PH / ROS double-response type drug-loaded hydrogel dressing for urethral repair and preparation process thereof

Abstract

The invention discloses a pH / ROS double-response type drug-loaded hydrogel dressing for urethral repair and a preparation process thereof, and belongs to the technical field of urethral injury repair. The CPT-Cel hydrogel dressing provided by the invention is effectively adhered to and integrated into the urethra, and immediately plays a role in stopping bleeding and resisting bacteria. The hydrogel can respond to an acid environment and high-concentration ROS, on-demand and controllable release of Cel is achieved, and damage of low pH and excessive ROS to cells is relieved. In addition, the sustained release of Cel in the CPT-Cel hydrogel can inhibit the excessive activation of urethral fibroblasts in a fibrosis promoting microenvironment, prevent the excessive hyperplasia of collagen in the urethral scar formation process, recover the smoothness of the urethra, and realize the scar-free healing and functional reconstruction of the urethra.

Description

TECHNICAL FIELD

[0001] The application relates to a pH / ROS dual-responsive drug-loaded hydrogel dressing for urethral repair and a preparation process thereof, and belongs to the technical field of urethral injury repair. BACKGROUND

[0002] Urethral injury is a common and complex clinical condition, which often leads to excessive deposition of collagen and tissue fibrosis, and further causes urethral scarring, and eventually develops into urethral stenosis, and finally progresses to dysuria or even atresia. After urethral injury or defect, timely urethral repair and reconstruction surgery is the primary treatment for patients. At present, the main urethral repair and reconstruction replacement grafts in clinical practice are patient's own tissues, such as penile flaps, oral mucosa, etc. However, they are limited in size and quantity, and bring many complications to the donor site. Tissue engineering materials, especially hydrogels, are a very promising alternative material and have shown great potential in the field of regenerative medicine.

[0003] The poor urethral microenvironment and its dynamic changes after urethral injury are the main factors hindering the rational regeneration of the urethra. Specifically, the deterioration of the urethral microenvironment in the early stage of urethral injury or the flow of urine will not only induce the aggregation and excessive production of inflammatory cells, leading to oxidative stress and even cell death, but also aggravate the acidic environment of the wound, affecting cell activity, proliferation and migration. In addition, bacteria (such as Escherichia coli) in urine often cause infection, leading to tissue necrosis and serious complications. These factors together weaken cell proliferation, epithelialization and vascularization, hindering the repair and healing of the damaged urethra. More importantly, to cope with the pathological stress caused by factors such as urine and inflammation, urethral fibroblasts will differentiate into activated myofibroblasts, which express alpha-smooth muscle actin (alpha-SMA), fibronectin (FN) and other matrix proteins, and secrete them into the extracellular matrix (ECM). Although the activation of myofibroblasts is considered a beneficial protective mechanism for urethral repair, the poor urethral environment promotes the sustained overactivation of fibroblasts, leading to excessive proliferation and deposition of collagen fibers and irreversible urethral scarring. The biomaterials currently used in the field of urethra are mostly static designs or only focus on the effect of material modification, and do not have the multifunctional requirements of anti-ROS, antibacterial and hemostatic, and cannot adapt to the changes in the urethral microenvironment and the transition of the regeneration stage, and cannot achieve efficient repair and scar prevention.

[0004] Intelligent responsive hydrogel, as an advanced biomaterial platform, provides a revolutionary solution to overcome the above challenges. It can perceive and respond to specific local microenvironment stimuli (such as pH change, specific enzyme, reactive oxygen species (ROS) level, temperature or mechanical signal), achieve precise intervention, such as on-demand, precise and controllable release of active substances. This feature is particularly important for the dynamic and complex urethral microenvironment, because the microenvironment during the repair process is changing all the time. For example, high ROS levels are often present in the early stages of urethral injury, and acidic urine often causes pH changes. In this case, a single intervention or release method cannot meet the needs. Therefore, it is urgent to design hydrogels using endogenous stimuli as trigger signals in order to achieve precise release and intervention of active substances at the injury site, maximize local efficacy, and reduce systemic exposure and side effects. SUMMARY

[0005] In view of the above problems of the prior art, the present application provides a pH / ROS dual-responsive drug-loaded hydrogel dressing for urethral repair and a preparation process thereof, aiming to solve the technical problems that the biomaterials in the prior art are mostly designed statically or only focus on the effect of material modification, do not have the multifunctional requirements of ROS resistance, antibacterial and hemostatic, and cannot adapt to the changes of the urethral microenvironment and the transition of the regeneration stage, and cannot achieve efficient repair and scar prevention.

[0006] The first technical solution provided by the present application is a preparation method of an intelligent responsive hydrogel. The method is to synthesize a pH / ROS responsive hydrogel by forming a borate ester bond between borax and carboxymethyl chitosan benzene boronic acid (CMCS-BA) polymer and polyvinyl alcohol (PVA), and adding tannic acid (TA) to the pH / ROS responsive hydrogel to obtain an intelligent responsive hydrogel.

