Preparation method and application of niacin-loaded flexible nanoparticle intelligent hydrogel

Abstract

The invention relates to a preparation method and application of niacin-loaded flexible nanoparticle intelligent hydrogel, and relates to the technical field of biological materials, the preparation method comprises the following steps: obtaining polymethyl methacrylate-butyl acrylate nanoparticles; the preparation method comprises the following steps: carrying out amidation reaction on silk fibroin, N-hydroxysuccinimide, carbodiimide and 3-aminophenylboronic acid to obtain a silk fibroin-3-aminophenylboronic acid compound; and carrying out an illumination cross-linking curing reaction on the polymethyl methacrylate-butyl acrylate nanoparticles, acrylamide, the silk fibroin-3-aminophenylboronic acid compound and nicotinic acid, so as to obtain the nicotinic acid-loaded flexible nanoparticle intelligent hydrogel. The niacin-loaded flexible nanoparticle intelligent hydrogel provided by the invention has the functions of wet tissue adhesion, stretchability, sustained release of niacin, pH response, photonic band gap effect, angiogenesis promotion and the like, can be used as a drug carrier in the fields of drugs for promoting angiogenesis and the like, and has wide practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a preparation method and application of a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid. Background Art

[0002] The cross-region perforator flap is a commonly used flap for repairing soft tissue defects of the extremities. However, distal necrosis of the flap remains a challenging problem. The perforator vascular body is the basic anatomical unit of the perforator flap and can be divided into three regions, including the anatomical region from the source artery, the dynamic region adjacent to the anatomical region, and the potential region adjacent to the dynamic region. The flap has relatively stable survival in the anatomical and dynamic regions, and necrosis often occurs in the potential region. The vessels with gradually decreasing caliber and anastomosing with each other between adjacent vascular body regions are called choke vessels, which are extremely important for the survival of the cross-region perforator flap. The anastomosis area of choke vessels between the anatomical region and the dynamic region is called the Chock I area, and the anastomosis area of choke vessels between the dynamic region and the potential region is called the Choke II area. The insufficient dilation and neovascularization of the vessels in the Choke II area are one of the important reasons for flap potential region necrosis. Improving the blood supply in the Choke II area is crucial for the survival of the flap.

[0003] In recent years, in order to improve the survival rate of cross-region perforator flaps, scholars have dilated and / or promoted neovascularization of the vessels in the Choke II area through surgical flap internal pressurization techniques and drug interventions to improve the blood supply in the Choke II area and increase the survival area of the flap. However, the surgical internal pressurization technique requires dissecting anastomosing vessels in the operative area, and the vessels here are often absent clinically. Drug intervention has gradually become a research hotspot for flap expansion due to its advantages such as high safety, small trauma, and little pain.

[0004] Many scholars at home and abroad have tried different vasoactive drugs to pre-treat cross-region perforator flaps, but all the studies conducted are systemic drug administrations. Systemic drug administration often requires a higher drug concentration compared with local drug administration. Local drug administration can reduce the side effects of drugs on the body, be more targeted, and at the same time avoid the interaction between drugs. Local delivery of drugs by hydrogel materials is one of the means to promote repair. Therefore, developing an efficient drug hydrogel delivery carrier to ensure the activity of drugs during transportation and controlled release, in order to show excellent tissue repair promotion ability, is the focus issue in this field. Summary of the Invention

[0005] To solve the above problems, the present invention provides a preparation method and application of a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid.

[0006] In a first aspect, the present invention provides a method for preparing a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid, and the preparation method includes the following steps:

[0007] Obtain polymethyl methacrylate-butyl acrylate nanoparticles;

[0008] Perform an amidation reaction on silk fibroin, N-hydroxysuccinimide, carbodiimide and 3-aminophenylboronic acid to obtain a silk fibroin-3-aminophenylboronic acid compound;

[0009] Perform a photo-crosslinking curing reaction on the polymethyl methacrylate-butyl acrylate nanoparticles, acrylamide, the silk fibroin-3-aminophenylboronic acid compound and nicotinic acid to obtain the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid.

