Preparation and application of a biphasic release supramolecular hydrogel

Abstract

The application discloses preparation and application of a biphasic release supramolecular hydrogel, which is made of alpha-cyclodextrin, pluronic F127, polydopamine nanoparticles loaded with a nitric oxide donor drug, L-arginine and water. The application crosslinks alpha-cyclodextrin and pluronic F127 to form a supramolecular hydrogel carrier, and mixes polydopamine nanoparticles loaded with a nitric oxide donor drug and L-arginine in the hydrogel carrier, so that a relatively effective healing effect can be achieved through two-phase release of NO and L-arginine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation and application of a biphasic release supramolecular hydrogel. Background Technology

[0002] The skin, the largest organ in the human body, plays an irreplaceable role in defending against invading pathogens, regulating body temperature, and sensing external stimuli. Once the skin is damaged, it impairs local defense against pathogens and tactile sensation, causing pain and other discomfort. Therefore, maintaining the integrity of the skin structure is crucial. Skin damage primarily leads to the accumulation and proliferation of bacteria at the wound site, often resulting in severe wound infection. Without medication, this can lead to further ulceration and delayed healing. The body's immune system also plays a role, recruiting a large number of macrophages at the wound site to clear ulcerated tissue and pathogens such as bacteria. However, its effectiveness is limited in severe bacterial infections. Long-term use of antibiotics in bacterial wounds can also lead to bacterial resistance.

[0003] Diabetes often causes serious complications, with approximately 15%-25% of diabetic patients developing foot ulcers (JAMA.2018;319(7):649.; N Engl J Med 2017;376:2367-2375). Long-term hyperglycemia can lead to peripheral vascular disease, causing local ischemic and neurological lesions, obstructing blood circulation in the legs and feet, further aggravating infection and ulceration (Clin Infect Dis.2012:54(12):e132-e173.). Currently, there are no particularly effective drugs or methods for treating diabetic foot ulcers (DFU). Clinically, treatment often involves controlling blood sugar, combined with tissue debridement, oral antibiotics, and hyperbaric oxygen therapy, but these methods are often ineffective, which is one of the reasons why diabetic foot ulcers often lead to high amputation and mortality rates. Therefore, finding a safe and effective treatment method is extremely urgent.

[0004] Traditional dressings like adhesive bandages and gauze tend to dry out and stick to the wound, requiring frequent dressing changes and potentially damaging newly formed tissue. Biocompatible hydrogels, which maintain moisture within their three-dimensional network, absorb wound exudate, keep the wound moist, and promote cell growth, have garnered significant attention in wound healing and have seen some clinical translation. However, their effectiveness in chronic wound care remains limited; aside from silver ion antibacterial dressings, most still require combined antibiotic treatment and blood sugar management. Most common wound hydrogel dressings are chemically cross-linked from biocompatible materials, resulting in high cross-linking density, small gel pores that hinder cell growth, and limited functionality, limiting their ability to promote healing. In contrast, supramolecular hydrogels, formed through physical cross-linking, are soft, have larger pores, are suitable for application to wounds, and are simple to prepare, thus attracting considerable attention in the wound dressing field.

[0005] Encapsulating photothermal agents in hydrogels and using the energy of near-infrared light to convert it into heat to kill bacteria can prevent bacterial resistance and effectively alleviate local bacterial infections in wounds. However, the effect of simple photothermal antibacterial therapy is still limited, with only about 70%-80% of bacteria killed at around 50°C. Nitric oxide (NO) has received increasing attention due to its effects of vasodilation, promoting angiogenesis, antibacterial activity, and antitumor activity. Micromolar concentrations of nitric oxide applied to wounds can effectively exert antibacterial and angiogenesis-promoting and normalizing effects (Adv. Funct. Mater. 2022, 32, 2202857; Chem. Soc. Rev., 2012, 41, 3753-3758; Adv. Mater. 2021, 33, 2103593), showing great potential in the development of high-end wound dressings. Summary of the Invention

[0006] To address the technical challenges of treating diabetic foot ulcers and the limited effectiveness of existing wound care dressings, this invention provides a biphasic release supramolecular hydrogel (BPLA gel). This hydrogel is soft in texture, suitable for application to wounds, and can effectively inhibit bacterial infection of the wound bed, shorten the inflammatory period, and promote rapid healing of chronic diabetic wounds.

