Intelligent responsive comb-like gelatin-based hydrogel and application thereof

Abstract

This invention discloses a smart, responsive comb-shaped gelatin-based hydrogel and its applications, belonging to the field of biomedical materials technology. The preparation method of the hydrogel is as follows: S1, Prussian blue is modified with tannic acid to obtain TA-coated Prussian blue core-shell nanoparticles, referred to as the PB-TA complex; S2, gelatin is dissolved in PBS solution, then methacrylic anhydride is added dropwise, and the reaction is carried out at 50-60℃ for 90-120 min, followed by dialyzing and freeze-drying to obtain acylated gelatin; S3, an initiator and NIPAM solution are added to the acylated gelatin solution, and the reaction is stirred for 10-14 h to obtain a comb-shaped gelatin solution; S4, zein solution and PB-TA complex solution are added to the comb-shaped gelatin solution to prepare a smart hydrogel exhibiting both temperature and near-infrared photothermal response. This hydrogel can be used for drug delivery, enabling controlled drug release.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a smart responsive comb-shaped gelatin-based hydrogel and its applications. Background Technology

[0002] Gelatin, as a hydrolysis product of collagen, possesses excellent biocompatibility and biodegradability, and is widely used in the biomedical field due to its abundant sources and low cost. Smart responsive hydrogels, capable of rapidly responding to environmental stimuli such as pH, current, and temperature, have become one of the fastest-growing research directions in polymer science. Temperature, in particular, is widely used as a trigger signal in pulsed drug delivery systems. Its advantage lies in the fact that human body temperature deviates from normal physiological temperature (37 °C) under the influence of pathogens or pyrogens. This deviation can activate the drug release function of temperature-sensitive drug delivery systems, providing treatment for diseases accompanied by fever. Constructing non-toxic, biodegradable temperature-sensitive hydrogels remains a key research direction in the biomedical field. Gelatin, as a natural polymer compound, possesses excellent biocompatibility and biodegradability, and its side chains contain numerous active groups. Therefore, many researchers have developed gelatin-based smart responsive hydrogels. However, traditional gelatin-based materials suffer from low mechanical strength, limited functionality, and a lack of dynamic responsiveness, which greatly restricts their application in smart tissue engineering and controlled drug release. Therefore, modifying gelatin to expand its application range is of great significance.

[0003] Prussian blue (PB) is a coordination polymer material composed of iron and cyanide ions, with the chemical formula Fe₄[Fe(CN)₆]₃. Its core structure is a cubic lattice, composed of Fe... 2+ and Fe 3+ Through cyanide (CN) Bridges form unique metal-organic frameworks (MOFs). Prussian blue (PB) possesses photothermal effects and holds promise for the preparation of smart responsive hydrogels. However, PB nanoparticles suffer from drawbacks such as easy aggregation, easy oxidation, poor photothermal stability, and poor biocompatibility. Poor biocompatibility directly leads to poor interfacial bonding between PB nanoparticles and the gelatin matrix. These defects limit the application of PB in gelatin-based hydrogels. Summary of the Invention To address the limitations of Prussian blue in gelatin-based hydrogels, this invention provides a smart, responsive, comb-like gelatin-based hydrogel. In the hydrogel preparation process, Prussian blue is modified with tannic acid (TA), which not only overcomes the problems of easy aggregation and oxidation of Prussian blue but also enhances its binding with the gelatin matrix, resulting in a smart, responsive, comb-like gelatin-based hydrogel.

[0004] This invention utilizes the amidation reaction of free amino groups on the side chains of gelatin with methacrylic anhydride (MA) to methacrylamide-modify gelatin, introducing polymerizable double bonds. Subsequently, a free radical initiator is used to initiate the graft copolymerization of N-isopropylacrylamide (NIPAM) monomers, constructing a comb-like polymer network with LCST characteristics on the gelatin side chains, endowing the material with temperature-responsive properties. To introduce photothermal functionality, Prussian blue is modified with tannic acid (TA) to obtain TA-coated Prussian blue core-shell nanoparticles (PB-TA complex). The polyphenolic hydroxyl groups of TA enhance the interfacial interaction with the gelatin matrix. Finally, through the blending and compounding of zein with the PB-TA complex, the physical cross-linking network of the gelatin matrix is ​​synergistically regulated, forming a smart responsive comb-like gelatin-based hydrogel exhibiting both temperature and near-infrared photothermal responses.

[0005] The preparation method of the intelligent responsive comb-shaped gelatin-based hydrogel is as follows: S1. Tannic acid is dissolved in deionized water to form a TA solution. Then, the TA solution is mixed with a Prussian blue solution, stirred evenly, and then ultrasonically dispersed to obtain TA-coated Prussian blue core-shell structured nanoparticles, referred to as PB-TA complex solution.

[0006] The Prussian blue solution contains Prussian blue nanoparticles, and the preparation method is as follows: Ferric chloride solution was added dropwise to potassium ferrocyanide solution under stirring. After the addition was complete, the reaction was stirred for 2-3 hours to obtain a colloidal solution. The colloidal solution was centrifuged and the supernatant was taken to obtain a solution containing Prussian blue nanoparticles, referred to as Prussian blue solution.

