Collagen bio-ink for glucose responsive DLP 3D printing, preparation method and application

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to collagen bio-ink for glucose responsive DLP 3D printing, a preparation method and application. The invention provides a glucose responsive collagen bio-ink for DLP (Digital Light Processing) 3D (Three Dimensional) printing. The bio-ink can be used for preparing hydrogel with good mechanical properties, fidelity and degradation resistance under the physiological condition of pH 7; the gel can be responsively released in a hyperglycemia environment; the released curcumin can remove ROS in cells, regulate and control expression of macrophage inflammatory factors and exert antioxidant and anti-inflammatory effects, and the gel material can promote proliferation, adhesion and migration of the cells and has good biocompatibility and biological activity; a personalized solution with intelligent drug release, mechanical adaptation and active healing promoting functions is provided for diabetes wound treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials technology and is aimed at repairing chronic diabetic wounds. Specifically, it relates to a glucose-responsive collagen bio-ink for DLP 3D printing, its preparation method, and its application. Background Art

[0002] As one of the most devastating complications of diabetes, the pathological mechanism of diabetic chronic wounds involves multi-dimensional interactions. The high-glucose microenvironment induces mitochondrial dysfunction and uncoupling of oxidative phosphorylation, leading to excessive production of reactive oxygen species (ROS), which in turn activates the NF-κB signaling pathway and drives the release of inflammatory factor cascades. This mutual reinforcement of high oxidative stress and chronic inflammation not only leads to immune microenvironmental imbalance by regulating the polarization balance of macrophages, but also destroys the function of vascular endothelial cells, hinders the formation of new blood vessels, and ultimately causes a vicious cycle of "oxidative damage-prolonged inflammation-repair dysfunction." Although traditional dressings can provide a physical barrier and a moist environment, they lack environmental responsiveness and are difficult to synchronously adapt to glucose fluctuations, changes in ROS levels, and dynamic evolution of pH values ​​in the wound microenvironment, resulting in a spatiotemporal mismatch between treatment strategies and pathological processes.

[0003] Stimuli-responsive hydrogels based on dynamic covalent chemistry provide a new direction for solving this problem. Photothermal responsive hydrogels achieve on-demand drug release through exogenous stimulation, but are limited by device dependence and the lack of autonomous microenvironment sensing capabilities. Phenylboronic acid groups are widely used in the construction of glucose-sensitive drug delivery systems due to their reversible binding properties with cis-diol structures. However, systems that rely solely on this mechanism often face the challenges of insufficient mechanical adaptability and uncontrollable degradation rates. More importantly, the matrix used in existing systems lacks the bioactive components of the natural extracellular matrix (ECM), resulting in low efficiency of material-tissue interface integration and difficulty in supporting directional cell migration and functional tissue reconstruction.

[0004] Digital light processing (DLP) 3D printing technology provides technical support for the construction of biomimetic structure and function-integrated dressings through precise layer-by-layer cross-linking of photosensitive materials. The core advantage of this technology is the ability to combine macroscopic structural design with microscopic component arrangement to achieve precise control of porosity, pore size distribution and mechanical properties. However, existing photocurable bio-inks generally face the problem of single function, or focus on structural biomimetic but lack environmental responsiveness, or have dynamic properties but sacrifice biocompatibility. This "structure-function" split phenomenon seriously limits the clinical application potential of the material. Collagen, as the main component of ECM, can induce cell signal transduction and participate in cell adhesion, proliferation, migration and differentiation. However, natural collagen has low photosensitivity and lacks environmental responsiveness, which limits its use in advanced DLP 3D printing technology and has the defect of insufficient functionality for complex diabetic wounds. Summary of the Invention

[0005] In response to the above technical problems, the purpose of the present invention is to provide a new type of collagen bio-ink for glucose-responsive DLP 3D printing for treating chronic diabetic wounds. When preparing glucose-responsive DLP 3D printing collagen bio-ink, the present invention found that the commonly used DLP 3D printing collagen ink - methacryloyl collagen (CMA) could not be dissolved after being functionalized with phenylboronic acid, and could not be 3D printed; the collagen was functionalized with phenylboronic acid, and it was found that when phenylboronic acid was directly reacted with collagen, the collagen functionalization was low; and when the collagen was first itaconylated and then phenylboronic acid was added, the modification phenomenon also occurred; further, the collagen was itaconylated and the phenylboronic acid was activated with EDC / NHS. It was unexpectedly found that compared with 3-aminophenylboronic acid, 4-carboxyphenylboronic acid could successfully modify collagen after EDC / NHS activation, and the obtained bio-ink had good printability. Specifically including the following contents:

[0006] In a first aspect, the present invention provides a glucose-responsive DLP 3D printing collagen bio-ink, the bio-ink comprising the following components: 0.5% to 2.0% m / v phenylboronic acid functionalized itaconylated collagen, 0.25% to 0.5% m / v photoinitiator, and 0 to 0.05% m / v UV absorber;

[0007] The preparation method of the phenylboronic acid functionalized itaconylated collagen comprises the following steps: adding itaconic anhydride to a collagen solution, adjusting the pH to 7-8, and reacting in an ice bath to obtain itaconylated collagen; and adding 4-carboxyphenylboronic acid to the itaconylated collagen solution, adjusting the pH to 5.5-6.0, and reacting in an ice bath to obtain the phenylboronic acid functionalized itaconylated collagen.

[0008] Preferably, the bio-ink further comprises 64 μg / mL to 128 μg / mL curcumin-functionalized gold nanoparticles.

[0009] Preferably, the preparation method of the curcumin-functionalized gold nanoparticles is as follows: 5.7 mg of curcumin is completely dissolved in 30 mL of 0.2 mg / mL NaOH solution, 480 μL of 50 mg / mL chloroauric acid trihydrate is slowly added dropwise to the curcumin solution within 5 minutes, and stirred at room temperature until the solution turns wine red, centrifuged, and washed with water to remove the chloroauric acid trihydrate that does not participate in the reaction; then, according to the molar ratio of curcumin to gold nanoparticles of 1:4, the reaction is carried out to modify the curcumin on the gold nanoparticles, centrifuged, washed with water to remove the curcumin that does not participate in the reaction, and resuspended in ultrapure water to obtain a curcumin-functionalized gold nanoparticle solution.

