Nanoparticle reinforced hydrogel with pH intelligent response and fluorescence labeling functions and preparation method thereof

Abstract

The invention belongs to the field of biomedical apparatuses and instruments, and discloses nano-particle reinforced hydrogel with pH intelligent response and fluorescence labeling functions and a preparation method of the nano-particle reinforced hydrogel. The hydrogel is constructed under the action of a photoinitiator by taking polyethylene glycol or a derivative thereof as a long chain, polysaccharide as a short chain and amino acid derivative quantum dot nanoparticles as a reinforcing phase. Wherein the amino acid derivative quantum dots endow the hydrogel with a pH response characteristic, a fluorescence labeling capability and an excellent antibacterial property; the polysaccharide forms a dynamic physical network through interaction of hydrogen bonds and static electricity, so that the flexibility and cytocompatibility of the hydrogel are improved. The hydrogel disclosed by the invention can be used for assisting the stent to open the narrow part of the urethra and reducing the damage to the urethra soft tissue caused when the stent is implanted. Meanwhile, the hydrogel shows excellent selective antibacterial performance in a complex urethra microenvironment, and the amino acid derivative quantum dots released by the hydrogel have high uptake performance on amino acid substances in endothelial cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical devices, and particularly relates to a nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions and a preparation method thereof. Background Art

[0002] The urethra, as an important part of the urinary system, bears the pressure of urine during urination and has unique expansion and contraction capabilities. Common urethral diseases include stenosis, injury, infection, and congenital malformations, which may lead to dysuria, urinary incontinence, and even affect reproductive function, severely reducing the quality of life and mental health of patients. Urethral repair has always been one of the major challenges in clinical surgery. How to achieve rapid and painless inflammation control and tissue reconstruction is an urgent problem to be solved. With the progress of materials science and medical technology, urethral stents, as a tissue engineering alternative material, have been widely used in urethral repair. Its porous structure not only provides the necessary mechanical support to keep the urethra unobstructed but also promotes cell adhesion, growth, and tissue regeneration.

[0003] Although some polymer or metal stents have played an important role in urethral stricture repair, they still face many challenges. For example, the mechanical properties of some stents are too strong and the biocompatibility is poor, which may scratch the soft urethral tissue, causing inflammation or rejection reactions. In addition, the mismatch between its degradation rate and the growth rate of urethral tissue may also affect the urethral repair process. To overcome these problems, hydrogels have gradually become an important development direction for urethral stent coatings due to their soft properties and adjustable degradation characteristics. Compared with single-component hydrogels, double-network hydrogels form a cross-linked network by combining two types of polymer molecules with different lengths and have higher strength and toughness in the form of "sacrificial bonds", which can better meet the mechanical property requirements during the repair of narrow urethral tissues.

[0004] During the urethral repair process, infection and inflammation are important factors affecting the treatment effect. Therefore, the controllable release strategy of drugs in drug-loaded hydrogels for the complex physiological environment requirements of the urethra has become one of the research focuses. Hydrogels can be used as carriers for antibacterial and anti-inflammatory drugs because there are abundant chemical groups in the internal polymer molecular chains. It achieves the purpose of slowly releasing drugs, killing bacteria, and reducing local inflammatory reactions by gradually weakening the intermolecular forces / coordination bonds between the chemical groups and the drugs. Among many traditional antibacterial agents, such as antibiotics or silver ions, although they have good bactericidal effects, they often have poor biocompatibility, and long-term use may lead to toxicity accumulation and increase the risk of bacterial drug resistance. Therefore, researchers are actively exploring new materials with good biocompatibility, low bacterial drug resistance, and appropriate degradation rates for the repair and regeneration of urethral soft tissues. Summary of the Invention

[0005] The object of the present invention is to provide a hydrogel suitable for urethral stents, which integrates pH intelligent response, fluorescence labeling, excellent antibacterial property and biocompatibility.

[0006] Another object of the present invention is to provide a preparation method of amino acid derivative quantum dots integrating pH intelligent response, fluorescence labeling and antibacterial property.

[0007] The present invention provides a nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions. The nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions uses polyethylene glycol or its derivative as the long chain, polysaccharide as the short chain, and amino acid derivative quantum dot nanoparticles as the reinforcing phase, and is constructed under the action of a photoinitiator.

[0008] The present invention also provides a preparation method of the above-mentioned nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions, including the following steps: adding amino acid derivative quantum dot nanoparticles into a mixed solution of polyethylene glycol or its derivative and polysaccharide, then adding a photoinitiator, and uniformly stirring to obtain a hydrogel precursor solution; standing the hydrogel precursor solution to form a physical network; exposing the physically cross-linked hydrogel to ultraviolet light and chemically cross-linking to obtain a nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions.

