A methacrylated chitosan hydrogel microneedle patch and its preparation method and application

Abstract

The invention discloses a methacrylylated chitosan hydrogel microneedle patch and a preparation method and application thereof, and belongs to the field of medical technology. The invention comprises the following steps: methacrylic anhydride MA reacts with chitosan acetic acid solution to prepare a methacrylylated chitosan CSMA hydrogel precursor; CSMA is dissolved in a photoinitiator solution to prepare a CSMA microneedle matrix; the microneedle matrix is ​​loaded on a microneedle mold, the mold microcavity and the mold backing layer are filled, and after drying and ultraviolet light irradiation, the microneedle patch is separated from the microneedle mold to obtain the hydrogel microneedle patch. The hydrogel microneedle patch of the invention has a reasonable formula composition, a simple preparation process, low cost, high transmission efficiency, good biocompatibility, good mechanical properties, and adjustable cross-linking properties and drug sustained release properties.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a methacrylylated chitosan hydrogel microneedle patch and a preparation method and application thereof. Background Art

[0002] The issue of drug delivery has always been a focus of scientific research, and the therapeutic effect of drugs is closely related to the method of drug delivery. Transdermal drug delivery can avoid the first-pass effect and gastrointestinal reactions of oral drugs, reduce the pain and fear of patients, and avoid the generation of a large amount of medical waste. Therefore, it is widely used in clinical practice. However, due to the obstruction of the stratum corneum of the human skin, the efficiency of transdermal delivery of traditional drugs is low. Therefore, it is of great significance to develop an efficient transdermal drug delivery system that can penetrate the stratum corneum of the skin.

[0003] Microneedle technology is a new drug delivery system that has emerged in recent years. Microneedles can penetrate the stratum corneum to create microchannels, thereby delivering the loaded drugs into the skin. The height of the microneedle is generally 25 to 1000 μm, which can penetrate the stratum corneum but cleverly avoid contact with the blood vessels and nerve fibers under the dermis, thus achieving a minimally invasive, painless, and bloodless application effect.

[0004] The existing types of microneedles include solid microneedles, hollow microneedles, coated microneedles, soluble microneedles, and hydrogel microneedles developed in recent years. The drug microchannels formed by the pretreatment of solid microneedles reduce the efficacy due to skin healing, and solid microneedles have safety issues caused by the needle body breaking and being retained in the human body. Hollow microneedles need to be prepared using a digitally controlled microelectromechanical system, which has complex manufacturing processes, high costs, and problems such as easy clogging of the needle body during use. Coated microneedles load drugs outside the needle body by infiltration and other methods, which have problems such as low drug loading, blunt needle tip, high friction, and reduced effective drug dosage. Dissolvable microneedles have problems such as low drug bioavailability and drug adsorption by the backing layer during use due to the soluble needle body. As a new type of microneedle developed in recent years, hydrogel microneedles avoid many problems existing in the preparation and application of microneedles, such as complex manufacturing processes, high costs, needle body breaking and retention in the human body, too fast drug release, and low drug bioavailability.

[0005] As an efficient transdermal drug delivery system, hydrogel microneedles need to have multiple properties such as high biocompatibility, sufficient mechanical strength and high drug loading capacity. However, most existing hydrogel materials cannot meet the above basic requirements. Therefore, it is of great significance to develop a matrix suitable for the preparation of hydrogel microneedles.

[0006] Hydrogels can be divided into two categories: natural hydrogels and synthetic hydrogels. Natural hydrogels such as gelatin have good biocompatibility, but the hydrogel microneedles prepared using them have weak mechanical strength and are not enough to penetrate the human epidermis. In addition, some natural hydrogels have poor water solubility and need to be dissolved in organic solvents before they can be used as microneedle preparation matrices, which reduces the biocompatibility of microneedles. Synthetic hydrogels such as methacrylic polymers and PVA-PVP crosslinkers are mostly modified products of natural hydrogels. This type of hydrogel not only retains the good biocompatibility of natural hydrogels, but is also endowed with strong mechanical properties and adjustable degradation properties. However, some synthetic hydrogel microneedles require high temperature and other processes for preparation, which is not conducive to the preservation of drugs.

[0007] At present, some teams have reported that methacrylic polymers have good biocompatibility and adjustable cross-linking properties, and can be used as the matrix of hydrogel microneedles, such as methacrylated gelatin and methacrylated hyaluronic acid. However, in the process of preparing microneedles, the microneedle matrix they used had a high concentration (Journal of Controlled Release, 2021, 336: 537-548), low water content, and low transmission efficiency. At the same time, some matrices need to maintain a high temperature during the preparation of microneedles to maintain the rheology of the matrix (ACS Nano, 2020, 14: 5901-5908), which complicates the microneedle preparation process. Therefore, it is very important to develop a hydrogel microneedle matrix with a simple preparation process, high transmission efficiency, good biocompatibility, good mechanical properties, adjustable cross-linking properties, and a high drug loading capacity.

[0008] In recent years, methacrylated materials have been widely used in the biomedical field, such as injectable hydrogels, microsphere materials, 3D cell printing matrices, drug-loaded hydrogels, etc. However, there are still no reports on the application of methacrylated chitosan materials in the preparation of hydrogel microneedles and drug delivery applications. Summary of the invention

[0009] Purpose of the invention: The purpose of the present invention is to provide a methacrylylated chitosan hydrogel microneedle patch and its preparation method and application in view of the deficiencies of the prior art. The hydrogel microneedle patch of the present invention has a reasonable formula composition, a simple preparation process, low cost, high transmission efficiency, good biocompatibility, good mechanical properties, adjustable cross-linking properties and drug sustained release properties.

[0010] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0011] The invention provides a methacrylated chitosan hydrogel microneedle patch. The microneedle patch is prepared from a microneedle matrix made of methacrylic anhydride MA, high-density chitosan CS with a density of ≥0.4 g / ml and a photoinitiator, which is loaded on a microneedle mold and cured by ultraviolet light.

