PH-responsive super-long dual-network hydrogel composite microneedle and preparation method thereof

Abstract

The invention discloses a pH-responsive super-long dual-network hydrogel composite microneedle and a preparation method thereof, and belongs to the technical field of hydrogel microneedles, methacrylated gelatin and an oxidized hyaluronic acid solution are placed in an EP tube, a photoinitiator is added, the mixture is placed in a 37 DEG C water bath or a thermostat until the photoinitiator is completely dissolved, and a hydrogel microneedle filling solution is obtained; injecting the hydrogel microneedle filling solution into a PDMS microneedle mold, carrying out vacuum negative pressure, drying, and irradiating with a blue light lamp to obtain a dual-network hydrogel microneedle; the pH-responsive dual-network cross-linked hydrogel microneedle has the characteristics of adjustable length in a range of 200-3000 [mu] m, adjustable mechanical properties and various shapes, not only can controllably degrade and release loaded drugs in a pathological environment with unbalanced pH, but also has the advantages of large microneedle length, simplicity in manufacturing, low cost, controllable biocompatibility and the like, and can be applied to the field of medical treatment. The method has a good application prospect in the field of biomedicine.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and in particular to a pH-responsive ultra-long double-network hydrogel composite microneedle and a preparation method thereof. Background Art

[0002] With the rapid development of modern medicine and biotechnology, drug delivery technology is increasingly used in the field of healthcare. As an emerging drug delivery method, microneedle technology has attracted great attention from academia and industry due to its unique advantages. Traditional drug delivery methods, such as oral, injection and topical drugs, have problems such as low bioavailability and poor patient compliance. Microneedle technology achieves efficient delivery and precise release of drugs in a painless and non-invasive way, showing huge market potential.

[0003] Microneedle technology uses fine needle-like biomaterials or instruments to puncture tissues such as the skin and organ fascia, forming a large number of microchannels on the punctured surface, thereby increasing drug penetration and promoting the repair and regeneration of tissues and organs. It is widely used in a variety of biomedical fields. Compared with traditional silicon and metal materials, cross-linked hydrogels are widely used in microneedle manufacturing due to their excellent biocompatibility and structural design advantages. However, the existing hydrogel microneedles mainly have the disadvantages of complicated preparation process, long time, high cost, short length of prepared microneedles, low strength and stiffness of hydrogels, and weak puncture ability on the surface of organs such as skin and fascia.

[0004] Microneedle drug delivery is a new way of drug delivery that can overcome barriers such as skin and organ fascia, and can greatly improve the efficiency of drug delivery. Microneedles are different from the needles we use in our daily lives and the traditional syringes. They have a unique size and shape. They are generally several hundred microns long and generally conical or pyramidal in shape. The single shape limits the puncture effect and drug delivery efficiency.

[0005] At present, most human diseases such as inflammation, infection, and tumors are related to the acidic environment in the body, and the occurrence of diseases is accompanied by changes in the pH value in tissues or organs. How to deliver drugs through microneedles and release therapeutic drugs in a smart and responsive manner with changes in the local pH value is another opportunity and challenge faced by microneedle development.

[0006] Therefore, there is an urgent need to develop a hydrogel microneedle with good mechanical properties, simple preparation process, adjustable length according to the thickness of tissues and organs, and pH responsiveness. Summary of the invention

[0007] Technical problem solved: The present invention provides a pH-responsive ultra-long double-network hydrogel composite microneedle and a preparation method thereof, which solves the technical problems of microneedles in the prior art, such as complicated preparation, limited microneedle length, low mechanical strength and rigidity, single microneedle shape, and inability to release intelligently.

[0008] Technical solution:

[0009] A method for preparing a pH-responsive ultra-long double-network hydrogel composite microneedle, the specific steps are as follows:

[0010] The first step is to prepare modified oxidized hyaluronic acid and methacrylic gelatin: first, weigh 1-2g of hyaluronic acid HA, dissolve it in 100-300ml deionized water, and stir at room temperature until it dissolves; add 0.5-1.2g of sodium periodate, stir in the dark for 6-12 hours, and add 0.5-2ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 36-48 hours, freeze-dry by freeze drying, and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 1-2g of gelatin and dissolve it in 10-40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 4-8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 3-6d, and then freeze-dry the reaction product to obtain light yellow solid methacrylic gelatin, that is, light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0011] Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition;