[0009] Further, the preparation method of the amino acid derivative quantum dot nanoparticles includes the following steps: performing a hydrothermal reaction on an aqueous solution of an amino acid derivative to obtain an aqueous solution of amino acid derivative quantum dots; centrifuging the aqueous solution of amino acid derivative quantum dots to extract the supernatant, filtering and then dialyzing and purifying to obtain amino acid derivative quantum dots; freeze-drying the amino acid derivative quantum dots to obtain amino acid derivative quantum dot nanoparticles.

[0010] Further, the temperature of the hydrothermal reaction is 140-200°C, and the reaction time is 4-16 h.

[0011] Further, the mass fraction of the solute in the aqueous solution of the amino acid derivative is 6-16 wt.%.

[0012] Further, the mass ratio of polyethylene glycol or its derivative to polysaccharide is 1:5-12.5.

[0013] Further, the mass fraction of the photoinitiator in the mixed solution of amino acid derivative quantum dot nanoparticles, polyethylene glycol or its derivative and polysaccharide is 0.2-1 wt.%.

[0014] Further, the mass ratio of the amino acid derivative quantum dots in the hydrogel precursor solution is 0.4-1.6 wt.%.

[0015] Furthermore, a method for preparing amino acid derivative quantum dot nanoparticles includes the following steps:

[0016] Step 1: Dissolve the amino acid derivative in deionized water and stir to fully dissolve it;

[0017] Step 2: Transfer the fully dissolved amino acid derivative solution into a hydrothermal reaction kettle and react at a temperature of 140 - 200 °C for 4 - 16 hours to obtain an amino acid derivative quantum dot aqueous solution;

[0018] Step 3: Centrifuge the amino acid derivative quantum dot aqueous solution to extract the supernatant, filter it, and then purify it by dialysis;

[0019] Step 4: Subject the purified amino acid derivative quantum dots to freeze-drying treatment to obtain amino acid derivative quantum dot nanoparticles.

[0020] Furthermore, the water content of the hydrogel system is 75 - 80%, and the thickness is 0.01 - 0.1 mm.

[0021] A method for preparing a double-network hydrogel integrating pH intelligent response, fluorescence labeling, excellent antibacterial property, and biocompatibility includes the following steps:

[0022] Step 1: Mix polyethylene glycol and its derivatives with polysaccharides to prepare a mixed solution;

[0023] Step 2: Add amino acid derivative quantum dots to the above solution and mix evenly;

[0024] Step 3: Add a photoinitiator to the mixed solution in Step 2 and stir evenly to form a hydrogel precursor solution;

[0025] Step 4: Let the hydrogel precursor solution in Step 3 stand to form a physical network;

[0026] Step 5: Expose the physically cross-linked hydrogel to ultraviolet light and obtain a stable composite double-network hydrogel after chemical cross-linking.

[0027] The present invention also provides the application of the above-mentioned nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions or the preparation method in medical devices.

[0028] In the present invention, an amino acid derivative rich in amino (-NH2) functional groups is used as a precursor. Through hydrothermal synthesis, its amino structure can be partially retained, and nitrogen-doped defects and functional modifications can be introduced on the surface of carbon dots. During the hydrothermal reaction process, the amino group can undergo oxidation, dehydration condensation, and molecular rearrangement to form stable sp 2Conjugated carbon cores and surface charged groups, thus endowing the amino acid derivative quantum dots with pH-dependent charge regulation ability. In an acidic environment, the -NH2 on the surface of the quantum dots is protonated to form -NH3 + , making its overall charge distribution positive.

[0029] The present invention utilizes the amino acid derivative quantum dots, with their uniform small size characteristics and surface positive charges, to selectively bind to negatively charged bacterial cell membranes, enabling them to have an efficient antibacterial effect in the urethral bacterial environment without damaging normal cells in the urethral environment. First, their accumulation inside bacteria can catalyze the Fenton reaction to generate reactive oxygen species (ROS), destroy the biological macromolecules of bacteria, and induce oxidative stress, causing the cells to lose their activity. In addition, it can disrupt the respiratory chain, inactivate proteins, inhibit cell functions by disrupting amino acid and DNA synthesis, and damage the bacterial cell membrane to reduce the risk of antibiotic resistance. In particular, for the urethral environment used in the present invention, due to the presence of urethral stenosis lesions and inflammatory reactions, the pH value in the urethral environment decreases. The amino acid derivative quantum dots can respond to the change of urethral pH value and have a stronger bactericidal effect under acidic conditions.