[0007] Preferably, the mass ratio of Prussian blue nanoparticles to tannic acid is 1:2 to 1:5.

[0008] S2. Dissolve gelatin in PBS solution, then add methacrylic anhydride dropwise, and react at a constant temperature of 50-60 ℃ for 90-120 min. After the reaction is completed, dialyze at 40 ℃ for 20-24 h, and finally freeze dry to obtain acylated gelatin.

[0009] The preferred ratio of methacrylic anhydride to gelatin is 0.6 mL of methacrylic anhydride per 1 g of gelatin.

[0010] S3. Dissolve N-isopropylacrylamide in deionized water to form NIPAM solution; add initiator to acylated gelatin solution, stir for 2-4 min, then add NIPAM solution, stir and react for 10-14 h to obtain comb-like gelatin solution.

[0011] The preferred mass ratio of acylated gelatin to N-isopropylacrylamide is 2:1.

[0012] S4. Dissolve zein in ethanol to form zein solution. Add zein solution and PB-TA complex solution to comb-shaped gelatin solution, stir and mix evenly, and obtain smart responsive comb-shaped gelatin-based hydrogel by freeze-thaw method.

[0013] In step S4, the ratio of comb-shaped gelatin, zein, and PB-TA complex is 750:1:0.6-750:1:1, preferably 750:1:0.8.

[0014] The method of this invention utilizes the exposed free amino groups of gelatin to react with methacrylic anhydride, introducing double bonds into the molecular chain and endowing it with free radical polymerization activity to form a crosslinkable network backbone. Then, through free radical copolymerization of NIPAM and GelMA, PNIPAM segments with a low critical solution temperature are grafted onto the gelatin backbone to form a comb-like structure. The thermosensitive properties of PNIPAM enable the hydrogel to exhibit reversible swelling-shrinkage behavior within the physiological temperature range (30-35 °C), which, combined with the thermal responsiveness of gelatin itself, achieves synergistic temperature regulation. Given the poor mechanical properties of gelatin itself, hydrophobic zein crosslinks with PNIPAM and the gelatin network through hydrophobic interactions, improving the mechanical strength of the hydrogel.

[0015] Core-shell nanoparticles (PB-TA complex) are formed through coordination between TA and PB. The TA coating not only reduces cyanide ion leakage from PB, improving biocompatibility, but also allows TA to interact with Fe on the PB surface. 3+ or Fe 2+ Ions coordinate to form a dense organic coating layer, improving the dispersibility of PB in aqueous solution and inhibiting PB aggregation. The photothermal effect of PB can convert near-infrared light (NIR) into heat energy, remotely triggering the PNIPAM phase transition and achieving photocontrolled drug release.

[0016] The present invention also provides an application of the above-mentioned smart responsive comb-shaped gelatin-based hydrogel, mainly for drug delivery, to achieve controlled release of drugs.

[0017] The intelligent responsive comb-shaped gelatin-based hydrogel has a multi-response synergistic mechanism. It induces gel contraction through body temperature or NIR irradiation, driving targeted drug release. By blending the PB-TA complex with natural polymers, the system achieves controlled drug release under physiological conditions, improving the utilization rate of drug molecules. It also has broad prospects in the fields of tumor targeted therapy, intelligent wound dressings, and tissue engineering.

[0018] Compared with the prior art, the advantages of the present invention are: (1) In this invention, tannic acid (TA) is used to modify PB nanoparticles to form TA-coated Prussian blue core-shell structured nanoparticles (PB-TA complex), endowing the PB-TA complex nanonodes with multiple functions such as nano-stabilization, interfacial coupling, and photothermal enhancement. This not only improves the dispersibility and oxidizability of PB nanoparticles, but also enhances photothermal stability and biocompatibility, strengthens the interfacial bonding between the PB-TA complex and the gelatin matrix, and prepares a smart responsive comb-shaped gelatin-based hydrogel.

[0019] (2) This invention grafts gelatin to construct a comb-like gelatin network with a critical phase transition temperature. The comb-like gelatin is side-linked with thermosensitive polymers, endowing the gelatin with a dynamically tunable physical cross-linking network and temperature responsiveness. Its unique topology not only enhances the mechanical strength of the hydrogel, but also regulates drug release through LCST, while retaining the inherent biocompatibility of gelatin. At the same time, the PB-TA complex participates in the construction of the comb-like gelatin composite network, thereby achieving a synergistic enhancement of thermosensitive response, photothermal response, and drug release.

[0020] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0021] Figure 1 This is the UV absorption spectrum of the solutions of PB, TA, and PB-TA complex.

[0022] Figure 2 This is the infrared spectrum of PB, TA, and the PB-TA complex.

[0023] Figure 3 These are SEM images of the PB and PB-TA complex under different magnifications. In the images, a, b, and c are SEM images of PB, and d, e, and f are SEM images of the PB-TA complex.