[0010] Preferably, the collagen is bovine collagen.

[0011] Preferably, the bovine collagen is type I bovine collagen, and / or type II bovine collagen, and / or type II bovine collagen.

[0012] Preferably, the ultraviolet absorber is selected from lemon yellow or brilliant blue.

[0013] Preferably, the ultraviolet absorber is lemon yellow.

[0014] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

[0015] Preferably, the preparation method of the phenylboronic acid functionalized itaconylated collagen is:

[0016] (1) dissolving collagen in acetic acid solution to prepare a collagen solution;

[0017] (2) adjusting the pH of the collagen solution in step (1) to 7-9, adding itaconic anhydride dropwise, maintaining the pH at 7-8, and reacting at 4° C. for 12-72 hours to obtain itaconic acid-ylated collagen;

[0018] (3) dissolving the itaconylated collagen obtained in step (2) to obtain an itaconylated collagen solution, adjusting the pH of the itaconylated collagen solution to 5.5-6.0, adding 4-carboxyphenylboronic acid dropwise, maintaining the solution pH at 5.5-6.0, and reacting at 4°C for 12-72h to obtain phenylboronic acid functionalized itaconylated collagen.

[0019] Preferably, the 4-carboxyphenylboronic acid is activated by EDC / NHS.

[0020] In a second aspect, the present invention provides the use of the bio-ink described in the first aspect in the preparation of tissue engineering materials, hemostatic materials, wound dressings, and drug delivery materials.

[0021] In a third aspect, the present invention provides a dressing for treating diabetic wounds, wherein the dressing is obtained by DLP 3D printing of the biomaterial ink described in the first aspect.

[0022] In a fourth aspect, the present invention provides a method for preparing the glucose-responsive DLP 3D printing collagen bio-ink described in the first aspect, the method comprising:

[0023] (1) Dissolve collagen in 0.1-0.5 M acetic acid solution, adjust the pH to 7-9, add itaconic anhydride dropwise, maintain the solution pH at 7-9, and react at 4°C for 12-72 hours to obtain itaconicated collagen;

[0024] (2) dissolving itaconylated collagen in water, sodium chloride solution, PBS or cell culture medium to obtain an itaconylated collagen solution, adjusting the pH of the itaconylated collagen solution to 5.5-6.0, adding 4-carboxyphenylboronic acid dropwise, maintaining the solution pH at 5.5-6.0 and reacting at 4°C for 12-72 hours to obtain phenylboronic acid functionalized itaconylated collagen;

[0025] (3) 0.5% to 2.0% m / v phenylboronic acid functionalized itaconylated collagen solution, 0.25% to 0.5% m / v phenyl-2,4,6-trimethylbenzoyl lithium phosphate, and 0 to 0.05% m / v ultraviolet absorber were mixed to obtain glucose-responsive DLP 3D printing collagen bioink.

[0026] Preferably, 64 μg / mL to 128 μg / mL curcumin-functionalized gold nanoparticles are further added in step (3).

[0027] Preferably, the preparation method of the curcumin-functionalized gold nanoparticles is as follows: 5.7 mg of curcumin is completely dissolved in 30 mL of 0.2 mg / mL NaOH solution, 480 μL of 50 mg / mL chloroauric acid trihydrate is slowly added dropwise to the curcumin solution within 5 minutes, and stirred at room temperature until the solution turns wine red, centrifuged, and washed with water to remove the chloroauric acid trihydrate that does not participate in the reaction; then, according to the molar ratio of curcumin to gold nanoparticles of 1:4, the reaction is carried out to modify the curcumin on the gold nanoparticles, centrifuged, washed with water to remove the curcumin that does not participate in the reaction, and resuspended in ultrapure water to obtain a curcumin-functionalized gold nanoparticle solution.

[0028] Preferably, the 4-carboxyphenylboronic acid is activated by EDC / NHS.

[0029] The beneficial effects of the present invention are:

[0030] (1) The present invention provides a phenylboronic acid functionalized itaconylated collagen (CIP) composite curcumin functionalized gold nanoparticles (C-AuNPs) ink; the ink realizes responsive release under hyperglycemia conditions after 3D printing to form a gel, thereby exerting anti-inflammatory and antioxidant effects; (2) the bio-ink is dissolved in a solution of physiological pH to form a stable and uniform solution, and can be used for DLP 3D printing to prepare the desired gel; (3) the bio-ink of the present invention can be used to prepare a hydrogel with good mechanical properties, fidelity and anti-degradation ability through 3D printing; (4) the bio-ink 3D printed hydrogel of the present invention can promote cell proliferation, adhesion and migration, and has good biocompatibility and bioactivity; the novel composite collagen bio-ink has great development prospects in repairing diabetic chronic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Preparation and characterization of phenylboronic acid-functionalized itaconylated collagen (CIP); (a) SDS-PAGE images of ColⅠ, CIA, and CIP; (b) 1H NMR characterization of ColⅠ and CIP; (c) FTIR characterization of ColⅠ, CIA, and CIP; (d) Degree of amino substitution of CIA and CIP; (e) Full-spectrum circular dichroism scan curves of ColⅠ, CIA, and CIP (190-260 nm); (f) First-order reciprocal of the derivative of the thermal curves of ColⅠ, CIA, and CIP at 221 nm (dMRE / dT); (g) Display of CIP before and after curing under 405 nm UV light irradiation; (h) Storage modulus and loss modulus curves of different concentrations of CIP bioink (5-15 mg / mL) in situ cured under UV light.

[0032] Figure 2 Characterization of the properties of CIP-C-AuNPs hydrogel; (a) FTIR curves of CIP and CIP-C-AuNPs; (b, c) SEM images of CIP-C-AuNPs; (d) EDS scan of CIP-C-AuNPs; (e) in situ curing curves of CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2; (f) release curves of CIP-C-AuNPs in PBS with and without glucose.