[0010] Further, the weight ratio of the polymethyl methacrylate-butyl acrylate nanoparticles, the acrylamide, the silk fibroin-3-aminophenylboronic acid compound and the nicotinic acid is (2-8):(15-25):(15-20):(2-8).

[0011] Further, the working parameters of the photo-crosslinking curing reaction include: the added weight of the photoinitiator is 0.1-0.3 times the added weight of the polymethyl methacrylate-butyl acrylate nanoparticles, the light source is ultraviolet light, the light irradiation time is 20s-45s, and the light irradiation dose is 70-90UV.

[0012] Further, the step of obtaining the polymethyl methacrylate-butyl acrylate nanoparticles includes the following process:

[0013] Add methyl methacrylate, butyl acrylate, ethylene glycol dimethacrylate, acrylic acid and acrylamide to water for mixing to obtain a first mixture;

[0014] Under an inert gas atmosphere, perform a first heating and stirring on the first mixture, and then add an aqueous solution containing ammonium persulfate and sodium polystyrene sulfonate for a second heating and stirring to obtain a second mixture;

[0015] Perform centrifugal separation and filtration on the second mixture, collect the solid product and wash it to obtain the polymethyl methacrylate-butyl acrylate nanoparticles.

[0016] Further, the weight ratio of methyl methacrylate, butyl acrylate, ethylene glycol dimethacrylate, acrylic acid, acrylamide, ammonium persulfate, and sodium polystyrene sulfonate is (1.5 - 3.5):(2 - 3):(0.04 - 0.07):(0.1 - 0.3):(0.1 - 0.35):(0.1 - 0.3):(0.02 - 0.08); the working parameters of the first heating and stirring include: the first heating and stirring temperature is 80 - 100 °C, and the first heating and stirring time is 0.3 - 0.7 hours; the working parameters of the second heating and stirring include: the second heating and stirring temperature is 80 - 100 °C, and the second heating and stirring time is 7 - 10 hours.

[0017] Further, the steps of subjecting silk fibroin, N-hydroxysuccinimide, carbodiimide, and 3-aminophenylboronic acid to an amidation reaction to obtain a silk fibroin-3-aminophenylboronic acid compound include the following process:

[0018] Dissolve silk fibroin in a fatty acid methyl ester sulfonate buffer solution, and then continue to add N-hydroxysuccinimide and carbodiimide at room temperature and stir to dissolve, obtaining a third mixture;

[0019] Under an inert gas atmosphere, add 3-aminophenylboronic acid to the third mixture for an amidation reaction, collect the product, and dialyze it in distilled water to obtain the silk fibroin-3-aminophenylboronic acid compound.

[0020] Further, the weight ratio of silk fibroin, N-hydroxysuccinimide, carbodiimide, and 3-aminophenylboronic acid is (0.8 - 1.2):(0.8 - 1.2):(0.4 - 0.7):(1.0 - 1.5); the working parameters of the amidation reaction include: the amidation reaction temperature is room temperature, and the amidation reaction time is 10 - 14 hours.

[0021] In a second aspect, the present invention provides a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid, and the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid is prepared by using the preparation method according to any one of the first aspect.

[0022] In a third aspect, the embodiments of the present application provide an application of the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid according to any one of the second aspect in the preparation of a drug. The flexible nanoparticle intelligent hydrogel loaded with nicotinic acid is used as a drug carrier, and / or, the nicotinic acid in the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid is used as a drug efficacy component.

[0023] In a fourth aspect, the embodiments of the present application provide a drug for promoting angiogenesis, and the drug includes the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid according to any one of the second aspect.

[0024] The above technical solutions provided by the embodiments of the present invention have at least the following advantages compared with the prior art:

