defective skin, the development of multifunctional dressings by mimicking skin structure and function has outstanding potential. In essence, the dense epidermis prevents water loss, electrolyte imbalance, and bacterial invasion, while the pores can close or expand according to the needs of material exchange to ensure the body's homeostasis. Although recent studies have emphasized the development of skin-inspired hydrogels, they usually involve the synthesis of multiple materials and cumbersome preparation, and few are targeted for the purpose of diabetic wound healing. Summary of the invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides a self-regulating skin biomimetic hydrogel, a preparation method and its application in the repair of diabetic infected wounds, which improves the case microenvironment of diabetic infected wounds and promotes wound healing by simulating the skin structure and self-regulating drug release.

[0006] A self-regulating skin biomimetic hydrogel is prepared by polymerization reaction of methyl acrylate and 3-acrylamidophenylboronic acid, and loaded with polyphenol hydrophobic drugs and antibiotics; methyl acrylate and 3-acrylamidophenylboronic acid are uniformly dispersed and dissolved in dimethyl sulfoxide (DMSO), a cross-linking agent and an initiator are added, and an organic gel is prepared by photoinitiation; the organic gel is loaded with polyphenol hydrophobic drugs and antibiotics by an immersion method, and then the drug-loaded gel is immersed in purified water, and the skin biomimetic hydrogel is obtained by solvent replacement.

[0007] Preferably, the crosslinking agent is 1,4-butanediol diacrylate, and the initiator is diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide.

[0008] Preferably, the organogel is photo-initiated by ultraviolet light irradiation, and the irradiation time is 5 min to 120 min; the mass fraction of methyl acrylate is 10% to 30%, the mass fraction of 1,4-butanediol diacrylate is 0.3% to 3%, the mass fraction of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide is 0.1% to 1%, and the feeding of 3-acrylamidophenylboric acid is carried out according to the molar ratio of 3-acrylamidophenylboric acid to methyl acrylate of 0.5:99.5 to 20:80.

[0009] Preferably, the polyphenol hydrophobic drug and the antibiotic are fully dissolved in DMSO to prepare a drug mixture with a mass fraction of 0.1% to 2%. The organogel is immersed in the drug mixture for 24h to 72h and the drug is loaded by a physical impregnation method.

[0010] Preferably, the polyphenol hydrophobic drug is a mixture of one or more of quercetin, ellagic acid, caffeic acid, luteolin, lithospermic acid, myricetin, baicalin, rutin and their derivatives; the antibiotic is a mixture of one or more of levofloxacin or its salt, ciprofloxacin or its salt, moxifloxacin or its salt, norfloxacin or its salt, amoxicillin, vancomycin or its salt, gentamicin or its salt, doxycycline or its salt.

[0011] Preferably, the gel is immersed in purified water at room temperature for 24 hours to 72 hours, and after sufficient solvent replacement, the DMSO in the gel cavity is replaced by purified water and swells, thereby obtaining a skin biomimetic hydrogel.

[0012] A method for preparing a self-regulating bionic hydrogel is obtained by the following steps:

[0013] Preparation of organic gel: Organic gel is prepared by ultraviolet light-induced free radical polymerization; the prescribed amount of methyl acrylate, 3-acrylamidophenylboronic acid and 1,4-butanediol diacrylate are weighed and dissolved in an appropriate amount of DMSO solution, the solution is fully dissolved by ultrasonic treatment and protected from light, the prescribed amount of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide is weighed and dissolved to obtain a gel solution, and then the gel solution is irradiated with ultraviolet light to obtain an organic gel, and then the organic gel is soaked in an excess of DMSO solvent to wash away unreacted monomers and fully swell to obtain a purified organic gel.

[0014] Preferably, the mass fraction of the methyl acrylate is 10% to 30%, the mass fraction of the 1,4-butanediol diacrylate is 0.3% to 3%, the mass fraction of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide is 0.1% to 1%, and the 3-acrylamidophenylboric acid is added according to a molar ratio of 3-acrylamidophenylboric acid to methyl acrylate of 0.5:99.5 to 20:80; the DMSO is an anhydrous ultra-dry DMSO solution, the wavelength of the ultraviolet lamp is 365nm, and the illumination time is 5min to 120min.