[0012] Preferably, the density of the high-density chitosan is ≥0.6 g / ml.

[0013] The present invention also provides a method for preparing a methacrylylated chitosan hydrogel microneedle patch, comprising the following steps:

[0014] (1) reacting methacrylic anhydride (MA) with high-density chitosan (CS) acetic acid solution to prepare methacrylylated chitosan hydrogel precursor (CSMA);

[0015] (2) dissolving the CSMA prepared in step (1) in a photoinitiator solution to obtain a CSMA microneedle matrix;

[0016] (3) Loading the CSMA microneedle matrix of step (2) onto the microneedle mold, filling the mold microcavity and the mold backing layer, and after drying and ultraviolet light irradiation, separating the microneedle patch from the microneedle mold to obtain the hydrogel microneedle patch.

[0017] Preferably, in step (1), the added amounts of methacrylic anhydride MA and high-density chitosan acetic acid solution are: (0.08-2.8):40 v / v, and the concentration of the high-density chitosan acetic acid solution is 0.1-5% w / v.

[0018] Preferably, in step (2), the photoinitiator is at least one of photoinitiator LAP, photoinitiator 907, photoinitiator IHT-PI659 or photoinitiator I2959.

[0019] More preferably, the concentration of the photoinitiator is 0.1-1% w / v. When the concentration of the photoinitiator is further increased and exceeds the maximum value of the present invention, although microneedles can be prepared, the cytotoxicity of the microneedles increases with the increase of the concentration of the photoinitiator.

[0020] Preferably, the CSMA microneedle matrix concentration is 2-5% w / v. When the CSMA microneedle matrix concentration is lower than the lower limit value of 2% given in the present invention, or higher than the upper limit value of 5% given in the present invention, the corresponding microneedles cannot be prepared.

[0021] Preferably, in step (3), the microneedle mold is a polydimethylsiloxane (PDMS) mold.

[0022] Further preferably, the polydimethylsiloxane (PDMS) mold has a pyramidal hole array, the bottom side length of the array pyramid is 20 to 1000 μm, and the height of each needle tip is 25 to 1000 μm.

[0023] In a preferred embodiment of the present invention, the microneedle body is in the form of a quadrangular pyramid, and the bottom is a square of 300 μm×300 μm.

[0024] Furthermore, the present invention adopts a vacuum method to fill the microcavity of the PDMS mold.

[0025] The present invention also provides application of the methacrylylated chitosan hydrogel microneedle patch in drug delivery.

[0026] Chitosan (CS) is a natural alkaline polysaccharide with good biocompatibility and antibacterial properties. Unmodified CS has low solubility due to the presence of intermolecular / intramolecular hydrogen bonds, which limits its application in physiological environments. When CS is modified with methacrylic anhydride (MA), it can not only increase its solubility by destroying its intermolecular / intramolecular hydrogen bonds, but also endow CS with the property of UV cross-linking.

[0027] At present, there is no report in the art on directly using CSMA hydrogel as a microneedle matrix to prepare a microneedle patch.

[0028] Chinese patent CN201811419778.0 "A method for preparing high-strength methacrylated chitosan hydrogel" mentions: In order to solve the water solubility of chitosan, chitosan acetic acid solution is reacted with methacrylic anhydride to obtain water-soluble methacrylated chitosan that can be cross-linked by UV light; in order to increase its cross-linking density, acrylated F127 is added as a cross-linking agent to prepare methacrylated chitosan hydrogel, ensuring the stability of the hydrogel structure. The invention points out that due to the structural instability of methacrylated chitosan hydrogel, acrylated F127 needs to be added as a cross-linking agent.

[0029] Chinese patent CN201810636701.2 "A self-adhesive microneedle patch that can swell rapidly and a method for preparing the same" mentions: chitosan is modified with methacrylic anhydride to synthesize an acrylated hydrophilic polymer, and then a catechol compound is reacted with it for a secondary reaction to synthesize a catechol-modified acrylated hydrophilic polymer, which is finally prepared into a microneedle patch. Since the microneedle patch needs to be irradiated with ultraviolet light during the preparation process, the microneedles made of catechol-modified acrylated hydrophilic polymers will not pose a risk of microneedle breakage and will not remain in the skin. The invention points out that methacrylylated chitosan hydrogel is directly used as a microneedle matrix to prepare a microneedle patch, which has the problem of being fragile, so catechol needs to be used for modification.

[0030] It can be seen that, considering the above problems, those skilled in the art will not choose to directly use CSMA hydrogel as a microneedle matrix to prepare a microneedle patch.

[0031] The present invention also encountered many difficulties in the preparation process of CSMA hydrogel microneedle patch. The product prepared by using ordinary chitosan (viscosity 100-200mPa·s) could not form a good needle shape and had poor mechanical properties. In the mechanical force test, when the force reached 0.2N, the microneedle was compressed by about 500μm from the height of 620μm, and almost all of them were bent.

[0032] Generally speaking, the bulk density of ordinary chitosan in powder form is 0.15-0.30 g / ml. After the inventors tried to replace ordinary chitosan with high-density chitosan with a density ≥ 0.4 g / ml, they unexpectedly found that not only could they successfully prepare CSMA hydrogel microneedle patches, but the prepared microneedle patches also had good stability and mechanical properties, overcoming the long-standing problems in this field.

[0033] The present invention proves that the microneedle has good mechanical properties through the mechanical strength experiment of the CSMA microneedle patch; proves that the microneedle can penetrate the skin well through the mouse skin penetration experiment; proves that the microneedle has good swelling properties through the swelling property determination; proves that the microneedle has high transmission efficiency, adjustable cross-linking properties and drug sustained release properties through the in vitro transdermal diffusion experiment of the drug-loaded microneedle; proves that CSMA has good biocompatibility through the hemolysis rate test and the cytotoxicity test. Therefore, the CSMA microneedle patch of the present invention can be applied to the delivery of drugs or macromolecular proteins to treat diseases, and to extract interstitial fluid for the detection of diseases or toxic substances.

