3D printing composite hydrogel microneedle as well as preparation method and application thereof

Abstract

The present invention relates to a 3D printing composite hydrogel microneedle, the 3D printing composite hydrogel microneedle comprises methacrylated chitosan, o-nitrobenzyl alcohol polyethylene glycol, resveratrol and a liposome, and the resveratrol is entrapped in the liposome. The preparation method comprises the following steps: preparing resveratrol-loaded lipidosome, and preparing the 3D printing composite hydrogel microneedle. Through organic combination of the pharmacological activity of resveratrol, the controllable skeleton of PEGNB, the adhesion and antibacterial characteristics of CSMA, the precise drug delivery capability of a microneedle and the precise preparation advantage of 3D printing, the innovative microneedle patch with long-acting drug delivery, excellent biocompatibility and myocardial repair function is provided; and a new solution is provided for myocardial injury treatment.

Description

Technical Field

[0001] This invention relates to the field of hydrogel microneedle technology, and in particular to a 3D printed composite hydrogel microneedle, its preparation method, and its application. Background Technology

[0002] Myocardial injury is accompanied by inflammatory responses, oxidative stress, and apoptosis, ultimately leading to an irreversible decline in cardiac function. To improve the microenvironment of the injured area and promote cardiac function recovery, various biomaterial-based strategies have been explored in recent years. For example, hydrogel scaffolds constructed using natural or synthetic polymers can form a protective barrier on the cardiac surface and achieve local drug release by incorporating active molecules; liposome-based drug delivery systems based on nanotechnology can encapsulate antioxidants or signaling molecules, improving drug stability and sustained-release performance to some extent; in addition, cardiac patches, as an exogenous support method, can improve mechanical support and drug accumulation in the myocardial injury area at a macroscopic level. In recent years, microneedle technology has been increasingly applied in myocardial repair research. Its arrayed structure can overcome surface barriers and precisely deliver drugs to the damaged myocardial surface, effectively improving local drug utilization. Meanwhile, the rise of 3D printing technology has provided a highly controllable molding method for the preparation of microneedles and cardiac patches, allowing for personalized design of needle height, density, and arrangement according to the specific circumstances of myocardial injury. In summary, current research has gradually evolved from single drug delivery methods to multifunctional composite systems, attempting to achieve precise intervention in myocardial injury through materials engineering and manufacturing technologies.

[0003] Despite some progress in myocardial injury repair, current research still has significant limitations. First, traditional hydrogel patches are mostly based on natural polymers (such as hyaluronic acid and gelatin). While they possess good biocompatibility, their mechanical strength and toughness are insufficient to withstand the high-frequency dynamic stress caused by continuous cardiac contraction, leading to detachment or deformation and reduced therapeutic efficacy. Furthermore, the degradation rate of these hydrogels is uncontrollable and often fails to match the myocardial repair process. Second, while liposomes are widely used in drug delivery, their stability in the dynamic cardiac environment is limited. They easily diffuse with blood flow, resulting in insufficient drug localization and difficulty in achieving a sustained and effective drug concentration in the damaged area. Moreover, using liposomes alone lacks fixation, limiting therapeutic efficacy. Third, while existing microneedle patches show potential for local drug delivery, their materials are mostly single-layer hydrogels with insufficient mechanical strength, making stable penetration of the myocardial fiber layer difficult. They are prone to breakage or deformation under tissue stress, and the height, density, and other structural parameters of the microneedles largely depend on mold forming, lacking controllability and failing to meet the personalized needs of different patients in different injury areas. Finally, although 3D printing technology has been applied in tissue engineering, its integration with cardiac repair materials is still in the exploratory stage. Most existing 3D-printed cardiac patches are primarily single-function, lacking synergistic design for drug delivery and tissue repair, and failing to simultaneously address key requirements such as biomechanical fit, precise positioning, sustained drug release, and long-term stability. Therefore, current technologies often only address one aspect of the myocardial repair process, lacking a systematic and holistic approach, making it difficult to achieve efficient, long-term, and personalized treatment of myocardial injury. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a 3D-printed composite hydrogel microneedle comprising methacryloyl chitosan (CSMA), o-nitrobenzyl glycol-modified polyethylene glycol (PEGNB), resveratrol, and liposomes (Lip), wherein the resveratrol is encapsulated within the liposomes.