[0007] In some embodiments, the method comprises the following steps: (1) Synthesizing benzene boronic acid grafted carboxymethyl chitosan (CMCS-BA) by using benzene boronic acid (BA) and carboxymethyl chitosan (CMCS); (2) Adding tannic acid (TA) to the CMCS-BA in (1) to prepare CMCS-BA / TA (CT); (3) Mixing polyvinyl alcohol (PVA) with the CMCS-BA / TA (CT) in (2), and then adding borax to obtain an intelligent responsive hydrogel.

[0008] In some embodiments, in step (1), 1 wt% carboxymethyl chitosan solution is added to 12 g / L benzene boronic acid, 4.0 g / L EDC·HCl and 3 g / L NHS, and stirred thoroughly for 48 hours. After dialysis and lyophilization, CMCS-BA powder is obtained.

[0009] In some embodiments, in step (2), 2% tannic acid (TA) is added to the 1 wt% CMCS-BA solution.

[0010] In some embodiments, in step (3), the CMCS-BA / TA solution is mixed with the polyvinyl alcohol solution at a ratio of 1:1, and 1% borax is then added.

[0011] The second technical solution provided by the present application is a smart responsive hydrogel prepared by the method of the first technical solution.

[0012] The third technical solution provided by the present application is a drug carrier containing the hydrogel of the second technical solution.

[0013] The fourth technical solution provided by the present application is a preparation method of a pH / ROS dual-responsive drug-loaded hydrogel for urethral repair. Cel is wrapped with poly(lactic-co-glycolic acid) (PLGA) into nanoparticles (PLGA@Cel NPs) and incorporated into the hydrogel of the second technical solution or the drug carrier of the third technical solution to obtain a CPT-Cel hydrogel.

[0014] In some embodiments, the method comprises the following steps: S1, benzene boronic acid (BA) is used to synthesize carboxymethyl chitosan (CMCS-BA) grafted with carboxymethyl chitosan (CMCS); S2, tannic acid (TA) is added to the CMCS-BA in S1 to prepare CMCS-BA / TA (CT); S3, Cel is wrapped with poly(lactic-co-glycolic acid) (PLGA) into nanoparticles PLGA@Cel NPs; S4, the nanoparticles PLGA@Cel in S3 are mixed with polyvinyl alcohol (PVA), then mixed with the CMCS-BA / TA (CT) in S3, and borax is added to obtain a pH / ROS dual-responsive drug-loaded hydrogel.

[0015] In some embodiments, in S1, 1 wt% carboxymethyl chitosan solution is added with 12 g / L benzene boronic acid, 4.0 g / L EDC·HCl, and 3 g / L NHS, and stirred thoroughly for 48 hours. After dialysis and lyophilization, CMCS-BA powder is obtained.

[0016] In some embodiments, in S2, 2% tannic acid (TA) is added to the 1 wt% CMCS-BA solution.

[0017] In some embodiments, the preparation of PLGA@Cel nanoparticles is as follows: (a) The dispersed phase was prepared by mixing 5 mg / mL polylactic acid-glycolic acid copolymer chloroform solution with 1 / 30 g / mL Cel and then uniformly ultrasonic treatment; (b) The continuous phase was prepared by adding a certain amount of SDS and PVA into deionized water and heating and stirring in a 60℃ water bath until completely dissolved; (c) The dispersed phase of (a) was added into a storage tank, and the dispersed phase was pressed into the continuous phase of S32 through an SPG microporous membrane under a pressure of 0.06 MPa to obtain a monodisperse water-in-oil (O / W) emulsion; (d) The water-in-oil emulsion of (c) was transferred into a 40℃ constant temperature bath, and the volatilization of chloroform and acetone was carried out by stirring at 500 r / min for 6~8 hours to obtain PLGA@Cel microspheres.

[0018] In some embodiments, in S4, the nanoparticle PLGA@Cel is mixed with a polyvinyl alcohol (PVA) solution, and the final concentration of Cel is 0.2 μM.

[0019] In some embodiments, in S4, the CMCS-BA / TA solution is mixed with a polyvinyl alcohol solution at a ratio of 1:1, and 1% borax is added.

[0020] The fifth technical solution provided by the application is a pH / ROS dual-responsive drug-loaded hydrogel for urethral repair prepared by the method of the fourth technical solution.

[0021] The sixth technical solution provided by the application is a dressing, which comprises the drug-loaded hydrogel of the fifth technical solution.

[0022] The seventh technical solution provided by the application is the use of the method of the first technical solution, the hydrogel of the second technical solution, the drug carrier of the third technical solution, the method of the fourth technical solution, the drug-loaded hydrogel of the fifth technical solution, or the dressing of the sixth technical solution in the preparation of a product for urethral repair.