[0034] Beneficial effects:

[0035] (1) The present invention uses high-density chitosan with a density of ≥0.4 g / ml, methacrylic anhydride and a photoinitiator to prepare a methacrylylated chitosan microneedle matrix, and then prepares a methacrylylated chitosan hydrogel microneedle patch. The product has good morphological characteristics, and neatly arranged pyramidal microneedle patches can be seen, with good stability and mechanical properties.

[0036] (2) When methacryloyl chitosan is used as the microneedle matrix in the preparation method of the present invention, the concentration used is only 2-5%. The microneedle preparation process is simple, does not need to be restricted by specific conditions, and has low cost.

[0037] (3) The CSMA hydrogel microneedle patch of the present invention has strong mechanical properties detected by a tensile tester. After piercing the skin, it shows efficient drug transdermal diffusion efficiency, slowly delivers the drug through the skin into the human body within 24 hours, and can be completely removed after the drug delivery is complete, with good biosafety. At the same time, this product has good biocompatibility, adjustable cross-linking properties and swelling properties, and can achieve a high drug release efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the infrared characterization image of the CSMA hydrogel precursor in Example 1;

[0039] Figure 2 This is the nuclear magnetic resonance spectroscopy characterization diagram of the CSMA hydrogel precursor in Example 1;

[0040] Figure 3 The scanning electron microscope (SEM) images of the CSMA hydrogel in Example 1 under different UV irradiation times;

[0041] Figure 4 is an optical microscope image of the CSMA hydrogel microneedle in Example 1;

[0042] Figure 5 This is an optical microscope image of the CSMA hydrogel microneedle in Comparative Example 1;

[0043] Figure 6 This is an optical microscope image of the drug-loaded CSMA hydrogel microneedle in Example 6;

[0044] Figure 7 It is a comparison diagram of mechanical force test of CSMA blank hydrogel microneedle of Example 1 and drug-loaded hydrogel microneedle of Example 6;

[0045] Figure 8 This is an optical microscope image of the CSMA hydrogel microneedles in Example 1 after being placed for 50 days;

[0046] Fig. 9 This is a comparison diagram of the mechanical force test of the CSMA hydrogel microneedles in Example 1 before and after 50 days of placement;

[0047] Fig.10 This is a diagram of the skin insertion of the CSMA hydrogel microneedle in Example 1;

[0048] Fig.11 This is a diagram showing the swelling results of the CSMA hydrogel microneedles in Example 1;

[0049] Fig.12 This is a graph showing the cumulative drug release results of the drug-loaded CSMA hydrogel microneedles in Example 6;

[0050] Fig.13This is a graph showing the CSMA hemolysis rate test results in Example 1;

[0051] Fig.14 This is a diagram showing the CSMA cell compatibility test results in Example 1. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0053] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0055] Example 1 Preparation of methacrylylated chitosan hydrogel microneedle patch

[0056] Materials: high-density chitosan CAS: 9012-76-4, deacetylation degree 85%, density ≥ 0.6g / ml, brand Macklin;

[0057] Methacrylic anhydride CAS: 760-93-0, brand: Macklin;

[0058] Instruments: Infrared spectrometer (Shimadzu Corp, Japan), nuclear magnetic hydrogen spectrometer (300 Ultrashied, Bruker)

[0059] (1) Preparation of methacryloyl chitosan (CSMA) hydrogel precursor

[0060] High-density chitosan (CS) was dissolved in 1% (v / v) acetic acid solution to obtain a 1% (w / v) CS solution. 0.7 ml of methacrylic anhydride (MA) was slowly added dropwise to 40 ml of 1% (w / v) CS solution, and stirred at 60°C for 6 hours to obtain a CSMA solution. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust the pH to neutral to terminate the reaction. The prepared solution was placed in a dialysis bag, dialyzed for 4-6 days, and placed in a freeze dryer to freeze-dry to obtain a white sponge-like product for standby use.

[0061] CSMA infrared characterization: Take 10 mg of CS and CSMA and dissolve them in 1 ml of water. Add the above solution drop by drop into potassium bromide solid and dry at 45℃. Add a small amount of potassium bromide and grind it into a fine powder, press it into a tablet, and measure it with an infrared spectrometer. Figure 1The following is a comparison of the infrared spectra of CS and CSMA. It can be seen from the figure that CSMA has a peak at 1654 cm -1 、1536cm -1 and 1315cm -1 There are peaks at wavelengths, but there are no peaks for CS. These three peaks are characteristic peaks of amide bonds. Therefore, it can be known from the infrared spectrum that new amide bonds are formed in CSMA.

[0062] CSMA H-NMR spectrum characterization: CS and CSMA were dissolved in a deuterated aqueous solution containing 2% deuterated hydrochloric acid and measured using a H-NMR spectrometer. Figure 2 The figure shows the comparison of the H NMR spectra of CS and CSMA. It can be seen from the figure that CSMA has a double peak at 5-6ppm, while CS has no peak, which is the hydrogen of the olefin in MA.

[0063] Combination Figure 1 , Figure 2 , it can be proved that CSMA is successfully synthesized.

[0064] (2) Preparation of CSMA microneedle matrix

[0065] Weigh the photoinitiator I2959 at room temperature in the dark, dissolve it in deionized water, and stir at 50°C to obtain a 0.5% (w / v) I2959 solution. Weigh 30 mg CSMA, dissolve it in 1 ml I2959 solution, and stir it evenly at room temperature to obtain a 3% (w / v) CSMA microneedle matrix.