[0015] Preparation of skin biomimetic hydrogel: Step 1, dissolving an appropriate amount of 10% to 30% methyl acrylate, 0.3% to 3% 1,4-butanediol diacrylate, 0.1% to 1% diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, and 3-acrylamide phenylboronic acid (3-acrylamide phenylboronic acid: methyl acrylate molar ratio 0.5:99.5 to 20:80) in a DMSO solution, and preparing an organic gel by ultraviolet light initiation, dissolving an appropriate amount of polyphenol hydrophobic drugs and antibiotics in a DMSO solution to form a drug mixture, and immersing the organic gel in the drug mixture for a period of time to obtain a drug-loaded organic gel;

[0016] Step 2: soaking the drug-loaded organogel in excess purified water and allowing it to stand for a period of time at room temperature to obtain a skin biomimetic hydrogel.

[0017] Preferably, the polyphenol hydrophobic drug used is quercetin, and the antibiotic is levofloxacin hydrochloride. The drug mixture is obtained by dissolving quercetin and levofloxacin hydrochloride in DMSO with a mass fraction of 0.1% to 2% respectively and mixing them. The gel is immersed in the drug mixture for 24 hours to 72 hours. The gel is immersed in purified water, and the ratio of the drug-loaded organic gel to the water solvent is 1:50 to 1:100, and the immersion time is 24 hours to 72 hours.

[0018] Skin biomimetic hydrogels are used to prepare dressings for diabetic infected wounds.

[0019] In this study, methyl acrylate (MA) and 3-acrylamidophenylboronic acid (AAPBA) were used as raw materials to prepare gels, 1,4-butanediol diacrylate was used as a crosslinker, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide was used as a photoinitiator, and ultraviolet light was used to initiate gelation to construct an organogel. At the same time, the organogel was immersed in a mixed solution of polyphenol hydrophobic drugs and antibiotic drugs for drug encapsulation, and the solvent was replaced by simple purified water immersion to obtain a self-regulating skin biomimetic hydrogel. The MA fragment of the skin biomimetic hydrogel can phase separate to form a dense epidermis-like layer, maximizing drug loading, drug release cycle and water retention. At the same time, the AAPBA fragment containing boronic acid groups can form boronate bonds with hydrophobic polyphenol drugs to simulate the pores that regulate secretion. Once stimulated by excessive glucose, the boronate bonds will cleave and produce pore-like sites, which can self-regulate the release kinetics of drugs.

[0020] At the same time, the synergistic effect of quercetin and levofloxacin hydrochloride can quickly debride, promote angiogenesis, hair follicle regeneration, promote re-epithelialization and extracellular matrix remodeling. Skin biomimetic hydrogel can self-regulate drug release kinetics under the stimulation of wound microenvironment, prolong drug action time, and reduce the frequency of gel replacement.

[0021] In the present invention, the drugs used are quercetin and levofloxacin hydrochloride. Studies have shown that quercetin has anti-inflammatory, anti-oxidative, hypoglycemic and diabetic complication-improving effects. Levofloxacin is a first-line antibiotic with a spectrum of antibacterial capabilities. Quercetin and levofloxacin hydrochloride are loaded on the skin biomimetic hydrogel to increase the sustained and self-regulated release of drugs in wound lesions and improve the wound healing efficacy.

[0022] The present invention intends to use methyl acrylate (MA) and 3-acrylamidophenylboronic acid (AAPBA) copolymerization to construct a new skin biomimetic hydrogel. In order to enhance the therapeutic potential of the hydrogel, polyphenol hydrophobic drugs with multiple biological activities and first-line antibiotics are loaded into the hydrogel by the immersion method. The skin biomimetic hydrogel is expected to utilize the synergistic gain effect of drugs and hydrogel matrix, effectively eliminate harmful factors such as bacteria and reactive oxygen in the wound site, downregulate inflammatory response, promote angiogenesis, and restore the normal function of the immune system, ultimately achieving the purpose of accelerating the healing of diabetic infected wounds.