[0005] In one embodiment, the mass concentrations of the methacryloylchitosan, the o-nitrobenzyl glycol, the resveratrol, and the liposomes in the 3D printed composite hydrogel microneedles are 10~20 mg / mL, 50~90 mg / mL, 0.5~1 mg / mL, and 1~2 mg / mL, respectively.

[0006] A second aspect of the present invention provides a method for preparing the above-mentioned 3D printed composite hydrogel microneedles, comprising the following steps:

[0007] Preparation of resveratrol-loaded liposomes: Hydrogenated soybean lecithin (HSPC), cholesterol (CHO-HP) and resveratrol were dissolved, sonicated, added dropwise to water, and evaporated to obtain the liposomes. Preparation of 3D printed composite hydrogel microneedles: Take a liposome solution loaded with resveratrol, add a photoinitiator and lemon yellow, mix well, add methacrylamide chitosan and o-nitrobenzyl alcohol-modified polyethylene glycol, print, and wash to obtain 3D printed composite hydrogel microneedles.

[0008] Integrating PEGNB photosensitive network, CSMA natural polysaccharide, and resveratrol liposomes for 3D printing to construct myocardial repair microneedle patches is not a simple matter of splicing. The key challenge lies in the fact that the photofracture process of PEGNB generates free radicals and acidic intermediates, which easily damage the liposome structure; the high viscosity of CSMA can also lead to uneven distribution of the material and liposome aggregation; and the ultrafast photocuring of PEGNB makes the 3D printing forming window extremely narrow. Continuing with conventional conditions in existing technologies would result in problems such as liposome rupture, drug leakage, and printed structure collapse. This invention overcomes the multiple technical obstacles of material compatibility and process window by precisely controlling the PEGNB / CSMA ratio, reducing the photoinitiator concentration, optimizing the printing light intensity, and improving the anti-free radical ability by adjusting the liposome membrane composition. This allows the liposomes to maintain structural integrity during photocuring and obtain a stable printable microneedle array, thus achieving a stable configuration that is impossible with existing technologies.

[0009] In this invention, to ensure the integrity and stability of resveratrol liposomes within the PEGNB / CSMA hydrogel microneedle structure, the preparation conditions were systematically optimized. Key protective conditions include the following: First, this invention employs a PEGNB photocrosslinking system instead of traditional thermal or strong oxidative crosslinking methods. The nitrobenzyl photocleavage reaction of PEGNB is a mild phototriggered mechanism, which does not generate high temperatures, release free radicals, or rely on vigorous oxidants. Therefore, it avoids thermal damage or chemical oxidation of the liposome lipid bilayer during crosslinking, a crucial condition for protecting the liposome structure. Second, this invention selects CSMA as the natural polysaccharide segment. Its dissolution and mixing occur under neutral aqueous phase and room temperature conditions. The system contains no strong acids or bases, nor does it contain surfactants, metal ions, or organic solvents, thus preventing dissolution, ion breakdown, or reduced membrane stability of the liposome membrane structure from the outset. Third, resveratrol liposomes are uniformly dispersed in a hydrogel precursor solution before photocrosslinking and curing. This invention controls the viscosity and osmotic pressure of the precursor solution to ensure it is isotonic or near-isootonic with the liposomes, effectively preventing liposome rupture or collapse due to abnormal osmotic pressure. Finally, this invention controls the light intensity and photocrosslinking time to achieve gelation without generating excessive light energy or localized temperature rise, thereby further ensuring the structural integrity of the liposomes during the hydrogel network formation process. Therefore, these mild photocrosslinking conditions, neutral aqueous phase conditions, and isotonic environment together constitute the decisive factors for protecting the liposome structure. These are not simply dependent on the entire method, but rather key process control points that have been specifically screened and validated.

[0010] In one embodiment, the method for preparing the methacrylamide chitosan includes the following steps: Chitosan was dissolved in acetic acid solution, diluted with ethanol, sonicated, and then methacrylic anhydride was added. The mixture was stirred at room temperature, dialyzed, and freeze-dried to obtain the final product. The stirring time was 6-8 hours. The chitosan to methacrylic anhydride ratio is 1:(1~2) by mass.

[0011] In one embodiment, the dilution with ethanol is a dilution with an equal volume of ethanol as the acetic acid solution.