[0030] The present invention utilizes the amino acid derivative quantum dots with n-π and π-π conjugated electron transitions, surface defect state luminescence, and edge state effects, which exhibit stable blue fluorescence under 365 nm ultraviolet light excitation. Exert the high-efficient cell uptake ability of the amino acid derivative quantum dots, especially the selective uptake characteristics by amine group transport-related cells (such as endothelial cells responsible for angiogenesis after urethral stenosis lesions). Therefore, the amino acid derivative quantum dots can be enriched in the urethral injury area and feedback the local cell metabolic activity, the process of vascularized tissue repair, and the inflammatory state through fluorescence labeling. In addition, for the urethral environment used in the present invention, the amino acid derivative quantum dots can also be used to monitor the residence time of the quantum dots under urethral flushing through the interaction with urethral epithelial cells and urine components. Combining with fluorescence imaging technology, doctors can accurately evaluate the urethral repair situation based on the fluorescence signal intensity and distribution, improve the accuracy of postoperative treatment and the ability of efficacy prediction, so as to further optimize the treatment plan.

[0031] The double-network hydrogel prepared by the present invention presents a uniform multi-layer porous structure inside. The incorporation of amino acid derivative quantum dots confers a nano-enhancement effect. By strengthening the hydrogen bonding and electrostatic interactions within the internal network, the crosslinking density is increased, thereby optimizing its mechanical properties. The enhanced mechanical properties provide a more solid support for the narrow urethra. Under acidic conditions, the hydrolysis rates of the ester bonds, ether bonds, and amino functional groups in polyethylene glycol and polysaccharides in the hydrogel are significantly accelerated, thereby accelerating the degradation of the hydrogel and promoting the release of the loaded quantum dots. This property enables the hydrogel to respond to the pathological acidic environment of the urethra and achieve the dynamically controllable release of amino acids and their derivative quantum dots to enhance the local repair and antibacterial effects.

[0032] The internal pore size of the quantum dot-enhanced double-network hydrogel prepared by the present invention is distributed within 2-15 μm, which is beneficial to cell penetration and nutrient exchange. The introduction of the natural organic matter polysaccharide significantly improves the biocompatibility of the hydrogel and can promote cell proliferation, migration, and tissue reconstruction. In addition, the good biocompatibility of polyethylene glycol and its derivatives has been approved by the US FDA. In the preliminary work of the present invention, the qualitative analysis of the antibacterial effect of the hydrogel has been carried out in accordance with the international standard of GB / T 31402-2015, and it has been confirmed that the hydrogel of the present invention has good antibacterial properties, and the antibacterial rate can reach 90%. According to the national standard of GB / T16886.5-2017 for the evaluation of the in vitro cytotoxicity test of medical devices, it has been confirmed that the hydrogel has good biocompatibility, and among them, the endothelial cell activity exceeds 80%.

[0033] Beneficial effects

[0034] To optimize the antibacterial properties of the hydrogel system and improve safety, the present invention introduces amino acid derivative quantum dots into the hydrogel system as a "drug". In terms of mechanical strengthening, the amino acid derivative quantum dots improve the crosslinking density and energy dissipation ability through physical and chemical interactions with the polymer network, enhance hydrogen bonding and electrostatic interactions, thereby significantly improving the mechanical properties, toughness, and stability of the hydrogel. In terms of physiological functions, the crosslinked network introduced by the amino acid derivative quantum dots can sense the changes in the acid-base environment of the diseased urethra and intelligently adjust the drug release rate, thereby achieving the effect of inhibiting the growth of pathogenic microorganisms on demand and improving the accuracy and efficiency of treatment. In addition, the amino acid derivative quantum dots also have tunable fluorescence emission, high quantum yield, and photostability, and can specifically identify and efficiently label cells, providing accurate and continuous fluorescence signals for bioimaging and real-time dynamic monitoring. Their good biocompatibility also helps to promote cell proliferation and tissue repair while reducing the risk of body inflammatory responses. Therefore, the hydrogel composite system based on amino acid derivative quantum dots provides a safer, more efficient, and intelligent solution for urethral repair, showing broad clinical application prospects.