[0007] The technical solution adopted in this invention is as follows:

[0008] A biphasic release supramolecular hydrogel is prepared from α-cyclodextrin, Pluronic F127, polydopamine nanoparticles loaded with a nitric oxide donor drug, L-arginine, and water. The α-cyclodextrin comprises 1%-20% of the hydrogel by mass, Pluronic F127 comprises 1%-18% by mass, BP comprises 0.1%-2% by mass, and L-arginine comprises 1%-2% by mass.

[0009] Furthermore, the nitric oxide donor drug is BNN6.

[0010] Further, the preparation method of the polydopamine nanoparticles loaded with nitric oxide donor drug is as follows: polydopamine nanoparticles and BNN6 are added to dimethyl sulfoxide, and the mixture is stirred and reacted in the dark. The reaction solution is centrifuged, washed three times with pure water, and freeze-dried to obtain polydopamine nanoparticles (BP) loaded with BNN6. The mass ratio of polydopamine nanoparticles to BNN6 is BNN6:polydopamine nanoparticles = (0.5-10):1. The stirring time is 5-48 h, and the centrifugation conditions are 6000-15000 rpm for 5-30 minutes. The preferred stirring time is 10-30 h, and the preferred centrifugation speed is 8000-13000 rpm.

[0011] In this invention, the polydopamine nanoparticles are black nanoparticles obtained by the self-polymerization of dopamine hydrochloride in an alkaline environment of pH 8-9, and can be prepared using conventional methods in the art. In one embodiment of this invention, the preparation method is as follows: 1.0 g of dopamine hydrochloride is weighed and dissolved in a mixed solvent of ethanol and water in 260 mL. The pH is adjusted to 8-9 with concentrated ammonia, and the reaction is stirred for a period of time. The reaction solution is centrifuged to obtain a polydopamine nanoparticle precipitate, which is washed with pure water, centrifuged again, and then freeze-dried to obtain the polydopamine nanoparticles. Wherein: the ratio of ethanol to water in the mixed solvent is ethanol:water = (1-8):9, the required stirring time is 5-48 h, the centrifugation speed of the reaction solution is 6000-15000 rpm, and the centrifugation time is 5-30 minutes.

[0012] The preparation method of the above-mentioned biphasic release supramolecular hydrogel includes the following steps:

[0013] Step 1: Dissolve Pluronic F127 in water to obtain an aqueous solution of Pluronic F127;

[0014] The mass fraction of the aqueous solution of Pranic F127 is 1-30%, preferably 5%-20%.

[0015] Step 2: Add polydopamine nanoparticles (BP) loaded with nitric oxide donor drug to water, disperse by ultrasonication, and then add α-cyclodextrin and L-arginine to obtain a suspension containing BP, α-cyclodextrin and L-arginine.

[0016] The concentration of BP in the suspension is 1-20 mg / mL, the concentration of α-cyclodextrin is 50-500 mg / mL, and the concentration of L-arginine is 10-100 mg / mL. Preferably, the concentration of BP is 1-10 mg / mL, the concentration of α-cyclodextrin is 100-320 mg / mL, and the concentration of L-arginine is 10-60 mg / mL.

[0017] Step 3: Mix the aqueous solution from Step 1 and the suspension from Step 2, and let it stand to obtain a biphasic release supramolecular hydrogel.

[0018] The final mass fraction of Pronnic F127 is 1%-18%, preferably 3%-14%; the final mass fraction of α-cyclodextrin is 1%-20%, preferably 3%-15%; the final concentration of BP is 1-10 mg / mL, preferably 1-6 mg / mL; and the final concentration of L-arginine is 5-30 mg / mL, preferably 10-20 mg / mL.

[0019] Furthermore, the number-average molecular weight of the selected Pronnick F127 is selected from one or more of the following: 8000-10000 Da, 13000 Da, and 15000 Da.

[0020] The above-mentioned biphasic release supramolecular hydrogel is used in the preparation of topical formulations for repairing diabetic ulcers.

[0021] Furthermore, the topical preparations include ointments, patches, and gels, etc.