[0012] In one embodiment, the preparation of the resveratrol-loaded liposomes includes the following steps: Hydrogenated soybean lecithin, cholesterol, and resveratrol are dissolved in anhydrous ethanol, sonicated, added dropwise to water, and evaporated to obtain the final product. The mass ratio of the hydrogenated soybean lecithin to the cholesterol to the resveratrol is 1:(2~3):(1~2).

[0013] In one embodiment, the preparation of the 3D-printed composite hydrogel microneedles includes the following steps: Take a liposome solution loaded with resveratrol, add a photoinitiator and lemon yellow, vortex mix evenly, add methacrylamide chitosan and o-nitrobenzyl glycol to dissolve, print, wash to obtain 3D printed composite hydrogel microneedles. The photoinitiator includes LAP, and the ratio of the resveratrol-loaded liposome solution to the photoinitiator to the lemon yellow is (1~2) mL: (1~2) mg: (0.5~1.2) mg.

[0014] In one embodiment, the printing parameters are set as follows: light intensity: 10mW / cm2, exposure time: 20s, number of substrate layers: 1, and substrate exposure time: 25s.

[0015] In one embodiment, the mass concentrations of the methacryloylchitosan, the o-nitrobenzyl glycol, the resveratrol, and the liposomes in the 3D printed composite hydrogel microneedles are 10~20 mg / mL, 50~90 mg / mL, 0.5~1 mg / mL, and 1~2 mg / mL, respectively.

[0016] A third aspect of the present invention also provides the application of the above-described 3D-printed composite hydrogel microneedles, or the 3D-printed composite hydrogel microneedles obtained by the above-described preparation method, in the preparation of products for myocardial repair.

[0017] In addition, the present invention also provides a product for myocardial repair, comprising the above-mentioned 3D printed composite hydrogel microneedles, or the 3D printed composite hydrogel microneedles obtained by the above preparation method.

[0018] The PEGNB / CSMA / resveratrol@liposome composite hydrogel microneedle patch proposed in this invention has significant advantages in myocardial injury repair. Firstly, resveratrol, as a natural polyphenol, possesses excellent antioxidant, anti-inflammatory, and angiogenesis-promoting effects, effectively clearing excess reactive oxygen species (ROS) after myocardial infarction, reducing inflammatory responses, and promoting functional recovery of myocardial tissue. However, its poor water solubility and low in vivo stability limit its application. This invention achieves efficient delivery and sustained release of resveratrol through liposome encapsulation. Secondly, o-nitrobenzyl alcohol-modified polyethylene glycol (PEGNB), as a hydrogel framework material, has good biocompatibility and photocrosslinking properties. It can form a stable three-dimensional network structure through a controllable crosslinking reaction, thereby ensuring the molding precision and mechanical properties of the microneedles and facilitating long-acting drug loading and release. Compared to the traditional methacryloyl hyaluronic acid (HAMA) system, this invention innovatively introduces methacryloyl chitosan (CSMA). Its cationic properties enhance the adhesion of the patch to myocardial tissue, ensuring stable attachment of the microneedles to the surface of the beating heart. Simultaneously, CSMA possesses natural antibacterial properties, effectively reducing the risk of infection in the inflammatory microenvironment of myocardial infarction. Furthermore, the microneedle structure enables non-invasive penetration of the fibrotic barrier on the myocardial surface, precisely delivering drugs to the damaged area, significantly improving drug bioavailability and avoiding the side effects caused by systemic administration. Finally, 3D printing technology allows for precise control of the microneedles' geometry and material distribution, ensuring sufficient mechanical strength while also possessing sustained drug release properties and good biodegradability, thus overcoming the limitations of traditional processes in achieving personalized preparation and structural optimization. This invention provides an innovative microneedle patch that combines the pharmacological activity of resveratrol, the controllable framework of PEGNB, the adhesion and antibacterial properties of CSMA, the precise drug delivery capability of microneedles, and the precision manufacturing advantages of 3D printing, offering a new solution for the treatment of myocardial injury by organically combining the pharmacological activity of resveratrol, the controllable framework of PEGNB, the adhesion and antibacterial properties of CSMA, the precise drug delivery capability of microneedles, and the precision manufacturing advantages of 3D printing.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. A hydrogel network was constructed using PEGNB and CSMA, which combines controllable photocrosslinking, mechanical strength, and good biocompatibility.