[0035] Amino acid derivative quantum dot-enhanced double-network hydrogels play an important role in urethral injury repair. First, due to the soft characteristics of the hydrogel, it can provide an auxiliary effect for local stenosis sites, helping the stent to expand the urethral stenosis site without damaging the urethral soft tissue. Second, the hydrogel exhibits excellent selective antibacterial properties in the complex urethral microenvironment. Especially in the acidic microenvironment created at the diseased urethral injury site, it can accelerate the release of amino acid derivative quantum dots, achieving the purpose of enhanced bactericidal effect. In addition, based on the good biosecurity of the hydrogel, the released amino acid derivative quantum dots have a high uptake of amino acid substances in endothelial cells. At the same time, with the fluorescence characteristics of quantum dots, endothelial cells can be labeled to achieve fluorescence tracing and real-time monitoring of the tissue repair process. Finally, the entire hydrogel system can achieve controllable degradation and quantum dot release according to the change of urethral pH value, ensuring that the hydrogel degrades harmlessly after tissue repair is completed. Brief Description of the Drawings

[0036] Here, taking the Arg-CQDs enhanced double-network hydrogel composed of L-arginine quantum dots (Arg-CQDs) as the reinforcing phase, carboxymethyl chitosan (CMCS) as the short chain, and polyethylene glycol diacrylate (PEGDA) as the long chain as an example, the beneficial effects of this kind of hydrogel and their causes are illustrated.

[0037] Figure 1 Schematic diagram for the preparation of the Arg-CQDs / CMCS / PEGDA double-network hydrogel of the present invention;

[0038] Figure 2 Transmission electron microscope images and particle size distributions of the Arg-CQDs nanoparticles prepared in Examples 2-3; where a is the transmission electron microscope image of the Arg-CQDs nanoparticles, and b is the particle size distribution diagram of the Arg-CQDs nanoparticles;

[0039] Figure 3 Ultraviolet-visible absorption spectra and photoluminescence spectra of the Arg-CQDs nanoparticles with different processes in Examples 2-3; where a is the ultraviolet-visible absorption spectrum of the Arg-CQDs with different processes, b is the photoluminescence excitation spectrum of the Arg-CQDs nanoparticles prepared by hydrothermal treatment for 4 h, c is the photoluminescence excitation spectrum of the Arg-CQDs nanoparticles prepared by hydrothermal treatment for 8 h, d is the photoluminescence excitation spectrum of the Arg-CQDs nanoparticles prepared by hydrothermal treatment for 12 h, and e is the photoluminescence excitation spectrum of the Arg-CQDs nanoparticles prepared by hydrothermal treatment for 16 h;

[0040] Figure 4Antibacterial performance evaluation of Arg-CQDs nanoparticles with different preparation processes for Example 2-3; among them, a is the culture dish of Escherichia coli with Arg-CQDs nanoparticles prepared by different processes, b is the number of Escherichia coli with Arg-CQDs nanoparticles prepared by different processes, and c is the antibacterial rate of Arg-CQDs nanoparticles prepared by different processes against Escherichia coli;

[0041] Figure 5 For Example 3, the surface potential of Arg-CQDs nanoparticles at different pH values;

[0042] Figure 6 Antibacterial performance evaluation of hydrogels against Escherichia coli at different pH values for Example 4; among them, a is the culture dish of Escherichia coli with Arg-CQDs nanoparticles at different pH values, b is the number of Escherichia coli with Arg-CQDs nanoparticles at different pH values, and c is the antibacterial rate of Arg-CQDs nanoparticles at different pH values against Escherichia coli;

[0043] Figure 7 Scanning electron microscopy images of hydrogels with different concentrations of Arg-CQDs nanoparticles for Example 5-7; among them, a is the scanning electron microscopy image of PEGDA hydrogel, b is the scanning electron microscopy image of CMCS / PEGDA hydrogel, c is the scanning electron microscopy image of 0.4% Arg-CQDs / CMCS / PEGDA hydrogel, d is the scanning electron microscopy image of 0.8% Arg-CQDs / CMCS / PEGDA hydrogel, e is the scanning electron microscopy image of 1.2% Arg-CQDs / CMCS / PEGDA hydrogel, and f is the scanning electron microscopy image of 1.6% Arg-CQDs / CMCS / PEGDA hydrogel;

[0044] Figure 8 Detection images of antibacterial performance of hydrogels with different concentrations of Arg-CQDs nanoparticles against Escherichia coli for Example 5-7; among them, a is the culture dish of Escherichia coli with different hydrogels, b is the number of Escherichia coli with different hydrogels, and c is the antibacterial rate of different hydrogels against Escherichia coli;

[0045] Figure 9 For Example 5-7, a is the culture dish of Staphylococcus aureus with different hydrogels, b is the number of Staphylococcus aureus with different hydrogels, and c is the antibacterial rate of different hydrogels against Staphylococcus aureus;

[0046] Figure 10 Detection images of biocompatibility of hydrogels with different concentrations of Arg-CQDs nanoparticles for Example 5-7; among them, a is the cell viability and death fluorescence labeling of different hydrogels, b is the MTT absorbance of cytotoxicity of different hydrogels, and c is the cell viability of different hydrogels; Detailed implementation manners

[0047] The present invention will be further explained below with reference to the accompanying drawings.