[0023] In some embodiments, the use at least includes one of the following effects: (1) Urethral blood vessel regeneration; (2) Promoting urethral epithelial regeneration; (3) Promoting urethral vascularization and cell proliferation; (4) Promoting scar-free healing of the urethra.

[0024] Compared with the prior art, the application has the following beneficial effects: The application provides a novel intelligent responsive hydrogel system, aiming at efficiently loading Cel and accurately delivering it to the urethral injury site on demand, and actively regulating the local microenvironment to achieve scarless regeneration. First, by forming a borate ester bond between carboxymethyl chitosan benzene boronic acid (CMCS-BA) polymer and polyvinyl alcohol (PVA) through borax, a pH / ROS responsive hydrogel (CP) is synthesized. The addition of tannic acid (TA), a natural source of polyphenol, endows the hydrogel with adhesion, hemostatic and antibacterial properties (referred to as CPT), so that it meets the basic requirements of urethral regeneration. Cel is wrapped into nanoparticles (PLGA@Cel NPs) by polylactic acid-glycolic acid copolymer (PLGA) and incorporated into the CPT hydrogel to obtain a multifunctional CPT-Cel hydrogel. After application, the multifunctional CPT-Cel hydrogel dressing effectively adheres and integrates into the urethra, immediately exerting hemostatic and antibacterial effects. The hydrogel can respond to an acidic environment and high concentration of ROS to achieve on-demand and controllable release of Cel, reducing the damage of low pH and excessive ROS to cells. In addition, the sustained release of Cel in the CPT-Cel hydrogel can inhibit the excessive activation of urethral fibroblasts in the pro-fibrotic microenvironment, preventing excessive proliferation of collagen during the process of urethral scar formation. In vitro experimental system verifies the multifunctional effects of CPT-Cel such as hemostasis, antibiosis, anti-inflammation and anti-fibrosis, and histological and functional evaluation shows that CPT-Cel can promote early epithelialization, vascularization and cell proliferation, reduce local ECM deposition, restore urethral patency, and achieve scarless healing and functional reconstruction of the urethra. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a schematic diagram for the preparation and application principle of the CPT double-responsive hydrogel of the application.

[0026] Figure 2 It is a synthesis and test diagram of CMCS-BA; wherein: a is the synthesis principle of CMCS-BA, b is its 1H nuclear magnetic resonance spectrum, and c is its Fourier transform infrared spectrum.

[0027] Figure 3 It is a characterization diagram of PLGA@Cel nanoparticles; wherein: a is the particle size distribution of PLGA and PLGA@Cel nanoparticles, b is their Zeta potential, and c is their transmission electron microscope image.

[0028] Figure 4 It is a characterization of CPT hydrogel; wherein: a is a schematic diagram of the hydrogel synthesis process, b is a hydrogel gelation photo, and c is a Fourier transform infrared spectrum of the CPT hydrogel.

[0029] Figure 5Figure 4 is a responsive degradation and responsive release chart of CPT-Cel hydrogel; wherein: a is the degradation rate of CPT-Cel hydrogel under each condition, b is the Cel release rate of CPT-Cel hydrogel under each condition.

[0030] Figure 6 Figure 5 is a hydrogel adhesion performance verification chart; wherein: a is a hydrogel adhesion organ photo, b is a hydrogel filling urethra photo, c is a hydrogel adhesion urethra interface scanning electron microscope chart, d is a hydrogel peeling pig skin strength test.

[0031] Figure 7 Figure 6 is a verification chart of in vitro biocompatibility; wherein: a is a hydrogel co-cultured dead cell staining, b is a cell proliferation rate.

[0032] Figure 8 Figure 7 is a hydrogel coagulation curve chart.

[0033] Figure 9 Figure 8 is a verification of in vitro hydrogel ROS, anti-inflammatory and antibacterial effect; wherein: a is the proportion of dead cells after hydrogen peroxide and hydrogel treatment, b is the free radical scavenging efficiency of each hydrogel, c is the fluorescence staining of macrophage CD86 under different treatments, d is the antibacterial experiment of hydrogel.

[0034] Figure 10 Figure 9 is a verification chart of anti-fibrosis performance; wherein: a, b are fluorescence charts and positive rates of ki67 staining under different hydrogel treatments, c is cell viability, d, g are cell migration pictures and rate quantification, f, g are Tanswell experiment pictures and cell migration quantity quantification, i is the average fluorescence intensity of each index, j is the wb result of each index.

[0035] Figure 11 Figure 10 is a verification of in vivo repair effect; wherein: a is the urethrogram picture and obstruction rate calculation of each group, b is the gross observation and histological staining of each group.

[0036] Figure 12 Figure 11 is an in vivo toxicity test. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application, and are not used to limit the present application.

[0038] Test method: 1. The loading efficiency (LE%) of Cel is calculated according to the following formula: loading efficiency (%) = (mass of loaded Cel) / (mass of loaded Cel + mass of PLGA in nanoparticles) x 100%.