[0066] (3) Preparation of CSMA hydrogel microneedle patch

[0067] Microneedle patches were prepared using polydimethylsiloxane (PDMS) molds with pyramidal hole arrays. All arrays contain 10×10 needles, the side length of the pyramid base is 300μm, the height of each needle tip is 620μm, and the spacing between each needle tip is 600μm. The microneedle body is in the form of a quadrangular pyramid with a 300μm×300μm square at the bottom.

[0068] 200 μL of the prepared CSMA microneedle matrix was dripped onto the PDMS mold, and vacuum was applied at 0.9 MPa for 10 min to fill the microcavities of the PDMS mold and remove the excess solution and bubbles remaining on the surface of the mold. 1 mL of the prepared CSMA microneedle matrix was then dripped onto the PDMS mold to fill the mold backing layer and dried for 24 h. After drying, the microneedle patch was separated from the PDMS mold by irradiation with ultraviolet light for 60 s and stored in a desiccator.

[0069] Figure 3The SEM images of CSMA hydrogel under different UV irradiation times are shown in Figure 1. As can be seen from the figure, the porosity of CSMA hydrogel is different with different UV irradiation times, and as the UV irradiation time increases, the porosity becomes smaller and smaller, and the cross-linking degree becomes stronger and stronger. This shows that the modification of CS by MA gives CS UV cross-linkability.

[0070] Figure 4 This is an optical microscope image of the CSMA hydrogel microneedle. It can be seen from the image that the microneedle patch is a neatly arranged pyramidal microneedle patch with good morphological characteristics.

[0071] Example 2 Preparation of methacrylylated chitosan hydrogel microneedle patch

[0072] Materials and instruments: Same as in Example 1.

[0073] (1) Preparation of methacryloyl chitosan (CSMA) hydrogel precursor

[0074] High-density chitosan (CS) was dissolved in 1% (v / v) acetic acid solution to obtain a 1% (w / v) CS solution. 0.35 ml of methacrylic anhydride (MA) was slowly added dropwise to 40 ml of 1% (w / v) CS solution, and stirred at 60°C for 6 hours to obtain a CSMA solution. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust the pH to neutral to terminate the reaction. The prepared solution was placed in a dialysis bag, dialyzed for 4-6 days, and placed in a freeze dryer to freeze-dry to obtain a white sponge-like product for standby use.

[0075] Infrared and nuclear magnetic resonance hydrogen spectrum detection proved that CSMA was successfully synthesized.

[0076] (2) Preparation of CSMA microneedle matrix

[0077] Weigh the photoinitiator LAP at room temperature in the dark, dissolve it in deionized water, and stir at 50°C to obtain a 0.1% (w / v) LAP solution. Weigh 20 mg CSMA, dissolve it in 1 ml LAP solution, and stir it evenly at room temperature to obtain a 2% (w / v) CSMA microneedle matrix.

[0078] (3) Preparation of CSMA hydrogel microneedle patch

[0079] Microneedle patches were prepared using polydimethylsiloxane (PDMS) molds with pyramidal hole arrays. All arrays contained 10×10 needles, the side length of the pyramid base was 200 μm, the height of each needle tip was 25 μm, and the spacing between each needle was 600 μm.

[0080] 200 μL of the prepared CSMA microneedle matrix was dripped onto the PDMS mold, and vacuum was applied at 0.9 MPa for 10 min to fill the microcavities of the PDMS mold and remove the excess solution and bubbles remaining on the surface of the mold. 1 mL of the prepared CSMA microneedle matrix was then dripped onto the PDMS mold to fill the mold backing layer and dried for 24 h. After drying, the microneedle patch was separated from the PDMS mold by irradiation with ultraviolet light for 60 s and stored in a desiccator.

[0081] Example 3 Preparation of methacrylylated chitosan hydrogel microneedle patch

[0082] Materials and instruments: Same as in Example 1.

[0083] (1) Preparation of methacryloyl chitosan (CSMA) hydrogel precursor

[0084] High-density chitosan (CS) was dissolved in 1% (v / v) acetic acid solution to obtain a 1% (w / v) CS solution. 1.4 ml of methacrylic anhydride (MA) was slowly added dropwise to 40 ml of 1% (w / v) CS solution, and stirred at 60°C for 6 hours to obtain a CSMA solution. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust the pH to neutral to terminate the reaction. The prepared solution was placed in a dialysis bag, dialyzed for 4-6 days, and placed in a freeze dryer to freeze-dry to obtain a white sponge-like product for standby use.

[0085] Infrared and nuclear magnetic resonance hydrogen spectrum detection proved that CSMA was successfully synthesized.

[0086] (2) Preparation of CSMA microneedle matrix

[0087] Weigh photoinitiator 907 at room temperature in the dark, dissolve it in deionized water, and stir at 50°C to obtain a 1% (w / v) 907 solution. Weigh 50 mg CSMA, dissolve it in 1 ml 907 solution, and stir it evenly at room temperature to obtain a 5% (w / v) CSMA microneedle matrix.

[0088] (3) Preparation of CSMA hydrogel microneedle patch

[0089] Microneedle patches were prepared using polydimethylsiloxane (PDMS) molds with pyramidal hole arrays. All arrays contained 10×10 needles, with a pyramid base side length of 500 μm, a needle tip height of 800 μm, and a needle spacing of 600 μm.

[0090] 200 μL of the prepared blank microneedle matrix was dripped onto the PDMS mold, and vacuum was applied at 0.9 MPa for 10 min to fill the microcavities of the PDMS mold and remove the excess solution and bubbles remaining on the surface of the mold. 1 mL of the prepared blank microneedle matrix and drug-loaded microneedle matrix were then dripped onto the PDMS mold to fill the mold backing layer and dried for 24 h. After drying, the microneedle patch was separated from the PDMS mold by irradiation with ultraviolet light for 60 s and stored in a desiccator.