[0025] The embodiments of the present invention provide a preparation method and application of a flexible nanoparticle intelligent hydrogel loaded with niacin. On the one hand, the present invention synthesizes polymethyl methacrylate-butyl acrylate nanoparticles (P(MMA-BA)) for the first time, enabling the hydrogel to have a photonic bandgap effect, that is, the hydrogel will change color with stretching, which enables the hydrogel to sense the change in flap tension. Or it can be said that the change in the position of the photonic bandgap of this photonic crystal hydrogel in response to external stimuli can be converted into a visible color change, thereby realizing semi-quantitative colorimetric detection of external stimuli; on the other hand, the introduction of borate bonds makes the hydrogel have a pH response and has a better drug release rate in an environment with a low pH value at the wound surface. At the same time, the flexible nanoparticle intelligent hydrogel loaded with niacin can locally deliver and release niacin. Conventional drug use is systemic administration, but there is insufficient angiogenesis in the Choke II area, and it is difficult for drugs administered systemically to reach the Choke II area. Therefore, niacin is loaded on the hydrogel and applied locally to release niacin locally, avoiding the disadvantage that drugs administered systemically are difficult to reach the lesion.

[0026] In summary, the flexible nanoparticle intelligent hydrogel loaded with niacin provided by the present invention has functions such as wet tissue adhesion, stretchability, slow release of niacin, pH response function, photonic bandgap effect, and angiogenesis promotion, and can be used as a drug carrier in fields such as drugs for promoting angiogenesis, and has broad practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention.

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is a schematic flow chart of a preparation method of a flexible nanoparticle intelligent hydrogel loaded with niacin provided by the embodiments of the present invention.

[0030] Figure 2 It is a schematic flow chart of a preparation method of a flexible nanoparticle intelligent hydrogel loaded with niacin provided by Embodiment 1 of the present invention.

[0031] Figure 3Physical photograph of the flexible nanoparticle intelligent hydrogel loaded with niacin provided in Example 1 of the present invention.

[0032] Figure 4 Infrared spectrum of the flexible nanoparticle intelligent hydrogel loaded with niacin provided in Example 1 of the present invention.

[0033] Figure 5 XPS spectrum of the flexible nanoparticle intelligent hydrogel loaded with niacin provided in Example 1 of the present invention.

[0034] Figure 6 SEM spectrum of the flexible nanoparticle intelligent hydrogel loaded with niacin provided in Example 1 of the present invention.

[0035] Figure 7 SEM spectrum of P(MMA-BA) in the flexible nanoparticle intelligent hydrogel loaded with niacin provided in Example 1 of the present invention.

[0036] Figure 8 Stress-strain test and rheology test results of the flexible nanoparticle intelligent hydrogel loaded with niacin provided in Example 1 of the present invention.

[0037] Figure 9 Test results in Test Example 2 of the present invention Figure 1 。

[0038] Figure 10 Test results in Test Example 2 of the present invention Figure 2 。

[0039] Figure 11 Test results in Test Example 2 of the present invention Figure 3 。

[0040] Figure 12 Test results in Test Example 2 of the present invention Figure 4 。

[0041] Figure 13 Test results in Test Example 2 of the present invention Figure 5 。 Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Unless otherwise specified, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or prepared by existing methods.

[0044] In a first aspect, the present invention provides a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid, as Figure 1 shown, and the preparation method includes the following steps:

[0045] Obtain polymethyl methacrylate-butyl acrylate nanoparticles;

[0046] Perform an amidation reaction on silk fibroin, N-hydroxysuccinimide, carbodiimide, and 3-aminophenylboronic acid to obtain a silk fibroin-3-aminophenylboronic acid compound;

[0047] Perform a photo-crosslinking curing reaction on the polymethyl methacrylate-butyl acrylate nanoparticles, acrylamide, the silk fibroin-3-aminophenylboronic acid compound, and nicotinic acid to obtain the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid.

[0048] The embodiment of the present invention provides a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid. This flexible nanoparticle intelligent hydrogel loaded with nicotinic acid has functions such as wet tissue adhesion, stretchability, slow release of nicotinic acid, pH response function, photonic bandgap effect, and angiogenesis promotion, and can be used as a drug carrier in fields such as drugs for promoting angiogenesis, having broad practical application value.