[0023] Compared with the prior art, the present invention has the following advantages and effects:

[0024] (1) The method for preparing the skin biomimetic hydrogel of the present invention is simple in process and low in cost. The outer layer thereof is dense and similar in structure and function to the skin. It has microenvironment adaptability, long-term moisturizing and self-regulating release of drugs as needed.

[0025] (2) The skin biomimetic hydrogel of the present invention has multiple biological activities, which can inhibit the growth of bacteria in the wound site for a long time, resist oxidation, and resist inflammation, and promote the reconstruction of the vascular network and matrix remodeling.

[0026] (3) The skin biomimetic hydrogel of the present invention can be individually designed according to the actual needs of the wound surface, and a drug load with complementary therapeutic effects can be reasonably selected to promote wound healing.

[0027] (4) The skin biomimetic hydrogel prepared by the present invention can be continuously applied to the wound surface to reduce the frequency of dressing changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Laser microscope images of the blank hydrogel (left) and the double-drug-loaded skin biomimetic hydrogel (right) in the present invention.

[0029] Figure 2 It is a water absorption and swelling curve of the hydrogel in the present invention.

[0030] Figure 3 It is a water loss rate curve diagram of the hydrogel in the present invention and the traditional hydrophilic gel.

[0031] Figure 4 is a release diagram of the drug in the hydrogel of the present invention, wherein: Figure 4 A is the release curve of quercetin (Q) in QL@MAB within 24 hours. Figure 4 B is the long-term release curve of quercetin (Q) in QL@MAB. Figure 4 C is the release curve of levofloxacin hydrochloride (L) in QL@MAB within 24 hours.

[0032] Figure 5The laser microscope image and 3D super depth image of the skin biomimetic hydrogel in the present invention after incubation with PBS and glucose solution, wherein: Figure 5 A is a cross-sectional view of a laser microscope. Figure 5 B is the laser microscope surface image, Figure 5 C is the 3D super depth image. Figure 5 D is the corresponding average roughness evaluation.

[0033] Figure 6 The in vitro antibacterial effect diagram of the hydrogel of the present invention, wherein: Figure 6 A is the actual picture of the antibacterial ring of the gel against Escherichia coli and Staphylococcus aureus. Figure 6 B is the statistical diagram of inhibiting the diameter of E. coli. Figure 6 C is the statistical graph of inhibition of Staphylococcus aureus diameter.

[0034] Figure 7 This is a diagram showing the effect of promoting endothelial cell migration in the skin biomimetic hydrogel of the present invention, wherein: Figure 7 A is an optical microscope photo of cell migration. Figure 7 B is the mobility curve.

[0035] Figure 8 This is a diagram showing the therapeutic effect of the present invention on diabetic infection wounds. Figure 8 A is the wound healing diagram at different time points. Figure 8 B is the heat map of wound healing. Figure 8 C is the wound healing curve.

[0036] Fig. 9 This is a diagram showing the antibacterial effect of the present invention on diabetic infected wounds. Fig. 9 A is the actual picture of bacteria coating at different time points. Fig. 9 B is the bacterial survival rate curve. DETAILED DESCRIPTION

[0037] In order to make the technical solution of the present invention easier to understand, the technical solution of the present invention is now clearly and completely described in the form of specific embodiments in conjunction with the accompanying drawings.