[0020] 2. Resveratrol is encapsulated in liposomes to achieve stable loading, sustained release, and controlled release functions, thereby improving bioavailability.

[0021] 3. It can continuously remove excess reactive oxygen species (ROS) after myocardial infarction, inhibit inflammatory responses, improve the myocardial microenvironment, and promote angiogenesis and tissue repair.

[0022] 4. The microneedle structure design enables precise delivery of drugs to the site of myocardial injury under non-invasive conditions, improving drug penetration depth and utilization efficiency.

[0023] 5. 3D printing technology ensures precise microneedle geometry and sufficient tip strength, supporting personalized design and highly repeatable fabrication.

[0024] 6. Compared with traditional cardiac patches, it can be fixed without sutures or injections, making the operation simple and reducing treatment risks.

[0025] 7. By integrating material systems and preparation processes, the system achieves dual functions of mechanical support and drug therapy, thereby enhancing myocardial repair effects and clinical application potential. Attached Figure Description

[0026] Figure 1 Image of a hydrogel microneedle patch; Figure 2 This is a graph showing the antioxidant capacity analysis. Figure 3 Release curve of the substance; Figure 4 This is a graph evaluating the anti-inflammatory effect. Detailed Implementation

[0027] This invention achieves efficient delivery and sustained release of resveratrol through liposome encapsulation. Liposome loading of resveratrol significantly improves its solubility and chemical stability, prolongs blood circulation time, enhances in vivo bioavailability, and achieves higher local drug concentrations and sustained-release effects, thereby enhancing antioxidant and anti-inflammatory effects while reducing systemic toxicity. A 3D-printed microneedle patch is designed, with a microneedle array that gently penetrates the epicardium, precisely delivering the resveratrol-loaded liposomes to the myocardial surface, improving local retention and significantly reducing systemic side effects. 3D printing technology ensures highly controllable microneedle geometry (length, taper, arrangement), allowing it to match the curvature of the heart surface, achieving good mechanical compliance, and reducing stress damage during cardiac pulsation. This invention prepares a hydrogel system composed of PEGNB and CSMA. The cationic properties of CSMA enhance the adhesion of the patch to myocardial tissue, ensuring stable adhesion of the microneedles to the surface of the beating heart. Simultaneously, CSMA possesses natural antibacterial properties, effectively reducing the risk of infection in the inflammatory microenvironment of myocardial infarction. The controllable cross-linking of PEGNB ensures the molding precision of the microneedles.

[0028] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0031] Example I. This invention provides a method for preparing 3D-printed composite hydrogel microneedles, comprising the following steps: 1. Preparation of CSMA 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After sonicating to remove all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacryloxychitosan (CSMA).

[0032] 2. Preparation of resveratrol-loaded liposomes Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Sonicate to dissolve the ethanol. Slowly add 200 μL of the solution to 12 mL of deionized water that has been stirred into a vortex. Remove the ethanol by rotary evaporation to obtain liposomes loaded with 2 mg / mL resveratrol (1 mg / mL).

[0033] 3. 3D printing of resveratrol-loaded microneedle patches Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds to mix thoroughly, and obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0034] II. This invention also provides a method for preparing blank liposomes, comprising the following steps: Weigh 7.4 mg HSPC and 16.7 mg CHO-HP and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water that has been stirred into a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL blank liposomes.

[0035] The microneedle patch tips of the embodiments and comparative examples of the present invention are prepared by modifying the following raw materials, which, by mass concentration, include PEGNB 50~90mg / mL, CSMA 10~20mg / mL, liposomes 1~2mg / mL, and resveratrol 0.5~1mg / mL.

[0036] The optimal formulation is: PEGNB 70mg / mL, CSMA 20mg / mL, liposomes 2mg / mL, and resveratrol 1mg / mL.