[0033] Figure 3 Rheological analysis and printing of CIP-C-AuNPs gel; (a) Storage modulus (G') and loss modulus (G") of each group of gels at different strains; (b) Storage modulus (G') and loss modulus (G") of each group of gels at different angular frequencies; (c) Viscosity characterization of each group of hydrogels in the shear rate range of 0.2 to 100s-1; (d1, e1, f1, g1) Heart, petals, five-pointed star, and leaf models made; (d2, e2, f2, g2) Printing renderings of hearts, petals, five-pointed stars, and leaves.

[0034] Figure 4 Biocompatibility of CIP-C-AuNPs; (a) Cell viability of L929 cells in different extracts (including Blank group, control group (CIA group), CIP group, CIP-C-AuNPs-1 group, and CIP-C-AuNPs-2 group); (b) Cell viability of L929 cells in the extracts of each group on the first, third, and fifth days; (c) Semi-quantitative analysis of the cell number of L929 cells in the extracts of each group on the first, third, and fifth days by live / dead staining; (d) Live / dead cell staining of fibroblasts in the extracts of each group on the first, third, and fifth days.

[0035] Figure 5Bioactivity of CIP-C-AuNPs. (a) Cell scratch assay. Migration of fibroblast monolayers after scratching (0 h) and after 48 h of culture. Scale bar: 100 μm. (b) Cytoskeleton immunofluorescence staining. Scale bar: 50 μm. Figure 7 CAA bioink-loaded cell printing.

[0036] Figure 6 Antioxidant properties of CIP-C-AuNPs; (a) DPPH free radical scavenging effect diagram of each group; (b) Quantification of DPPH free radical scavenging effect of each group; (c) DCFH-DA staining of L929 cells in vitro; (d) Quantification of DCFH-DA fluorescence; (e) Live / dead cell staining of L929 cells induced by H2O2 after culture in each group (live); (f) Counting of live cells based on the staining results; (g) Live / dead cell staining of L929 cells induced by H2O2 after culture in each group (dead); (h) Counting of dead cells based on the staining results.

[0037] Figure 7 Anti-inflammatory property of CIP-C-AuNPs; (a, b) DAPI (blue) and CD163 and iNOS (green) immunofluorescence staining of Blank group on day 0 after polarization, CIP group on day 1 after polarization, CIP-C-AuNPs-1 group, and CIP-C-AuNPs-2 group, scale bar 50 μm; (c) Semi-quantitative analysis of the fluorescence area ratio of each group stained with CD 163; (d) Semi-quantitative analysis of the fluorescence area ratio of each group stained with iNOS. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings. Obviously, the described embodiments are only part of the present invention, not the entire invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0039] It should be noted that, in the following examples, unless otherwise specified, the methods used are all conventional methods; the reagents used are all conventional reagents and can be purchased from the market.

[0040] In the following examples, the collagen used was extracted in our laboratory. The extraction method is described in invention patent CN113520900B. However, the collagen described in the present invention is not limited to the above-mentioned collagen.

[0041] In the following examples, the CIA ink was prepared by dissolving CIP in PBS to prepare a 10 mg / mL CIA solution, and then adding 5% LAP solution to the solution to make the final concentration 2.5 mg / mL to prepare CIP ink;

[0042] Preparation of CIP and CIP-AuNPs inks: CIP was dissolved in PBS to prepare a 10 mg / mL CIP solution, and then 5% LAP solution was added to it to make the final concentration of 2.5 mg / mL. 68 μL of PBS solution or 1.8824 mg / mL C-AuNPs solution was added to the prepared CIP ink to prepare CIP and CIP-C-AuNPs inks, respectively.

[0043] Preparation of CIP, CIP-AuNPs-1, and CIP-AuNPs-2 bio-inks: CIP was dissolved in PBS to prepare a 10 mg / mL CIP solution, and then 5% LAP solution was added thereto to make the final concentration of 2.5 mg / mL. 1 mL of the above solution was taken and added to three centrifuge tubes, and 0 μL, 34 μL, and 68 μL of 1.8824 mg / mL C-AuNPs solution and 68 μL, 34 μL, and 0 μL of PBS solution were added to the centrifuge tubes, respectively, to prepare CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 bio-inks.

[0044] Preparation of CIA, CIP, and CIP-C-AuNPs DLP 3D printing inks: CIA was dissolved in PBS to prepare a 10 mg / mL CIA solution, and then 5% LAP solution and 5% UV absorber solution were added to make the final concentrations of 2.5 mg / mL and 0.5 mg / mL, and 68 μL PBS solution was added to prepare CIA printing ink; CIP was dissolved in PBS to prepare a 10 mg / mL CIP solution, and then 5% LAP solution and 5% UV absorber solution were added to make the final concentrations of 2.5 mg / mL and 0.5 mg / mL, and 68 μL PBS solution was added to prepare CIP DLP 3D printing ink; CIP was dissolved in PBS to prepare a 10 mg / mL CIP solution, and then 5% LAP solution and 5% UV absorber solution were added to make the final concentrations of 2.5 mg / mL and 0.5 mg / mL, and 68 μL 1.8824 mg / mL C-AuNPs solution was prepared as CIP-C-AuNPsDLP 3D printing ink.

[0045] Example 1 Preparation and Characterization of Phenylboronic Acid Functionalized Itaconylated Collagen (CIP)

[0046] Prepare a 0.3% m / v collagen solution in a 0.5M acetic acid solution. After complete dissolution, add sodium hydroxide solution to adjust the pH to 8.0. Add ten times the equivalent of itaconic anhydride (IA) to the collagen lysine, and maintain the solution pH at 7-8. After reacting in an ice bath for 24 hours, dialyze in ultrapure water, freeze-dry, and prepare itaconylated collagen (CIA). Dissolve CIA in a neutral solution, adjust the pH to 6.0 using acetic acid or sodium hydroxide solution, and add 4-carboxyphenylboronic acid activated by EDC / NHS dropwise, and maintain the solution pH at 5.5-6.0. After reacting in an ice bath for 24 hours, dialyze in ultrapure water, freeze-dry, and prepare phenylboronic acid functionalized itaconylated collagen (CIP).

[0047] The purity of CIP was verified by SDS-PAGE gel electrophoresis. Figure 1 As shown in (a), SDS-PAGE results show that the α1 chain and α2 chain bands of unmodified collagen (Col I) are distributed in the range of 130-180 kDa, and the β dimer band is higher than 180 kDa. After modification with itaconic anhydride (IA) and 4-carboxyphenylboronic acid (4-CPBA), the α1, α2 chain and β dimer bands of collagen are significantly higher than those of Col I. This increase in molecular weight indicates that IA and PBA have been grafted onto the collagen skeleton.