[0038] Example 1

[0039] Preparation of organogel:

[0040] 10% to 30% of methyl acrylate, 0.3% to 3% of 1,4-butanediol diacrylate and 0.1% to 1% of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide are weighed and dissolved in anhydrous dimethyl sulfoxide (DMSO) solution, 3-acrylamidophenylboric acid is weighed according to a molar ratio of 3-acrylamidophenylboric acid to methyl acrylate of 0.5:99.5 to 20:80, added into the DMSO solution to fully dissolve, and irradiated under a 365nm ultraviolet lamp for 5min to 120min to obtain an organic gel. Among them, 10% methyl acrylate, 0.3% 1,4-butanediol diacrylate, 0.1% diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 3-acrylamidophenyl boronic acid (3-acrylamidophenyl boronic acid: methyl acrylate molar ratio 1:99) were weighed, and the organic gel collapsed after 365nm ultraviolet light exposure for 5 minutes. At the same time, the prescription is 20% methyl acrylate, 3% 1,4-butanediol diacrylate, 0.5% diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 3-acrylamidophenyl boronic acid, and the molar ratio of 3-acrylamidophenyl boronic acid to methyl acrylate is 1:99. The organic gel after ultraviolet light exposure for 60 minutes is too hard and brittle. At the same time, the formula is 20% by mass of methyl acrylate, 1% of 1,4-butanediol diacrylate, 0.2% of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 3-acrylamidophenylboric acid, the molar ratio of methyl acrylate to 3-acrylamidophenylboric acid is 1:99, and the organic gel is strong and tough after ultraviolet light exposure for 60 minutes. The optimal formula selected is 20% by mass of methyl acrylate, 1% of 1,4-butanediol diacrylate, 0.2% of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 3-acrylamidophenylboric acid, the molar ratio of 3-acrylamidophenylboric acid to methyl acrylate is 1:99, and ultraviolet light exposure is 60 minutes.

[0041] Preparation of skin biomimetic hydrogel:

[0042] As described above, according to the optimal recipe, 20% methyl acrylate, 1% 1,4-butanediol diacrylate, 0.2% diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide and 3-acrylamidophenylboronic acid were weighed, and the molar ratio of 3-acrylamidophenylboronic acid to methyl acrylate was 1:99, dissolved in DMSO solution, and irradiated with ultraviolet light for 60 minutes to form an organic gel. The organic gel was placed in excess DMSO to fully swell and store.

[0043] Quercetin and levofloxacin hydrochloride with a mass fraction of 0.1% to 2% were weighed and dissolved in DMSO solution to obtain a drug mixture. The organogel was immersed in the drug mixture for 24h to 72h, and then transferred to excess purified water for immersion for 24h to 72h to obtain a skin biomimetic hydrogel. Among them, the drug loading rates of the organogel immersed in the drug mixture with a mass fraction of 1% and 2%, and immersed in the drug mixture for 24h and 72h were not much different. In addition, there was still a lot of organic solvent remaining in the organogel after immersion in purified water for 24h, while 72h would cause excessive drug loss. Considering the cost of hydrogel, the residual rate of organic solvent and the drug loading rate, the optimal prescription was selected as a drug mixture with a mass fraction of 1%, the organogel was immersed in the drug mixture for 24h, and immersed in purified water for 48h. The preparation of the blank hydrogel was the same as the drug-loaded skin biomimetic hydrogel except that the drug was not immersed. Cross-sectional laser microscopy images of blank hydrogel (MAB) and dual-drug loaded skin biomimetic hydrogel (QL@MAB) are shown in Figure 1 , it can be observed that QL@MAB has a multilayer structure similar to skin.

[0044] Example 2

[0045] Water absorption and swelling of skin biomimetic hydrogel:

[0046] The coordination of the polyphenol hydrophobic drug quercetin with 3-acrylamidophenylboronic acid containing a boronic acid group will affect the hydrophobicity of the gel network and thus its swelling properties. Therefore, the water absorption and swelling behavior of the hydrogel was evaluated by recording the mass change of the hydrogel in phosphate buffer over time. Simply put, the initial mass of the organogel was recorded in advance, and then the organogel was immersed in an excess of phosphate buffer and incubated in a constant temperature shaker at 37°C. At the specified time point, the gel was taken out, the excess buffer on the surface was gently wiped off, and the weight was recorded. The water absorption and swelling ratio was calculated as follows:

[0047] Swelling ratio = time period weight / initial weight × 100%

[0048] The water absorption swelling curve is shown in Figure 2 The results showed that the swelling rates of the quercetin-loaded hydrogel group (Q@MAB) and the quercetin and levofloxacin hydrochloride-loaded hydrogel group (QL@MAB) were significantly stronger than those of the blank hydrogel group (MAB) and the levofloxacin hydrochloride-loaded hydrogel group (L@MAB) due to the complexation with quercetin.