[0037] Table 1

[0038] Example 1 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0039] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0040] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.09 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0041] Example 2 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0042] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0043] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0044] Example 3 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0045] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0046] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.05 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0047] Example 4 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0048] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0049] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.01 g of CSMA and 0.09 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0050] Example 5 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0051] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0052] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 s, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.01 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0053] Example 6 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0054] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0055] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.01 g of CSMA and 0.05 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0056] Comparative Example 1 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After sonicating to remove all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0057] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of deionized water, add 1 mg of LAP, 0.6 mg of lemon yellow, and 1 mg of resveratrol, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated resveratrol solution. Dissolve 0.02 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0058] Comparative Example 2 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0059] Preparation of resveratrol-loaded liposomes: Weigh 3.7 mg HSPC, 8.35 mg CHO-HP, and 12 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 1 mg / mL resveratrol-loaded (1 mg / mL) liposomes.

[0060] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0061] Comparative Example 3 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0062] Preparation of blank liposomes: Weigh 7.4 mg HSPC and 16.7 mg CHO-HP and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water that has been stirred into a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL blank liposomes.

[0063] 3D Printing Microneedle Patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds to mix evenly, and obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0064] Comparative Example 4 Preparation of CSMA: 3.0 g of chitosan was dissolved in 100 mL of 2% (v / v) acetic acid solution, and then the solution was diluted with an equal volume of ethanol. After ultrasonic removal of all air bubbles, 3 g of methacrylic anhydride was slowly added to the chitosan solution, and the mixture was stirred continuously at room temperature for 7 h. The solution was dialyzed using an 8000 Da dialysis bag, and finally freeze-dried to obtain methacrylated chitosan (CSMA).

[0065] Preparation of resveratrol-loaded liposomes: Weigh 7.4 mg HSPC, 16.7 mg CHO-HP, and 6 mg resveratrol and dissolve them in 1 mL of anhydrous ethanol. Dissolve by sonication. Slowly add 200 μL of the solution dropwise to 12 mL of deionized water stirred in a vortex. Remove the ethanol by rotary evaporation to obtain 2 mg / mL resveratrol-loaded (0.5 mg / mL) liposomes.

[0066] 3D printing resveratrol-loaded microneedle patches: Take 1 mL of liposome solution, add 1 mg of LAP and 0.6 mg of lemon yellow, vortex for 30 seconds, mix thoroughly to obtain a photo-initiated liposome solution. Dissolve 0.02 g of CSMA and 0.07 g of PEGNB in ​​the above solution to obtain ink. Then, pour the prepared bio-ink into the printer, select the microneedle model and model size, and print (light intensity: 10 mW / cm²). 2 Exposure time: 20s, number of base layers: 1, base layer exposure time: 25s). Finally, wash the printed hydrogel 2-3 times with sterile PBS to obtain the hydrogel microneedle scaffold.

[0067] Implementation effect evaluation 1. Appearance of hydrogel microneedles from Figure 1 It can be seen that microneedles with intact tips and good morphology can be prepared.

[0068] 2. Single needle mechanical properties Test method: Place the microneedle sample on the sample stage with the tip pointing upwards, and adjust the height of the sample stage so that both the upper and lower surfaces are in contact with the fixture. Compress at a constant rate of 0.05 mm / s, and record the compression displacement (L) and load (P) until the set displacement endpoint is reached. The mechanical strength of a single needle is the ratio of load to the number of needle tips. The test results are shown in Table 2.

[0069] Table 2 Single needle mechanical strength values

[0070] Table 2 shows that the amount of resveratrol added does not significantly affect the tip strength of the microneedles; however, too low an amount cannot achieve good antioxidant and anti-inflammatory effects. Literature research indicates that a single needle with a mechanical strength greater than 0.04 N can pierce the stratum corneum and epidermis. Examples 1-6 and Comparative Examples 1-4 all meet this requirement.

[0071] 3. Antioxidant capacity test Test Method: The antioxidant performance of different examples and comparative examples was evaluated using a method for scavenging 1,1-diphenyl-2-pyridylhydrazide (DPPH) free radicals. Mixtures of the different examples and comparative examples with DPPH reagent were incubated in the dark with stirring for 30 minutes, and the remaining DPPH was analyzed using UV-Vis spectroscopy. The formula for determining the DPPH scavenging rate is: D VC (%) = [(A) 空白 -A 阳性对照 ) / A 空白 ]×100% D (%) = {[A 空白 -(A 测定 -A对照 )] / A 空白}×100% like Figure 2 As shown, the efficiency of free radical scavenging increases with increasing concentrations of resveratrol and CSMA. Resveratrol exhibits a greater antioxidant effect on hydrogel microneedles, while CSMA has a slight impact on the antioxidant effect. This indicates that resveratrol can effectively scavenge free radicals and thus has a better antioxidant effect. Meanwhile, the antioxidant effect of resveratrol encapsulated in liposomes is slightly weaker than that of unencapsulated resveratrol within 30 minutes, indicating that the release rate of resveratrol is slowed down after encapsulation in liposomes. This suggests that liposomes can improve the stability of resveratrol and play a synergistic role in free radical scavenging.