[0048] Example 1

[0049] (1) L-arginine was weighed at mass fractions of 6, 8, 10, 12, 14, and 16 wt.% respectively and dissolved in deionized water. Then, under a constant stirring speed of 600 rpm / min, stirring was continued for 30 min to explore the maximum saturation of L-arginine in deionized water.

[0050] Example 2

[0051] (1) L-arginine was accurately weighed at a mass fraction of 12 wt.% and added to deionized water. Then, under a constant stirring speed of 600 rpm / min, stirring was continued for 30 min to ensure that L-arginine was completely dissolved to form a uniform transparent solution.

[0052] (2) The obtained L-arginine solution was transferred to a reaction autoclave and hydrothermally treated at 140, 160, 180, and 200 °C for 8 h to cause the carbonization reaction of L-arginine at high temperature to form Arg-CQDs nanoparticles.

[0053] (3) The obtained aqueous solution containing Arg-CQDs was placed in a centrifuge, the centrifugation speed was set at 12000 rpm / min, and centrifugation was carried out for 20 min to remove larger unreacted solid substances and impurities, and the supernatant was obtained. Then, the supernatant was further filtered using a filter with a pore size of 0.22 um to remove finer particulate matter. The filtered solution was transferred to a dialysis bag, and a dialysis membrane with a molecular weight cut-off value of 1.0 kDa was used and placed in a large amount of deionized water for dialysis for 24 h. During this process, the water was changed every 2 h to effectively remove low-molecular-weight impurities and by-products in the aqueous solution to obtain a purer Arg-CQDs solution.

[0054] (4) The purified aqueous solution of Arg-CQDs was placed in a freeze dryer and freeze-dried for 48 h. During this process, the water was completely removed, and the remaining Arg-CQDs were deposited in the form of a dry powder.

[0055] Example 3

[0056] (1) L-arginine was accurately weighed at a mass fraction of 12 wt.% and added to deionized water. Then, under a constant stirring speed of 600 rpm / min, stirring was continued for 30 min to ensure that L-arginine was completely dissolved to form a uniform transparent solution.

[0057] (2) Transfer the obtained L-arginine solution to a reaction kettle and raise the reaction temperature to 180 °C. Maintain the reaction time at 4 h, 8 h, 12 h, and 16 h respectively at this temperature to cause the carbonization reaction of L-arginine at high temperature to form Arg-CQDs nanoparticles.

[0058] (3) Place the obtained aqueous solution containing Arg-CQDs in a centrifuge, set the centrifugation speed to 12000 rpm / min, and centrifuge for 20 min to remove larger unreacted solid substances and impurities, and obtain the supernatant. Then further filter the supernatant using a filter with a pore size of 0.22 μm to remove finer particulate matter. Transfer the filtered solution to a dialysis bag, use a dialysis membrane with a molecular weight cut-off value of 1.0 kDa, and place it in a large amount of deionized water for dialysis for 24 h. During this process, change the water every 2 h to effectively remove low-molecular-weight impurities and by-products in the aqueous solution and obtain a purer Arg-CQDs solution. The microscopic morphology of the prepared Arg-CQDs nanoparticles is as Figure 2 shown. The Arg-CQDs nanoparticles are evenly distributed, and the particle size is about 3.5 nm, which meets the particle size of quantum dots.

[0059] (4) Place the purified aqueous solution of Arg-CQDs in a freeze dryer and perform freeze-drying for 48 h. During this process, the water is completely removed, and the remaining Arg-CQDs are deposited in the form of a dry powder. Evaluate the ultraviolet-visible absorption spectrum and photoluminescence spectrum of Arg-CQDs nanoparticles with different processes, as Figure 3 shown. At 180 °C and a reaction time of 8 h, the conjugated structure of the Arg-CQDs nanoparticles is the best, and an appropriate amount of surface functional groups is retained, which enhances the π → π* transition at 215 nm, and the n → π* electronic transition at 290 nm is still obvious, and the overall absorption intensity is the highest. At the same time, due to the best balance between the carbon core structure and surface functional groups of the Arg-CQDs nanoparticles prepared by this process, the number of luminescence centers increases, and high fluorescence intensities are exhibited in multiple excitation bands. Especially under 360 nm excitation, the fluorescence intensity at 420 nm reaches the maximum, and the energy level transition of this process is the best.