[0049] Example 3

[0050] Study on water retention rate of skin biomimetic hydrogel in vitro

[0051] The room temperature water retention capacity of the hydrogel was evaluated by the specific gravity method. In short, the organogel was immersed in purified water to fully swell for 48 hours, then the excess water on the surface was wiped off, the initial mass of the hydrogel was weighed and recorded, and then the gel was placed in a ventilated environment at room temperature. The remaining weight of the gel was measured and recorded at the specified time point. The formula for calculating the water retention rate of the gel is as follows:

[0052] Water retention rate = time period weight / initial weight × 100%

[0053] Gel dehydration curve Figure 3 The results showed that all hydrogels had similar water loss characteristics and still retained a certain amount of water after standing for 6 days, showing the excellent water retention properties of the hydrogel. Under the same conditions, conventional hydrophilic hydrogel (methacrylated gelatin, GelMA) was completely dehydrated after air drying for 24 hours.

[0054] Example 4

[0055] In vitro drug release from skin-mimetic hydrogels:

[0056] First, the standard concentration curve equations of quercetin and levofloxacin hydrochloride were established. Then, the skin biomimetic hydrogel (QL@MAB) was placed in phosphate buffer (or containing 4 mg / mL glucose) to investigate the in vitro release behavior of the drug. It was immersed in a 50 mL centrifuge tube and placed in a 37°C constant temperature shaker. 2 mL of the release solution was taken out at a fixed point to measure the released drug, and 2 mL of fresh release solution was added at the same time. The absorbance value was measured using a UV spectrophotometer, and the drug release percentage was determined based on the standard curve. Figure 4 A, the release of quercetin under glucose stimulation for 24 h was more than twice that incubated in phosphate buffer, e.g. Figure 4 B, As time goes by, the skin biomimetic hydrogel shows a quercetin release cycle of up to 16 days, which shows that the skin biomimetic hydrogel has the purpose of self-regulating drug release and sustained drug delivery. The release curve of levofloxacin hydrochloride within 24 hours is shown in Figure 4 C, indicating that the release rate of levofloxacin hydrochloride was also improved after the cleavage of the boronate group.

[0057] Example 5

[0058] Study on the self-regulating mechanism of skin-mimetic hydrogel

[0059] First, the biomimetic hydrogel (QL@MAB) was immersed in phosphate buffer or 4 mg / mL glucose solution and incubated in a 37°C constant temperature shaker for 3 days. The surface and cross section of the hydrogel were scanned using a 3D laser microscope, and the three-dimensional morphology of the hydrogel surface was scanned using a 3D ultra-depth microscope.

[0060] The cross-section and surface of the 3D laser microscope scans are shown in Figure 2. Figure 5 A and 5B, 3D ultra-depth scanning of the gel surface is shown in Figure 5 C, and the average roughness statistics are shown in Figure 5 D. The results show that after glucose stimulation, the surface of the skin biomimetic hydrogel (QL@MAB) of the present invention is notched, forming a penetration channel similar to pore dilation. At the same time, the 3D super depth of field image shows that QL@MAB presents a more irregular surface. This indicates that after glucose stimulation, the borate group breaks and the gel morphology becomes rougher.