[0024] Figure 4 These are particle size distribution diagrams of PB and PB-TA composite particles. In the diagram, a represents the particle size distribution of PB particles, and b represents the particle size distribution of the PB-TA composite particles.

[0025] Figure 5 These are thermal images of the PB and PB-TA complex solution during a photothermal experiment.

[0026] Figure 6 These are the temperature change curves of the PB and PB-TA complex solution under near-infrared 808 nm laser irradiation followed by natural cooling for one cycle. Where a is the temperature change curve of the PB solution, and b is the temperature change curve of the PB-TA complex solution.

[0027] Figure 7 The curve shows the temperature change of the PB and PB-TA complex solution under near-infrared 808 nm laser irradiation for 10 min.

[0028] Figure 8 These are infrared spectra of different hydrogels.

[0029] Figure 9 This is a DSC diagram of a comb-shaped gelatin solution.

[0030] Figure 10 These are graphs showing the mechanical properties of different hydrogels. Among them, (a) is the tensile stress-strain curve of different hydrogels, (b) is the tensile strength graph of different hydrogels, (c) is the elongation graph of different hydrogels, and (d) is the elastic modulus graph of different hydrogels.

[0031] Figure 11 These are SEM images of different hydrogels under different magnifications. In the images, a and d are blank hydrogels, b and e are composite hydrogels, and c and f are 1 mg-composite hydrogels.

[0032] Figure 12 These are comparison graphs of the BET specific surface area and pore volume of the freeze-dried blank hydrogel and the composite hydrogel. In graph a, we show the comparison of the BET specific surface area of ​​the freeze-dried blank hydrogel and the composite hydrogel. In graph b, we show the comparison of the pore volume of the freeze-dried blank hydrogel and the composite hydrogel.

[0033] Figure 13 These are experimental diagrams showing the bacterial inhibition loops of different hydrogels. Diagram a shows the results of the bacterial inhibition loop experiment against Staphylococcus aureus using hydrogels. Diagram b shows the results of the bacterial inhibition loop experiment against Escherichia coli. In the diagrams, ① is the blank hydrogel, ② is the composite hydrogel, ③ is the 1 mg blank hydrogel, and ④ is the 1 mg composite hydrogel. Detailed Implementation

[0034] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0035] Example 1 A smart, responsive comb-shaped gelatin-based hydrogel is prepared using the following method: (1) Preparation of Prussian blue nanoparticles: Weigh 270 mg FeCl3·6H2O and dissolve it in 100 mL of deionized water to obtain a 0.01 M ferric chloride solution.

[0036] Weigh 422 mg of K4[Fe(CN)6]·3H2O and dissolve it in 100 mL of deionized water to obtain a 0.01 M potassium ferrocyanide solution.

[0037] Under stirring at 750 rpm, 30 mL of ferric chloride solution was slowly added dropwise to 30 mL of potassium ferrocyanide solution at a rate of 1 mL / min. After the addition was complete, the reaction was stirred for another 2 h to obtain a blue colloidal solution. The blue colloidal solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain a Prussian blue nanoparticle solution (PB), which was stored at 4 ℃ for later use.

[0038] (2) Preparation of Prussian blue-tannic acid complex (PB-TA complex): Weigh 40 mg TA into a centrifuge tube, add 20 mL of deionized water, and sonicate until completely dissolved to obtain a TA solution of 2 mg / mL.

[0039] Equal volumes of Prussian blue solution and TA solution were mixed and magnetically stirred until homogeneous. The mixture was then ultrasonically dispersed at 25 °C for 5 min (ultrasonic power 100 W, ultrasonic frequency 40 kHz) to form TA-coated Prussian blue core-shell structured nanoparticles (PB-TA complex), thus obtaining the PB-TA complex solution.

[0040] (3) Preparation of acylated gelatin: 9 g of gelatin was dissolved in 51 mL of PBS solution (15% w / v) under a 50 ℃ water bath. After complete dissolution, 5.4 mL of methacrylic anhydride (MA) was slowly added dropwise. The mixture was stirred at 50 ℃ for 90 min. After the reaction was completed, the reaction solution was dialyzed at 40 ℃ for 1 day, with the deionized water replaced every 4 h to obtain acylated gelatin (GelMA). The dialyzed product was freeze-dried for 1 day, and the dried sample was stored at -80 ℃ for later use.

[0041] (4) Preparation of comb-shaped temperature-responsive gelatin: Weigh 3 g of N-isopropylacrylamide and dissolve it in 17 mL of deionized water. Vortex and sonicate for a certain period of time to completely dissolve the solid and obtain NIPAM solution.

[0042] Dissolve 1.5 g of lyophilized acylated gelatin in 8.5 mL of deionized water and stir to obtain an acylated gelatin solution. Take 10 mL of the acylated gelatin solution into a reaction flask, bubble with nitrogen for 10 min, add 0.5 mL of ammonium persulfate solution (4% by mass) and stir for 2 min. Then slowly add 5 mL of NIPAM solution and stir for 12 h to obtain a comb-like gelatin solution with a GelMA to NIPAM mass ratio of 2:1.