[0060] (5) Evaluate the antibacterial properties of Arg-CQDs nanoparticles with different processes, as Figure 4 shown. At 180 °C and a reaction time of 8 h, the carbonization degree of the Arg-CQDs nanoparticles reaches the best, the conjugated structure develops fully, the separation efficiency of electron-hole pairs is high, and the ROS production is the largest. Appropriate surface functional groups (such as C=NH, -COOH) enhance the interaction with water or oxygen molecules, promote the superoxide anion (O2• -)and the generation of hydroxyl radicals (•OH), making its antibacterial performance the strongest. Therefore, the Arg-CQDs nanoparticles prepared by this process have the best comprehensive performance.

[0061] Example 4

[0062] (1) Weigh L-arginine accurately at a mass fraction of 12 wt.% and add it to deionized water. Then, under a constant stirring speed of 600 rpm / min, stir continuously for 30 min to ensure that L-arginine is completely dissolved to form a uniform transparent solution.

[0063] (2) Transfer the obtained L-arginine solution to a reaction autoclave and raise the reaction temperature to 180 °C. Maintain the reaction time at this temperature for 12 h to cause the carbonization reaction of L-arginine at high temperature to form Arg-CQDs nanoparticles.

[0064] (3) Place the obtained aqueous solution containing Arg-CQDs in a centrifuge, set the centrifugation speed to 12000 rpm / min, and centrifuge for 20 min to remove larger unreacted solid substances and impurities, and obtain the supernatant. Then use a filter with a pore size of 0.22 um to further filter the supernatant to remove finer particulate matter. Transfer the filtered solution to a dialysis bag, use a dialysis membrane with a molecular weight cut-off value of 1.0 kDa, and place it in a large amount of deionized water for dialysis for 24 h. During this process, change the water every 2 h to effectively remove low-molecular-weight impurities and by-products in the aqueous solution and obtain a purer Arg-CQDs solution.

[0065] (4) Place the purified aqueous solution of Arg-CQDs in a freeze dryer and perform freeze-drying for 48 h. During this process, the water is completely removed, and the remaining Arg-CQDs are deposited in the form of dry powder.

[0066] (5) Dissolve 40 mg of Arg-CQDs nanoparticles in 1 g of nutrient broth, and adjust the pH of the solution to 7.4, 6.7, and 6.0 respectively with 1 wt.% dilute hydrochloric acid. Prepare the culture medium according to the standard, and evaluate the antibacterial performance of Arg-CQDs nanoparticles against Escherichia coli at different pH values, as Figure 6As shown in the figure. The antibacterial rate of 0.4%wt.% Arg-CQDs nanoparticles against Escherichia coli under different pH conditions increased significantly with the decrease of pH. The antibacterial rate was 78.98% at pH 7.4, increased to 84.74% at pH 6.7, and further rose to 92.71% at pH 6.0. This phenomenon is mainly related to the change of the Zeta potential on the surface of Arg-CQDs, which affects its electrostatic interaction with bacteria. As the pH decreases, the amino groups on the surface of Arg-CQDs are gradually protonated, increasing the overall Zeta potential and resulting in enhanced positive charge, as Figure 5 shown. The strong electrostatic attraction enables Arg-CQDs to exhibit a higher antibacterial rate under lower pH conditions. Therefore, as the pH value decreases, the interaction between Arg-CQDs nanoparticles and bacteria is enhanced, thus significantly improving their antibacterial performance. These nanoparticles are expected to be used as a new type of antibacterial material with the ability to respond to pH changes caused by urinary tract inflammation for enhanced bactericidal treatment under environmental stimuli.

[0067] Example 5

[0068] (1) Put PEGDA into an oven at 60 °C for 30 min until it turns into a liquid state. Take 2 g of the PEGDA liquid and add it to 10 ml of deionized water. Stir at 50 °C and 600 rpm / min for 30 min to obtain a PEGDA solution.

[0069] (2) Weigh 0.2, 0.4, 0.6, 0.8, 1 wt.% of Irgacure 2959 photoinitiator respectively and add them to the PEGDA solution. Stir at 50 °C and 600 rpm / min for 30 min to obtain a PEGDA hydrogel precursor solution.

[0070] (3) Spray the PEGDA hydrogel precursor solution evenly onto the biological scaffold using an aerosol spraying technique.

[0071] (4) Irradiate with ultraviolet light at a wavelength of 365 nm for 30 s to form stable chemical bonds, enabling the PEGDA hydrogel precursor to solidify into a PEGDA hydrogel. Prepare the culture medium according to the standard and evaluate the antibacterial performance of the hydrogel against Escherichia coli and Staphylococcus aureus. At the same time, evaluate the in vitro endothelial cell toxicity of the hydrogel according to the national standard.