[0012] Step 3: dissolving the hydrogel composition with 1-10 ml ultrapure water or PBS at 37° C. according to the mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-20 mg: 1-10 mL to prepare a hydrogel solution;

[0013] Step 4: pouring the hydrogel solution prepared in the third step into the polydimethylsiloxane PDMS mold microneedle array model, evacuating the air at the needle tip of the polydimethylsiloxane PDMS mold microneedle array model in a vacuum negative pressure device, and maintaining the temperature at 30-50° C. The polydimethylsiloxane PDMS mold microneedle array model is an n*m rectangular array of microneedles, the center spacing between adjacent microneedles is 500-1200 μm, the height of each microneedle is 200-3000 μm, m and n are both integers ≥2, the needle diameter of each microneedle is 5-10 μm, and the bottom width of each microneedle is 250-400 μm;

[0014] Step 5: After the vacuum negative pressure device is deflated, quickly remove the bubbles on the surface of the hydrogel solution in the PDMS mold microneedle array model, and repeat this step 3-5 times;

[0015] Step 6: Drying, blue light cross-linking for 1-5 minutes, and finally demolding to obtain hydrogel microneedles; the wavelength of the blue light source is 405nm.

[0016] Furthermore, the hydrogel composition in the second step is a double cross-linked hydrogel composition, which is obtained by batch mixing or direct mixing. The batch mixing method is to first mix the photoinitiator and the photocurable hydrogel monomer evenly to obtain a hydrogel matrix, and then mix the hydrogel matrix with the modified oxidized hyaluronic acid evenly to obtain a double cross-linked hydrogel composition; the direct mixing method is to mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid at the same time to obtain a double cross-linked hydrogel composition.

[0017] Furthermore, in the fourth step, the dimethylsiloxane PDMS mold microneedle array model is a 20*20 microneedle rectangular array, the center spacing between adjacent microneedles is 550 μm, the needle diameter of each microneedle is 5 μm, and the bottom width of each microneedle is 250 μm.

[0018] Furthermore, in the fourth step, the method for preparing the polydimethylsiloxane PDMS mold microneedle array model is to pour the polydimethylsiloxane solution into the microneedle array mold and perform a curing treatment to obtain the polydimethylsiloxane PDMS mold microneedle array model, and the curing treatment time is 40-80 minutes.

[0019] Furthermore, the drying time in the sixth step is 6-12 hours.

[0020] A pH-responsive ultra-long double-network hydrogel composite microneedle, the raw materials of which include a photocrosslinking part and a self-crosslinking part, the photocrosslinking part is a photoinitiator and a photocurable hydrogel monomer, the photocurable hydrogel monomer is methacrylated gelatin, and the self-crosslinking part is modified oxidized hyaluronic acid, and the CHO bond of the modified oxidized hyaluronic acid undergoes a Schiff base reaction with the amino group of the methacrylated gelatin.

[0021] Furthermore, according to different ratios of oxidized hyaluronic acid and methacryloyl gelatin and different polydimethylsiloxane (PDMS) mold microneedle array models, a pH-responsive ultra-long double-network hydrogel microneedle with adjustable mechanical strength and length in the range of 200-3000 μm was prepared. The pH-responsive ultra-long double-network hydrogel microneedle has variable shapes such as conical, pyramidal, cylindrical, and hollow, and can respond to pH intelligently and release loaded drugs.

[0022] Beneficial effects: 1. The present application provides a pH-responsive ultra-long double-network hydrogel composite microneedle with a variety of shape designs: The microneedle of the present invention not only adopts a variety of typical shape designs, such as cone, rocket, pencil, bullet and ice cone, but also can select a suitable shape according to specific application requirements to optimize the puncture effect and drug delivery efficiency. These shape designs not only improve the functionality of the microneedle, but also expand its applicability in different medical fields;

[0023] 2. The present application provides a pH-responsive ultra-long double-network hydrogel composite microneedle, which is mainly composed of oxidized hyaluronic acid and methacrylated gelatin materials, wherein the hyaluronic acid is modified by sodium periodate to obtain oxidized hyaluronic acid, and the gelatin is modified by methacrylic acid to obtain methacrylated gelatin. The primary crosslinking is a Schiff base reaction between the CHO bond of the modified oxidized hyaluronic acid and the amino group of the modified gelatin. The second-level crosslinking is photocured under blue light. The double crosslinking increases the stability of the hydrogel system. The hydrogel is then reacted to obtain a hydrogel, which is poured into a mold and then demolded to obtain a hydrogel microneedle.