[0039] 2、Chemical structure of CMCS-BA polymer was determined by hydrogen nuclear magnetic resonance (1H NMR) spectrometer (Bruker Avance II, 400 MHz). Characteristic Fourier transform infrared (FTIR) spectra of different samples were detected on a Nicolet iS20 instrument. Morphological characteristics were observed by scanning electron microscopy (Zeiss Sigma SEM 300). A universal material testing machine (INSTRON-5542) was used to test the adhesion performance. Laser confocal microscopy (LASAF SP5) and fluorescence microscopy (Olympus IX70-S1F2) were used to observe cell behavior.

[0040] 3、Degradation rate and drug release rate: First, weigh the hydrogel and record it as w0. Then place it in different conditions (PBS pH=7.4, PBS pH=6.0 or PBS+0.5 mM H2O2 pH=7.4) for incubation. After the predetermined time, take out the sample and weigh it, and record the weight as wt. The degradation rate is recorded as Aw, calculated according to the formula: Aw=(w0 wt) / w0 x 100%. To investigate the release behavior of Cel, CPT-Cel (molecular weight cutoff value: 50 kDa) was incubated with PBS solution (10 mL, pH=7.4), PBS solution (10 mL, pH=6.0) or PBS solution containing H2O2 (0.5 mM) (10 mL, pH=7.4), respectively, at 37°C. At the predetermined time point, a quantitative solution was taken, and the Cel concentration was detected and calculated using a UV-visible spectrophotometer at a wavelength of 425 nm.

[0041] 4、Cell viability assay In drug toxicity tests, 0.01 μM, 0.1 μM, 1 μM and 10 μM Cel were added to the culture medium of each group, and the cell viability was measured after 24 hours. In hydrogel toxicity experiments, different hydrogels (CPT, CPT and CPT-Cel) were immersed in cell culture medium (1 g / 10 mL) for 24 hours to obtain the extract. The cytotoxicity and proliferation of cells incubated in different extracts for 1, 3 and 5 days were determined. In the anti-fibrosis experiment, TGF-β1 (15 μg / ml) and TGF-β1+0.1 μM Cel were added to the culture medium of each group except the control group, and the cell viability was measured after 48 hours. 10 μL CCK-8 reagent was added at the predetermined time point, and incubated at 37°C for 2 hours. The absorbance value was measured at a wavelength of 450 nm using a microplate reader.

[0042] 5、Thrombin inhibition experiment Take 0.1 g CPT, CPT and CPT-Cel hydrogel in the centrifuge tube, drop 100 μL anticoagulant whole blood (containing 10 μM CaCl2) on the material surface, incubate at 37℃. Then add 1 mL deionized water to each centrifuge tube at different time points (1, 5 and 10 minutes), after shaking, centrifuge at 116 g for 10 minutes, take the supernatant to the microwell plate reader to detect the absorbance value of hemoglobin A1 at 540 nm. Take 100 μL blood (containing 10 μM CaCl2), add 1 mL deionized water, measure the absorbance value at 540 nm (A2), calculate the coagulation index BCI of each group according to the formula BCI=A1 / A2 and draw the BCI curve.

[0043] 6、Live / dead cell detection In vitro endpoint (3 days) to evaluate the survival rate of fibroblasts in different extract medium. Cells were washed three times with PBS, then treated with propidium iodide (PI, 4 x 10 -6 M) and calcein-AM (2 x 10 -6 M), incubated in the dark for 20 minutes. Then use fluorescence microscope to take pictures. After 3 days of direct co-culture of CPT-Cel hydrogel and fibroblasts, live and dead staining was performed, and the spatial distribution of fibroblasts was observed by image three-dimensional reconstruction. To evaluate the protective effect of hydrogel on cells, cells were placed in different hydrogel extracts and treated with 50 μM H2O2 for 24 hours. Then Calcein-AM / PI double staining was performed using the above experimental procedure. After Image J analysis, the percentage of cell survival and death was calculated.

[0044] 7、DPPH scavenging effect Take 5 μL of different groups of hydrogel extract and add to each well of the 96-well plate, then add 195 μL DPPH working solution. The mixed solution was incubated in the dark for 30 minutes, and detected using a microwell plate reader at a wavelength of 515 nm. PBS solution was used as a blank control group. The DPPH scavenging rate was calculated according to the following formula: DPPH scavenging rate = (sample absorbance - blank absorbance) / blank absorbance x 100%.

[0045] 8、Effect of hydrogel on macrophage polarization First, activate Thp-1 cells to M0 type macrophages by treating with PMA (200 ng / mL) for 24 hours. Induce M1 type macrophages by using culture medium containing LPS (100 ng / mL) and IFN-γ (20 ng / mL). The experimental group uses culture medium containing LPS and IFN-γ and hydrogel extract, and the control group uses culture medium without adding hydrogel. Macrophages were seeded in 6-well plates and cultured in the above culture medium for 36 hours. Analyze the expression of CD86 (M1 marker) by immunofluorescence staining.