[0048] CIP and Col were dissolved in deuterated DMSO and analyzed by H-NMR spectroscopy. Figure 1 As shown in b, 1 The H-NMR spectrum showed that the modified collagen CIP had vinyl protons (CH2=CH - ), indicating the successful introduction of itaconic anhydride (IA) groups. Simultaneously, multiple peaks were detected in the 7.3-8.3 ppm range, attributed to the aromatic proton multiplets (C6H4-B(OH)2) of the monosubstituted benzene ring of 4-carboxyphenylboronic acid (PBA). NMR results indicate that both IA and PBA were successfully covalently grafted onto the collagen backbone.

[0049] CIP, CIA and Col were freeze-dried and ground together with potassium bromide, and thin slices were prepared by tableting for infrared spectroscopy. Figure 1 As shown in middle c, the FTIR spectrum results show that the intensity of the CH bond stretching vibration peak at 2851 cm-1 is significantly enhanced, corresponding to the methylene (-CH2-) introduced by itaconic anhydride (IA) modification; at the same time, the BO bond symmetric vibration peak and the CH bond bending vibration band broadening characteristics appear at 1020 cm-1 and 708 cm-1, respectively, both of which are derived from the introduction of 4-carboxyphenylboronic acid (PBA) groups.

[0050] The modification degree of CIP was quantitatively analyzed by 2,4,6-trinitrobenzenesulfonic acid (TNBS). Figure 1 As shown in Figure d, the amino substitution degree of CIA is 74%, while the substitution degree of the final product CIP is increased to 95% compared with CIA, further indicating the successful grafting of PBA.

[0051] The thermal stability of phenylboronic acid functionalized itaconylated collagen, itaconylated collagen and collagen was characterized by circular dichroism spectroscopy. Figure 1 As shown in Figure e, the unmodified collagen shows a negative peak near 200nm and a positive peak near 222nm, representing a typical triple helical structure. CIA and CIP also show corresponding negative and positive peaks at the same wavelength, indicating that neither IA nor 4-CPBA modification destroys the triple helical structure of collagen. Figure 1 As shown in (f), the thermal transition temperatures of CIA and CIP decreased slightly but remained around 30°C, indicating that they still had high thermal stability. These results indicate that both CIA and CIP retain the typical triple helical structure of collagen and maintain good thermal stability.

[0052] The curing properties of CIP bio-ink were characterized by UV cross-linking and rheological analysis. Figure 1 As shown in Figures g and h, in a system containing the photoinitiator LAP, the CIP ink forms a stable gel that does not flow after being inverted after being irradiated with 405nm UV light for 10 seconds, indicating that it has the ability to quickly photocuring. Figure 1 As shown in Figure g, the storage modulus (G') of the 5, 10, and 15 mg / mL CIP inks increases over time under illumination. The G' of the 10 and 15 mg / mL groups exceeds 1000 Pa within 4 seconds, demonstrating significantly higher curing rates than the 5 mg / mL group. After 60 seconds of curing, G' reaches 2341 Pa (10 mg / mL) and 3354 Pa (15 mg / mL), respectively, demonstrating a concentration-dependent enhancement pattern, indicating a positive correlation between the crosslink density and concentration. The G' values ​​of all systems remain consistently higher than the loss modulus (G"), demonstrating the high mechanical stability of the gel network and meeting the functional requirements of biomaterials.

[0053] Example 2 Preparation and Characterization of CIP-C-AuNPs Hydrogel

[0054] Curcumin-functionalized gold nanoparticles (C-AuNPs) were prepared according to a reference (Curcumin-functionalized gold nanoparticles attenuate AAPH-induced acute cardiotoxicity via reduction of lipid peroxidation and modulation of antioxidant parameters in a chicken embryo model). Specifically, 5.7 mg of curcumin was completely dissolved in 30 mL of 0.2 mg / mL NaOH solution and stirred for 20 minutes. Then, 480 μL of 50 mg / mL chloroauric acid trihydrate was slowly added dropwise to the curcumin solution over 5 minutes. The solution was stirred at room temperature until it turned wine red. The solution was then centrifuged at 10,000 rpm for 15 minutes and washed three times with water to remove any unreacted chloroauric acid trihydrate. The solution was then stirred again overnight at a curcumin-to-gold nanoparticle molar ratio of 1:4, successfully coating the gold nanoparticles with curcumin. The solution was then centrifuged at 10,000 rpm for 15 minutes and washed twice with water and twice with ethanol to remove any unreacted curcumin. Then, 15 mL of ultrapure water was added to resuspend and dilute to obtain a 1.8824 mg / mL C-AuNPs solution.

[0055] CIP was dissolved in PBS to prepare a 10 mg / mL CIP solution, to which 5% LAP solution was then added to a final concentration of 2.5 mg / mL to prepare CIP DLP 3D printing ink. CIP and CIP-C-AuNPs DLP 3D printing inks were prepared by adding 68 μL of PBS or 1.8824 mg / mL of C-AuNPs solution to the prepared CIP DLP 3D printing ink, respectively. These inks were then added to vials for curing to produce the gels.

[0056] CIP and CIP-C-AuNPs were freeze-dried and then co-ground with potassium bromide. Thin sheets were prepared by tableting for infrared spectroscopy. Figure 2 As shown in middle a, the infrared spectrum shows that the CIP-C-AuNPs gel has a -1 The characteristic peak of borate ester bond (BOC) at 1161 cm -1 The COC peak signal at 400 nm was weakened, indicating that curcumin was bound to the phenylboronic acid group of CIP through a dynamic boronate ester bond.

