Hydrogel microneedle with adjustable mechanical property based on methacrylic acid chitosan as well as preparation method and application of hydrogel microneedle

Abstract

The invention discloses a methacrylic acid chitosan (CSMA)-based hydrogel microneedle with adjustable mechanical properties and a preparation method and application thereof, the microneedle is based on CSMA hydrogel and constructed through an ultraviolet light crosslinking curing technology, the preparation process is simple and efficient, and the material stability is excellent. Polyethylene glycol diacrylate (PEGDA) is introduced into a micro-needle system to form a double-crosslinking structure, accurate adaptation of mechanical properties of the micro-needle can be realized by flexibly regulating and controlling crosslinking parameters, the mechanical strength and tissue penetrating power of the micro-needle are remarkably improved, and the mechanical requirements of different administration scenes are met. The three-dimensional porous network in the microneedle can efficiently load various therapeutic drugs, and has different release rates for drugs with different properties. In addition, experimental verification also shows that the microneedle system has excellent biocompatibility and no biocompatibility, the material can be naturally degraded, and the risk of secondary taking out is avoided. The invention provides a novel solution with customizable mechanical properties and flexible drug loading for personalized drug delivery.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and drug delivery, and specifically relates to a hydrogel microneedle with adjustable mechanical properties based on methacrylated chitosan, and a preparation method and application thereof. Background Art

[0002] Drug delivery technology is the key to improving the efficacy of tumor, chronic disease and gene therapy, but its development is limited by the delivery efficiency and stability of biomacromolecule drugs (such as proteins and nucleic acids). Traditional oral or injection administration faces problems such as poor penetration of biological barriers, high off-target toxicity and uncontrollable release. It is urgent to develop new carriers that have both efficient delivery, targeted sustained release and mechanical adaptability.

[0003] Microneedle technology breaks through physiological barriers through minimally invasive punctures and delivers drugs directly to target tissues, but the existing system has significant limitations: metal / silicon-based microneedles are non-degradable and the process is complex, making it difficult to load sensitive drugs; degradable polymer microneedles (such as polylactic acid) have a narrow mechanical regulation range, are easily broken in high-toughness tissues, and have a mismatch between degradation and release kinetics. Although hydrogel microneedles are degradable and flexible in drug loading, they generally face problems such as swelling and disintegration (such as gelatin-based) and environmental sensitivity (such as sodium alginate ion cross-linking), which lead to sudden release of drugs or loss of activity. More importantly, the existing systems are mostly limited to a single mechanical property and cannot adapt to a variety of tissues. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the prior art, and to construct copolymerized hydrogel microneedles based on methacrylated chitosan (CSMA) and polyethylene glycol diacrylate (PEGDA), dynamically regulate mechanical properties through photocrosslinking technology, and be compatible with multi-drug efficient loading. The present invention utilizes methacrylated natural polysaccharides to impart photocrosslinking ability, and utilizes PEGDA copolymerization to enhance performance, providing an innovative solution for precise delivery.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing hydrogel microneedles with adjustable mechanical properties based on methacrylated chitosan (CSMA), comprising the following steps: S1. Preparation of methacrylated chitosan (CSMA): The chitosan solution is prepared by dissolving chitosan in an acetic acid solution, and the chitosan solution is mixed with methacrylic anhydride by stirring, and then reacted under the catalysis of triethylamine (TEA); the reaction solution is dialyzed and freeze-dried to obtain methacrylated chitosan freeze-dried powder; S2. Microneedle preparation: The methacrylated chitosan (CSMA) freeze-dried powder is re-dissolved in deionized water to prepare the methacrylated chitosan prepolymer solution, polyethylene glycol diacrylate (PEGDA) and a photoinitiator are added, and the therapeutic drug is mixed; the mixed solution is injected into the microneedle mold, and the excess solution is removed after the injection is completed. After curing, the mold is demolded, washed, and dried to obtain the microneedle mold.

[0006] Specifically, in step S1, chitosan is dissolved in a 1-2% (v / v) acetic acid aqueous solution to prepare a 3-5% (w / v, g / ml) chitosan solution.

[0007] Specifically, in step S1, methacrylic anhydride and chitosan are mixed at a mass ratio of 1:1 to 3:1 under stirring at room temperature, and catalyzed by 1% to 3% (v / v) triethylamine of the total volume of the reaction system for 3 to 5 hours.

[0008] Specifically, in step S1, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3000 Da, and dialyzed with ultrapure water with the same pH value at room temperature for 24-48 hours, and the dialysate is replaced every 2 hours; the dialyzed solution is pre-frozen at -40°C and freeze-dried for 24-48 hours to obtain methacrylated chitosan freeze-dried powder.