[0046] 9. Antibacterial test The filter paper infiltrated with different groups of hydrogel extract was placed on the solid medium coated with uniform bacterial suspension and cultured at 37°C for 24 hours. Photographs were taken to record the size of the inhibition zone. The bacterial suspension of a specific concentration (10 6 CFU / mL) was co-cultured with different gels for 24 hours. Then the different groups of bacterial suspension were inoculated on the surface of solid medium, and the number of colonies was evaluated after 24 hours of culture at 37°C by taking photographs.

[0047] 10. Cell migration In the scratch test, the over-proliferated fibroblasts in the six-well plate were manually scraped along the ruler guide using a 1000 μL special pipette tip. Then photographs of the damaged area of different treatment groups were taken at the predetermined time point using an optical microscope, and the area of cell-free region was measured using Image J software. In the Transwell test, HUF cell suspension was inoculated in the upper chamber containing serum-free DMEM medium, and serum-containing DMEM medium, serum medium containing 15 μg / ml TGF-β1, serum medium containing 15 μg / ml TGF-β1 and CPT-Cel hydrogel were added to the lower chamber, respectively, to detect the cell migration ability. After 12 or 24 hours of culture, the bottom cells were stained with crystal violet staining solution, and quantitative analysis was performed under an optical microscope.

[0048] Raw materials used in the examples: 1. Carboxymethyl chitosan (CMCS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl), N-hydroxysuccinimide (NHS), 3-aminobenzoic acid, polyvinyl alcohol (PVA), tannic acid (TA), borax and celastrol (Cel) were purchased from Aladdin Biochemical Technology Co., Ltd.

[0049] 2. Rat tissues: SD rats were purchased from Shanghai Jiagan Biotechnology Co., Ltd., and were sacrificed to obtain various organs of rats.

[0050] 3. New Zealand rabbits: New Zealand rabbits were purchased from Shanghai Jiagan Biotechnology Co., Ltd., and were used for urethral repair and obtaining rabbit urethra.

[0051] Example 1 Preparation of hydrogel Reference Figure 1 The specific steps are as follows: 1. Synthesis of benzene boronic acid grafted carboxymethyl chitosan (CMCS-BA) (reference Figure 2 a): Carboxymethyl chitosan (CMCS) was dissolved in deionized water to prepare a 500 mL 1 wt% CMCS solution. BA (6.0 g), EDC·HCl (4.0 g), and NHS (1.5 g) were added sequentially, and the mixture was stirred thoroughly for 48 hours. After dialyzing and lyophilization, CMCS-BA powder was obtained. 1 ¹H NMR and FTIR spectra confirmed the successful preparation of the synthesized CMCS-BA polymer. CMCS-BA 1 H NMR spectrum ( Figure 2 Image b) shows a proton peak corresponding to the benzene ring at 7.3-7.7 ppm, confirming that BA has been successfully grafted onto the side chain of CMCS. The FTIR spectrum of CMCS-BA (…) Figure 2 c) In the middle, 1500-1600 cm -1 The vibrational peaks within the range are attributed to the C=C bonds of the benzene ring, while the 1340 cm⁻¹ peak is... -1 The vibrational peak at 700 cm⁻¹ is attributed to the BO bond in boric acid. Furthermore, the peak at 700 cm⁻¹... -1 The vibrational peak at the specified location is attributed to the CH bond extension vibration of the phenylboronic ester. These results confirm the successful preparation of CMCS-BA.

[0052] 2. Preparation and characterization of PLGA@Cel nanoparticles: The dispersed phase was prepared by dissolving 100 mg PLGA and 10 mg Cel in 20 mL chloroform and 3 mL acetone, respectively, and then subjecting the mixture to homogeneous ultrasonic treatment. The continuous phase was prepared by adding a certain amount of SDS and PVA to 500 mL deionized water and heating in a water bath (60°C) with stirring until completely dissolved. The dispersed phase was added to a storage tank, and then pressed into the continuous phase through an SPG microporous membrane at a pressure of 0.06 MPa to obtain a monodisperse water-in-oil (O / W) emulsion. The O / W emulsion was transferred to a 40°C constant temperature bath and stirred at 500 r / min for 6–8 hours. As the organic solvents chloroform and acetone evaporated, the PLGA@Cel microspheres eventually solidified. The particle size distribution and zeta potential of the PLGA@Cel nanoparticles (NPs) were determined by dynamic light scattering (DLS, Malvern Zetasizer). Figure 3 (a, b). The morphology of PLGA@Cel NPs was observed by transmission electron microscopy (TEM). Figure 3 c). The Cel mass of the load was detected and calculated using a standard curve (425 nm) based on Cel using a UV-Vis spectrophotometer.