[0057] The surface microstructure of freeze-dried CIP-C-AuNPs was observed by scanning electron microscopy (SEM) to explore its microstructure. At the same time, X-ray (EDX) spectroscopy was used to analyze the chemical composition of CIP-C-AuNPs hydrogel. SEM images show that Figure 2 As shown in b and c, the CIP-C-AuNPs gel scaffold has a three-dimensional interpenetrating porous network structure, providing a bionic three-dimensional environment for cell growth. Figure 2 As shown in middle d, the characteristic peak of Au element (L α 9.71keV, M α 2.12keV, M β 2.20 keV), indicating that C-AuNPs were successfully integrated into the CIP gel network, and the remaining element peaks corresponded to collagen and curcumin (C, N, O), PBS (P, Cl, K, Na), components and conductive agent Pt.

[0058] An Anton Paar rheometer was used in a time sweep oscillation test with a 15 mm diameter parallel plate and a portable curing light source (wavelength 405 nm, light intensity 25 mW / cm 2 ) 5, 10, and 15 mg / mL CIP and CIP-C-AuNPs-1 and CIP-C-AuNPs-2 inks were irradiated for up to 60 seconds (strain γ = 1%, angular frequency ω = 1 rad / s, gap 0.7 mm) to measure the storage modulus (G') and loss modulus (G") of each group of inks during in situ gelation. The analysis results are shown in Figure 2. Figure 2 As shown in Figure e, the introduction of C-AuNPs prolonged the photocuring time of the CIP ink system by 2-3 seconds, but significantly increased the storage modulus from 1938 Pa to 2522 Pa, indicating that the formation of dynamic boronate bonds between C-AuNPs and CIP further enhanced the mechanical properties of the CIP gel system. These results collectively demonstrate that C-AuNPs achieve chemical crosslinking with CIP via boronate bonds.

[0059] Add 1 mL of CIP-C-AuNPs-2 hydrogel to 20 mL of PBS containing 5 mg / mL glucose or sugar-free solution, respectively, and shake at 37°C, 220 rpm. Take 300 μL of the supernatant and replenish with 300 μL of the corresponding PBS. Compare the absorbance of the supernatant at 428 nm at each time point. Calculate the absorbance according to formula (1):

[0060]

[0061] Among them, Abs n Indicates the absorbance measured in the supernatant at each time period, Abs tIt represents the absorbance of 128 μg / mL C-AuNPs at 428 nm.

[0062] In vitro release experiments were conducted to evaluate the glucose response characteristics of CIP-C-AuNPs gel. Figure 2 As shown in Figure f, UV monitoring based on the characteristic absorption peak of curcumin (λmax = 280nm) revealed a two-stage release behavior in both PBS with and without glucose: a rapid release phase from 0 to 7 hours (cumulative release >50% in the glucose-containing group), a sustained release phase from 7 to 24 hours (cumulative release reached 65% in the glucose-containing group), and a plateau phase after 24 hours. The cumulative release of the glucose-containing group over 24 hours was twice that of the glucose-free group. This phenomenon is attributed to the competitive binding of the CIP phenylboronic acid group to the dynamic reversible ester bond between glucose molecules, triggering gel network relaxation and accelerating C-AuNPs release. These results demonstrate the glucose-responsiveness of the CIP-C-AuNPs gel system and provide strong validation for its use as a smart drug delivery system, particularly for intelligent delivery of polyphenolic compounds in the context of chronic hyperglycemia in diabetic wounds.

[0063] Example 3 Preparation and screening of CIP-C-AuNPs bio-ink

[0064] The freeze-dried CIP sponge was added to water, PBS, or cell culture medium, and the C-AuNPs solution was added dropwise. The solution was dissolved at low temperature, and a blocking reagent was added to stabilize the pH. The photoinitiator and UV absorber components were then added, mixed thoroughly, and centrifuged to remove bubbles to prepare the CAA bio-ink. The formulations of bio-inks 1-8 are shown in Table 1.

[0065] Table 1 Formulations of different bio-inks

[0066]

[0067] CIP-C-AuNPs crosslink under the action of a photoinitiator and UV light to form a gel. Bioinks 1-3 were used to screen C-AuNP concentrations. Experiments showed that Bioink 1, containing LAP, cured in 20 seconds under 365nm light, while Bioinks 2 and 3 took several minutes and exhibited low gel strength, making them unsuitable for 3D printing. LAP was selected as the photoinitiator.

[0068] Bio-inks 3-5 contain varying concentrations of CIP to suit different printing requirements. Bio-inks 6-7 contain varying concentrations of UV absorbers, and Bio-inks 7-8 contain varying concentrations of photoinitiators. By screening these two types, the most suitable working concentration for DLP printing is selected to optimize printing time, biocompatibility, and precision. A variety of bio-ink formulations can be formulated based on specific needs.

[0069] Example 4 CIP-C-AuNPs gel properties and ink 3D printing

[0070] CIP was dissolved in PBS to prepare a 10 mg / mL CIP solution, and then 5% LAP solution was added to it to make the final concentration of 2.5 mg / mL to prepare CIP DLP 3D printing ink; 0 μL, 34 μL, and 68 μL of 1.8824 mg / mL C-AuNPs solution and 68 μL, 34 μL, and 0 μL of PBS solution were added to the prepared CIP DLP 3D printing ink, respectively, to prepare CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 bio-inks and added to penicillin bottles for solidification.

[0071] The same instrument as in Example 2 was used to measure the G' and G" (fixed angular frequency of 1 rad / s) of the three groups of gels, CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2, in oscillation mode (0.4%-100% strain). Still in this mode, the G' and G" (fixed at 1% strain) of 5, 10, and 15 mg / mL CIP gels at angular frequencies of 10-100 rad / s and CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 at angular frequencies of 1-420 rad / s were measured. The dynamic viscosity of the CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 gels was also measured (shear rate of 0.2 to 100 / s, fixed angular frequency of 1 rad / s, and strain of 1%).