[0009] Specifically, in step S2, the prepared methacrylated chitosan prepolymer solution has a concentration of 3-5% (w / v, g / ml), and 1-6% (w / w) polyethylene glycol diacrylate and 0.1% (w / v, g / ml) 2-hydroxy-2-methylpropiophenone photoinitiator are added.

[0010] Specifically, in step S2, the therapeutic drug includes protein, peptide, antibiotic or anti-inflammatory drug, and the concentration of the therapeutic drug in the mixed solution is 10-100 μg / mL.

[0011] Specifically, in step S2, the mixed solution is injected into the microneedle mold that has been cleaned and dried with ethanol, and the perfusion is completed by centrifugation at 1000-3000 rpm for 10 minutes; after removing the excess solution, it is irradiated under 300-400 nm ultraviolet light for 30 seconds to cure, and after demolding, the microneedles are washed with physiological saline and dried to obtain.

[0012] Furthermore, the present invention also claims protection for the mechanically adjustable hydrogel microneedles based on methacrylated chitosan prepared by the above preparation method.

[0013] Preferably, the microneedle tip height is 500-1000 μm, the tip spacing is 300-500 μm, and the tip array is arranged in a regular geometric pattern; the compression modulus of the microneedle is 10-100 kPa, and the fracture compression rate is 20%-60%, which is suitable for different tissue mechanical environments.

[0014] Furthermore, the present invention also claims to protect the use of the above-mentioned methacrylated chitosan-based hydrogel microneedles with adjustable mechanical properties in the preparation of a drug delivery system.

[0015] The microneedles can load and continuously release drugs, with a release period of 1-10 days.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Compared with traditional oral administration, the microneedle system of the present invention delivers drugs directly to the dermis by penetrating the stratum corneum of the skin, thus avoiding gastrointestinal degradation and first-pass effect in the liver. It is particularly suitable for polypeptide drugs (such as insulin) that are easily destroyed by gastric acid, significantly improving bioavailability while reducing side effects caused by systemic distribution, thus solving the limitation of oral administration for the delivery of biomacromolecule drugs.

[0017] (2) Compared with conventional injections, subcutaneous or intramuscular injections require frequent punctures, poor patient compliance, and the drug release rate cannot be controlled. The microneedle patch of the present invention is designed to release the drug slowly, and a single application can maintain the therapeutic effect for several days, reducing the number of dosing times; its minimally invasive characteristics (needle tip height 500-1000 μm) only act on the epidermis, avoiding the stimulation of deep nerves and blood vessels by traditional injection needles, significantly reducing pain and infection risks, and is suitable for the management of chronic diseases that require long-term administration.

[0018] (3) Compared with transdermal patches and creams, traditional transdermal preparations rely on passive diffusion and have extremely low penetration efficiency for drugs with larger molecular weights (such as antibodies and growth factors). The microneedles of the present invention form microchannels through physical puncture, allowing large molecular weight drugs to reach the target tissue directly, breaking through the molecular weight limit of transdermal delivery, and the drug loading capacity is significantly higher than that of traditional patches. At the same time, the hydrogel matrix can maintain a moist environment for the drug, avoiding the loss of activity of cream preparations due to dry skin.

[0019] (4) Compared with existing hydrogel microneedles, traditional chitosan microneedles are difficult to penetrate dense tissues due to insufficient mechanical strength, and they swell too quickly, resulting in sudden release of drugs. The present invention introduces PEGDA copolymer cross-linking to significantly improve the hardness and toughness of microneedles while retaining the biological activity of chitosan. It can penetrate high-resistance barriers such as scarred skin, and achieves sustained drug release by regulating the cross-linking density to avoid the problem of sudden release. For example, in in vitro release experiments, the drug release period can be extended to more than twice that of traditional chitosan microneedles.

[0020] (5) Compared with synthetic polymer microneedles (such as PLGA), the preparation process relies on high-temperature injection molding, which is easy to damage the activity of drugs. The present invention adopts room-temperature UV curing technology to avoid high temperature and organic solvents, and completely retains the activity of drugs such as proteins and peptides; at the same time, the degradation products of CSMA / PEGDA are biocompatible oligosaccharides, which have no inflammatory risks and are significantly safer than synthetic materials.

[0021] (6) In terms of preparation process and cost control, traditional microneedles rely on complex photolithography molds and multi-step centrifugal filling, with high production costs and poor batch consistency. The present invention uses PDMS soft molds and "centrifugal-photocuring" integrated process, which is easy to operate and has high repeatability. The single-batch production cost is more than 60% lower than that of silicon-based microneedles, and has the potential for large-scale production, providing a solid foundation for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0023] Figure 1 The present invention is a flow chart of the preparation of hydrogel microneedles.

[0024] Figure 2 This is a scanning electron microscope (SEM) image of the microneedle of the present invention.