[0022] Furthermore, the biphasic release supramolecular hydrogel is used as an active ingredient in combination with near-infrared light.

[0023] This invention provides a biphasic release supramolecular hydrogel (BPLA gel). The hydrogel's carrier is formed by inclusion complexation of α-cyclodextrin (α-CD) and the polymeric material Pluronic F127, creating a PPRX structure as crosslinking points. The hydrogel carrier contains polydopamine nanoparticles (PDA-NPs) loaded with the nitric oxide (NO) donor drug BNN6 and L-arginine. Upon irradiation with near-infrared light (808 nm), the drug-loaded PDA-NPs responsively release a large amount of NO gas, which, in conjunction with the photothermal effect of the PDA-NPs, rapidly kills bacteria at the wound site, inhibiting bacterial infection and wound inflammation. L-arginine is slowly released from the hydrogel and utilized by cells, leading to the production of endogenous NO and promoting vascular endothelial cell proliferation, migration, and angiogenesis. This biphasic release of NO and L-arginine achieves a relatively effective healing-promoting effect.

[0024] While Pluronic F127 can function as a gelling material, it requires a relatively high concentration (approximately 20-30%) to form a gel, and its viscosity is also quite high. This poses certain risks to wound dressing changes and debridement, potentially damaging new tissue and hindering the growth of wound bed cells. This invention utilizes α-CD to form a supramolecular hydrogel with Pluronic F127, significantly reducing the concentration of Pluronic F127 needed for gel formation and further improving the rheological properties of the gel, thus benefiting its application in wound care.

[0025] BNN6 is a nitric oxide donor drug that can degrade and release NO under 365nm ultraviolet light irradiation, or it can be loaded into photothermal materials and release NO after being irradiated by near-infrared laser.

[0026] This invention designs and successfully prepares a biphasic release supramolecular hydrogel that can accelerate the repair of skin wounds in chronic diabetes. This supramolecular hydrogel has a soft texture as a drug carrier, making it very suitable for wound cleaning without damaging new skin tissue during wound application and dressing changes. The addition of BP and L-arginine endows it with highly efficient antibacterial and skin vascular regeneration capabilities, which is conducive to granulation tissue growth and achieves efficient and accelerated healing of diabetic ulcer wounds.

[0027] The beneficial effects of this invention are:

[0028] (1) The supramolecular hydrogel described in this invention has larger pores than most hydrogels. Compared with existing gauze, bandages, nano-silver fiber dressings, etc., it can absorb wound exudate and maintain a moist healing environment for the wound, which is conducive to cell proliferation and migration.

[0029] (2) The supramolecular hydrogel described in this invention can exert a very efficient antibacterial effect. It can kill 99% of bacteria in the skin infection caused by diabetes and bacterial invasion, thereby shortening the inflammatory period of wound healing and quickly entering the proliferation period.

[0030] (3) The supramolecular hydrogel described in this invention can use L-arginine as an intracellular NO donor to promote the proliferation and migration of local vascular endothelial cells and the rapid regeneration of blood vessels by NO osmosis, thereby providing nutrients and oxygen for the growth of new tissue and alleviating local microcirculation disorders.

[0031] (4) This invention provides a simple and efficient method for preparing supramolecular hydrogels. The prepared hydrogels are safe and non-toxic and have great potential for biomedical applications. Attached Figure Description

[0032] Figure 1 The image shows the particle size and transmission electron microscopy (TEM) image of polydopamine nanoparticles.

[0033] Figure 2 Image of the supernatant of BNN6-loaded polydopamine nanoparticles (BP) after UV irradiation.

[0034] Figure 3 The results show the drug loading, drug loading rate, and surface potential under different BNN6 and polydopamine nanoparticle mass ratios.

[0035] Figure 4 The images show the gelation process of the biphasic supramolecular hydrogel, scanning electron microscopy images of the lyophilized gel, and the release of NO and L-arginine.

[0036] Figure 5 The rheological properties of the biphase release supramolecular hydrogel are shown.

[0037] Figure 6 The photothermal effect curve of the near-infrared responsive biphase release supramolecular hydrogel is shown.

[0038] Figure 7 The results show the cytotoxicity of the hydrogel.