[0049] In the present invention, the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid can locally deliver and release nicotinic acid. Conventional drug use is systemic administration, but there is insufficient angiogenesis in the Choke II area, and it is difficult for drugs administered systemically to reach the Choke II area. Therefore, nicotinic acid is loaded on the hydrogel and applied locally to release nicotinic acid locally, avoiding the disadvantage that drugs administered systemically are difficult to reach the lesion. At the same time, "intelligent" in this application means that on the one hand, polymethyl methacrylate-butyl acrylate nanoparticles (P(MMA-BA)) make the hydrogel have a photonic bandgap effect, that is, the hydrogel will change color with stretching, which enables the hydrogel to sense the change in flap tension. Or it can be said that the change in the photonic bandgap position of this photonic crystal hydrogel in response to external stimuli can be converted into a visible color change, thereby realizing semi-quantitative colorimetric detection of external stimuli; on the other hand, the introduction of borate bonds makes the hydrogel have a pH response and has a better drug release rate in an environment with a low pH value at the wound surface.

[0050] In the present invention, polymethyl methacrylate-butyl acrylate nanoparticles (abbreviated as P(MMA-BA) in English).

[0051] In some specific embodiments, the weight ratio of the polymethyl methacrylate-butyl acrylate nanoparticles, the acrylamide, the silk fibroin-3-aminophenylboronic acid compound, and the nicotinic acid is (2-8):(15-25):(15-20):(2-8).

[0052] In some specific embodiments, the working parameters of the photo-crosslinking curing reaction include: the added weight of the photoinitiator is 0.1-0.3 times the added weight of the polymethyl methacrylate-butyl acrylate nanoparticles, the light source is ultraviolet light, the light irradiation time is 20s-45s, and the light irradiation dose is 70-90UV.

[0053] In some specific embodiments, the step of obtaining the polymethyl methacrylate-butyl acrylate nanoparticles includes the following process:

[0054] Methyl methacrylate (abbreviated as MMA in English), butyl acrylate (abbreviated as BA in English), ethylene glycol dimethacrylate (abbreviated as EGDMA in English), acrylic acid (abbreviated as AA in English), and acrylamide (abbreviated as AAM in English) are added to water and mixed to obtain a first mixture;

[0055] In an inert gas atmosphere, the first mixture is subjected to first heating and stirring, and then an aqueous solution containing ammonium persulfate (abbreviated as APS in English) and sodium polystyrene sulfonate (abbreviated as NaPSS in English) is added for second heating and stirring to obtain a second mixture;

[0056] The second mixture is centrifuged and filtered, and the solid product is collected and washed to obtain the polymethyl methacrylate-butyl acrylate nanoparticles.

[0057] In some specific embodiments, the weight ratio of the methyl methacrylate, the butyl acrylate, the ethylene glycol dimethacrylate, the acrylic acid, the acrylamide, the ammonium persulfate, and the sodium polystyrene sulfonate is (1.5-3.5):(2-3):(0.04-0.07):(0.1-0.3):(0.1-0.35):(0.1-0.3):(0.02-0.08); the working parameters of the first heating and stirring include: the first heating and stirring temperature is 80-100°C, and the first heating and stirring time is 0.3-0.7 hours; the working parameters of the second heating and stirring include: the second heating and stirring temperature is 80-100°C, and the second heating and stirring time is 7-10 hours.

[0058] In some specific embodiments, the steps of performing an amidation reaction on silk fibroin (abbreviated as SF in English), N-hydroxysuccinimide (abbreviated as NHS in English), carbodiimide (abbreviated as EDC in English), and 3-aminophenylboronic acid (abbreviated as PBA in English) to obtain a silk fibroin-3-aminophenylboronic acid compound include the following processes:

[0059] Dissolve silk fibroin in a fatty acid methyl ester sulfonate buffer solution, and then continue to add N-hydroxysuccinimide and carbodiimide at room temperature and stir to dissolve, obtaining a third mixture;

[0060] Under an inert gas atmosphere, add 3-aminophenylboronic acid to the third mixture for an amidation reaction, collect the product, and dialyze it in distilled water to obtain the silk fibroin-3-aminophenylboronic acid compound.

[0061] In some specific embodiments, the weight ratio of the silk fibroin, the N-hydroxysuccinimide, the carbodiimide, and the 3-aminophenylboronic acid is (0.8 - 1.2):(0.8 - 1.2):(0.4 - 0.7):(1.0 - 1.5); the working parameters of the amidation reaction include: the amidation reaction temperature is room temperature, and the amidation reaction time is 10 - 14 hours.