[0061] Example 6

[0062] Study on the antibacterial activity of skin-mimetic hydrogel in vitro

[0063] First, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) strains frozen in glycerol tubes were taken out and gradually adapted to room temperature. Then, they were cultured overnight in LB broth in a constant temperature incubator at 37°C and 220rpm. When the bacterial solution became turbid, the bacterial activity was evaluated with an ELISA instrument to ensure that the bacteria resumed reproduction. Then, the antibacterial ability of the four gels was evaluated using the inhibition zone method. Specifically, the bacterial suspension (1×10 6 CFU / mL, 100μL) was spread on LB agar plate and evenly coated, the gel was dispersed on the agar plate and placed in a 37℃ constant temperature incubator for culture. The diameter of the inhibition ring was measured on the 1st, 4th, 7th and 10th day, and the diameter of the inhibition ring was counted and recorded by ImageJ software. Figure 6 As shown in A, gels 1, 2, 3 and 4 are blank hydrogel group (MAB), quercetin single hydrogel group (Q@MAB), levofloxacin hydrochloride single hydrogel group (L@MAB), quercetin and levofloxacin hydrochloride dual hydrogel group (QL@MAB), and the curve of the change of the diameter of the inhibition zone is shown in Figure 6 B inhibits the diameter of E. coli and Figure 6 C inhibition of Staphylococcus aureus diameter statistics. The results showed that L@MAB and QL@MAB groups had long-term antibacterial activity against Staphylococcus aureus and Escherichia coli.

[0064] Example 7

[0065] Study on the migration of endothelial cells in vitro using skin-mimetic hydrogels

[0066] 5 × 10 4 HUVECs cells were placed in 5% CO 2The cells were cultured overnight in a cell culture incubator. The cells were then scraped with a 200 μL pipette tip and washed with phosphate buffer solution to remove cell debris. DMEM medium containing 100 ng / mL lipopolysaccharide (LPS) was then added for culture, and the group without LPS stimulation was named Control (-) group. At the same time, a transwell chamber was placed in the culture medium and a blank hydrogel group (MAB), a quercetin single-loaded hydrogel group (Q@MAB), a levofloxacin hydrochloride single-loaded hydrogel group (L@MAB), and a quercetin and levofloxacin hydrochloride double-loaded hydrogel group (QL@MAB) were added, and photographed and observed under an inverted microscope at 12h, 18h, 24h, and 36h. The cell migration rate was calculated according to the formula:

[0067] Migration rate = (initial wound area - timed wound area) / initial wound area × 100%

[0068] Cell scratch test Figure 7 A, and the cell migration rate change curve is shown in Figure 7 B. The results show that the skin biomimetic hydrogel (QL@MAB) of the present invention has significant cell migration promoting activity.

[0069] Example 8

[0070] Therapeutic effect of skin-mimetic hydrogel on diabetic infected wounds

[0071] Establishment of diabetic rat model:

[0072] 200-240g SD rats were fasted overnight after adaptive feeding, and 65mg / kg streptozotocin (STZ) was intraperitoneally injected in the fasting state of the rats. Blood was collected from the tail vein on days 0, 3, 7, 10 and 14 to measure the fasting blood glucose level of the rats. When the blood glucose level was maintained at 16.7 mmol / l, the diabetic rat model was successfully established.

[0073] Construction of diabetic infection wound model:

[0074] Diabetic rats were anesthetized by intraperitoneal injection of 2 ml / kg sodium pentobarbital; after anesthesia, the back of the rat was used as the modeling area and hair removal was performed. The skin in the modeling area was fully excised with surgical scissors, with a diameter of 8 mm, deep to the subcutaneous layer without damaging the muscle layer, and a steel ring was sutured around the wound with 2-0 surgical sutures, and bandaged with bandages and gauze to make a diabetic full-thickness defect infection model.

[0075] Rats were randomly divided into 6 groups, each with 6 rats. After the diabetic model was established, they were randomly divided into no treatment group (Control), blank hydrogel group (MAB), quercetin single hydrogel group (Q@MAB), levofloxacin hydrochloride single hydrogel group (L@MAB), quercetin and levofloxacin hydrochloride dual hydrogel group (QL@MAB). The gels of each group were replaced on the 3rd, 7th, 10th and 12th days, and representative pictures of wound healing were taken. The wound area was analyzed using Image J analysis software (n=6). The wound healing rate was calculated as follows:

[0076] Healing rate = (initial wound area - timed wound area) / initial wound area × 100%

[0077] Comparison of wound healing:

[0078] like Figure 8 A shows that the quercetin and levofloxacin hydrochloride dual-loaded hydrogel group (QL@MAB) significantly accelerated the healing of wounds covered by hair after 12 days of treatment, while unhealed wounds were still visible in other groups. In order to better visualize the wound healing rate of each group, Figure 8 B depicts the trajectory of the healing area at each time point. Figure 8 C plotted the corresponding healing curve. After 12 days of treatment, the wound healing of the untreated group and the blank hydrogel group (MAB) was delayed. In contrast, the wound healing rates of the quercetin single hydrogel group (Q@MAB), the levofloxacin hydrochloride single hydrogel group (L@MAB), and the QL@MAB group were effectively improved, among which QL@MAB had the most ideal effect on the repair of diabetic infected wounds. In summary, QL@MAB can significantly improve the efficiency of diabetic infected wound healing, and the effect is better than other groups. It is an ideal dressing for promoting the healing of diabetic infected wounds.

[0079] Example 9

[0080] Study on the sustained antibacterial ability of skin-mimetic hydrogel on wounds

[0081] The survival rate of Staphylococcus aureus in the wound infection of diabetic rats after treatment with different dressings was determined using the spread plate method. Specifically, on the 3rd, 7th and 10th days after surgery, the skin and subcutaneous tissue around the wound were gently wiped with a cotton swab to extract the bacteria from the wound. The cotton swab was then immersed in LB broth and incubated at 37°C for 7 hours. Next, the incubated bacterial extract was spread on the LB agar plate and incubated upside down in a constant temperature incubator at 37°C for 24 hours. Representative photos of the bacterial spread culture were taken, and bacterial growth was evaluated by colony counting. See the actual picture of bacterial spread. Fig. 9 A. Statistical analysis of antibacterial rates during different wound healing processes. Fig. 9B. The results show that the skin biomimetic hydrogel (QL@MAB) of the present invention has a significant and sustained antibacterial effect in vivo.

[0082] It should be noted that the embodiments described herein are only partial embodiments of the present invention, rather than all implementations of the present invention, and the embodiments are only exemplary, and their role is only to provide a more intuitive and clear way to understand the content of the present invention, rather than to limit the technical solutions described in the present invention. Without departing from the concept of the present invention, all other implementation methods that can be thought of by ordinary technicians in this field without creative work, and other simple replacements and various changes to the technical solutions of the present invention, all belong to the protection scope of the present invention.

Claims

1. A self-regulating skin biomimetic hydrogel, characterized by: The self-regulating skin biomimetic hydrogel is prepared by polymerization reaction of methyl acrylate and 3-acrylamidophenylboronic acid, and loaded with polyphenol hydrophobic drugs and antibiotics; methyl acrylate and 3-acrylamidophenylboronic acid are uniformly dispersed and dissolved in dimethyl sulfoxide (DMSO), a cross-linking agent and an initiator are added, and an organic gel is prepared by photoinitiation; the organic gel is loaded with polyphenol hydrophobic drugs and antibiotics by an immersion method, and then the drug-loaded gel is immersed in purified water, and the skin biomimetic hydrogel is obtained by solvent replacement.

2. The self-regulating skin biomimetic hydrogel according to claim 1, characterized in that: The crosslinking agent is 1,4-butanediol diacrylate, and the initiator is diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide.

3. The self-regulating skin biomimetic hydrogel according to claim 1, characterized in that: The organic gel is photo-initiated by ultraviolet light irradiation, and the irradiation time is 5 minutes to 120 minutes; the mass fraction of methyl acrylate is 10% to 30%, the mass fraction of 1,4-butanediol diacrylate is 0.3% to 3%, the mass fraction of diphenyl (2,4,6-trimethylbenzoyl) phosphorus oxide is 0.1% to 1%, and the feeding of 3-acrylamidophenylboric acid is carried out according to the molar ratio of 3-acrylamidophenylboric acid to methyl acrylate of 0.5:99.5 to 20:

80.

4. The self-regulating skin biomimetic hydrogel according to claim 1, characterized in that: The polyphenol hydrophobic drug and antibiotic are dissolved in DMSO to prepare a drug mixture with a mass fraction of 0.1% to 2%. The organogel is immersed in the drug mixture for 24h to 72h and the drug is loaded by a physical impregnation method.