[0039] Figure 8 The results of plate coating of Escherichia coli and Staphylococcus aureus after gel treatment in each group.

[0040] Figure 9 Images and statistical results showing changes in wound area.

[0041] Figure 10 HE staining results for the wound. Detailed Implementation

[0042] This invention designs and successfully prepares a biphasic release supramolecular hydrogel (BPLA gel). This supramolecular hydrogel is cross-linked by physical interactions between Pluronic F127 and α-cyclodextrin. Using this hydrogel as a carrier, drug-loaded nanoparticles with near-infrared responsive release and L-arginine are encapsulated within it. The hydrogel of this invention exhibits highly efficient antibacterial activity and effectively promotes the repair of chronic diabetic wounds.

[0043] In one embodiment of this invention, a diabetes model was constructed using SD rats, and a full-thickness skin injury model was established based on this model to evaluate the therapeutic effect of the biphasic release supramolecular hydrogel of this invention.

[0044] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0047] In the following examples, the number-average molecular weight of Pluronic F127 used was 13000 Da.

[0048] Example 1

[0049] 1. Synthesis of polydopamine nanoparticles (PDA-NPs)

[0050] (1) Add 80 mL of ethanol and 180 mL of pure water (v / v = 4:9) to a 500 mL round bottom flask and mix well. Add 1.0 g of dopamine hydrochloride to dissolve and obtain a dopamine solution.

[0051] (2) Adjust the pH of the solution in step (1) to 8.5 with concentrated ammonia, stir in a water bath at 30°C for 30 min, and then transfer to room temperature and stir for 24 h to obtain a black solution.

[0052] (3) Centrifuge the black solution from step (2) at 11,000 rpm for 20 min to obtain a precipitate of polydopamine nanoparticles. Wash the precipitate three times with pure water and centrifuge to obtain polydopamine nanoparticles, which are then freeze-dried for 24 h to obtain the final product.

[0053] Figure 1 The images show the particle size and transmission electron microscopy (TEM) images of polydopamine nanoparticles. The nanoparticles exhibit a relatively round spherical shape and uniform particle size, with a particle size of 285.4 ± 8.854 nm and a PDI of 0.058 ± 0.039.

[0054] 2. Preparation of BNN6-loaded polydopamine nanoparticles (BP)

[0055] (1) Weigh polydopamine nanoparticles into DMSO and disperse them by ultrasonication to a concentration of 1 mg / mL.

[0056] (2) Weigh out BNN6 powder and dissolve it in the solution of step (1) so that the mass ratio of BNN6 to polydopamine nanoparticles is 0.5, 1, 2, 4. Stir for 20 h at room temperature and in the dark, and let stand for 2 h to obtain the reaction solution.

[0057] (3) The reaction solution from step (2) was centrifuged at 11,000 rpm for 20 min to obtain drug-loaded polydopamine nanoparticle precipitate and supernatant. The drug-loaded polydopamine nanoparticle precipitate was washed three times with pure water and centrifuged, and then freeze-dried for 24 h to obtain the final product.

[0058] The absorbance of the supernatant at 360 nm was measured using a UV spectrophotometer, and the concentration of the remaining free BNN6 in the supernatant was calculated based on the standard curve. The drug loading capacity and drug loading rate of polydopamine nanoparticles for BNN6 were then calculated.

[0059] Figure 2 This image shows the supernatant of drug-loaded nanoparticles (BP) after UV irradiation. BP was dispersed in PBS and the supernatant was obtained by centrifugation after irradiation with 365 nm UV light for 10 min. Since BNN6 decomposes under UV irradiation to produce a red substance called BPA, the supernatant of BP showed the same color as BPA after UV irradiation, indicating successful loading of BNN6.

[0060] Figure 3 Figure A shows the drug loading and loading rate of BNN6 by polydopamine nanoparticles under different mass ratios of BNN6 and polydopamine nanoparticles (m / m = 0.5, 1, 2 or 4). Figure 3 When B represents BNN6 and the mass ratio of polydopamine nanoparticles is m / m = 4, a comparison of the surface potential of BP and the surface potential of blank polydopamine nanoparticles shows that the absolute value of the surface potential of BP increases. In the following examples, all BP samples were prepared with a mass ratio of BNN6 and polydopamine nanoparticles of 4.