[0024] 3. The hydrogel microneedles prepared in this application have a simple preparation process, and the microneedle length is adjustable within the range of 25-3000 μm. They can be prepared according to specific usage scenarios, have adjustable mechanical properties, and have variable shapes such as conical, pyramidal, cylindrical, and hollow. They can respond to pH intelligently and release loaded drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron microscope image of the conical hydrogel microneedle prepared in Example 1 of the present invention;

[0026] Figure 2 This is a scanning electron microscope image of the columnar hydrogel microneedle prepared in Example 2 of the present invention;

[0027] Figure 3 The scanning electron microscope images of hydrogels with different ratios and concentrations according to the present invention are shown;

[0028] Figure 4 This is a biomechanical test diagram of hydrogels with different ratios and concentrations implemented in the present invention;

[0029] Figure 5 Live-dead staining images of the present invention after co-culturing hydrogels with different ratios and concentrations with annulus fibrosus cells;

[0030] Figure 6 This is a cytoskeleton diagram after co-culturing of hydrogels with different ratios and concentrations and annulus fibrosus cells according to the present invention.

[0031] Figure 7 This is a diagram of an in vitro porcine skin transdermal experiment performed in accordance with the present invention. DETAILED DESCRIPTION

[0032] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively with reference to the relevant drawings. The drawings show preferred embodiments of the present invention, and the principles and features of the present invention are described in combination with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention; on the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0033] Example 1

[0034] See attached Figure 1 , a method for preparing a pH-responsive ultra-long double-network hydrogel composite microneedle, the specific steps are as follows:

[0035] The first step is the preparation of modified oxidized hyaluronic acid and methacryloyl gelatin: first, weigh 1g of hyaluronic acid HA, dissolve it in 100ml of deionized water, and stir at room temperature until it dissolves; add 0.5g of sodium periodate, stir for 12 hours in the dark, and add 1ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 36 hours, freeze-dry by freeze drying, and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 1g of gelatin and dissolve it in 10ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 4-8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 3d, and then freeze-dry the reaction product to obtain light yellow solid methacryloyl gelatin, i.e., light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0036] Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition;

[0037] Step 3: dissolving the hydrogel composition with 1 ml of ultrapure water or PBS at 37° C. according to the mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-20 mg: 1 ml, to prepare a hydrogel solution;

[0038] Step 4: Pour the hydrogel prepared in the third step into a polydimethylsiloxane (PDMS) mold, evacuate the air at the needle tip by vacuum negative pressure, and maintain the temperature at 30°C; the polydimethylsiloxane (PDMS) mold microneedle array model is a 20*20 microneedle rectangular array, the center spacing between adjacent microneedles is 550μm, the height of each microneedle is 600μm, the needle diameter of each microneedle is 5μm, and the bottom width of each microneedle is 250μm;

[0039] Step 5: After the vacuum negative pressure device is deflated, quickly remove the bubbles on the surface of the hydrogel solution in the PDMS mold microneedle array model, and repeat this step 3 times;

[0040] Step 6: Dry for 6-12 hours, crosslink with blue light for 1-5 minutes, and finally demold to obtain hydrogel microneedles; the wavelength of the blue light source is 405nm. Use tweezers to gently remove the dried patch and carefully store it for subsequent experiments. Figure 1 shown.

[0041] The double network hydrogel composite microneedles prepared in this embodiment integrate oxidized hyaluronic acid and methacryloyl gelatin, and the hydrogel microneedles undergo primary crosslinking (Schiff base reaction between oxidized hyaluronic acid CHO bonds and modified gelatin amino groups) and secondary crosslinking (photo-crosslinking of methacryloyl gelatin in the microneedle patch by ultraviolet irradiation at the optimal time), which enables the obtained microneedle patch to exhibit good mechanical strength, thereby being able to penetrate the stratum corneum of the skin minimally invasively; and retaining a certain amount of uncrosslinked oxidized hyaluronic acid CHO bonds can also load amino-containing drugs for sustained release. It is further applied to and transformed into the field of biomedicine.

[0042] In the second step, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate; the hydrogel composition is a double cross-linked hydrogel composition, which is obtained by batch mixing or direct mixing. The batch mixing method is to first mix the photoinitiator and the photocurable hydrogel monomer evenly to obtain a hydrogel matrix, and then mix the hydrogel matrix with the modified oxidized hyaluronic acid evenly to obtain a double cross-linked hydrogel composition; the direct mixing method is to mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid at the same time to obtain a double cross-linked hydrogel composition.