[0091] Example 4 Preparation of methacrylylated chitosan hydrogel microneedle patch

[0092] Materials: High-density chitosan CAS: 9012-76-4, deacetylation degree 85%, density ≥ 0.4 g / ml, Zhejiang Jinke Biochemical Co., Ltd.

[0093] Instrument: Same as Example 1.

[0094] (1) Preparation of methacryloyl chitosan (CSMA) hydrogel precursor

[0095] High-density chitosan (CS) was dissolved in 1% (v / v) acetic acid solution to obtain a 0.1% (w / v) CS solution. 0.08 ml of methacrylic anhydride (MA) was slowly added dropwise to 40 ml of 0.1% (w / v) CS solution, and stirred at 60°C for 6 hours to obtain a CSMA solution. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust the pH to neutral to terminate the reaction. The prepared solution was placed in a dialysis bag, dialyzed for 4-6 days, and placed in a freeze dryer to freeze-dry to obtain a white sponge-like product for standby use.

[0096] Infrared and nuclear magnetic resonance hydrogen spectrum detection proved that CSMA was successfully synthesized.

[0097] (2) Preparation of CSMA microneedle matrix

[0098] Weigh photoinitiator 907 at room temperature in the dark, dissolve it in deionized water, and stir at 50°C to obtain a 0.5% (w / v) 907 solution. Weigh 40 mg CSMA, dissolve it in 1 ml 907 solution, and stir it evenly at room temperature to obtain a 4% (w / v) CSMA microneedle matrix.

[0099] (3) Preparation of CSMA hydrogel microneedle patch

[0100] Microneedle patches were prepared using polydimethylsiloxane (PDMS) molds with pyramidal hole arrays. All arrays contained 10×10 needles, the side length of the pyramid base was 20 μm, the height of each needle tip was 80 μm, and the spacing between each needle was 600 μm.

[0101] 200 μL of the prepared blank microneedle matrix was dripped onto the PDMS mold, and vacuum was applied at 0.9 MPa for 10 min to fill the microcavities of the PDMS mold and remove the excess solution and bubbles remaining on the surface of the mold. 1 mL of the prepared blank microneedle matrix and drug-loaded microneedle matrix were then dripped onto the PDMS mold to fill the mold backing layer and dried for 24 h. After drying, the microneedle patch was separated from the PDMS mold by irradiation with ultraviolet light for 60 s and stored in a desiccator.

[0102] Example 5 Preparation of methacrylylated chitosan hydrogel microneedle patch

[0103] Materials: high-density chitosan CAS: 9012-76-4, deacetylation degree 85%, density ≥ 0.8g / ml, brand Macklin;

[0104] Methacrylic anhydride CAS: 760-93-0, brand: Macklin

[0105] Instrument: Same as Example 1.

[0106] (1) Preparation of methacryloyl chitosan (CSMA) hydrogel precursor

[0107] High-density chitosan (CS) was dissolved in 1% (v / v) acetic acid solution to obtain a 5% (w / v) CS solution. 2.8 ml of methacrylic anhydride (MA) was slowly added dropwise to 40 ml of 1% (w / v) CS solution, and stirred at 60°C for 6 hours to obtain a CSMA solution. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust the pH to neutral to terminate the reaction. The prepared solution was placed in a dialysis bag, dialyzed for 4-6 days, and placed in a freeze dryer to freeze-dry to obtain a white sponge-like product for standby use.

[0108] Infrared and nuclear magnetic resonance hydrogen spectrum detection proved that CSMA was successfully synthesized.

[0109] (2) Preparation of CSMA microneedle matrix

[0110] Weigh photoinitiator 907 at room temperature in the dark, dissolve it in deionized water, and stir at 50°C to obtain a 1% (w / v) 907 solution. Weigh 50 mg CSMA, dissolve it in 1 ml 907 solution, and stir it evenly at room temperature to obtain a 5% (w / v) CSMA microneedle matrix.

[0111] (3) Preparation of CSMA hydrogel microneedle patch

[0112] Microneedle patches were prepared using polydimethylsiloxane (PDMS) molds with pyramidal hole arrays. All arrays contained 10×10 needles, the side length of the pyramid base was 1000 μm, the height of each needle tip was 1000 μm, and the spacing between each needle was 600 μm.

[0113] 200 μL of the prepared blank microneedle matrix was dripped onto the PDMS mold, and vacuum was applied at 0.9 MPa for 10 min to fill the microcavities of the PDMS mold and remove the excess solution and bubbles remaining on the surface of the mold. 1 mL of the prepared blank microneedle matrix and drug-loaded microneedle matrix were then dripped onto the PDMS mold to fill the mold backing layer and dried for 24 h. After drying, the microneedle patch was separated from the PDMS mold by irradiation with ultraviolet light for 60 s and stored in a desiccator.

[0114] Comparative Example 1 CSMA hydrogel microneedle patch prepared from ordinary chitosan

[0115] Materials: Ordinary chitosan CAS: 9012-76-4, deacetylation degree ≥ 95%, viscosity 100-200mpa·s, brand Macklin;

[0116] Methacrylic anhydride CAS: 760-93-0, brand: Macklin;

[0117] The preparation method is the same as that of Example 1.

[0118] Experimental results: The CSMA hydrogel microneedle patch prepared with ordinary chitosan has poor morphology and cannot form a good needle shape. The optical microscope image is shown in Figure 5 The mechanical force test found that when the force reached 0.2N, the height of the microneedles was compressed from 620μm to about 500μm, and almost all of them were bent.

[0119] From Examples 1-5 and Comparative Example 1, it can be seen that after the inventors replaced the ordinary chitosan with a high-density chitosan with a density of ≥0.4 g / ml, they unexpectedly found that a CSMA hydrogel microneedle patch with good morphology was successfully prepared. In addition, the inventors found that the preparation of microneedles is related to the microneedle matrix that can be retained after the final drying. The higher the density of chitosan, the greater the retention rate of the prepared microneedle matrix.