[0072] Strain sweep tests showed that within the strain range of 0.1-20%, the storage modulus (G') of all gel systems was significantly higher than the loss modulus (G"), among which the G' value of the unmodified CIA control group was about 460Pa, while the G' of the phenylboronic acid functionalized CIP group increased to 500Pa, preliminarily verifying that the phenylboronic acid group had a certain enhancing effect on the viscoelasticity of the gel; when CIP and C-AuNPs were cross-linked through dynamic covalent bonds, the G' values ​​of CIP-C-AuNPs-1 and CIP-C-AuNPs-2 reached 800Pa and 900Pa, respectively, indicating that the dynamic cross-linking formed by gold nanoparticles and phenylboronic acid groups significantly enhanced the viscoelasticity of the gel ( Figure 3 The frequency sweep results further revealed the dynamic response characteristics of the material in the range of 0.1-400 rad / s: the G' values ​​of all systems in the low-frequency region (0.1-10 rad / s) and the high-frequency region (10-400 rad / s) were higher than G", indicating that the gel network has good mechanical stability and can meet the application requirements in complex physiological environments ( Figure 3 (b) The shear rate-viscosity curve shows that when the shear rate is increased from 0.2s -1 Gradually increase to 100s -1When the initial viscosity of the system drops sharply from 8000-16000 mPa·s to about 1 mPa·s, it shows significant shear thinning behavior and is injectable.

[0073] The printability of the CIP-C-AuNPs ink system was further characterized. CIP-C-AuNPs printing ink was prepared and DLP 3D printing was used to prepare the heart ( Figure 3 d2), flowers (e2 in 3 in the picture), three-dimensional five-pointed star ( Figure 3 f2), and leaves ( Figure 3 Gels of different shapes such as g2) were prepared. By comparison, it can be seen that all four shapes of gels successfully replicated the corresponding CAD 3D models ( Figure 3 In images d1-g1), the gel structure is intact, with good support and sharp edges. Details such as the corners of the three-dimensional five-pointed star are clearly visible, demonstrating that the CIP-C-AuNPs ink has good printability and is suitable for DLP 3D printing.

[0074] These results demonstrate that the dynamic crosslinking strategy of C-AuNPs and phenylboronic acid significantly enhances the rheological properties of the hydrogel, ensuring excellent structural integrity and mechanical stability after printing. As a novel printable ink and responsive release gel system, the CIP-C-AuNPs system has significant application potential in biomedicine, 3D printing, tissue engineering, and other fields.

[0075] Example 5 Biocompatibility of CIP-C-AuNPs Biomaterial Ink

[0076] 100 μL of CIA, CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 hydrogels were prepared and immersed in DMEM at 37 °C. After 72 h, the supernatant was removed and used for subsequent experiments.

[0077] Cytotoxicity was determined by the CCK-8 method. L929 mouse fibroblasts were seeded into 96-well plates at a density of 1×105 cells / mL, and then incubated for one day with complete culture medium (89% DMEM, 10% FBS and 1% penicillin-streptomycin) to allow the cells to be seeded in the well plates. After 24 hours, the culture medium was aspirated, and 100 μL of hydrogel extract was added and cultured for another 24 hours. The extract was discarded, washed three times with PBS, and 100 μL of CCK-8 solution diluted with DMEM was added to each well plate. The well plates were placed in a 37°C incubator in the dark for one hour, and the OD values ​​of DMEM, blank group and each group of samples at 450 nm were measured using a multifunctional microplate reader to calculate the cell activity.

[0078] The CCK-8 assay was also used to investigate cell proliferation. Cells were seeded as in the cytotoxicity experiments, with the extracts added to three 96-well plates. Cell viability was measured using a multifunctional microplate reader at three time points (days 1, 3, and 5). A blank control (with DMEM) on day 1 was used as a control for subsequent calculations.

[0079] The biocompatibility of the hydrogel was qualitatively investigated by live / dead staining of L929 cells using the Calcein-AM / PI kit. Following the instructions, samples from each group were stained and imaged using a laser confocal microscope on days 1, 3, and 5 after treatment with the extract.

[0080] The cytotoxicity results showed that compared with Blank (100%), the cell viability of CIA, CIP, CIP-C-AuNPs-1 and CIP-C-AuNPs-2 were significantly greater than 100%, indicating that the four groups of gels had no cytotoxicity to fibroblasts ( Figure 4 The relative proliferation rates of the four groups of gels after 1, 3, and 5 days of culture were further characterized. The results showed that the CIP-C-AuNPs-1 and CIP-C-AuNPs-2 gel groups showed higher proliferation rates than Blank, CIA, and CIP ( Figure 4 (As shown in Figure 2b). The cell proliferation rate in the CIP-C-AuNPs-1 group reached 336.7% on Day 3 and 480.1% on Day 5, significantly higher than that of CIP. This indicates that the C-AuNPs / CIP composite not only exhibits good biocompatibility but also further promotes cell proliferation. Furthermore, increasing the concentration of C-AuNPs further boosted cell proliferation. The cell proliferation rate in the CIP-C-AuNPs-2 gel group reached 504% on Day 5, demonstrating a significant effect in promoting fibroblast proliferation.

[0081] The cell proliferation results showed that the cell proliferation rate in each experimental group was significantly higher than that in the blank control group, which was consistent with the CCK-8 test results. The cell density of the composite gel group with C-AuNPs (such as CIP-C-AuNPs-2) increased significantly. The number of cells in the field of view reached 3361 on the 5th day of culture, which was significantly higher than the 2571 in the CIP group. The quantitative data were consistent with the proliferation curve trend ( Figure 4 (b, c). Furthermore, staining results showed that all groups maintained high viability after 5 days of culture, indicating that the material lacked significant cytotoxicity. Cell viability, proliferation kinetics, and live / dead staining demonstrated the excellent biocompatibility of CIP and its composite gel system, providing key safety evidence for its biomedical applications.

[0082] Example 6 Bioactivity of CIP-C-AuNPs Biomaterial Ink

[0083] The bioactive cell migration ability of the hydrogel was investigated by a scratch test. L929 cells were seeded into 6-well plates at a density of 1×106 cells / mL and incubated with complete medium until the cell density reached 80%. The complete medium was discarded, DMEM was added, and scratches were made using a 10 μL pipette tip and photographed using an inverted fluorescence microscope (0 hours). The DMEM was discarded, the sample extract was added, and the sample was photographed after incubation for 48 hours. The photographs were processed using Image J software to measure the migration area and calculate the migration rate.

[0084] The cytoskeleton structure of L929 cells was observed using a rhodamine-phalloidin staining kit. L929 cells were plated at 1×10 6 Cells were seeded at a density of 10 cells / mL in a laser confocal microscope dish and incubated for 48 hours. The culture medium was discarded and the sample extract was added. The cells were stained with rhodamine-phalloidin (labeling actin fibers, red) and Hoechst 33342 (labeling cell nuclei, blue). Fluorescence images of the cells were observed and recorded using a laser confocal microscope (Olympus). The staining steps were performed according to the kit instructions.