[0072] 4. Drug release test of hydrogel Test method: Resveratrol solutions were prepared according to a certain concentration gradient, and the absorbance was measured at 306 nm using a UV spectrophotometer to construct a standard curve. Then, the corresponding example and comparative samples were placed in PBS and incubated at 37°C. The supernatant was collected on days 1, 3, 5, 7, 14, 21, and 28, and the absorbance was measured using a UV spectrophotometer. The drug release was calculated based on the standard curve.

[0073] from Figure 3 It is known that encapsulating resveratrol with liposomes can achieve a sustained-release effect; however, excessively low liposome concentrations cannot effectively control the release of resveratrol. The drug release rate can better match the rate of myocardial tissue repair, which is beneficial for the treatment of myocardial injury.

[0074] 5. In vitro cytotoxicity test Test method: According to GB / T 16886.5-2017 Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test. After the colloid was prepared and completely cured according to the implementation method, it was extracted with culture medium for 24 hours according to the standard method; the extract was then used to treat L929 cells for 24 hours, and the absorbance was detected by CCK-8 assay and the viability was calculated. Normal cultured cells were used as controls; if the viability was <70%, it was considered to have potential cytotoxicity (n=3).

[0075] Table 3 Potential cytotoxicity

[0076] As can be seen from Table 3, the cell survival rate of each group is ≥90%, with no potential cytotoxicity, proving that the system of the present invention has good cell compatibility.

[0077] 6. Anti-inflammatory ability test of hydrogel Rats were anesthetized by intraperitoneal injection of sodium pentobarbital and then divided into sham-operated group, I / R model group, implementation group, and control group according to the experimental design. Surgery was performed with the heart exposed through thoracic incision. In the I / R group and each patch treatment group, a myocardial ischemia-reperfusion model was established by ligating the left anterior descending coronary artery. After ischemia was maintained for a certain period, the ligation was released to restore blood flow, simulating the myocardial injury-reperfusion process. Subsequently, PEGNB / CSMA / resveratrol@liposome hydrogel microneedle patches were implanted into the ischemic area. The control group received patches without drugs or containing only empty liposomes. The sham-operated group underwent only thoracotomy without ligation. Mice were sacrificed on postoperative day 7, and myocardial ischemia tissue was harvested, homogenized thoroughly in a buffer containing protease inhibitors, and centrifuged to collect the supernatant. The supernatant was used for ELISA to detect inflammatory factor levels, thereby evaluating the effect of the microneedle hydrogel patch in improving the inflammatory response of myocardial injury.

[0078] from Figure 4 It can be seen that, compared with the IS group, the hydrogel loaded with resveratrol can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6, and IL-β, and can effectively exert an anti-inflammatory effect, providing a stable microenvironment for myocardial tissue repair. Comparative Example 1 (without liposome encapsulation of resveratrol) and Comparative Example 2 (with lower liposome concentration), although showing some anti-inflammatory effects, suffered from poorer later treatment efficacy compared to Example 2 due to the ease with which the drug is degraded.

[0079] 7. PEGNB's photo-triggered tissue integration capability PEGNB's unique photo-triggered tissue integration capability is achieved through the photocleavage of nitrobenzyl groups to generate new aldehyde groups, enabling in-situ chemical integration with the amino groups of epicardial proteins (Schiff base or similar condensation reaction). While methacrylate materials (PEGDA, GelMA, etc.) can be photocrosslinked and cured, they are essentially chain-like free radical polymers and do not expose aldehyde groups on the tissue surface. Therefore, they can only form "physical adhesion" on the tissue surface, which is the core reason for PEGNB's irreplaceable nature. Therefore, this invention determines whether PEGNB and other materials possess photo-triggered tissue integration capability by detecting whether they generate new aldehyde groups after being exposed to light.