[0043] (5) Preparation of composite hydrogel: Weigh 40 mg of zein into a centrifuge tube, add 20 mL of 75% ethanol solution, and sonicate until completely dissolved to obtain a 2 mg / mL zein solution.

[0044] Take 5 mL of comb-shaped gelatin solution into a centrifuge tube, add 0.5 mL of zein solution and 0.5 mL of PB-TA complex solution, place the mold containing the mixed solution into an ultra-low temperature freezer at -80 ℃ for 20 hours, then take it out and thaw it completely at room temperature (about 25 ℃). Repeat this "freeze-thaw" process 3 times to obtain the composite hydrogel, which is the smart responsive comb-shaped gelatin-based hydrogel.

[0045] The following performance tests were performed on the products prepared in different steps of Example 1: (1) Figure 1 This is the UV absorption spectrum of a solution of PB, TA, and a PB-TA complex. According to the UV absorption spectrum of PB, there is a strong absorption peak at 690 nm, corresponding to Fe... 2+ To Fe 3+ The strong absorption in the near-infrared region, resulting from intermetallic charge transfer, is a hallmark of PB, consistent with its photothermal properties. TA shows no absorption in the near-infrared region, but its strong absorption peak at 275 nm is characteristic of the electronic transition of the phenolic hydroxyl group. The UV absorption spectrum of the PB-TA complex still shows an absorption band near 690 nm, indicating that the core structure of PB remains intact and its characteristic peaks are preserved, thus retaining its photothermal properties. Furthermore, the presence of a TA absorption peak at 275 nm verifies that TA successfully encapsulates PB. Simultaneously, the absorbance of the PB-TA complex at 690 nm is slightly higher than that of PB, indicating that TA encapsulation improves the dispersibility of PB and reduces scattering losses. In addition, comparing the UV absorption spectra of the PB-TA complex and TA, the enhanced absorption peak at 275 nm indicates improved light absorption in the UV region by TA. Combined with the strong near-infrared absorption of PB, the PB-TA complex can achieve full-spectrum light capture, improving the overall photothermal conversion efficiency.

[0046] (2) Figure 2This is the Fourier transform infrared spectrum of PB, TA, and the PB-TA complex. TA is in the 3200-3500 cm⁻¹ range. -1 The broad absorption peaks present are due to the OH stretching vibrations of the numerous phenolic hydroxyl groups in TA. At 1710 cm⁻¹... -1 The vibrational absorption peak at that location corresponds to the carbonyl group in the ester group. Furthermore, due to skeletal vibrations of the benzene ring, a peak appears at 1650-1420 cm⁻¹ in the figure. -1 Multiple peaks within the range, at 1200 cm⁻¹ -1 The presence of CO stretching vibration in phenol also resulted in an absorption peak at 2080 cm⁻¹. Meanwhile, PB showed an absorption peak at 2080 cm⁻¹. -1 The sharp, strong peaks are CN - Stretching vibrations indicate that the cyanide-bridged structure is intact; 490-710 cm -1 The weak peaks within the range are related to metal-cyano coordination bonds. In the infrared spectrum of the PB-TA complex, the peaks in the 3200-3500 cm⁻¹ range are... -1 The absorption peak intensity at this point decreased significantly, indicating that some of the phenolic hydroxyl groups in TA coordinated with PB, reducing the number of free phenolic hydroxyl groups, further proving the formation of the PB-TA complex structure. Furthermore, CN... - The peak is at 2080 cm⁻¹ in PB. -1 Moved to 2070 cm -1 A blue shift occurred, indicating that TA interacts with cyanide ions through hydrogen bonds, affecting CN. - The electron cloud density. Compared to the TA spectrum, the PB-TA complex also showed a metal-oxygen coordination peak at 500 cm⁻¹. -1 The appearance of the Fe-O vibration peak directly proves that the phenolic hydroxyl group of TA is related to the Fe of PB. 3+ / Fe 2+ Coordination confirmed the successful preparation of the PB-TA complex.

[0047] (3) The surface potentials of PB and the PB-TA complex were measured using a Zeta potential meter. Based on multiple Zeta potential measurements, the average surface potential of PB was found to be -17.19 ± 1.34 mV, and the average surface potential of the PB-TA complex was found to be -20.41 ± 1.60 mV. The PB-TA complex contains a large number of phenolic hydroxyl groups, which will ionize in solution to form -O - The TA exists in the form of a substance directly coating the PB surface, increasing the negative charge density of the complex. The negative charge of PB itself may originate from the partial hydrolysis of surface cyano groups, thus PB itself exhibits a negative charge in solution. The higher absolute value of the zeta potential of the PB-TA complex indicates better dispersion stability, which also proves the successful modification of the PB surface by TA.