[0053] 3. Preparation of CPT-Cel hydrogel A 7 wt% PVA solution was prepared by stirring polyvinyl alcohol (PVA 1799) at 90 °C for 2 h. The CMCS-BA solution concentration was 1 wt%; a 2% tannic acid (TA) was added to the 1 wt% CMCS-BA solution to prepare the CMCS-BA / TA (CT) solution. Subsequently, the CMCS-BA solution or CT solution was mixed with the PVA solution at a ratio of 1:1, and 1% borax was added and stirred to obtain the CMCS-BA / PVA (CP) or CT / PVA (CPT) hydrogel. Figure 4 Fig. 2c shows the FTIR spectra of the CPT hydrogel. Figure 4 Fig. 2c shows the FTIR spectra of the CPT hydrogel. -1 Fig. 2c shows the FTIR spectra of the CPT hydrogel. -1 Fig. 2c shows the FTIR spectra of the CPT hydrogel. -1 Fig. 2c shows the FTIR spectra of the CPT hydrogel. -1 Fig. 2c shows the FTIR spectra of the CPT hydrogel. Figure 4 For PLGA@Cel microsphere loading, it was mixed with PVA solution in advance, and the CPT-Cel hydrogel was obtained after the above steps.

[0054] Example 2 1. Responsive degradation and responsive release of CPT-Cel hydrogel The responsive degradation and responsive release of the CPT-Cel hydrogel were tested: Figure 5 Fig. 2a shows the degradation behavior of the hydrogel in a low-pH or high- reactive oxygen environment; the results show that the degradation rate of CPT-Cel in phosphate buffered saline (PBS, pH 7.4) is significantly lower than that in an acidic environment (pH 6.0) or a high- reactive oxygen environment (0.5 mM H2O2). Figure 5 Fig. 2b shows the release behavior of cellulose in the hydrogel. After 1 day of release, the release amount in PBS was 10.09 ± 1.39%, and after 7 days, the release amount reached 27.30 ± 2.20%, which was slightly lower than the degradation rate of the hydrogel.

[0055] 2. Verification of hydrogel adhesion performance Rat tissues or organs (including heart, liver, kidney, spleen and muscle) were used to qualitatively evaluate the tissue adhesion. The adhesion strength of hydrogels was tested by lap shear test: CPT-Cel was placed between two pieces of pig skin for 1 minute, then clamped into a universal testing machine to apply a tensile load at a strain rate of 10 mm / s. The adhesion strength of hydrogels was measured at the point of separation. After the hydrogels were adhered to the rabbit urethra, they were either freeze-dried or dehydrated by frozen section, and the interface was observed by scanning electron microscopy or the interface integration between the hydrogel and the urethra was observed by H&E staining.

[0056] The CPT-Cel hydrogel was tested for adhesion: the CPT-Cel hydrogel adhered immediately when it came into contact with fresh organs and could bear their weight ( Figure 6 In the middle a), importantly, the CPT-Cel hydrogel could quickly fill and adhere to the urethral defect, and remain stable in flowing PBS ( Figure 6 In the middle b), which is crucial for the hydrogel to adapt to the moist and dynamic urethral environment. The results of tissue section (H&E staining) and SEM clearly showed that a tight and seamless interface was formed between the CPT hydrogel and the urethra ( Figure 6 In the middle c). The adhesion performance of CPT hydrogel was quantitatively evaluated using pig skin shear, with a peak bonding strength of 25.9 ± 2.9 kPa ( Figure 6 In the middle d), which was significantly higher than that of commercially available fibrin glue (12.2 ± 1.7 kPa). These results showed that the hydrogel had good adhesion and could be an ideal wound dressing for urethral repair.

[0057] 3. Verification of biocompatibility, hemostatic, anti-inflammatory and antibacterial performance The cell compatibility of the hydrogel was evaluated using fibroblasts. The results of live / dead cell staining test also showed that all cells in the hydrogel treatment group were basically in a living state (green dots), with only negligible dead cells (red dots), and grew well ( Figure 7 In the middle a) compared with the control group, the cell viability assay at 1, 3 and 5 days showed that all hydrogel groups did not affect the activity of cells ( Figure 7 In the middle b). We further evaluated the hemostatic effect of each group of hydrogels using a coagulation test (BCI). The dynamic coagulation curve ( Figure 8 ) showed that the hemostatic effect of CPT was significantly improved after the addition of TA compared with CP. While due to the anti-coagulation effect of Cel, the BCI index of CPT-Cel decreased slightly, but still reached 16.1% after 10 minutes, which was not much different from CPT (13.3%). This showed that both CPT and CPT-Cel could meet the basic requirements of urethral wound hemostasis.