[0072] Example 6

[0073] (1) Place PEGDA in an oven at 60 °C for 30 min until it turns into a liquid state. Take 2 g of the PEGDA liquid and add it to 5 ml of deionized water. Stir for 30 min at 50 °C and 600 rpm / min to obtain a PEGDA solution. Respectively, take 0.16 g, 0.2 g, 0.267 g, and 0.4 g of CMCS powder and add them to 5 ml of deionized water. Stir for 3 h at 50 °C and 600 rpm / min to obtain CMCS solutions. Mix the PEGDA solution with the CMCS solutions and stir for 1 h at 50 °C and 600 rpm / min to obtain CMCS / PEGDA solutions, and the mass ratios of the CMCS / PEGDA mixed solutions are 1:12.5, 1:10, 1:7.5, and 1:5 respectively.

[0074] (2) Weigh 0.6 wt.% Irgacure2959 and add it to the CMCS / PEGDA solution. Stir for 30 min at 50 °C and 600 rpm / min to obtain a CMCS / PEGDA hydrogel precursor solution.

[0075] (3) Uniformly spray the CMCS / PEGDA hydrogel precursor solution onto the biological scaffold using an aerosol spraying technique. Let it stand for 12 h to complete physical cross-linking and then peel off the mold.

[0076] (4) Irradiate it with ultraviolet light with a wavelength of 365 nm for 30 s to form stable chemical bonds, so that the CMCS / PEGDA hydrogel precursor cures into a CMCS / PEGDA hydrogel. Prepare the culture medium according to the standard configuration and evaluate the antibacterial properties of the hydrogel against Escherichia coli and Staphylococcus aureus. At the same time, evaluate the in vitro endothelial cell toxicity of the hydrogel according to the national standard.

[0077] Example 7

[0078] (1) Place PEGDA in an oven at 60 °C and heat for 30 minutes until it turns into a liquid state. Then take 2 g of the PEGDA liquid and add it to 4.9 ml of deionized water. Stir for 30 minutes at 50 °C and 600 rpm / min to obtain a PEGDA solution. Next, add 0.267 g of CMCS powder to 5 ml of deionized water and stir for 3 hours at 50 °C and 600 rpm / min to obtain a CMCS solution. Finally, mix the PEGDA solution with the CMCS solution and stir for 1 hour at 50 °C and 600 rpm / min to obtain a CMCS / PEGDA solution.

[0079] (2) Weigh 0.6 wt.% of Irgacure 2959 and add it to the CMCS / PEGDA solution. Stir at 50 °C and 600 rpm / min for 30 min to obtain the CMCS / PEGDA hydrogel precursor solution.

[0080] (3) Add 1.97 g of freeze-dried Arg-CQDs nanoparticles to 4 ml, 2 ml, 1.67 ml, and 1 ml of deionized water respectively. Stir uniformly at 37 °C and 600 rpm / min for 12 h to obtain a uniform Arg-CQDs solution. Take 100 μl of the Arg-CQDs solution and add it to the CMCS / PEGDA hydrogel precursor solution. Stir uniformly at 50 °C and 600 rpm / min for 10 min to obtain 0.4, 0.8, 1.2, and 1.6 wt.% Arg-CQDs / CMCS / PEGDA hydrogel precursor solutions.

[0081] (4) Uniformly spray the Arg-CQDs / CMCS / PEGDA hydrogel precursor solution onto the biological scaffold using the aerosol spraying technique and let it stand for 12 h to obtain the Arg-CQDs / CMCS / PEGDA hydrogel with a fully formed physical network.

[0082] (5) Irradiate it with ultraviolet light with a wavelength of 365 nm for 30 s to form stable chemical bonds, so that the Arg-CQDs / CMCS / PEGDA hydrogel forms a tight chemical network, thereby forming a double-network hydrogel. The microscopic morphology is as Figure 7 shown. During the photo-crosslinking process, PEGDA can form a three-dimensional network structure with a relatively high crosslinking density, making its pore structure relatively dense. After introducing CMCS, the overall pore size of the CMCS / PEGDA hydrogel increases, while the number of pores decreases. Compared with Examples 5 and 6, after further doping with Arg-CQDs nanoparticles, the pore structure of the hydrogel tends to be uniform and the pore size decreases. This is because Arg-CQDs provide additional physical crosslinking points in the hydrogel network, thereby increasing the overall crosslinking density and making the network structure more dense. However, as the concentration of Arg-CQDs further increases, the pore size of the hydrogel begins to increase and the pore structure also tends to be non-uniform. This is mainly attributed to the aggregation of high-concentration Arg-CQDs inside the hydrogel, resulting in a decrease in the local crosslinking density. From the microscopic morphology, an appropriate amount of Arg-CQDs nanoparticles can effectively improve the crosslinking degree of the hydrogel, thereby enhancing its overall macroscopic mechanical properties.