[0062] It should be noted that for the operation steps involved in the preparation method of the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid provided in the embodiments of the present invention, if there is no special description, they can be carried out according to the conventional operations in the art or using existing equipment according to the operation manual; at the same time, for the raw materials used in the preparation method, if there is no special limitation or description, each component can directly adopt commercially available products or be prepared by oneself according to the existing preparation process.

[0063] In a second aspect, based on the same inventive concept, the present invention provides a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid, and the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid is prepared by using the preparation method according to any one of the first aspect.

[0064] The flexible nanoparticle intelligent hydrogel loaded with nicotinic acid provided by the present invention has functions such as wet tissue adhesion, stretchability, slow release of nicotinic acid, pH response function, photonic band effect, and angiogenesis promotion, and can be used as a drug carrier in fields such as drugs for promoting angiogenesis, and has wide practical application value.

[0065] In a third aspect, based on the same inventive concept, the embodiments of the present application provide an application of the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid according to any one of the second aspect in the preparation of drugs, and the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid is used as a drug carrier, and / or, nicotinic acid in the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid is used as a drug efficacy component.

[0066] In a fourth aspect, based on the same inventive concept, embodiments of the present application provide a drug for promoting angiogenesis, and the drug includes the flexible nanoparticle intelligent hydrogel loaded with nicotinic acid according to any one of the second aspect.

[0067] In some specific embodiments, the drug efficacy components loaded on the drug carrier may also be selected from existing drugs such as Danhong and tanshinone IIA.

[0068] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually determined according to national standards. If there is no corresponding national standard, they are carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0069] Example 1

[0070] This example provides a flexible nanoparticle intelligent hydrogel loaded with nicotinic acid, and its preparation schematic diagram is as Figure 2 shown, and specifically includes the following steps:

[0071] Step (1) Preparation of P(MMA-BA) particles: Mix 3 mL of MMA, 3 mL of BA, 50 μL of EGDMA, 0.2 mL of AA, and 0.2 g of AAm monomers with 60 mL of deionized water; heat the mixture to 90 °C and stir for 0.5 hours under a nitrogen atmosphere; then, add 4 mL of an aqueous solution containing 180 mg of APS and 50 mg of NaPSS to the mixture and continue to react for 8 hours; centrifuge to collect the product, wash it 3 times with deionized water, prepare P(MMA-BA) particles, and then disperse them in 20 mL of deionized water for standby;

[0072] Step (2) Preparation of SF-PBA (i.e., silk fibroin-3-aminophenylboronic acid compound): Dissolve 1.00 g of SF in a solution with pH 5.5 in MES buffer; then, add 0.95 g of NHS and 0.58 g of EDC at room temperature and stir for 30 minutes; subsequently, add 1.20 g of PBA under a N2 atmosphere and stir at a constant temperature for 12 hours; dialyze the obtained product in distilled water (MWCO, cut-off molecular weight is 3500) for 3 days and freeze-dry to obtain SF-PBA;

[0073] Step (3) Preparation of nicotinic acid-loaded flexible nanoparticle intelligent hydrogel: Mix the SF-PBA obtained in step (2), AM, the P(MMA-BA) particles obtained in step (1), nicotinic acid, and photoinitiator 2959, and carry out a photo-crosslinking curing reaction under ultraviolet light irradiation to form a hydrogel, denoted as PPSB-1; among them, based on weight percentage, the addition amounts of each raw material are: 20% SF-PBA, 5% P(MMA-BA) colloidal nanoparticles, 20% PAM, 1% 2959, 5% nicotinic acid; the irradiation time of the photo-crosslinking curing reaction is 30 s, and the irradiation dose is 80 UV.

[0074] Test Example 1

[0075] In this example, the nicotinic acid-loaded flexible nanoparticle intelligent hydrogel obtained in Example 1 was characterized. The physical picture, infrared spectrum, and XPS of the obtained nicotinic acid-loaded flexible nanoparticle intelligent hydrogel are respectively as Figure 3 , Figure 4 and Figure 5 shown.