[0043] The preparation method of the polydimethylsiloxane PDMS mold microneedle array model is to pour the polydimethylsiloxane solution into the microneedle array mold and perform a curing treatment to obtain the polydimethylsiloxane PDMS mold microneedle array model, and the curing treatment time is 40-80 minutes.

[0044] In the step of cross-linking methacryloyl gelatin by blue light irradiation, the irradiation time is 2 minutes. If the irradiation time is less than 1 minute, the cross-linking degree of methacryloyl gelatin is insufficient, and the mechanical strength of the prepared hydrogel microneedle patch is low and it is not easy to penetrate the stratum corneum of the skin; if the irradiation time is greater than 5 minutes, the cross-linking degree of methacryloyl gelatin is too high, so that the swelling rate of the prepared hydrogel microneedle patch is low. Therefore, considering the mechanical strength, 1-5 minutes is selected as the UV curing time to ensure that the prepared microneedle patch has both better mechanical properties and better swelling rate.

[0045] Example 2

[0046] See attached Figure 2 , a method for preparing a pH-responsive ultra-long double-network hydrogel composite microneedle, the specific steps are as follows:

[0047] The first step is the preparation of modified oxidized hyaluronic acid and methacrylic gelatin: first, weigh 2g of hyaluronic acid HA, dissolve it in 300ml of deionized water, and stir at room temperature until it dissolves; add 1.2g of sodium periodate, stir in the dark for 6-12 hours, and add 2ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 48 hours, freeze-dry by freeze drying, and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 2g of gelatin and dissolve it in 40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 6d, then freeze-dry the reaction product to obtain light yellow solid methacrylic gelatin, i.e., light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0048] Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition;

[0049] Step 3: according to the mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-200 mg: 1-10 mL, dissolving the hydrogel composition with 1 ml of ultrapure water or PBS at 37° C. to prepare a hydrogel solution;

[0050] Step 4: Pour the hydrogel prepared in the third step into a polydimethylsiloxane (PDMS) mold, perform vacuum extraction, exhaust the air at the needle tip, and maintain the temperature at 50°C; the polydimethylsiloxane (PDMS) mold microneedle array model is an 8*8 microneedle rectangular array, the center spacing between adjacent microneedles is 1200μm, the height of each microneedle is 1500μm, the needle diameter of each microneedle is 10μm, and the bottom width of each microneedle is 360μm. ;

[0051] Step 5: After the vacuum negative pressure device is deflated, maintain the temperature at 30-50°C, quickly remove the bubbles on the surface of the hydrogel solution in the PDMS mold microneedle array model, and repeat this step 3-5 times;

[0052] Step 6: Dry for 6-12 hours, crosslink with blue light for 1-5 minutes, and finally demold to obtain hydrogel microneedles; the wavelength of the blue light source is 405nm. Gently remove the dried patch with tweezers and store it carefully for subsequent experiments. Figure 2 shown.

[0053] Example 3

[0054] The ultrastructure and biomechanics of a hydrogel microneedle system, the specific steps are as follows:

[0055] The first step is the preparation of modified oxidized hyaluronic acid and methacryloyl gelatin: first, weigh 1g of hyaluronic acid HA, dissolve it in 100ml of deionized water, and stir at room temperature until it dissolves; add 0.5g of sodium periodate, stir for 12 hours in the dark, and add 1ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 48 hours, freeze-dry and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 2g of gelatin and dissolve it in 40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 6d, then freeze-dry the reaction product to obtain light yellow solid methacryloyl gelatin, i.e., light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0056] Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition;

[0057] Step 3: Dissolve the hydrogel composition and ultrapure water / PBS at a mass volume ratio of 5-200 mg: 1-10 mL at 37° C. to prepare hydrogel solutions with concentrations of 0% HG, 2% HG, 4% HG, 8% HG, and 10% HG;

[0058] Step 4: The composite hydrogel solutions of different proportions and concentrations were respectively drawn and added into a 1.mL syringe, and photocrosslinked for 1 minute using a blue light source with a wavelength of 405nm to obtain a cylindrical composite hydrogel;

[0059] Step 5: freeze-dry a portion of the hydrogel, crack the hydrogel with liquid nitrogen after freeze-drying, and observe the cross-sectional morphology of the hydrogel using a scanning electron microscope; place a portion of the prepared composite hydrogel sample on the platform of a universal mechanical testing machine, adjust the height of the compression platform to just contact the composite hydrogel, then reset all parameters, set the compression speed to 5mm / min, start the mechanical compression test, and end the test when the mechanical curve shows a downward trend. Figure 3 , Figure 4 shown.