[0080] Microneedles were prepared according to the proportions in Example 1, with PEGNB replaced by GelMA, HAMA, and PEGDA, while keeping the other components unchanged. A 2,4-dinitrophenylhydrazine (DNPH) precipitation test was used for testing. First, 0.2% DNPH was prepared in a 2M HCl solution. The microneedle sample, after being exposed to light, was placed flat on a clean ceramic plate, and 2-3 drops of DNPH solution were added to the sample surface. The plate was left at room temperature for 1-5 minutes. Excess liquid was gently tilted or absorbed with anhydrous paper, and the presence of a yellow / orange or yellow precipitate was observed. If a yellow precipitate appeared, the material could produce aldehyde groups after light exposure; if no yellow precipitate appeared, the material could not produce aldehyde groups after light exposure.

[0081] Table 4

[0082] 8. Adhesion properties of CSMA The unique adhesion of CSMA was demonstrated using a pull-off method. Microneedles were prepared according to the proportions in Example 1, with CSMA replaced by CMCMA, AlgMA, and GelMA, while keeping the other components unchanged. The prepared microneedles were attached to the surface of fresh pigskin, and after contact for 30 seconds under constant pressure, they were vertically pulled off. The maximum required separation force was recorded, and the adhesion was scored from 1 to 5 points based on relative strength. Each experiment was repeated three times, and the average value was taken to evaluate the influence of different materials on the adhesion performance of microneedles.

[0083] The adhesion scoring criteria are as follows: 1 point: Separation force ≤ 0.05 N / cm 2 The pigskin has no visible residue on its surface, falls off easily to the touch, and offers almost no resistance; this indicates "virtually no adhesion," making it impossible to fix clinically.

[0084] 2 points: Separation force 0.05~0.15 N / cm 2 Less than 10% residue remains, and there is a slight dragging sensation when pulling it away; this indicates "extremely weak adhesion," meaning it will fall off with the slightest touch.

[0085] 3 points: Separation force 0.15~0.30 N / cm 2 10-30% residue remains, requiring continuous force to pull it off; this represents "medium adhesion," which can be fixed for a short time but has average reliability.

[0086] 4 points: Separation force 0.30~0.50 N / cm 2 30-60% residue remains, requiring significant and continuous force during the pull-off process; this indicates "good adhesion" and is acceptable for clinical use.

[0087] 5 points: Separation force > 0.50 N / cm 2The residue exceeds 60% and may even tear off the pigskin, making it extremely difficult to pull off. It is often accompanied by hydrogel cohesion destruction; this indicates "super strong adhesion" and may pose an excessive risk.

[0088] Table 5

[0089] The PEGNB, CSMA, and resveratrol liposomes in this invention exhibit a significant synergistic effect, resulting in comprehensive performance unpredictable by existing technologies: the photocleavage of PEGNB's nitrobenzyl groups exposes aldehyde groups on the tissue surface, enabling rapid chemical integration of the microneedle patch onto the epicardium; the natural polysaccharide segments provided by CSMA endow the microneedles with stronger tissue affinity and flexibility, and its cationic properties enhance the adhesion of the patch to myocardial tissue, maintaining stable adhesion under cardiac pulsation; while resveratrol, after being encapsulated in liposomes and embedded in the PEGNB / CSMA network, exhibits stable sustained-release, antioxidant, and cell migration-promoting capabilities far exceeding those of simple liposomes or ordinary hydrogels. This multi-layered synergistic effect is a composite effect unforeseen by existing technologies. Furthermore, PEGNB would lose its rapid tissue integration capability if replaced by other acrylates; CSMA would not provide the same biocompatibility and adhesion if replaced by other polysaccharides; and resveratrol would struggle to achieve sustained efficacy locally in the myocardium without being loaded in a liposome manner. Therefore, each component is irreplaceable.