[0076] In this example, the surface morphology of the obtained nicotinic acid-loaded flexible nanoparticle intelligent hydrogel was also analyzed. The hydrogel was frozen at -20 °C for 12 hours, and then the water in the gel was removed using a freeze dryer. The cross-sectional morphology of the hydrogel was observed using a scanning electron microscope (SEM) to study the internal cross-linked network structure. The SEM map of the nicotinic acid-loaded flexible nanoparticle intelligent hydrogel is as Figure 6 shown, and the SEM map of P(MMA-BA) is as Figure 7 shown. From Figure 6 and Figure 7 , it can be seen that: the particle size of P(MMA-BA) is about 150 nm, Figure 7 the PPSB hydrogel shows a porous 3D structure.

[0077] In this example, the stress-strain test and rheological test of the obtained nicotinic acid-loaded flexible nanoparticle intelligent hydrogel were also carried out, and the results are respectively as Figure 8 ( Figure 8 where e in Figure 8 is the stress-strain test result, and h in Figure 8 is the rheological test result) shown. From Figure 8 , it can be seen that: (1) The PPSB hydrogel has better mechanical properties compared with PAM and PAM / SF-PBA; (2) The storage modulus G' of the hydrogel is greater than the loss modulus G", indicating that the hydrogel is in a liquid state.

[0078] Test Example 2 Detection of the swelling ratio, degradation rate, and drug release of PPSB hydrogel

[0079] 2.1 Detection of swelling ratio

[0080] Freeze-dry the coating and record it as the initial weight, labeled as WI. Then place the sample in PBS buffer. Every 1 hour, remove the water on the surface of the hydrogel with filter paper, weigh it and label it as WS. The swelling rate (SR) is calculated according to the following formula:

[0081] Swelling rate = (WS - WI) / WI × 100%.

[0082] 2.2 Degradation rate detection

[0083] Measure the release effect of the drug by ultraviolet absorbance. Take 0.1 g of hydrogel, dissolve it in 10 mL of PBS at a temperature of 37 °C and 100 rpm / min. Collect 1 mL of the supernatant every 1 hour and replace it with 1 mL of PBS. Measure the absorbance at λ = 280 nm using an ultraviolet spectrophotometer and calculate it through a standard curve. To characterize the pH slow-release efficacy of the coating, place the hydrogel in buffers with pH 4.5 and 7.2 for 30 minutes respectively. Subsequently, detect the pH response of the coating according to the above method.

[0084] Adhesion experiment of PPSB hydrogel

[0085] A tensile shear force test was carried out on a tensile testing machine (FLR-303, Flora Automatic Technology, China) at a test speed of 10 mm / min according to the ASTM F2255-05(2015) standard to study the adhesion performance of the hydrogel. The tensile shear joint [length (L) × width (W) = 25 × 20 mm] was completely prepared by adding 500 mL of pre-gel solution between two glass slides. Then, all samples were pressed together with a 100 g heavy object and placed for 30 minutes.

[0086] Tensile response of PPSB hydrogel

[0087] Clamp both ends of the sheet-like gel respectively, and place a graduated steel ruler below. Subsequently, start stretching. After stretching, gradually release both hands to remove the stress. Record the whole process as a video. According to the video frame corresponding to a certain moment, calculate the tensile strain corresponding to that moment, and record the structural color change at the corresponding moment through an optical fiber spectrometer.

[0088] Cytotoxicity detection of PPSB hydrogel

[0089] The cytotoxicity of the hydrogel was evaluated using a CCK-8 assay kit. BMSC cells were resuscitated and passaged three times. The culture medium was discarded, and after washing several times with PBS, 3 mL of trypsin was added, and the cells were incubated at 37 °C for 3 minutes. The digestion was terminated with DMEM containing 10% FBS. After washing once, the cell suspension was seeded in a 12-well plate and cultured in a 5% CO2 incubator at 37 °C. Subsequently, 100 cell suspensions were transferred to a new 96-well plate and incubated at 37 °C for 24 hours and 48 hours. Then, 10 μL of CCK-8 was added to each well and incubated for another 4 hours. The absorbance of the solution was measured at 450 nm using a Multiskan FC microplate reader (Thermo, US). The cell viability was calculated using the following formula:

[0090] Cell viability = (AT - A0) / (AC - A0) × 100% (2)

[0091] where AT, A0, and AC represent the absorbance of the sample group, control group, and blank group, respectively.