[0048] (4) A suitable amount of the PB and PB-TA complex solution was dropped onto the silicon wafer, dried in a vacuum oven overnight, sputtered with gold, and its morphology was observed using a scanning electron microscope. The results are as follows: Figure 3 As shown, a, b, and c are SEM images of PB, and d, e, and f are SEM images of the PB-TA complex. It can be seen that uncoated PB exhibits significant aggregation due to high surface energy driving particle aggregation to reduce energy, making it difficult to observe clearly defined individual particles in the images. In contrast, the PB-TA complex shows a clear cubic shape because TA molecules adsorb onto the PB surface, forming a dense coating layer that increases the distance between particles, rendering van der Waals forces ineffective. Simultaneously, the free phenolic hydroxyl groups of TA carry a negative charge in solution, directly coating the PB surface and increasing the negative charge density of PB, consistent with the results obtained from Zeta potential characterization. Therefore, TA molecules inhibit PB aggregation, and the size of the PB-TA complex particles is smaller than that of the PB particles.

[0049] (5) Figure 4 The figures show the particle size distributions of PB and PB-TA composite particles, where a represents the particle size distribution of PB particles and b represents the particle size distribution of the PB-TA composite particles. It can be seen that the size of the PB-TA composite particles is significantly smaller than that of the PB particles. Due to the effect of the TA molecules, the PB-TA composite improves dispersibility and inhibits PB aggregation, with the main particle size being within 100 nm. This demonstrates the successful modification of PB particles by the TA molecules.

[0050] (6) Photothermal performance test: Take an appropriate amount of PB solution in a cuvette, insert the temperature sensing probe into the solution, and then adjust the position of the probe of the near-infrared irradiation device to control the intensity per unit area to be the same during the irradiation process. Turn on the near-infrared irradiation device with a power of 1.0 W / cm². 2 The sample was irradiated with near-infrared light at a wavelength of 808 nm. A timer was simultaneously activated, and the thermocouple thermometer readings were recorded at 30-second intervals, continuously recording the temperature change over 10 minutes. To further determine the stability of its photothermal performance, the infrared irradiation device was removed after 10 minutes of irradiation, allowing the sample to cool naturally, with temperature changes still recorded every 30 seconds. The same procedure was repeated for the PB-TA composite solution. Finally, a temperature change curve was plotted with near-infrared irradiation time on the x-axis and the sample temperature during irradiation or cooling on the y-axis. To more intuitively express the temperature change effect of the nanoparticle system during the photothermal effect experiment, a near-infrared thermal imager was used to record infrared images, maximum and minimum temperatures of the sample at different times during the photothermal experiment. Furthermore, the system's temperature and color change scales were set using software to observe the corresponding heating and cooling processes. Specific test results are available in [link to test results]. Figure 5-7 . Figure 5These are thermal images (using 1.0 W / cm²) of the PB and PB-TA complex solution during a photothermal experiment. 2 Irradiation with near-infrared 808 nm laser light for 10 min). Figure 6 The solution of PB and PB-TA complex under near-infrared 808 nm laser light (1.0 W / cm²) was tested. 2 Temperature change curves for one cycle of "irradiation-natural cooling". Where a is the temperature change curve of the PB solution and b is the temperature change curve of the PB-TA complex solution. Figure 7 The temperature change curve of the PB / PB-TA complex solution under near-infrared 808 nm laser irradiation for 10 min (1.0 W / cm²) is shown. 2 ).

[0051] It can be seen that both PB and the PB-TA complex possess certain photothermal conversion capabilities. In the near-infrared light at 808 nm (1.0 W / cm²), [the following parameters are observed]. 2 After 10 minutes of irradiation, the temperature of the PB aqueous solution increased by approximately 12 °C, while the PB-TA complex aqueous solution increased by approximately 23 °C under the same conditions. This indicates that TA molecules can improve the photothermal conversion effect of PB. On the one hand, TA forms coordination bonds with the PB surface, broadening the light absorption range and achieving full-spectrum light capture; on the other hand, the encapsulation of TA molecules reduces the aggregation of PB particles and lowers light scattering loss. Therefore, TA modification enhances the light capture efficiency of PB. Under the same natural cooling time, the PB-TA complex solution cooled faster than the PB solution. This is because the nanoscale dispersion of the PB-TA complex increases the contact area with the solvent, accelerating heat diffusion. At the same time, the hydrophilic groups of TA promote heat exchange between water molecules and the PB surface, indicating that the thermal conductivity of the PB-TA complex is improved.

[0052] (7) Acylated gelatin (GelMA), blank hydrogel, zein-hydrogel, composite hydrogel, and 1 mg-composite hydrogel were pre-frozen and then freeze-dried. The changes in chemical groups of gelatin before and after modification were observed using ATR reflection to determine the hydrogel composite situation. The blank hydrogel was prepared as follows: 5 mL of comb-shaped gelatin solution was placed in a centrifuge tube, 1 mL of deionized water solution was added, and the mixture was stirred thoroughly. The comb-shaped gelatin-based hydrogel was obtained by freeze-thaw method and designated as the blank hydrogel. The zein-hydrogel was prepared as follows: 5 mL of comb-shaped gelatin solution was placed in a centrifuge tube, 1 mL of zein solution was added, and the mixture was stirred thoroughly. The zein-hydrogel was obtained by freeze-thaw method. The 1 mg-composite hydrogel was prepared as follows: 1 mg of amoxicillin was dissolved in 0.5 mL of PB-TA complex solution, and the mixture was subjected to ultrasonic loading. The composite hydrogel was obtained using the same method as in Example 1 and designated as the 1 mg-composite hydrogel. The test results are shown in […]. Figure 8 .