[0058] Subsequent experiments were performed using an appropriate concentration (50 mM) of H2O2, and the results showed that the H2O2 (50 mM) group induced significant cell death (18.9 ± 2.1%), and each group reduced the proportion of dead cells to varying degrees, while the CPT-Cel group significantly reduced the number of dead cells (2.3 ± 0.98%), which was not significantly different from the control group (2.37 ± 0.60%) Figure 9 a). The results showed that the addition of TA and the loading of PLGA@Cel NPs could effectively improve the oxidative damage of cells. Next, the antioxidant capacity of the hydrogel was evaluated by measuring the scavenging efficiency of the hydrogel on stable free radicals DPPH. As shown in Figure 9 b, CPT and CPT-Cel hydrogels both showed certain DPPH scavenging efficiency, which removed 13.8% (CP hydrogel), 32.0% (CPT hydrogel), and 38.9% (CPT-Cel hydrogel) of DPPH. These results showed that the hydrogel based on boron ester bond had certain antioxidant capacity, which could effectively scavenge exogenous ROS, and was further enhanced in hydrogel CPT and hydrogel CPT-Cel, which may be attributed to the excellent antioxidant activity of TA and Cel. Therefore, CPT-Cel hydrogel loaded with PLGA@Cel NPs can effectively neutralize the complex oxidative stress at the wound site on demand, and protect the wound resident and regenerative cells. In addition, we further explored the immunomodulatory capacity of the hydrogel using macrophages Figure 9 c). Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were added to the culture medium to simulate the inflammatory microenvironment at the early stage of urethral wound, and then the extract of each group of hydrogel was added. The results showed that the expression of CD86 in macrophages was significantly increased in the inflammatory environment, indicating that it was converted to M1 (pro-inflammatory type), and the expression of CD86 decreased to varying degrees after the addition of each group of hydrogel, and the CPT-Cel group was the lowest. This further proves that the CPT-Cel hydrogel has good immunomodulatory function, which can suppress the exacerbation of inflammation at the early stage of urethral wound.

[0059] The antibacterial performance of the hydrogel is very important for the prevention of urethral wound infection. Further use of Escherichia coli and Staphylococcus aureus to perform bacterial inhibition ring test to preliminarily evaluate the antibacterial capacity of the hydrogel, and it can be seen that compared with the CP hydrogel, the CPT and CPT-Cel hydrogels formed more obvious bacterial inhibition rings Figure 9 d). The bacterial suspension was co-cultured with the corresponding hydrogel, and then evenly smeared on the bacterial culture plate after 24 h. As shown in Figure 9 d, the bactericidal capacity of the hydrogel gradually increased with the addition of TA and Cel, and the CPT-Cel hydrogel reached the best bactericidal effect, which could basically meet the antibacterial requirements of the urethral wound.

[0060] 4. Verification of anti-fibrosis performance Although Cel has been proven to play an anti-fibrotic role in other fields such as heart, its role in urethral fibroblast activation and urethral scar formation needs further exploration. HUFs were activated into myofibroblasts using 15 ng / mL TGF-β1 for 48 h. Ki67 fluorescein staining was used to detect the inhibitory effect of Cel on the cell proliferation of activated HUFs. It can be found that when Cel was co-incubated, it can significantly reduce the Ki67 positive rate of activated myofibroblasts, close to the control group level (P < 0.05) Figure 10 Figure 10 Figure 10 Figure 10 Figure 10

[0061] Example 3 In vivo repair effect and toxicity verification The in vivo biological effects of CPT-Cel functionalized hydrogel dressing were further studied in a rabbit urethral injury model. After making a urethral defect of a certain size, the appropriate amount of hydrogel was completely covered and adhered to the urethral defect.

[0062] ​​​​​​​Twenty-four male New Zealand rabbits (2-2.5 kg) were divided into four groups: control group, CPT group, CPT-Cel group and sham operation group to verify the regenerative potential of hydrogel dressing. Except for the sham operation group (no urethral injury during operation), the rest of the groups were all implemented urethral injury modeling. Subsequently, the control group was not treated, the CPT group was applied with CPT hydrogel, and the CPT-Cel group was applied with CPT-Cel hydrogel. At the 4th and 8th week after operation, the experimental rabbits were sacrificed, and the urethra was taken out to take photos of the dorsal injury site, then the tissue was immersed in 4% formaldehyde solution for 24 hours, and then dehydrated and fixed. After paraffin embedding, the tissue was sectioned (4 μm), and histological observation was performed by H&E staining, Masson's trichrome staining and Sirius red staining. The organs of the CPT-Cel group were taken for H&E staining and Masson's trichrome staining to observe the toxicity of the material in vivo.

[0063] Urethrography and urine flow rate determination were performed before the rabbits of the 8th week group were sacrificed. The urethrography operation process was as follows: an 8F catheter was placed in the urethra, and after the injection of iodinated contrast medium, an X-ray film was taken, and the degree of urethral stenosis was evaluated by the ratio of the width of each group to the total width of the urethra. During the urine flow rate test, 200 ml of normal saline was injected into the rabbit bladder, and after the rabbit naturally urinated, the maximum urination flow rate was determined by a urine flow rate detector.