[0092] The culture medium in the 12-well plate was discarded, washed once with PBS, the gel was added to the well plate to form a film, and incubated at 37 °C under 5% CO2 conditions. The well plates were taken out at preset time intervals (24, 48 hours), and the gels with cells were collected in centrifuge tubes. Then, 1 mL of PBS was added to each well, and then 5 μL of fluorescein diacetate (FDA) and propidium iodide (PI) were added to each well to stain the cell membrane. After staining for 3 minutes in the dark, the stained cells were observed under a fluorescence microscope.

[0093] Test results:

[0094] As Figure 9 shown, Figure 9 in: (a) is the swelling test result of each group of hydrogels; (b) is the degradation test result of each group of hydrogels; (c) is that under different pH conditions, due to different forms of borate bonds, the pH response of PPSB hydrogels is also different; (d) is the drug release result of the hydrogel obtained in Example 1 at different pH levels. It can be Figure 9 seen that: the PPSB hydrogel released 70% of the drug at 32 hours in a pH 4.5 buffer solution and 20% of the drug in a pH 7.2 buffer solution.

[0095] As Figure 10 shown, Figure 10 in: (a) is the adhesion test result of the hydrogel; (b) is the adhesion strength test result of the hydrogel on different substrates; (c) is the adhesion diagram of the hydrogel on different substrates. It can be Figure 10It can be seen that the PPSB hydrogel exhibits a bonding strength at the MPa level on different substrates, ensuring that it does not fall off during the wound healing process.

[0096] As Figure 11 shown, Figure 11 in: a is the test result of the PPSB hydrogel for wound management; b is the spectral test result of the hydrogel; c is the test result graph of the continuous movement of the hydrogel. From Figure 11 it can be seen that: as the joint bends, the color of the hydrogel changes, which can be used to prompt wound information. As the hydrogel is continuously stretched; after several stretching cycles, the color change can still be maintained. The hydrogel exhibits a uniform structural color change during continuous stretching, and can maintain a rapid color transition and recovery even after 20 cycles.

[0097] As Figure 12 shown, Figure 12 in: a is the test result of the cell viability of the hydrogel; b is the live-dead staining result of the hydrogel. From Figure 12 it can be seen that: all hydrogels did not show any inhibitory effect on the growth of fibroblasts after co-culture for 1 day and 3 days. These results indicate that the hydrogel has good biocompatibility in biomedical applications.

[0098] As Figure 13 shown, Figure 13 in: A is the test result of the hydrogel promoting the survival of rat skin flaps; B is the result of the increased blood flow of the skin flap; C is the increased expression of CD31; D is the result of the increased vascular density. From Figure 13 it can be seen that: PPSB promotes the survival of rat distal skin flaps, which is related to promoting the increase of blood flow in the Choke II area and angiogenesis.

[0099] Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and individual values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, which applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0100] In the present invention, unless otherwise specified, the orientation terms such as "upper" and "lower" specifically refer to the drawing directions in the attached drawings. Additionally, in the description of the specification of the present invention, terms such as "comprising" and "including" mean "including but not limited to". In this text, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this text, "and / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural. In this text, "at least one" means one or more, and "multiple" means two or more. "At least one kind", "at least one (item) of the following" or similar expressions refer to any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or "at least one (item) of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0101] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a niacin-loaded flexible nanoparticle smart hydrogel, characterized in that: The preparation method comprises the following steps: obtaining polymethyl methacrylate-butyl acrylate nanoparticles; The silk fibroin, N-hydroxysuccinimide, carbodiimide and 3-aminophenylboronic acid are subjected to an amidation reaction to obtain a silk fibroin-3-aminophenylboronic acid compound; The polymethyl methacrylate-butyl acrylate nanoparticles, acrylamide, the silk fibroin-3 aminophenylboronic acid compound and nicotinic acid are subjected to a light cross-linking and curing reaction to obtain the nicotinic acid-loaded flexible nanoparticle intelligent hydrogel.