[0053] Comparing the infrared spectra of the blank hydrogel and GelMA, the NIPAM free radicals copolymerized on the side chains of the gelatin showed a higher concentration at 2970 cm⁻¹. -1 and 2870 cm -1 A peak appeared, which is the CH peak of isopropyl, and at 1385 cm⁻¹... -1 and 1365 cm -1 The presence of a characteristic double peak for isopropyl groups at [value missing] cm⁻¹ confirms the successful grafting of PNIPAM. Further comparison of the blank hydrogel and the zein-hydrogel reveals a glutamine side-chain peak (1450 cm⁻¹) in the IR spectrum of the zein-hydrogel. -1 Meanwhile, due to hydrogen bond recombination or zein hydrophobic interactions reducing free hydroxyl groups, at 3300 cm⁻¹ -1 The peak shape is sharper at this point. Furthermore, due to the stretching vibration of the CH bond, the peak shape is more pronounced at 2950 cm⁻¹. -1 -2850 cm -1 The enhanced peak intensity at 2080 cm⁻¹ is due to the hydrophobic side chains of zein. Comparing zein-hydrogels and composite hydrogels, the composite hydrogel incorporates a PB-TA complex; the addition of PB results in a higher peak intensity at 2080 cm⁻¹. -1 The new peak is due to the C≡N stretching vibration of the cyano group in PB, and it also appears at 1260 cm⁻¹. -1 -1220 cm -1 At this point, a new peak appears due to the CO stretching vibration in the TA molecule. Finally, the infrared spectrum of the 1 mg amoxicillin-loaded composite hydrogel shows a peak at 1770 cm⁻¹. -1 With 1540 cm -1 The presence of characteristic peaks for amoxicillin at the specified locations is due to the C=O stretching vibration of the β-lactam ring and the CN stretching vibration of the β-lactam ring, respectively, indicating successful loading of amoxicillin.

[0054] (8) Figure 9 This is a DSC chromatogram of a comb-shaped gelatin solution. The two characteristic peak temperatures in the chromatogram are: 30.4 ℃, which may be due to the acylation treatment reducing the molecular chain rigidity of the gelatin, resulting in a slightly lower Tg than unmodified gelatin; and 32.5 ℃, which is close to the phase transition temperature of PNIPAM. This means that after grafting NIPAM and GelMA, the original temperature sensitivity is maintained, and the phase transition temperatures are close, demonstrating that the PNIPAM grafted chains endow the gelatin group with precise temperature response.

[0055] (9) To study the mechanical properties of the composite hydrogels, tensile tests were conducted on different hydrogels. The hydrogels were cut into 10 mm wide cuboids and subjected to tensile testing using a servo-controlled computer system at a tensile rate of 10 mm / min. Stress-strain curves were plotted after the tests to analyze the mechanical properties. The results are shown in […]. Figure 10 In the figure, (a) is the tensile stress-strain curve of different hydrogels, (b) is the tensile strength graph of different hydrogels, (c) is the elongation graph of different hydrogels, and (d) is the elastic modulus graph of different hydrogels. The tensile strength of zein-hydrogel is 0.7 MPa, and the tensile strength of composite hydrogel is 1.43 MPa, both of which are better than gelatin and blank hydrogel. This indicates that the addition of zein improves the network structure of hydrogel. This is because zein is rich in hydrophobic amino acids, and its hydrophobic groups form physical cross-linking points with the hydrophobic microdomains of gelatin through van der Waals forces, thus strengthening the network structure. With the addition of PB-TA complex, the tensile strength of hydrogel is further enhanced, because PB-TA complex enhances the stability and structural strength of hydrogel through electrostatic and physical adsorption. At the same time, due to the enhanced physical cross-linking density and network structure of hydrogel, the elastic modulus of composite hydrogel and zein-hydrogel are much higher than those of gelatin and blank hydrogel. Furthermore, compared to zein-hydrogel, the composite hydrogel exhibits a slightly lower elastic modulus, gradually weakening its elasticity. This indicates that the composite hydrogel possesses moderate elasticity while maintaining a certain degree of rigidity. The addition of the zein-PB-TA complex resulted in a certain reduction in the elongation of the hydrogel. Zein, rich in hydrophobic amino acids, binds to the hydrophobic regions of gelatin through van der Waals forces, forming additional physical cross-linking points. This significantly increases the cross-linking density, restricting the slippage and extension of molecular chains, leading to a decrease in elongation. Meanwhile, the PB-TA complex forms nanoscale particles within the gel, acting as a physical filler to hinder molecular chain extension and reduce the stretchability of chain segments. Therefore, by adjusting the ratio of the PB-TA complex to zein, the balance between tensile strength, elastic modulus, and elongation can be improved, overcoming the poor mechanical properties of traditional gelatin while retaining its biocompatibility and biodegradability, making it more valuable for biomedical applications.