[0090] This invention not only achieves a first-ever synergistic combination of PEGNB, CSMA, and resveratrol liposomes in its material system, but more importantly, it successfully applies this combined system to a 3D-printable myocardial repair microneedle platform, achieving molding precision, tissue integration capability, and drug retention efficiency unattainable by existing technologies. Existing microneedles or hydrogels do not simultaneously possess the unified integration of four functions: rapid photochemical tissue integration, natural polysaccharide flexibility, stable sustained release of hydrophobic drug liposomes, and deep delivery via microneedles. Furthermore, no feasible pathway for protecting the structural integrity of liposomes in a PEGNB photocrosslinking system has been reported. The ternary system of this invention enables the microneedle patch to stably adhere to the surface of the frequently pulsating myocardium, achieving long-lasting, deep, and uniform resveratrol delivery, effectively improving myocardial oxidative stress and tissue repair capabilities, forming a novel comprehensive performance that existing technologies cannot provide.

[0091] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0092] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A 3D-printed composite hydrogel microneedle, characterized in that, The 3D-printed composite hydrogel microneedles comprise methacrylamide chitosan, o-nitrobenzyl glycol, resveratrol, and liposomes, with the resveratrol encapsulated within the liposomes.

2. The 3D-printed composite hydrogel microneedles according to claim 1, characterized in that, In the 3D printed composite hydrogel microneedles, the mass concentrations of the methacrylamide chitosan, the o-nitrobenzyl glycol, the resveratrol, and the liposomes are 10~20 mg / mL, 50~90 mg / mL, 0.5~1 mg / mL, and 1~2 mg / mL, respectively.

3. The method for preparing 3D-printed composite hydrogel microneedles according to any one of claims 1-2, characterized in that, Includes the following steps: Preparation of resveratrol-loaded liposomes: Hydrogenated soybean lecithin, cholesterol and resveratrol were dissolved, sonicated, added dropwise to water, and evaporated to obtain the liposomes. Preparation of 3D printed composite hydrogel microneedles: Take a liposome solution loaded with resveratrol, add a photoinitiator and lemon yellow, mix well, add methacrylamide chitosan and o-nitrobenzyl alcohol-modified polyethylene glycol, print, and wash to obtain 3D printed composite hydrogel microneedles.

4. The preparation method according to claim 3, characterized in that, The method for preparing the methacrylated chitosan includes the following steps: Chitosan was dissolved in acetic acid solution, diluted with ethanol, sonicated, and then methacrylic anhydride was added. The mixture was stirred at room temperature, dialyzed, and freeze-dried to obtain the final product. The stirring time was 6-8 hours. The chitosan to methacrylic anhydride ratio is 1:(1~2) by mass.

5. The preparation method according to claim 3, characterized in that, The preparation of the resveratrol-loaded liposomes includes the following steps: Hydrogenated soybean lecithin, cholesterol, and resveratrol are dissolved in anhydrous ethanol, sonicated, added dropwise to water, and evaporated to obtain the final product. The mass ratio of the hydrogenated soybean lecithin to the cholesterol to the resveratrol is 1:(2~3):(1~2).

6. The preparation method according to claim 3, characterized in that, The preparation of the 3D-printed composite hydrogel microneedles includes the following steps: Take a liposome solution loaded with resveratrol, add a photoinitiator and lemon yellow, vortex mix evenly, add methacrylamide chitosan and o-nitrobenzyl glycol to dissolve, print, wash to obtain 3D printed composite hydrogel microneedles. The photoinitiator includes LAP, and the ratio of the resveratrol-loaded liposome solution to the photoinitiator to the lemon yellow is (1~2) mL: (1~2) mg: (0.5~1.2) mg.

7. The preparation method according to claim 6, characterized in that, The printing parameters were set to a light intensity of 10 mW / cm². 2 Exposure time: 20s, Number of base layers: 1, Base layer exposure time: 25s.

8. The preparation method according to claim 3, characterized in that, In the 3D printed composite hydrogel microneedles, the mass concentrations of the methacrylamide chitosan, the o-nitrobenzyl glycol, the resveratrol, and the liposomes are 10~20 mg / mL, 50~90 mg / mL, 0.5~1 mg / mL, and 1~2 mg / mL, respectively.

9. The application of the 3D-printed composite hydrogel microneedles as described in claims 1-2, or the 3D-printed composite hydrogel microneedles obtained by the preparation method described in claims 3-8, in the preparation of products for myocardial repair.

10. A product for myocardial repair, characterized in that, This includes 3D-printed composite hydrogel microneedles as described in claims 1-2, or 3D-printed composite hydrogel microneedles obtained by the preparation method described in claims 3-8.

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