[0061] 3. Preparation of biphasic release supramolecular hydrogels (BPLA gels)

[0062] (1) Weigh 180mg of Pronic F127 into 1.580mL of pure water and heat in a 50℃ water bath until dissolved.

[0063] (2) Weigh 6.0 mg of BNN6-loaded polydopamine nanoparticles (BP) and redissolve them in 1.0 mL of pure water, then disperse them by sonication. Weigh 240 mg of α-cyclodextrin and 45 mg of L-arginine into the resulting solution, vortex to dissolve them, and obtain a suspension containing BP, L-arginine and α-cyclodextrin.

[0064] (3) Mix the solution from step (1) and the suspension from step (2) thoroughly, and vortex to dissolve them so that the final BP concentration is 2 mg / mL, the L-arginine concentration is 15 mg / mL, the mass fraction of Prönnick F127 is 6%, and the mass fraction of α-cyclodextrin is 8%. Let it stand for 1-2 hours to obtain a biphase release supramolecular hydrogel.

[0065] 4. Release Research

[0066] Take 300 μL of the mixed solution from step (4) into a centrifuge tube, let it stand for 2 hours to form a gel, add 1 mL of PBS solution, take 50 μL of the supernatant for later use, and add 50 μL of PBS. Irradiate with a 1.5W 808nm laser for 10 min, take a 50 μL sample and add PBS, take a 50 μL sample after 10 min, and add PBS. Then irradiate again for 10 min, take a 50 μL sample and add PBS, and repeat this cycle. Use parallel samples placed under natural light throughout the process as controls. Use a NO detection kit to measure the collected samples and calculate the NO release.

[0067] Figure 4 Figure A shows a comparison of the mixture before and after gelation. The results indicate that a stable hydrogel was formed after standing. Figure 4 Image B is a scanning electron microscope image of the hydrogel after freeze-drying, which shows that the hydrogel has relatively large pores and a relatively large porosity.

[0068] Figure 4 In the figure, C represents the NO release from the hydrogel under near-infrared light irradiation. The results show that, compared to no laser irradiation, the hydrogel exhibits a burst release of NO under 808 nm laser irradiation. Under natural light throughout the entire process, only a very small amount of NO is released from the hydrogel.

[0069] Figure 4 Figure D shows the release of L-arginine from the hydrogel in PBS. It can be seen that L-arginine in the gel exhibits a slow release behavior over 6-7 days in PBS.

[0070] 5. Rheological properties

[0071] The rheological properties of the two-phase release supramolecular hydrogel were investigated using a rheometer.

[0072] Figure 5 In Figure A, the changes in elastic modulus and storage modulus of the hydrogel were observed by scanning the strain from 0.1% to 100% using a rheometer at a frequency of 1Hz. The results showed that the linear viscoelastic region of the hydrogel was relatively narrow, indicating that the supramolecular hydrogel was soft.

[0073] Figure 5 With B as a fixed strain of 0.28%, frequency scanning (0.1-100Hz) was performed to observe the changes in elastic modulus and storage modulus. The results showed that the state was gel-like within the range of 0.1-100Hz.

[0074] Figure 5C represents the shear-thinning properties of the hydrogel. The strain was fixed at 0.1% and 600%, the frequency was 1 Hz, the time was 60 seconds each, and the cycle was repeated three times. It can be seen that the hydrogel has excellent shear-thinning properties. Under large strain, it can transform into a fluid, which is convenient for application and debridement of wounds, while under small strain, it can return to the hydrogel state.

[0075] 6. Investigation of photothermal effect

[0076] The surface of the hydrogel was irradiated with an 808nm laser. The laser power was set to 0.5W, 1W, 1.5W and 2W respectively. The hydrogel was irradiated for 10 minutes, and the temperature of the gel was recorded every minute.

[0077] Figure 6 The photothermal effect curves of the near-infrared responsive supramolecular hydrogel were obtained. The results showed that when the laser power was 1.5W, the temperature of the hydrogel could rise to about 55℃, which can play a relatively effective photothermal role.