[0060] Example 4

[0061] Cytocompatibility test: Calcein-AM / PI live / dead cell staining to detect cell apoptosis:

[0062] The first step is the preparation of modified oxidized hyaluronic acid and methacryloyl gelatin: first, weigh 1g of hyaluronic acid HA, dissolve it in 100ml of deionized water, and stir at room temperature until it dissolves; add 0.5g of sodium periodate, stir for 12 hours in the dark, and add 1ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 48 hours, freeze-dry and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 2g of gelatin and dissolve it in 40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 6d, then freeze-dry the reaction product to obtain light yellow solid methacryloyl gelatin, i.e., light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0063] Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition;

[0064] Step 3: dissolving the hydrogel composition with 1-10 ml ultrapure water or PBS at 37° C. according to the mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-20 mg: 1-10 mL to prepare a hydrogel solution;

[0065] Step 4: All hydrogel solutions before cross-linking were sterilized by filtration using a sterile filter with a filter diameter of 0.22 μm. A certain amount of composite hydrogel solution was drawn into a 5 mL syringe without a head, and photocrosslinked for 1 minute using a blue light source with a wavelength of 405 nm. After being pushed out, it was placed in a sterile 24-well plate and washed three times with sterile PBS. Annular fiber cells were plated at 3×10 per well. 4 The cells were seeded on the surface of the hydrogel at a density of 100 μg / ml. Each well was added with 1 mL of DMEM / F12 complete medium containing 1% penicillin-streptomycin and 10% fetal bovine serum, and cultured in a 37°C, 5% CO2 incubator.

[0066] Step 5: When the cells grow to about 70%, aspirate the culture medium and wash three times with phosphate buffer solution (PBS);

[0067] Step 6: Add the prepared live-dead staining solution (200 μL per well), incubate at 37°C for 15 minutes in the dark (method for preparing live-dead staining solution: a. Prepare Calcein-AM and PI storage solutions according to the instructions; b. Add 10 μL Calcein-AM storage solution and 15 μL PI storage solution to 5 mL PBS, mix and prepare the live-dead staining solution, so that the concentration of Calcein-AM is 2 μM and the concentration of PI is 4.5 μM);

[0068] Step 7: Aspirate the live-dead staining solution and wash three times with PBS (add slowly while the cells adhere to the wall);

[0069] Step 8: Observe the cell growth status by inverted fluorescence microscope. Figure 5 shown.

[0070] Example 5

[0071] Cytocompatibility testing: Cytoskeleton staining:

[0072] The first step is the preparation of modified oxidized hyaluronic acid and methacryloyl gelatin: first, weigh 1g of hyaluronic acid HA, dissolve it in 100ml of deionized water, and stir at room temperature until it dissolves; add 0.5g of sodium periodate, stir for 12 hours in the dark, and add 1ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 48 hours, freeze-dry and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 2g of gelatin and dissolve it in 40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 6d, then freeze-dry the reaction product to obtain light yellow solid methacryloyl gelatin, i.e., light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0073] Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition;

[0074] Step 3: dissolving the hydrogel composition with 1-10 ml ultrapure water or PBS at 37° C. according to the mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-20 mg: 1-10 mL to prepare a hydrogel solution;

[0075] Step 4: All hydrogel solutions before cross-linking are sterilized by filtration using a sterile filter with a filter diameter of 0.22 μm. A certain amount of composite hydrogel solution is drawn separately, added to a 5 mL syringe without a head, and photocrosslinked for 1 minute using a blue light source with a wavelength of 405 nm. After being pushed out, it is placed in a sterile 24-well plate, washed three times with sterile PBS, and the annulus fibrosus cells are inoculated on the surface of the hydrogel at a cell density of 3×104 per well. 1 mL of DMEM / F12 complete medium containing 1% penicillin-streptomycin and 10% fetal bovine serum is added to each well, and cultured in a 37°C, 5% CO2 incubator;

[0076] Step 5: When the cells grow to about 70%, aspirate the culture medium and wash three times with phosphate buffer solution (PBS);