[0120] Example 6 Preparation of drug-loaded methacrylylated chitosan hydrogel microneedle patch

[0121] Materials and instruments are the same as in Example 1.

[0122] (1) Preparation of methacryloyl chitosan (CSMA) hydrogel precursor

[0123] High-density chitosan (CS) was dissolved in 1% (v / v) acetic acid solution to obtain a 1% (w / v) CS solution. 1.4 ml of methacrylic anhydride (MA) was slowly added dropwise to 40 ml of 1% (w / v) CS solution, and stirred at 60°C for 6 hours to obtain a CSMA solution. After the reaction was completed, a saturated sodium bicarbonate solution was added to adjust the pH to neutral to terminate the reaction. The prepared solution was placed in a dialysis bag, dialyzed for 4-6 days, and placed in a freeze dryer to freeze-dry to obtain a white sponge-like product for standby use.

[0124] Infrared and nuclear magnetic resonance hydrogen spectrum detection proved that CSMA was successfully synthesized.

[0125] (2) Preparation of drug-loaded microneedle matrix

[0126] Weigh the photoinitiator I2959 at room temperature in the dark, dissolve it in deionized water, and stir at 50°C to obtain a 0.5% (w / v) I2959 solution. Add 4 mg of methotrexate and 10 mg of nicotinamide, stir evenly at room temperature, weigh 30 mg of CSMA, dissolve it in 1 ml of I2959 solution, and stir evenly at room temperature to obtain 1 mL of drug-loaded CSMA microneedle matrix.

[0127] (3) Preparation of drug-loaded hydrogel microneedle patches

[0128] Microneedle patches were prepared using polydimethylsiloxane (PDMS) molds with pyramidal hole arrays. All arrays contain 10×10 needles, the side length of the pyramid base is 300μm, the height of each needle tip is 620μm, and the spacing between each needle tip is 600μm. The microneedle body is in the form of a quadrangular pyramid with a 300μm×300μm square at the bottom.

[0129] 200 μL of the prepared drug-loaded CSMA microneedle matrix was dripped onto the PDMS mold, and vacuumed at 0.9 MPa for 10 min to fill the microcavities of the PDMS mold and remove excess solution and bubbles remaining on the surface of the mold. 1 mL of the prepared drug-loaded microneedle matrix was then dripped onto the PDMS mold to fill the mold backing layer and dried for 24 h. After drying, the microneedle patch was separated from the PDMS mold by irradiation with ultraviolet light for 60 s and stored in a desiccator.

[0130] Figure 6 This is an optical microscope image of drug-loaded CSMA hydrogel microneedles. It can be seen from the image that the microneedle patch is a neatly arranged yellow pyramid-shaped microneedle patch, and yellow is the color of the drug.

[0131] Example 7 Mechanical Strength Test of Methacrylylated Chitosan Hydrogel Microneedle Patch

[0132] The performance test objects of this example are the blank methacryloyl chitosan CSMA hydrogel microneedle patch prepared in Example 1 and the drug-loaded methacryloyl chitosan hydrogel microneedle patch prepared in Example 6.

[0133] The microneedle patch was placed with the needle tip facing upward on the test platform of a tensile tester (TH-82033, Suzhou Tuobo). The sensor probe moved at a speed of 0.1 mm / s in the direction perpendicular to the microneedle tip. The force and displacement measurement started from the first contact of the sensor with the microneedle tip and continued until the force reached 70 N.

[0134] Figure 7 This is a comparison chart of the mechanical force test of CSMA blank hydrogel microneedles and drug-loaded hydrogel microneedles. As shown in the figure, the blank CSMA hydrogel microneedle and the drug-loaded microneedle needle tips did not show a breaking point under a pressure of 0.7N, which shows that the microneedle patch prepared by the present invention has good mechanical strength, far exceeding the mechanical force of 0.2N required for the microneedle to penetrate the skin (Design and evaluation of dissolving microneedles for enhancing mal delivery of propranolol hydrochloride. Pharmaceutics. 2021, 13, 579).

[0135] Example 8 Stability test of methacrylated chitosan hydrogel microneedle patch

[0136] This example demonstrates the stability of the product by comparing the morphology and mechanical force of the product before and after 50 days of storage. The performance test object of this example is the methacrylylated chitosan hydrogel microneedle patch prepared in Example 1.

[0137] The inventors placed the microneedle patch prepared in Example 1 for 50 days and took an optical microscope image of it. Figure 8 As can be seen from the figure, the morphology of the microneedle patch is good and similar to that of the newly prepared microneedle patch.

[0138] The inventors placed the microneedle patch prepared in Example 1 for 50 days and tested its mechanical strength. By comparing the mechanical strength test with that of the newly prepared microneedle patch (see Fig. 9 ). The test results showed that after 50 days, the microneedle still had good mechanical properties.

[0139] It can be seen that the methacrylylated chitosan hydrogel microneedle patch prepared by the present invention has good stability.

[0140] Example 9: Methacrylylated chitosan hydrogel microneedle patch skin penetration experiment

[0141] The performance test object of this example is the methacrylylated chitosan CSMA hydrogel microneedle patch prepared in Example 1.

[0142] In order to verify the real skin insertion ability of the microneedle of the present invention, mouse skin (mice purchased from Henan Sikebes Biotechnology Co., Ltd.) was used in the experiment to simulate human skin, and the microneedle patch to be tested was pressed on the mouse skin by hand and kept for 2 minutes. The microneedle patch was pulled out, and the pinholes in the mouse skin were photographed with a mobile phone for observation and recording. Fig.10 This is a picture of CSMA hydrogel microneedle inserted into the skin. As can be seen from the picture, there is a complete array of microneedle holes on the mouse skin, indicating that the microneedle patch prepared by the present invention has a good skin puncture effect.