[0085] The cell migration experiment showed that after 48 hours of culture, the central scratch area of ​​the Blank control group was more obvious and the number of cell migration was small. In contrast, the migration area of ​​cells treated with CIA, CIP, CIP-C-AuNPs-1 and CIP-C-AuNPs-2 gels was significantly larger than that of the control group. Further quantitative analysis showed that the migration rate of the blank control group was 31.4%, the migration rate of CIA was 59.4%, the migration rate of CIP was 60.7%, and the migration rates of CIP-C-AuNPs-1 and CIP-C-AuNPs-2 reached 69.0% and 85.2%, respectively. Figure 5 The results of cell migration indicate that CIP-C-AuNPs-1 and CIP-C-AuNPs-2 have enhanced bioactivity and significant potential to accelerate wound healing.

[0086] Rhodamine-phalloidin (red)-labeled F-actin and DAPI (blue)-labeled cell nuclei showed that compared with the blank group, the cells in each experimental group were well spread, with no significant decrease in the cytoplasm area surrounding individual cell nuclei, indicating that the material had no negative impact on cell adhesion and cytoskeletal spreading. Cellular experiments demonstrated that CIP and its composite gel system exhibit dual biological activities in regulating the wound microenvironment by maintaining cytoskeletal morphology and promoting migration and proliferation.

[0087] Example 7 Antioxidant properties of CIP-C-AuNPs gel

[0088] The antioxidant properties of CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 were evaluated using the 2,2-diphenyl-1-trinitrophenylhydrazine (DPPH) method. DPPH was dissolved in anhydrous ethanol to a final concentration of 8 mg / mL. The treated sample suspensions were mixed with the DPPH solution at a volume ratio of 1:1. After standing for half an hour, the supernatant was centrifuged and the absorbance at 517 nm was measured using a multifunctional microplate reader to further explore its antioxidant properties.

[0089] The DCFH-DA kit was used to investigate its ROS scavenging ability in cells. L929 cells were cultured at a rate of 1×10 5 and 1×10 6 Cells were seeded at 100 cells / mL in a 96-well plate and a laser confocal microplate dish, cultured in complete medium for 24 hours. The medium was then discarded and the sample extract was added for 24 hours. The extract was then discarded and treated with 88 μM H₂O₂ for one hour to create a ROS environment. The H₂O₂ solution was then discarded and the DCF-DA solution prepared according to the manufacturer's instructions was added for half an hour. The ROS scavenging ability of the cells was quantified and observed using a multi-function microplate reader and laser confocal microscopy.

[0090] In addition, L929 cells were stained for live / dead cells after each sample group and H2O2 treatment. The samples and H2O2 treatment procedures were the same as above. Each sample group was discarded and washed with diluted Assay Buffer. Calcein and PI dye were then added according to the manufacturer's instructions. Each sample group was then incubated at 37°C in the dark for half an hour. Live / dead cell images were captured using a confocal laser, and semi-quantitative analysis of live and dead cells was performed using Image J.

[0091] The images of the DPPH free radical scavenging experiment showed that the DPPH solution of the untreated Blank group showed a typical dark purple color, while the color of the DPPH solution of the CIP, CIP-C-AuNPs-1 and CIP-C-AuNPs-2 hydrogels became lighter after 30 minutes of treatment. Among them, the color of the CIP-C-AuNPs-2 group solution was the lightest, which intuitively demonstrated the good antioxidant activity of the CIP-C-AuNPs-2 gel ( Figure 6UV quantitative analysis further showed that the free radical scavenging rates of the CIP group, CIP-C-AuNPs-1 group, and CIP-C-AuNPs-2 group were 49.8%, 56.7%, and 86.6%, respectively. The scavenging efficiency increased significantly with the increase of C-AuNPs concentration ( Figure 6 The clearance rate of the CIP-C-AuNPs-2 group was 1.73 times that of the CIP group, indicating excellent free radical scavenging performance and significant potential in improving the oxidative stress microenvironment of diabetic wounds.

[0092] DCFH-DA ROS scavenging experiments showed that when the Blank group showed significant green fluorescence, indicating a significant increase in intracellular ROS levels, the cell fluorescence intensity of the CIP-C-AuNPs-1 and CIP-C-AuNPs-2 groups was significantly weaker, lower than that of the CIP control group and the Blank group ( Figure 6 This result was further confirmed by quantitative analysis of fluorescence intensity ( Figure 6 This indicates that the CIP-C-AuNPs composite gel system has a high efficiency in scavenging ROS and can effectively reduce the ROS level in cells.

[0093] Based on the H2O2-induced cell oxidative damage model, live / dead cell staining imaging was performed to further observe cell survival (shown in 6e). A large number of cell deaths were observed in the Blank group and the CIP control group. However, in the CIP-C-AuNPs-1 and CIP-C-AuNPs-2 groups, almost no red dead cells were observed. Quantitative data showed that the number of dead cells in the Blank group reached 221 per field of view, which was significantly higher than that in the experimental group (CIP group: 122; composite gel group: 23 and 9, respectively). This is attributed to the efficient ROS scavenging ability of its continuously released C-AuNPs, which effectively protected cells from free radical damage. In summary, the CIP-C-AuNPs hydrogel has excellent antioxidant properties.

[0094] Example 8 Antioxidant properties of CIP-C-AuNPs gel

[0095] Add 1×10 5 Cells were suspended in RAW 264.7 cells at 100 cells / mL and cultured for 24 hours. The culture medium was discarded and the cells were washed three times with PBS for 5 minutes each. The cells were then incubated with 0.1 mg / mL LPS diluted in complete culture medium for 12 hours to polarize M0 macrophages to an M1 phenotype. The extracts from each group were then added to small dishes and cultured for 24 hours before immunofluorescence staining.