[0077] Step 6: Add 500 μL 4% paraformaldehyde fixative to fix the cells at room temperature for 15 minutes;

[0078] Step 7: Aspirate the 4% paraformaldehyde fixative and wash three times with PBS;

[0079] Step 8: Add 2% bovine serum albumin (BSA) containing 0.1% Triton X-100 and block at room temperature for 1 hour;

[0080] Step 9: Aspirate the blocking solution, add skeleton staining solution (200 μL per well), and incubate at room temperature in the dark for 40 minutes (skeleton staining solution preparation method: prepare phalloidin storage solution according to the instructions, 1000×, and dilute it according to the ratio of PBS: phalloidin storage solution = 1000:1 when using to obtain skeleton staining solution); aspirate the skeleton staining solution, and wash three times with PBS;

[0081] Step 10: Add 4',6-diamidino-2-phenylindole (DAPI) staining solution to stain the cell nucleus and incubate at room temperature for 15 minutes in the dark;

[0082] Step 11: Aspirate the DAPI staining solution and wash three times with PBS;

[0083] Step 12: Use an inverted fluorescence microscope to observe the adhesion and spreading of cells. Figure 6 shown.

[0084] Example 6

[0085] See attached Figure 7 , a pH-responsive ultra-long double-network hydrogel composite microneedle pig skin puncture experiment, the specific steps are as follows:

[0086] The first step is the preparation of modified oxidized hyaluronic acid and methacryloyl gelatin: first, weigh 1g of hyaluronic acid HA, dissolve it in 100ml of deionized water, and stir at room temperature until it dissolves; add 0.5g of sodium periodate, stir for 12 hours in the dark, and add 1ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 48 hours, freeze-dry and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 2g of gelatin and dissolve it in 40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 6d, then freeze-dry the reaction product to obtain light yellow solid methacryloyl gelatin, i.e., light-cured hydrogel monomer, and seal and store it at 4°C for later use;

[0087] Step 2: uniformly mix the photoinitiator, photocurable hydrogel monomer and modified oxidized hyaluronic acid at a mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition; Step 3: dissolve the hydrogel composition and 1 ml of ultrapure water or PBS at 37° C. at a mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-20 mg: 1-10 mL to obtain a hydrogel solution;

[0088] Step 4: pouring the hydrogel solution prepared in the third step into the polydimethylsiloxane PDMS mold microneedle array model, and exhausting the air at the needle tip of the polydimethylsiloxane PDMS mold microneedle array model in a vacuum negative pressure device;

[0089] Step 5: After the vacuum negative pressure device is deflated, quickly remove the bubbles on the surface of the hydrogel solution in the PDMS mold microneedle array model, and repeat this step 3-5 times;

[0090] Step 6: Dry for 6-24 hours, crosslink with blue light for 1 minute, and finally demold to obtain hydrogel microneedles; the wavelength of the blue light source is 405nm. Gently remove the dried microneedle patch with tweezers.

[0091] Step 7: Puncture the pig skin with the prepared microneedle. The effect of puncturing the pig skin after coloring with methylene blue is as follows: Figure 7 As shown,

[0092] The pH-responsive ultra-long double-network hydrogel composite microneedles of the present invention not only overcome the shortcomings of existing hydrogel microneedles in terms of preparation process, microneedle length, mechanical properties, etc., but also achieve precise drug release by intelligently responding to changes in pH value. With the growing demand for personalized medicine and precision treatment, this innovative technology has broad market application prospects. The pH-responsive ultra-long double-network hydrogel composite microneedles of this application can be widely used in the following fields:

[0093] (1) Medical beauty and skin care field:

[0094] Microneedle technology has broad application prospects in the fields of beauty and skin care. By promoting the skin's absorption of active ingredients, it can achieve anti-aging, whitening, freckle removal and other effects, meeting consumers' demand for high-efficiency skin care products.

[0095] (2) Vaccination and disease prevention and control:

[0096] Traditional vaccination methods are painful and fearful, but microneedle technology can deliver vaccines painlessly, increasing vaccination rates and coverage, and is particularly suitable for sensitive groups such as children and the elderly.

[0097] (3) Chronic disease management:

[0098] For chronic diseases such as diabetes that require long-term management, non-invasive blood glucose monitoring and insulin delivery through microneedle technology can greatly improve patients' quality of life and treatment compliance.