[0143] Example 10 Determination of the swelling performance of the microneedle of the methacrylylated chitosan hydrogel microneedle patch at different ultraviolet irradiation times The performance test object of this example is the methacrylylated chitosan CSMA hydrogel microneedle patch prepared in Example 1.

[0144] The prepared CSMA hydrogel microneedle patch was weighed directly, and its dry weight was recorded as W0. It was swollen by absorbing water in 37°C phosphate buffer (PBS) (pH=7.4), taken out at different time points (0, 10, 60, 180, 360 min), and the water stains were absorbed with filter paper, and Wt was weighed at different time points. Ws=Wt / W0×100%, s is the abbreviation of Swellingrate, swelling rate. Ws is the swelling rate measured by the weight of the water absorbed by the microneedle. The larger the Ws, the more water the microneedle absorbs and the greater the swelling rate.

[0145] Fig.11 The figure shows the swelling results of the CSMA hydrogel microneedle patch. As can be seen from the figure, the swelling performance of the microneedle decreases with the increase of UV irradiation time, which indicates that the longer the UV irradiation time, the stronger the CSMA cross-linking degree, and the smaller the swelling performance of the microneedle patch. Fig.11 It can be seen that the swelling rate of the microneedles irradiated with UV for 60s and 90s is 200%, the swelling rate of the microneedles irradiated with UV for 30s is 400%, and the swelling rate of the microneedles irradiated with UV for 0s is 1600%, which indicates that the microneedles have good swelling properties.

[0146] Example 11 In vitro transdermal diffusion experiment of drug-loaded microneedles

[0147] The performance test object of this example is the drug-loaded methacrylylated chitosan hydrogel microneedle patch prepared in Example 6.

[0148] A Franz diffusion cell (TK-24, Shanghai Kaikai) was used to conduct in vitro transdermal diffusion experiments of drug-loaded microneedles.

[0149] The microneedle tip was inserted into the ex vivo skin of a rabbit (rabbits were purchased from Henan Sikebes Biotechnology Co., Ltd.) and pressed with fingers for 1 minute. The rabbit skin with the microneedle patch was transferred to a receiving pool with a stirrer, with the stratum corneum facing the supply pool and fixed. Among them, the receiving pool contained 3 ml of PBS (pH = 7.4) solution. Subsequently, the transdermal diffusion cell was placed on a transdermal diffusion instrument (TK-24, Shanghai Kaikai), the temperature was set to 37.0°C, the speed was set to 200rpm / min, and 300μl of the receiving solution was taken out as the sample solution to be tested at predetermined time intervals (2h, 4h, 6h, 8h, 10h and 24h), and 300μl of PBS (pH = 7.4) solution was immediately supplemented. Three parallels were set for each group. The sample solution was filtered with a 0.22μm filter membrane for testing.

[0150] The contents of nicotinamide (NIC) and methotrexate (MTX) were detected by HPLC-UV.

[0151] NIC chromatographic conditions: reverse phase C18 column, mobile phase ratio of acetonitrile: pure water = 7:3, column temperature of 37°C, detection wavelength: 260nm, injection sample volume of 20μL, mobile phase flow rate of 1mL / min.

[0152] MTX chromatographic conditions: reverse phase C18 column, mobile phase ratio of 7% sodium dihydrogen phosphate: 2% citric acid: acetonitrile = 8:1:1, triethylamine adjusted pH to 6, column temperature of 37°C, detection wavelength: 302nm, injection sample volume of 20μL, mobile phase flow rate of 1mL / min.

[0153] Samples were taken at different time points, the peak areas of the samples were measured, and the drug concentration was calculated by comparing with the standard curve; the drug release curve was obtained using the standard curve method. Fig.12 The figure is a result diagram of drug cumulative release of CSMA drug-loaded hydrogel microneedle patch. As can be seen from the figure, the microneedle patch of the present invention shows a high efficiency of drug transdermal diffusion. The diffusion rate within 24 hours is as high as 80%, which indicates that the microneedle patch can successfully deliver different loaded drugs (MTX, NIC) slowly into the human body through the skin.

[0154] Example 12 Hemolysis Rate Test

[0155] The performance test object of this example is the methacrylylated chitosan CSMA hydrogel microneedle patch prepared in Example 1.

[0156] Blood was collected from the eyeballs of live mice (purchased from Henan Sikebes Biotechnology Co., Ltd.), and the fresh blood obtained was subjected to a hemolysis test. About 500 μL of fresh mouse blood was taken, placed in an anticoagulant tube, centrifuged at 3000 rpm for 10 min, and red blood cells were collected; washed three times with PBS (pH 7.4) solution, and the red blood cells were diluted with 0.9% saline (first diluted with saline: red blood cell solution volume ratio of 8:2, shaken well, and then diluted with saline: red blood cell solution volume ratio of 9:1) to obtain a red blood cell suspension. The red blood cell suspension was mixed evenly with different concentrations of CSMA (31.25, 62.5, 125, 250, 500, 1000 μg / ml), and incubated in an incubator at 37°C for 6 h. The positive control group and the negative control group were pure water group and saline group, respectively. The absorbance of all samples was measured at 540 nm. If hemolysis occurs, the hemolysis rate at 540 nm will increase.

[0157] Fig.13 This is the CSMA hemolysis rate test result diagram. Fig.13 As shown, when the concentration of CSMA is as high as 1 mg / ml, the hemolysis rate of the material is less than 5%, which proves the biosafety of CSMA (a hemolysis rate of less than 5%, which proves the biosafety, see the preparation of soybean lysophospholipids and its biosafety analysis).

[0158] Example 13 Cytotoxicity Test

[0159] The performance test object of this example is the methacrylylated chitosan CSMA hydrogel microneedle patch prepared in Example 1.