[0056] (10) Hydrogels of different components were freeze-dried, subjected to liquid nitrogen brittle fracture treatment, and characterized by SEM. The results are as follows: Figure 11As shown in the figure, a and d are blank hydrogels, b and e are composite hydrogels, and c and f are 1 mg-composite hydrogels. It can be seen that the surface and pores of the composite hydrogels are very smooth, with almost no impurities, indicating that the added PB-TA complex and zein are uniformly distributed in the polymer network of the hydrogel. Simultaneously, the hydrogels exhibit a compact microporous structure, displaying a uniform and tightly connected network structure. The composite hydrogels have larger pores than the blank hydrogels. This is because in the composite hydrogels, the cubic PB-TA complex acts as a filler, promoting the formation of micron-sized pores. At the same time, TA stabilizes the dispersion of PB through hydrogen bonds and coordination bonds, preventing pore blockage caused by its aggregation. Furthermore, the hydrophobic amino acids of zein form micro-phase separation in the hydrophilic gelatin matrix. During freeze-drying, the interfacial tension difference between the hydrophobic micro-regions and the hydrophilic regions causes ice crystals to preferentially grow in the hydrophilic regions, ultimately forming a larger and continuous pore structure. The hydrophobic interaction and synergistic hydrogen bonding between zein and gelatin enhance network rigidity and reduce the collapse of the pore structure during freeze-drying, thus preserving a larger pore size. In the 1 mg composite hydrogel, amoxicillin particles are uniformly dispersed on the surface and inside the hydrogel pores through adsorption, without aggregation. This indicates that the composite hydrogel provides a large number of drug loading sites for amoxicillin, and the uniform drug dispersion also reduces the risk of burst release due to excessively high local concentrations, improving release stability.

[0057] (11) After degassing the blank hydrogel and the composite hydrogel at 40 °C for 12 h, the BET specific surface area was measured by N2 absorption-desorption test. The results are shown in […]. Figure 12 Figure 1 shows a comparison of the BET specific surface area of ​​the freeze-dried blank hydrogel and the composite hydrogel. Figure 2 shows a comparison of the pore volume of the freeze-dried blank hydrogel and the composite hydrogel. The results show that the BET specific surface area of ​​the freeze-dried composite hydrogel is significantly higher than that of the blank hydrogel, from 5.2386 m². 2 / g increased to 8.1289 m of the composite hydrogel 2 / g. This is because the introduction of the PB-TA complex and zein improves pore connectivity, exposing more internal surfaces. Simultaneously, the pore volume of the composite hydrogel is more than twice that of the blank hydrogel.

[0058] (12) Using Staphylococcus aureus and Escherichia coli as representatives of Gram-positive and Gram-negative bacteria respectively, the antibacterial effects of blank hydrogel and composite hydrogel, as well as the antibacterial effects of blank hydrogel and composite hydrogel under the same drug loading, were compared and studied. Experimental methods and steps: Step 1, Sterilization and plate pouring: Prepare solid culture medium (4.5% w / v) with NaCl, tryptone, yeast extract, agar powder and deionized water, and sterilize it in a high temperature and high pressure autoclave (120℃, 30min). After cooling, remove it. When the temperature is suitable, pour plates in a multi-functional clean bench, solidify and store in a refrigerator at 4℃ for later use. Step 2, Bacterial activation: Take 100 µL of bacterial stock solution (CFU = 10) of Escherichia coli and Staphylococcus aureus respectively. 7 Add 900 µL of culture medium to each centrifuge tube (CFU / mL), and incubate at 37 °C for 12 h in a shaker. Step 3, serial dilution: Take 100 µL of Escherichia coli and Staphylococcus aureus culture medium into centrifuge tubes, add 900 µL of PBS to each, and repeat this operation once to obtain CFU=10. 5 / mL and CFU = 10 4 Step 4: Spreading and observation: Label each agar medium, and take 100 µL of Escherichia coli and Staphylococcus aureus bacterial suspension into the agar medium respectively, and spread it evenly using a sterile spreader; select blank hydrogel, 1 mg-blank hydrogel, composite hydrogel, and 1 mg-composite hydrogel discs of the same shape and size as samples, inoculate them on the spread agar plates, and incubate them in a bacterial incubator at 37 ℃ for 24 h, and observe the size of the inhibition zone.

[0059] Experimental results are as follows Figure 13 As shown in the figure. Figure a shows the bacterial inhibition zone experiment results of the hydrogel against Staphylococcus aureus. Figure b shows the bacterial inhibition zone experiment results against Escherichia coli. In the figure, ① is the blank hydrogel, ② is the composite hydrogel, ③ is the 1 mg-blank hydrogel, and ④ is the 1 mg-composite hydrogel. Preparation method of the 1 mg-blank hydrogel: Take 5 mL of comb-shaped gelatin solution in a centrifuge tube, add 1 mL of deionized water and 1 mg of amoxicillin, stir thoroughly to mix, and obtain the comb-shaped gelatin-based hydrogel by freeze-thaw method, denoted as the 1 mg-blank hydrogel. The inhibition zones generated by each sample are circled.