[0078] Test Example 1

[0079] Cytotoxicity studies

[0080] Specifically, it is divided into four groups, and the preparation methods are as follows (all are performed under aseptic conditions):

[0081] 1. Blank gel group

[0082] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0083] (2) Weigh 240 mg of α-cyclodextrin and disperse it in 1 mL of pure water;

[0084] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Let stand for 2 hours to obtain a blank gel. Freeze-dry the blank gel.

[0085] 2. Arginine gel group

[0086] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0087] (2) Weigh 240 mg of α-cyclodextrin and 45 mg of L-arginine and disperse them in 1 mL of pure water;

[0088] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Let stand for 2 hours to obtain arginine gel, and then freeze-dry it.

[0089] 3. BP gel group

[0090] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0091] (2) Weigh 6 mg of BP and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and vortex to disperse it evenly.

[0092] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Let stand for 2 hours to obtain BP gel, and then freeze-dry it.

[0093] 4. BPLA gel group

[0094] The BPLA gel assembly, also known as the biphasic release supramolecular hydrogel assembly, contains BP and L-arginine.

[0095] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0096] (2) Weigh 6 mg of BP and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and 45 mg of L-arginine and vortex to disperse evenly.

[0097] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Let stand for 2 hours to obtain a biphase release supramolecular hydrogel, and then freeze-dry it.

[0098] Under aseptic conditions, 5120 μg of the four groups of prepared and freeze-dried hydrogels were weighed, added to 1 mL of cell culture medium, and extracted by shaking for 24 hours to obtain the corresponding hydrogel extracts for cytotoxicity experiments.

[0099] The cytotoxicity of gel extracts against mouse skin fibroblast L929 cells was determined using the MTT assay. Specifically, L929 cells in the logarithmic growth phase were digested and seeded into 96-well plates, with sterile PBS added to the edge wells. After culturing the 96-well plates at 37°C and 5% CO2 for 24 h, a series of concentration gradient hydrogel extracts (5120 μg / mL, 2560 μg / mL, 1280 μg / mL, 640 μg / mL, 320 μg / mL, and 160 μg / mL) were added, with blank culture medium and PBS used as the control group. After culturing the 96-well plates for another 24 h, 20 μL of 5 mg / mL MTT solution was added and the plates were incubated for another 4 h. Subsequently, the supernatant was removed from the plates, and 150 μL of DMSO solution was added and gently shaken to dissolve any crystals. The absorbance of each well was measured using a microplate reader at a detection wavelength of 490 nm. The relative viability of the cells was calculated using the following formula:

[0100]

[0101] Wherein, ODc: absorbance of the control group, OD T : Absorbance of the experimental group, OD0: Absorbance of the blank control group.

[0102] Figure 6 The study investigated the effects of different concentrations of extracts from four hydrogels on the viability of L929 cells. The results showed that at relatively high concentrations and below, the relative viability of L929 cells remained at 90% or higher, indicating that none of the hydrogels produced significant cytotoxicity to the cells. Therefore, the hydrogels exhibited low cytotoxicity and high biosafety.

[0103] Test Example 2

[0104] Antibacterial properties study

[0105] Specifically, the group is divided into the following six groups, and the preparation method for each group is as follows:

[0106] 1. Saline group

[0107] Simply take a certain amount of sterile saline solution and dilute the bacterial solution directly.

[0108] 2. Blank gel group

[0109] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0110] (2) Weigh 240 mg of α-cyclodextrin and disperse it in 1 mL of pure water;

[0111] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Take 300 μL of the mixed solution and let it stand in a centrifuge tube for 2 hours to obtain a blank gel.

[0112] 3. Polydopamine nanoparticle gel assembly

[0113] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0114] (2) Weigh 6 mg of freeze-dried polydopamine nanoparticle powder and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and vortex disperse it.

[0115] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Take 300 μL of the mixed solution and let it stand in a centrifuge tube for 2 hours to obtain polydopamine nanoparticle gel.

[0116] 4. BP gel group

[0117] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0118] (2) Weigh 6 mg of BP and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and vortex to disperse it evenly.

[0119] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Take 300 μL of the mixed solution and let it stand in a centrifuge tube for 2 hours to obtain BP gel.