[0160] MTT assay was used to detect the effect of CSMA on the growth of 293T cells. MTT assay method Source: Master's thesis of Guangzhou University of Chinese Medicine, a preliminary study on the effect of egg yolk oil on the psoriasis vulgaris model induced by LPS HaCaT cell proliferation and Bcl-2 expression.

[0161] 293T cells (cell bank of Chinese Academy of Sciences) were seeded in 96-well plates and cultured in a 37°C, 5% CO2 cell culture incubator with DMEM medium containing 5% fetal bovine serum (Gibco) for 24 h. Then, the same volume of CSMA (31.25, 62.5, 125, 250, 500, 1000 μg / ml) and fresh cell culture medium (5% fetal bovine serum (g ibco)-95% DMEM medium (Senbega) (CSMA: cell culture medium mass ratio is 1:10, which is more conducive to cell growth). After culturing for 12 hours, the medium was discarded and medium containing MTT (Keygen Biotechnology) was added. After 4 hours, blue-purple crystals could be seen. 100 μl of dimethyl sulfoxide was added and shaken for 5 minutes. The cells were detected using a microplate reader (SpectraMax M5 multimode microplate reader, Molecular Devices, Ltd.) at a wavelength of 490 nm.

[0162] The succinate dehydrogenase in the mitochondria of living cells can reduce MTT thiazolyl blue to water-insoluble blue-purple crystalline formazan, which is soluble in DMSO and can be detected at a wavelength of 490nm on an ELISA reader. Dead cells, however, do not have succinate dehydrogenase and cannot generate formazan.

[0163] Cell survival rate = (experimental group OD-blank group OD) / (CSMA group OD-blank group OD)*100%

[0164] A cell death rate of less than 20% is considered low toxicity.

[0165] Fig.14 Figure 2 is the result of CSMA cell compatibility test. Fig.14 As shown, when the concentration of CSMA is as high as 1 mg / ml, the cell survival rate is about 90%, which proves that CSMA has low cytotoxicity and good biosafety.

[0166] The present invention performs a performance test on the methacrylylated chitosan CSMA hydrogel microneedle patch prepared in Examples 1-5. The CSMA hydrogel microneedle patch has strong mechanical properties and shows high drug transdermal diffusion efficiency after piercing the skin. The drug is slowly delivered into the human body through the skin within 24 hours, and can be completely pulled out after the drug is fully delivered, with good biosafety. At the same time, by comparing the examples with the comparative examples, it can be seen that the present invention not only successfully prepares the CSMA hydrogel microneedle patch, but also the prepared microneedle patch has good stability and mechanical properties, overcoming the long-standing problems in the field.

[0167] The methacryloyl chitosan of the present invention has high biological safety and the property of being UV cross-linkable. When used as a microneedle matrix, the concentration used is only 2-5%. Compared with the existing methacryloyl gelatin and methacryloyl hyaluronic acid, the microneedles prepared by the matrix have good mechanical properties, the matrix concentration used is low, the microneedle preparation process is simple, and does not need to be restricted by specific conditions.

[0168] In summary, the hydrogel microneedle patch of the present invention has a reasonable formulation, a simple preparation process, low cost, high transmission efficiency, good biocompatibility, stability, and mechanical properties, and has adjustable cross-linking properties and drug sustained release properties. The CSMA microneedle patch of the present invention can be applied to the delivery of drugs or macromolecular proteins to treat diseases, and to extract interstitial fluid for the detection of diseases or toxic substances.

[0169] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A methacrylated chitosan hydrogel microneedle patch, characterized in that: The microneedle patch is formed by loading a microneedle matrix made of methacrylic anhydride MA, high-density chitosan CS with a density of ≥0.4 g / ml and a photoinitiator on a microneedle mold and curing it with ultraviolet light.

2. The hydrogel microneedle patch according to claim 1, characterized in that: The high-density chitosan has a density of ≥0.6 g / ml.

3. The method for preparing the hydrogel microneedle patch according to claim 1 or 2, characterized in that: The following steps are involved: (1) Methacrylic anhydride (MA) is reacted with high-density chitosan (CS) acetic acid solution to prepare methacrylylated chitosan hydrogel precursor (CSMA); (2) dissolving the CSMA prepared in step (1) in a photoinitiator solution to obtain a CSMA microneedle matrix; (3) The CSMA microneedle matrix of step (2) is loaded onto the microneedle mold, the mold microcavity and the mold backing layer are filled, and after drying and ultraviolet light irradiation, the microneedle patch is separated from the microneedle mold to obtain the hydrogel microneedle patch.

4. The preparation method according to claim 3, characterized in that: In step (1), the addition amount of the methacrylic anhydride MA and the high-density chitosan acetic acid solution is: (0.08~2.8): 40 v / v, and the concentration of the high-density chitosan acetic acid solution is 0.1~5% w / v.

5. The preparation method according to claim 3, characterized in that: In step (2), the photoinitiator is at least one of photoinitiator LAP, photoinitiator 907, photoinitiator IHT-PI659 or photoinitiator I2959.

6. The preparation method according to claim 5, characterized in that: The concentration of the photoinitiator is 0.1-1% w / v.

7. The preparation method according to claim 3, characterized in that: In step (2), the CSMA microneedle matrix concentration is 2-5% w / v.

8. The preparation method according to claim 3, characterized in that: In step (3), the microneedle mold is a polydimethylsiloxane (PDMS) mold.

9. The preparation method according to claim 8, characterized in that: The polydimethylsiloxane (PDMS) mold has a pyramid-shaped hole array, the bottom side length of the array pyramid is 20-1000 μm, and the height of each needle tip is 25-1000 μm.

10. Use of the methacrylylated chitosan hydrogel microneedle patch according to claim 1 in preparing a drug delivery product.

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