[0083] (6) Prepare the culture medium according to the standard and evaluate the antibacterial properties of the hydrogel against Escherichia coli and Staphylococcus aureus. As Figure 8 and 9As shown, PEGDA hydrogel, due to its good hydrophilicity and biocompatibility, easily provides a suitable environment for bacteria to attach and grow, resulting in an increase in the number of bacteria. By introducing CMCS, the hydrogel can be endowed with certain antibacterial properties, which is mainly attributed to the rich amino groups in its molecules, which can electrostatically attract the negatively charged components on the surface of the bacterial cell membrane, destroy the integrity of the cell membrane, and cause bacterial death. Compared with Examples 5 and 6, the further introduction of Arg-CQDs nanoparticles significantly enhanced the antibacterial ability of the hydrogel. When the concentration of Arg-CQDs was 2 wt.%, the antibacterial rates of the hydrogel against Escherichia coli and Staphylococcus aureus reached 85.51% and 97.01%, respectively.

[0084] (7) At the same time, the in vitro endothelial cell toxicity of the hydrogel was evaluated according to national standards. Figure 10 As shown, compared with Examples 5 and 6, after the introduction of Arg-CQDs nanoparticles in the range of 0.4 wt.%–1.2 wt.%, the activity of human umbilical vein endothelial cells cultured in the hydrogel was higher than that of the control group under complete medium conditions, indicating that the appropriate amount of Arg-CQDs nanoparticles not only did not show cytotoxicity, but effectively promoted the proliferation of human umbilical vein endothelial cells, further confirming its good biocompatibility and tissue repair potential. Combined with the results of the antibacterial test, it shows that Arg-CQDs nanoparticles have the ability to be selective for bacteria and cells. It can maintain efficient sterilization and promote cell proliferation, giving the hydrogel excellent biological efficacy. At the same time, its unique optical properties become a potential solution for cell fluorescence labeling.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions, characterized in that, The nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions is constructed under the action of a photoinitiator, using polyethylene glycol or its derivatives as the long chain, polysaccharide as the short chain, and amino acid derivative quantum dot nanoparticles as the reinforcing phase.

2. The nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 1, characterized in that, The nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions has a water content of 75 - 80%.

3. The preparation method of the nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 1 or 2, characterized in that, It includes the following steps: Add amino acid derivative quantum dot nanoparticles into the mixed solution of polyethylene glycol or its derivatives and polysaccharide, and then add a photoinitiator. After uniformly stirring, a hydrogel precursor solution is obtained; Let the hydrogel precursor solution stand to form its physical network; Expose the physically cross-linked hydrogel to ultraviolet light. After chemical cross-linking, a nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions is obtained.

4. The preparation method of the nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 3, characterized in that, The preparation method of the amino acid derivative quantum dot nanoparticles includes the following steps: Carry out a hydrothermal reaction on the amino acid derivative aqueous solution to obtain an amino acid derivative quantum dot aqueous solution; Centrifuge the amino acid derivative quantum dot aqueous solution to extract the supernatant, filter it and then purify it by dialysis to obtain amino acid derivative quantum dots; Freeze-dry the amino acid derivative quantum dots to obtain amino acid derivative quantum dot nanoparticles.

5. The preparation method of the nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 4, wherein, The temperature of the hydrothermal reaction is 140 - 200 °C, and the reaction time is 4 - 16 h.

6. The preparation method of the nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 4, characterized in that, The mass fraction of the solute in the amino acid derivative aqueous solution is 6 - 16 wt.%.

7. The preparation method of the nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 3, characterized in that, The mass ratio of polyethylene glycol or its derivatives to polysaccharide is 1:5 - 12.

5.

8. The preparation method of the nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 3, characterized in that, The mass fraction of the photoinitiator in the mixed solution of amino acid derivative quantum dot nanoparticles, polyethylene glycol or its derivatives and polysaccharide is 0.2 - 1 wt.%.

9. The preparation method of the nanoparticle-enhanced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 3, characterized in that, The mass ratio of the amino acid derivative quantum dots in the hydrogel precursor solution is 0.4 - 1.6 wt.%.

10. Application of the nanoparticle-reinforced hydrogel with both pH intelligent response and fluorescence labeling functions according to claim 1 or 2 or the preparation method according to any one of claims 3 - 9 in medical devices.

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