[0064] At the 8th week after operation, urethrography was used to evaluate the degree of urethral obstruction in each group Figure 11 The Control group showed a very narrow lumen due to excessive scar formation, with an obstruction rate of 52.6 ± 7.0%. The CPT group showed a certain degree of relatively wide urethral lumen, while the CPT-Cel group showed a smooth lumen similar to that of the sham operation group, with an obstruction rate of only 7.7 ± 2.0%. Gross morphology Figure 11 The Control group showed a very narrow lumen due to excessive scar formation, with an obstruction rate of 52.6 ± 7.0%. The CPT group showed a certain degree of relatively wide urethral lumen, while the CPT-Cel group showed a smooth lumen similar to that of the sham operation group, with an obstruction rate of only 7.7 ± 2.0%. Gross morphology Figure 11 The Control group showed a very narrow lumen due to excessive scar formation, with an obstruction rate of 52.6 ± 7.0%. The CPT group showed a certain degree of relatively wide urethral lumen, while the CPT-Cel group showed a smooth lumen similar to that of the sham operation group, with an obstruction rate of only 7.7 ± 2.0%. Gross morphology Figure 12 The Control group showed a very narrow lumen due to excessive scar formation, with an obstruction rate of 52.6 ± 7.0%. The CPT group showed a certain degree of relatively wide urethral lumen, while the CPT-Cel group showed a smooth lumen similar to that of the sham operation group, with an obstruction rate of only 7.7 ± 2.0%. Gross morphology

[0065] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. A method for preparing a smart responsive hydrogel, characterized in that, The method includes the following steps: (1) Using phenylboronic acid and carboxymethyl chitosan to synthesize phenylboronic acid-grafted carboxymethyl chitosan; (2) Add tannic acid to the phenylboronic acid grafted carboxymethyl chitosan in (1) to prepare CMCS-BA / TA; (3) Mix polyvinyl alcohol with CMCS-BA / TA in (2), and then add borax to obtain a smart responsive hydrogel.

2. The method according to claim 1, characterized in that, In step (1), 12 g / L of phenylboronic acid, 4.0 g / L of EDC·HCl and 3 g / L of NHS were added to 1 wt% carboxymethyl chitosan solution and stirred thoroughly for 48 hours. After dialysis and lyophilization, CMCS-BA powder was obtained. In step (2), 2% tannic acid is added to 1 wt% CMCS-BA solution; In step (3), the CMCS-BA / TA solution and the polyvinyl alcohol solution are mixed in a 1:1 ratio, and then 1% borax is added.

3. A smart responsive hydrogel prepared by the method according to any one of claims 1 to 2.

4. A drug carrier, characterized in that, The drug carrier contains the hydrogel according to claim 3.

5. A method for preparing a pH / ROS dual-responsive drug-loaded hydrogel, characterized in that, Cel is encapsulated as nanoparticles PLGA@Cel by polylactic acid-glycolic acid copolymer, and incorporated into the hydrogel of claim 3 or the drug carrier of claim 4 to obtain CPT-Cel hydrogel.

6. The method according to claim 5, characterized in that, The method includes the following steps: S1, using phenylboronic acid and carboxymethyl chitosan to synthesize phenylboronic acid-grafted carboxymethyl chitosan; S2, add tannic acid to phenylboronic acid grafted onto carboxymethyl chitosan in S1 to prepare CMCS-BA / TA; S3, Cel is encapsulated as nanoparticles PLGA@Cel NPs by polylactic acid-glycolic acid copolymer; S4: After mixing the PLGA@Cel nanoparticles from S3 with polyvinyl alcohol, it is then mixed with the CMCS-BA / TA from S3, and borax is added to obtain a pH / ROS dual-responsive drug-loaded hydrogel.

7. The method according to claim 5 or 6, characterized in that, The specific preparation of PLGA@Cel nanoparticles is as follows: (a) A chloroform solution of 5 mg / mL polylactic acid-glycolic acid copolymer was mixed with 1 / 30 g / mL Cel and then subjected to uniform ultrasonic treatment to prepare a dispersed phase; (b) Add a certain amount of SDS and PVA to deionized water and heat and stir in a 60°C water bath until completely dissolved to prepare a continuous phase; (c) The dispersed phase of (a) is added to the storage tank, and the dispersed phase is pressed into the continuous phase of S32 through the SPG microporous membrane at a pressure of 0.06 MPa to obtain a monodisperse water-in-oil (O / W) emulsion. (d) The water-in-oil emulsion from (c) was transferred to a 40°C constant temperature bath and stirred at 500 r / min for 6-8 hours to allow chloroform and acetone to evaporate, thus obtaining PLGA@Cel microspheres.

8. A pH / ROS dual-responsive drug-loaded hydrogel prepared by the method according to any one of claims 5 to 7.

9. A dressing, characterized in that, The dressing comprises the drug-loaded hydrogel of claim 8.

10. The use of the method according to any one of claims 1 to 2, the hydrogel according to claim 3, the drug carrier according to claim 4, the method according to any one of claims 5 to 7, the drug-loaded hydrogel according to claim 8, or the dressing according to claim 9 in the preparation of products for urethral repair.

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