[0025] Figure 3 The figure shows the fracture compression rate of the microneedle of the present invention at different ratios, where the mass volume ratio of CSMA is 3% and the volume ratio of PEGDA is 0%-6%. C3P0 corresponds to 3% CSMA, 0% PEGDA; C3P3 corresponds to 3% CSMA, 3% PEGDA; and C3P6 corresponds to 3% CSMA, 6% PEGDA.

[0026] Figure 4 The Young's modulus diagram of the microneedle of the present invention at different ratios, wherein the mass volume ratio of CSMA is 3%, and the volume ratio of PEGDA is 0%-6%. C3P0 corresponds to 3% CSMA, 0% PEGDA; C3P3 corresponds to 3% CSMA, 3% PEGDA; C3P6 corresponds to 3% CSMA, 6% PEGDA.

[0027] Figure 5 The drug release curves of the microneedles of the present invention when loaded with different drugs are shown, wherein FITC-BSA and rhodamine B are used respectively.

[0028] Figure 6 This is an in vitro biocompatibility experiment of the microneedle of the present invention. DETAILED DESCRIPTION

[0029] The present invention can be better understood with reference to the following examples.

[0030] Combination Figure 1 The preparation process of the hydrogel microneedle with adjustable mechanical properties of the present invention is as follows: S1. Preparation of methacrylated chitosan (CSMA): The chitosan solution is prepared by dissolving chitosan in an acetic acid solution, and the chitosan solution is mixed with methacrylic anhydride by stirring, and then reacted under the catalysis of triethylamine (TEA); the reaction solution is dialyzed and freeze-dried to obtain methacrylated chitosan freeze-dried powder; S2. Microneedle preparation: The methacrylated chitosan (CSMA) freeze-dried powder is re-dissolved in deionized water to prepare the methacrylated chitosan prepolymer solution, polyethylene glycol diacrylate (PEGDA) and a photoinitiator are added, and the therapeutic drug is mixed; the mixed solution is injected into the microneedle mold, and the excess solution is removed after the injection is completed. After curing, the mold is demolded, washed, and dried to obtain the microneedle mold.

[0031] Example 1: Preparation of CSMA and microneedle molding S1: Synthesis of CSMA Chitosan was dissolved in 2% (v / v) acetic acid solution to prepare a 3% (w / v, g / ml) chitosan solution. Methacrylic anhydride was slowly added at a chitosan to methacrylic anhydride mass ratio of 1:1 under stirring at room temperature, and 1% (v / v) triethylamine (TEA) of the total volume of the reaction system was used to catalyze the reaction for 4 hours. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed with ultrapure water of pH 5.0 for 24 hours at room temperature (the dialysate was replaced every 2 hours). The dialyzed solution was pre-frozen at -40℃ and freeze-dried for 24 hours to obtain CSMA freeze-dried powder (degree of substitution ≥80%, intrinsic viscosity 90-120 mL / g).

[0032] S2: Microneedle Preparation The CSMA lyophilized powder was re-dissolved in deionized water to prepare a 3% (w / v, g / ml) CSMA prepolymer solution, and 3% (w / w) polyethylene glycol diacrylate (PEGDA) and 0.1% (w / v, g / ml) 2-hydroxy-2-methylpropiophenone photoinitiator were added, and FITC-BSA (50 μg / mL) was mixed. The mixture was injected into the ethanol-cleaned and dried PDMS microneedle mold, and the perfusion was completed by centrifugation at 2000 rpm for 10 minutes. After removing the excess solution, it was cured under 365 nm ultraviolet light for 30 seconds. After demolding, it was washed and dried with saline to obtain microneedles with a tip height of 800±50 μm and a tip spacing of 400±30 μm ( Figure 2 ).

[0033] Example 2: Mechanical properties test of microneedles with different PEGDA ratios Microneedle preparation: Three groups of microneedles were prepared according to the method of Example 1: C3P0: 3% CSMA, 0% PEGDA; C3P3: 3% CSMA, 3% PEGDA; C3P6: 3% CSMA, 6% PEGDA.

[0034] S2: Mechanical properties characterization The compression modulus and fracture compression rate of the microneedle were tested using a universal material testing machine (loading rate 0.5 mm / min). Figure 3 As shown, the compression rate at break was 20±3% in the C3P0 group, increased to 35±4% in the C3P3 group, and reached 50±5% in the C3P6 group. Figure 4 It shows that the Young's modulus increases significantly with the increase of PEGDA ratio (C3P0: 10±2 kPa, C3P6: 80±10 kPa), verifying the regulatory effect of the double cross-linking structure on the mechanical properties.