[0120] 5. Arginine gel group

[0121] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0122] (2) Weigh 240 mg of α-cyclodextrin and 45 mg of L-arginine and disperse them in 1 mL of pure water;

[0123] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Take 300 μL of the mixed solution and let it stand in a centrifuge tube for 2 hours to obtain arginine gel.

[0124] 6. BPLA gel group

[0125] The BPLA gel assembly, also known as the biphasic release supramolecular hydrogel assembly, contains BP and L-arginine.

[0126] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0127] (2) Weigh 6 mg of BP and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and 45 mg of L-arginine and vortex to disperse evenly.

[0128] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Take 300 μL of the mixed solution and let it stand in a centrifuge tube for 2 hours to obtain a biphase release supramolecular hydrogel.

[0129] The bactericidal effect of each group of gels on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was observed using the dilution plate application method. Specifically, 100 μL of gel with a concentration of 1×10⁻⁶ was used. 6CFU / mL of *E. coli* or *S. aureus* bacterial suspension was added to centrifuge tubes of each hydrogel group. The polydopamine nanoparticle gel group, BP gel group, and near-infrared responsive supramolecular hydrogel group were irradiated with an 808 nm laser for 10 min, then transferred to a 37°C incubator for 1 h. The other three groups were directly incubated at 37°C after adding bacterial suspension. After incubation, 100 μL of the supernatant bacterial suspension from each group was diluted 300-fold with sterile physiological saline, and 100 μL was spread onto an agar plate and incubated upside down at 37°C for 24 h.

[0130] Figure 8 The results of plate coating of Escherichia coli and Staphylococcus aureus after gel treatment in each group show that the biphasic release supramolecular hydrogel group and BP gel group have the best killing effect on Escherichia coli and Staphylococcus aureus, which is significantly better than the polydopamine nanoparticle gel group with only photothermal effect.

[0131] Test Example 3

[0132] Application of hydrogels in full-thickness skin wound repair in diabetic rats

[0133] Specifically, it is divided into six groups, and the preparation method for each group is as follows:

[0134] 1. Saline group

[0135] Prepare a 0.9% sodium chloride solution and filter it through a 0.22μm microporous membrane for sterilization.

[0136] 2. Blank gel group

[0137] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0138] (2) Weigh 240 mg of α-cyclodextrin and disperse it in 1 mL of pure water;

[0139] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Aspirate the solution into a 5 mL sterile syringe and let it stand for 2 hours to obtain a blank gel.

[0140] 3. Polydopamine nanoparticle gel assembly

[0141] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0142] (2) Weigh 6 mg of freeze-dried polydopamine nanoparticle powder and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and vortex disperse it.

[0143] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Then, aspirate the solution into a 5 mL sterile syringe and let it stand for 2 hours to obtain polydopamine nanoparticle gel.

[0144] 4. BP gel group

[0145] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0146] (2) Weigh 6 mg of BP and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and vortex to disperse it evenly.

[0147] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Then, aspirate the solution into a 5 mL sterile syringe and let it stand for 2 hours to obtain BP gel.

[0148] 5. Arginine gel group

[0149] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0150] (2) Weigh 240 mg of α-cyclodextrin and 45 mg of L-arginine and disperse them in 1 mL of pure water;

[0151] (3) Mix the solutions from steps (1) and (2) and vortex until completely dissolved. Then, aspirate the solution into a 5 mL sterile syringe and let it stand for 2 hours to obtain arginine gel.

[0152] 6. BPLA gel group

[0153] The BPLA gel assembly, also known as the biphasic release supramolecular hydrogel assembly, contains BP and L-arginine.

[0154] (1) Weigh 180mg of Pronic F127 into 1580μL of pure water and dissolve it in a 50℃ water bath.

[0155] (2) Weigh 6 mg of BP and ultrasonically disperse it in 1 mL of pure water. Add 240 mg of α-cyclodextrin and 45 mg of L-arginine and vortex to disperse evenly.

[0156] (3) Mix and vortex the solutions from steps (1) and (2) until completely dissolved, aspirate into a 5 mL sterile syringe and let stand for 2 hours to obtain a biphasic release supramolecular hydrogel.