5. The self-regulating skin biomimetic hydrogel according to claim 4, characterized in that: The polyphenol hydrophobic drug is a mixture of one or more of quercetin, ellagic acid, caffeic acid, luteolin, lithospermic acid, myricetin, baicalin, rutin and their derivatives; the antibiotic is a mixture of one or more of levofloxacin or its salt, ciprofloxacin or its salt, moxifloxacin or its salt, norfloxacin or its salt, amoxicillin, vancomycin or its salt, gentamicin or its salt, doxycycline or its salt.

6. The self-regulating skin biomimetic hydrogel according to claim 1, characterized in that: The gel is immersed in purified water at room temperature for 24 to 72 hours. After sufficient solvent replacement, the DMSO in the gel cavity is replaced by purified water and swells, thereby obtaining a skin biomimetic hydrogel.

7. A method for preparing the self-regulating skin biomimetic hydrogel according to any one of claims 1 to 6, characterized in that: A method for preparing a self-regulating bionic hydrogel is obtained by the following steps: Preparation of organic gel: The organic gel is prepared by ultraviolet light-induced free radical polymerization; the prescribed amount of methyl acrylate, 3-acrylamidophenylboronic acid and 1,4-butanediol diacrylate are weighed and dissolved in a DMSO solution, the solution is fully dissolved by ultrasonic treatment and protected from light, the prescribed amount of diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide is weighed and dissolved to obtain a gel solution, and then the gel solution is irradiated with ultraviolet light to obtain an organic gel, and then the organic gel is soaked in an excess of DMSO solvent to wash away unreacted monomers and fully swell to obtain a purified organic gel; Preparation of skin biomimetic hydrogel: Step 1, dissolving 10% to 30% methyl acrylate, 0.3% to 3% 1,4-butanediol diacrylate, 0.1% to 1% diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, and 3-acrylamidophenylboronic acid in a DMSO solution, wherein the molar ratio of 3-acrylamidophenylboronic acid to methyl acrylate is 0.5:99.5 to 20:80, and preparing an organic gel by ultraviolet light initiation, dissolving a polyphenol hydrophobic drug and an antibiotic in a DMSO solution to form a drug mixture, and immersing the organic gel in the drug mixture for 24 hours to 72 hours to obtain a drug-loaded organic gel; Step 2: Soak the drug-loaded organogel in purified water with a ratio of the drug-loaded organogel to the water solvent of 1:50 to 1:100, and let it stand at room temperature for 24 hours to 72 hours to obtain a self-regulating skin biomimetic hydrogel.

8. The method for preparing the self-regulating skin biomimetic hydrogel according to claim 7, characterized in that: The mass fraction of the methyl acrylate is 10% to 30%, the mass fraction of the 1,4-butanediol diacrylate is 0.3% to 3%, the mass fraction of the diphenyl (2,4,6-trimethylbenzoyl) phosphorus oxide is 0.1% to 1%, and the 3-acrylamidophenylboric acid is added according to a molar ratio of 3-acrylamidophenylboric acid to methyl acrylate of 0.5:99.5 to 20:80; the DMSO is an anhydrous ultra-dry DMSO solution, the wavelength of the ultraviolet lamp is 365nm, and the illumination time is 5min to 120min.

9. The method for preparing the self-regulating skin biomimetic hydrogel according to claim 7, characterized in that: The drug mixture is obtained by dissolving a polyphenol hydrophobic drug and an antibiotic in DMSO at a mass fraction of 0.1% to 2% and mixing them, and the gel is immersed in the drug mixture for 24 hours to 72 hours; the gel is immersed in purified water, the ratio of the drug-loaded organic gel to the water solvent is 1:50 to 1:100, and the immersion time is 24 hours to 72 hours.

10. A use of the self-regulating skin biomimetic hydrogel according to any one of claims 1 to 6, characterized in that: Self-regulating skin-mimetic hydrogels are used to prepare dressings for diabetic infected wounds.

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