[0035] Example 3: Synergistic release of multiple drugs The microneedles prepared in Example 1 were loaded with FITC-BSA (50 μg / mL) and rhodamine B (100 μg / mL) and immersed in pH 7.4 PBS buffer (37°C, 100 rpm). Figure 5 As shown in the figure, FITC-BSA exhibits sustained release characteristics due to its large molecular weight (the cumulative release rate in 2 days is about 60%), while rhodamine B has a 2-day release rate of more than 80% due to its fast diffusion rate. The release kinetics conform to the Higuchi model, proving that microneedles can regulate the release rate by drug properties.

[0036] Example 4: In vitro biocompatibility evaluation The microneedle extract (0.2 g / mL) was co-cultured with L929 fibroblasts for 24 hours, and the cell survival rate was expressed by live-dead staining. Figure 6 As shown in the figure, the cell survival rate of each group was ≥95%, with no significant difference from the negative control group (p>0.05). The microneedle extract had no effect on cell morphology, confirming its excellent biocompatibility.

[0037] The present invention provides a mechanically adjustable hydrogel microneedle based on methacrylated chitosan and a method for preparing and applying the same. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. A method for preparing hydrogel microneedles with adjustable mechanical properties based on methacrylated chitosan, characterized in that: The steps include: S1. Preparation of methacrylated chitosan: The chitosan solution is prepared by dissolving chitosan in an acetic acid solution, and the chitosan solution is mixed with methacrylic anhydride by stirring, and then reacted under the catalysis of triethylamine; the reaction solution is dialyzed and freeze-dried to obtain methacrylated chitosan freeze-dried powder; S2. Microneedle preparation: The methacrylated chitosan freeze-dried powder is redissolved in deionized water to prepare a methacrylated chitosan prepolymer solution, polyethylene glycol diacrylate and a photoinitiator are added, and a therapeutic drug is mixed; the mixed solution is injected into a microneedle mold, and after the injection is completed, the excess solution is removed, and after curing, the mold is demolded, washed, and dried to obtain the microneedle mold.

2. The method for preparing the mechanically adjustable hydrogel microneedle based on methacrylated chitosan according to claim 1, characterized in that: In step S1, chitosan is dissolved in a 1-2% (v / v) acetic acid aqueous solution to prepare a 3-5% (w / v) chitosan aqueous solution.

3. The method for preparing the mechanically adjustable hydrogel microneedle based on methacrylated chitosan according to claim 1, characterized in that: In step S1, methacrylic anhydride and chitosan are mixed at a mass ratio of 1:1 to 3:1 under stirring at room temperature, and catalyzed by 1% to 3% (v / v) triethylamine of the total volume of the reaction system for 3 to 5 hours.

4. The method for preparing the mechanically adjustable hydrogel microneedle based on methacrylated chitosan according to claim 1, characterized in that: In step S1, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3000 Da, and dialyzed with ultrapure water with the same pH value at room temperature for 24-48 hours, and the dialysate is replaced every 2 hours; the dialyzed solution is pre-frozen at -40°C and freeze-dried for 24-48 hours to obtain methacrylated chitosan freeze-dried powder.

5. The method for preparing mechanically adjustable hydrogel microneedles based on methacrylated chitosan according to claim 1, characterized in that: In step S2, the prepared methacrylated chitosan prepolymer solution has a concentration of 3-5% (w / v), and 1-6% (w / w) polyethylene glycol diacrylate and 0.1% (w / v) 2-hydroxy-2-methylpropiophenone photoinitiator are added.

6. The method for preparing mechanically tunable hydrogel microneedles based on methacrylated chitosan according to claim 1, characterized in that: In step S2, the therapeutic drug includes protein, peptide, antibiotic or anti-inflammatory drug, and the concentration of the therapeutic drug in the mixed solution is 10-100 μg / mL.

7. The method for preparing mechanically adjustable hydrogel microneedles based on methacrylated chitosan according to claim 1, characterized in that: In step S2, the mixed solution is injected into the ethanol-cleaned and dried microneedle mold, and the injection is completed by centrifugation at 1000-3000 rpm for 10 minutes; After removing the excess solution, the samples were cured under 300-400 nm ultraviolet light for 30 seconds. After demolding, the samples were washed with saline and dried to obtain microneedles.

8. A hydrogel microneedle with adjustable mechanical properties based on methacrylated chitosan prepared by the preparation method according to any one of claims 1 to 7.

9. The mechanically tunable hydrogel microneedle based on methacrylated chitosan according to claim 8, characterized in that: The microneedle tip height is 500-1000 μm, the tip spacing is 300-500 μm, and the tip array is regularly geometrically arranged; the compression modulus of the microneedle is 10-100 kPa, and the fracture compression rate is 20%-60%.

10. Use of the mechanically adjustable hydrogel microneedle based on methacrylated chitosan according to claim 8 in preparing a drug delivery system.

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