[0096] Antibody staining was performed on each group of M0 cells after 24 hours of culture. First, the samples were washed three times with PBS for five minutes each time, and then 0.5% Triton diluted in PBS was added for permeabilization for ten minutes. Washed three times with PBS for five minutes each time, blocked with 5% goat serum for one hour. Then washed for five minutes, and CD163 and iNOS antibody markers diluted 250 times were added, and incubated at 4°C for 16 hours. The same operation was performed by washing with PBS and adding diluted secondary antibodies for two hours. After two hours, staining was performed for ten minutes according to the DAPI staining instructions. Fluorescence photography of each group was performed using a laser confocal microscope under the same parameters.

[0097] The results of immunofluorescence staining experiments showed that after 12 hours of LPS stimulation, the expression of iNOS in macrophages increased significantly, while CD163 was almost not expressed, indicating that M0 cells successfully differentiated into M1 phenotype ( Figure 7 (Figure 2a). Subsequently, after treatment with CIP, CIP-C-AuNPs-1, and CIP-C-AuNPs-2 hydrogels for 24 hours, a significant increase in CD163 fluorescence signal was observed in all three groups, indicating a transition from the M1 phenotype to the M2 phenotype. Fluorescence image analysis showed that the CD163 expression intensity in the CIP group was significantly higher than that in the blank control group, and the introduction of C-AuNPs further enhanced this effect: Figure 7 As shown in Figure c, the average CD163 fluorescence intensity of individual cells in the CIP-C-AuNPs-2 group was significantly higher than that in the CIP group and the CIP-C-AuNPs-1 group. This gradient difference indicates that the release concentration of curcumin-functionalized gold nanoparticles (C-AuNPs) is positively correlated with the M2 polarization efficiency, confirming that it enhances the anti-inflammatory properties of the material by delivering active ingredients.

[0098] In contrast to the expression trend of CD163, the iNOS fluorescence signal intensity in each group decreased significantly after treatment ( Figure 7 b, d). Semi-quantitative analysis showed that ( Figure 7 As shown in (d), in the blank control group (LPS treatment only without extract addition), the iNOS-positive area accounted for 53.8% of the total cell area in the field of view, while in the CIP group it dropped to 44.8%, and in the CIP-C-AuNPs-1 group it further dropped to 35.0%, with the lowest being in the CIP-C-AuNPs-2 group (29.7%). This data indicates that the CIP hydrogel itself has a certain ability to inhibit the maintenance of the M1 phenotype, and the introduction of C-AuNPs significantly amplifies this effect. Among them, the iNOS expression in the CIP-C-AuNPs-2 group was 44.8% lower than that in the blank group, proving that it inhibits the pro-inflammatory phenotype by synergistically regulating the inflammatory signaling pathway.

Claims

1. A glucose-responsive DLP 3D printing collagen bio-ink, characterized in that: The bio-ink comprises the following components: 0.5% to 2.0% m / v phenylboronic acid functionalized itaconylated collagen, 0.25% to 0.5% m / v photoinitiator, and 0 to 0.05% m / v ultraviolet absorber; The preparation method of the phenylboronic acid functionalized itaconylated collagen comprises the following steps: adding itaconic anhydride to a collagen solution, adjusting the pH to 7-8, and reacting in an ice bath to obtain itaconylated collagen; and adding 4-carboxyphenylboronic acid to the itaconylated collagen solution, adjusting the pH to 5.5-6.0, and reacting in an ice bath to obtain the phenylboronic acid functionalized itaconylated collagen.

2. The bio-ink according to claim 1, wherein The biological ink also includes 64 μg / mL to 128 μg / mL curcumin functionalized gold nanoparticles.

3. The bio-ink according to claim 1, wherein The ultraviolet absorber is selected from lemon yellow or brilliant blue; the photoinitiator is selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphate.

4. The bio-ink according to claim 1, wherein The preparation method of the phenylboronic acid functionalized itaconylated collagen is as follows: (1) dissolving collagen in acetic acid solution to prepare a collagen solution; (2) adjusting the pH of the collagen solution in step (1) to 7-9, adding itaconic anhydride dropwise, maintaining the pH at 7-8, and reacting at 4° C. for 12-72 hours to obtain itaconic acid-ylated collagen; (3) dissolving the itaconylated collagen obtained in step (2) to obtain an itaconylated collagen solution, adjusting the pH of the itaconylated collagen solution to 5.5-6.0, adding 4-carboxyphenylboronic acid dropwise, maintaining the solution pH at 5.5-6.0, and reacting at 4°C for 12-72h to obtain phenylboronic acid functionalized itaconylated collagen.

5. The bio-ink according to claim 4, wherein: The 4-carboxyphenylboronic acid was activated by EDC / NHS.

6. Use of the bio-ink according to any one of claims 1 to 5 in the preparation of tissue engineering materials, hemostatic materials, wound dressings, and drug delivery materials.

7. A dressing for treating diabetic wounds, wherein the dressing is obtained by DLP 3D printing of the biomaterial ink according to any one of claims 1 to 5.

8. The method for preparing glucose-responsive DLP 3D printing collagen bio-ink according to claim 1, wherein: The method comprises: (1) Dissolve collagen in 0.1-0.5 M acetic acid solution, adjust the pH to 7-9, add itaconic anhydride dropwise, maintain the solution pH at 7-9, and react at 4°C for 12-72 hours to obtain itaconic acid-ylated collagen; (2) dissolving itaconylated collagen in water, sodium chloride solution, PBS or cell culture medium to obtain itaconylated collagen solution, adjusting the pH of the itaconylated collagen solution to 5.5-6.0, adding 4-carboxyphenylboronic acid dropwise, maintaining the solution pH at 5.5-6.0 and reacting at 4°C for 12-72 hours to obtain phenylboronic acid functionalized itaconylated collagen; (3) 0.5% to 2.0% m / v phenylboronic acid functionalized itaconylated collagen solution, 0.25% to 0.5% m / v phenyl-2,4,6-trimethylbenzoyl lithium phosphate, and 0 to 0.05% m / v ultraviolet absorber were mixed to obtain glucose-responsive DLP 3D printing collagen bioink.

9. The preparation method according to claim 8, wherein In the step (3), 64 μg / mL to 128 μg / mL of curcumin-functionalized gold nanoparticles are also added.

10. The preparation method according to claim 9, characterized in that The 4-carboxyphenylboronic acid was activated by EDC / NHS.

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