[0157] A full-thickness skin defect model of diabetes was established in rats. Specifically, SD rats weighing 220-250g were selected and fed a 45% high-fat diet throughout the course of treatment. One week later, the rats were injected intraperitoneally with streptozotocin (65mg / Kg). Blood glucose levels and body weight were monitored for five consecutive days. If blood glucose was higher than 16.7mol / L for two consecutive days and body weight decreased significantly, the diabetes model was considered successfully established. After five days of monitoring, a circular full-thickness skin defect model with a diameter of 8mm was established on the back of the rats, and treatment was administered according to the experimental groups. The saline group received 0.5mL of saline injected into the wound and surrounding area, followed by a sterile dressing. The other five gel treatment groups received 200μL of gel injected into the wound, followed by a sterile dressing. The polydopamine nanoparticle gel group, BP gel group, and biphasic release supramolecular hydrogel group were irradiated with a 1.5W 808nm laser for 10 minutes after the sterile dressing was applied.

[0158] Wound changes were recorded by photographs on days 0, 3, 6, 9, and 12. Skin samples from the wound were taken on days 6, 12, and 20 for HE staining.

[0159] Figure 9 (A) Photographs of the wounds in each treatment group on days 0, 3, 6, 9, and 12. The results showed that the biphasic release supramolecular hydrogel group healed faster than the saline group and other gel treatment groups.

[0160] Figure 9 (B) is a statistical graph showing the wound area of ​​each treatment group on days 0, 3, 6, 9, and 12. The wound area decreased the fastest in the biphasic release supramolecular hydrogel group, while the other gel treatment groups also showed some degree of healing-promoting effect.

[0161] Figure 10 HE staining results of wound skin in each treatment group on days 6, 12, and 20. The results show that the biphasic-release supramolecular hydrogel group has the best effect on promoting wound healing. The saline group still has a large amount of skin defects on day 6. On day 12, except for the biphasic-release supramolecular hydrogel group and the arginine gel group, which have been completely covered by new epidermis and have generated some skin glands, the wounds in other groups still have blood crusts or incomplete new epidermis. By day 20, the new glands in the wound skin of the biphasic-release supramolecular hydrogel group are very close to the mature glands of normal skin tissue.

Claims

1. The application of biphasic release supramolecular hydrogels in the preparation of topical formulations for repairing diabetic ulcers, characterized in that, The biphasic release supramolecular hydrogel is composed of α-cyclodextrin, Pluronic F127, polydopamine nanoparticles loaded with a nitric oxide donor, L-arginine, and water; wherein the mass fraction of α-cyclodextrin is 1%-20%, the mass fraction of Pluronic F127 is 1%-18%, the mass fraction of polydopamine nanoparticles loaded with a nitric oxide donor is 0.1%-2%, the mass fraction of L-arginine is 1%-2%, and water is the balance, with the sum of the mass fractions of all components being 100%. The nitric oxide donor drug is BNN6; The preparation method of this biphasic release supramolecular hydrogel includes the following steps: Step 1: Dissolve Pluronic F127 in water to obtain an aqueous solution of Pluronic F127; Step 2: Add polydopamine nanoparticles loaded with nitric oxide donor drug to water, disperse them, and then add α-cyclodextrin and L-arginine to obtain a suspension containing polydopamine nanoparticles loaded with nitric oxide donor drug, α-cyclodextrin and L-arginine. Step 3: Mix the aqueous solution from Step 1 and the suspension from Step 2, and let stand to obtain a biphasic release supramolecular hydrogel.

2. The application according to claim 1, characterized in that, The preparation method of the polydopamine nanoparticles loaded with nitric oxide donor drug is as follows: polydopamine nanoparticles and BNN6 are added to dimethyl sulfoxide, stirred and reacted in a dark environment, the reaction solution is centrifuged, washed with pure water and then polydopamine nanoparticles loaded with BNN6 are obtained.

3. The application according to claim 1, characterized in that, When preparing the polydopamine nanoparticles loaded with the nitric oxide donor drug, the mass ratio of polydopamine nanoparticles to BNN6 is (0.5-10):1, the stirring time is 5-48 h, and the reaction solution is centrifuged at 6000-15000 rpm for 5-30 minutes.

4. The application according to claim 1, characterized in that, The number-average molecular weight of the Prunic F127 is selected from 8000-10000 Da, 13000 Da, or 15000 Da.

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