2. The method for preparing the niacin-loaded flexible nanoparticle smart hydrogel according to claim 1, characterized in that: The weight ratio of the polymethyl methacrylate-butyl acrylate nanoparticles, the acrylamide, the silk fibroin-3 aminophenylboronic acid compound and the nicotinic acid is (2-8):(15-25):(15-20):(2-8).

3. The method for preparing the niacin-loaded flexible nanoparticle smart hydrogel according to claim 1, characterized in that: The working parameters of the photocrosslinking curing reaction include: the added weight of the photoinitiator is 0.1 to 0.3 times the added weight of the polymethyl methacrylate-butyl acrylate nanoparticles, the light source is ultraviolet light, the illumination time is 20s to 45s, and the illumination dose is 70 to 90UV.

4. The method for preparing the niacin-loaded flexible nanoparticle smart hydrogel according to claim 1, characterized in that: The step of obtaining polymethyl methacrylate-butyl acrylate nanoparticles comprises the following process: Methyl methacrylate, butyl acrylate, ethylene glycol dimethacrylate, acrylic acid, and acrylamide are added into water and mixed to obtain a first mixture; Under an inert gas atmosphere, the first mixture is subjected to a first heating and stirring process, and then an aqueous solution containing ammonium persulfate and sodium polystyrene sulfonate is added thereto and subjected to a second heating and stirring process to obtain a second mixture; The second mixture is centrifuged and filtered, and the solid product is collected and washed to obtain the polymethyl methacrylate-butyl acrylate nanoparticles.

5. The method for preparing the niacin-loaded flexible nanoparticle smart hydrogel according to claim 4, characterized in that: The weight ratio of the methyl methacrylate, the butyl acrylate, the ethylene glycol dimethacrylate, the acrylic acid, the acrylamide, the ammonium persulfate and the sodium polystyrene sulfonate is (1.5-3.5):(2-3):(0.04-0.07):(0.1-0.3):(0.1-0.35):(0.1-0.3):(0.02-0.08); the working parameters of the first heating and stirring include: a first heating and stirring temperature of 80-100°C, and a first heating and stirring time of 0.3-0.7 hours; the working parameters of the second heating and stirring include: a second heating and stirring temperature of 80-100°C, and a second heating and stirring time of 7-10 hours.

6. The method for preparing the niacin-loaded flexible nanoparticle smart hydrogel according to claim 1, characterized in that: The step of subjecting silk fibroin, N-hydroxysuccinimide, carbodiimide and 3-aminophenylboronic acid to an amidation reaction to obtain a silk fibroin-3-aminophenylboronic acid compound comprises the following steps: Dissolving silk fibroin in fatty acid methyl ester sulfonate buffer, and then adding N-hydroxysuccinimide and carbodiimide at room temperature and stirring to dissolve, to obtain a third mixture; Under an inert gas atmosphere, 3-aminophenylboronic acid was added to the third mixture to carry out an amidation reaction. The product was collected and dialyzed in distilled water to obtain the silk fibroin-3-aminophenylboronic acid compound.

7. The method for preparing the niacin-loaded flexible nanoparticle smart hydrogel according to claim 6, characterized in that: The weight ratio of the silk fibroin, the N-hydroxysuccinimide, the carbodiimide and the 3-aminophenylboronic acid is (0.8-1.2):(0.8-1.2):(0.4-0.7):(1.0-1.5); the working parameters of the amidation reaction include: the amidation reaction temperature is room temperature, and the amidation reaction time is 10-14 hours.

8. A niacin-loaded flexible nanoparticle smart hydrogel, characterized in that: The niacin-loaded flexible nanoparticle smart hydrogel is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the niacin-loaded flexible nanoparticle smart hydrogel according to claim 8 in the preparation of medicines, characterized in that: The niacin-loaded flexible nanoparticle smart hydrogel serves as a drug carrier, and / or niacin in the niacin-loaded flexible nanoparticle smart hydrogel serves as a drug efficacy component.

10. A drug for promoting angiogenesis, characterized in that: The drug comprises the niacin-loaded flexible nanoparticle smart hydrogel according to claim 8.

Images