[0060] The results showed that the gelatin-based hydrogel itself had almost no antibacterial activity, while the hydrogel with the PB-TA complex and zein exhibited certain antibacterial activity. This is because the added TA is rich in multiple catechol and gallic acid groups, which can insert into the bacterial membrane structure. Through hydrophobic interactions and hydrogen bonds, these groups insert into the phospholipid bilayer of the bacterial cell membrane, disrupting membrane integrity and thus inhibiting bacterial growth. Furthermore, because the thick peptidoglycan layer of Gram-positive bacteria has low permeability to TA, TA showed a more significant inhibitory effect on Gram-negative bacteria, namely *Escherichia coli* used in the experiment, with a larger inhibition zone, consistent with the experimental results. PB can release Fe... 2+ / Fe 3+ These metal ions can interfere with the enzyme system of bacteria, inhibiting their growth and metabolism. Therefore, composite hydrogels themselves have a certain antibacterial effect.

[0061] According to the principles of drug diffusion, the higher the cumulative release of a drug, the larger the diameter of the inhibition zone formed after diffusion and spread, and the stronger the inhibitory effect on bacteria. Observing the size of the inhibition zone of 1 mg blank hydrogel and 1 mg composite hydrogel, it was found that under the same drug loading, the composite hydrogel not only achieved successful drug loading but also achieved a certain degree of sustained drug release. This is because the PB-TA complex increases the cross-linking density of the hydrogel to a certain extent, and the enhanced cross-linking network prolongs the diffusion path of drug molecules, slowing down their release rate. At the same time, for drug burst release, the composite hydrogel can achieve uniform drug dispersion, avoiding burst release due to excessively high local concentrations, improving release stability, and potentially prolonging treatment time, thus reducing the development of drug resistance to some extent. The PB-TA complex plays a synergistic role in antibacterial activity, achieving antibacterial effects through multiple mechanisms. It can also be combined with the photothermal effect of the PB-TA complex, achieving local temperature rise through NIR irradiation, and combined with the temperature responsiveness of comb-like gelatin, to achieve precise controlled drug release, potentially realizing phototherapy-chemical drug combination, providing new ideas for addressing antibiotic resistance and the field of antibacterial therapy.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A smart responsive comb-like gelatin-based hydrogel, characterized in that, Made by the following method: S1. Dissolve tannic acid in deionized water to form a TA solution, then mix the TA solution with a Prussian blue solution, stir evenly, and then ultrasonically disperse to obtain TA-coated Prussian blue core-shell structured nanoparticles, referred to as PB-TA complex solution. S2. Dissolve gelatin in PBS solution, then add methacrylic anhydride dropwise, and react at 50-60 ℃ for 90-120 min. After the reaction is complete, dialyze at 40 ℃ for 20-24 h, and finally freeze dry to obtain acylated gelatin. S3. Dissolve N-isopropylacrylamide in deionized water to form NIPAM solution; add initiator to acylated gelatin solution, stir for 2-4 min, then add NIPAM solution, stir and react for 10-14 h to obtain comb-like gelatin solution; S4. Dissolve zein in ethanol to form zein solution. Add zein solution and PB-TA complex solution to comb-shaped gelatin solution, stir and mix evenly, and obtain smart responsive comb-shaped gelatin-based hydrogel by freeze-thaw method.

2. The smart responsive comb-shaped gelatin-based hydrogel as described in claim 1, characterized in that, The Prussian blue solution contains Prussian blue nanoparticles.

3. The smart responsive comb-shaped gelatin-based hydrogel as described in claim 2, characterized in that, The Prussian blue solution is prepared as follows: Ferric chloride solution was added dropwise to potassium ferrocyanide solution under stirring. After the addition was complete, the reaction was stirred for 2-3 hours to obtain a colloidal solution. The colloidal solution was centrifuged and the supernatant was taken to obtain Prussian blue solution.

4. The smart responsive comb-shaped gelatin-based hydrogel as described in claim 3, characterized in that, The mass ratio of Prussian blue nanoparticles to tannic acid is 1:2 to 1:

5.

5. The smart responsive comb-shaped gelatin-based hydrogel as described in claim 1, characterized in that, In step S2, the ratio of gelatin to methacrylic anhydride is: 0.6 mL of methacrylic anhydride is added to every 1 g of gelatin.

6. The smart responsive comb-shaped gelatin-based hydrogel as described in claim 1, characterized in that, In step S3, the mass ratio of acylated gelatin to N-isopropylacrylamide is 2:

1.

7. The smart responsive comb-shaped gelatin-based hydrogel as described in claim 1, characterized in that, In step S4, the ratio of comb-shaped gelatin, zein, and PB-TA complex is 750:1:0.6-750:1:

1.

8. An application of the smart responsive comb-shaped gelatin-based hydrogel as described in any one of claims 1-7, characterized in that, Used for drug delivery to achieve controlled release of drugs.

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