[0099] The pH-responsive ultra-long double-network hydrogel composite microneedle prepared in this application has the following effects:

[0100] (1) Improve treatment effect:

[0101] pH-responsive microneedles can intelligently release therapeutic drugs according to changes in the acid-base environment of diseased tissues, thereby improving drug utilization efficiency and therapeutic effects.

[0102] (2) Microneedle length and strength are adjustable:

[0103] The length and strength of the microneedles of the present invention are adjustable, and can be adjusted individually according to different treatment needs and patient conditions. In particular, it breaks through the problem of limited length of hydrogel microneedles and can ensure the effectiveness and safety of drug delivery. This flexibility not only increases the scope of application of microneedles, but also enhances their practicality in various clinical applications.

[0104] (3) Various shape designs:

[0105] The microneedles of the present invention adopt a variety of typical shape designs, such as rocket shape, pencil shape, bullet shape and ice cone shape, etc., and can select the appropriate shape according to specific application requirements to optimize the puncture effect and drug delivery efficiency. These shape designs not only improve the functionality of the microneedles, but also expand their applicability in different medical fields.

[0106] (4) Expand the scope of application:

[0107] This technology is not only suitable for the treatment of common skin diseases, inflammation and other diseases, but can also be used for the precise treatment of chronic and complex diseases such as intervertebral disc degeneration and tumors, and has broad application potential.

[0108] (5) Simplify the preparation process:

[0109] The present invention simplifies the preparation process of hydrogel microneedles, reduces production costs, and helps promote the industrialization and large-scale application of microneedle technology.

[0110] According to market research reports, the global microneedle drug delivery market is expected to achieve significant growth in the next few years. With the continuous advancement of medical technology and the improvement of people's health awareness, the demand for microneedle technology will further expand. In particular, the pH-responsive ultra-long double-network hydrogel composite microneedle of the present invention, with its unique technical advantages and broad application prospects, is expected to achieve breakthroughs in the following aspects:

[0111] (1) Clinical application: It has broad application prospects in clinical treatment, especially in the fields of chronic disease management and cancer treatment, and has important clinical value and economic benefits.

[0112] (2) Home medical care: Microneedle technology is suitable for home self-management, which is convenient for patients to operate by themselves, improving the convenience and accessibility of medical services.

[0113] (3) Global market: Due to its non-invasive, painless and highly effective characteristics, microneedle technology has broad market demand in both developed and developing countries, and the future market size is huge.

[0114] The pH-responsive ultralong double-network hydrogel composite microneedle of the present invention, with its innovative technical advantages and broad application prospects, is expected to achieve important breakthroughs in the fields of drug delivery and biomedicine, and promote the further development and popularization of microneedle technology.

[0115] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a pH-responsive ultralong double-network hydrogel composite microneedle, characterized in that: The specific steps are as follows: The first step is to prepare modified oxidized hyaluronic acid and methacrylic gelatin: first, weigh 1-2g of hyaluronic acid HA, dissolve it in 100-300ml deionized water, and stir at room temperature until it dissolves; add 0.5-1.2g of sodium periodate, stir in the dark for 6-12 hours, and add 0.5-2ml of ethylene glycol to the reaction solution to terminate the reaction; dialyze at room temperature for 36-48 hours, freeze-dry by freeze drying, and place the obtained modified oxidized hyaluronic acid HA-CHO in a 50mL centrifuge tube; weigh 1-2g of gelatin and dissolve it in 10-40ml of ultrapure water or phosphate buffer with pH=7.5 at 50°C to form a uniform solution, add methacrylic anhydride under vigorous stirring, react for 4-8h, dilute the reaction mixture and dialyze it with deionized water at 40°C for 3-6d, and then freeze-dry the reaction product to obtain light yellow solid methacrylic gelatin, that is, light-cured hydrogel monomer, and seal and store it at 4°C for later use; Step 2: uniformly mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid according to the mass ratio of photoinitiator: photocurable hydrogel monomer: modified oxidized hyaluronic acid = 0.25-0.5: 5-10: 1-10 to obtain a hydrogel composition; Step 3: dissolving the hydrogel composition with 1-10 ml ultrapure water or PBS at 37° C. according to the mass volume ratio of hydrogel composition to ultrapure water / PBS = 5-20 mg: 1-10 mL to prepare a hydrogel solution; Step 4: pouring the hydrogel solution prepared in the third step into the polydimethylsiloxane PDMS mold microneedle array model, evacuating the air at the needle tip of the polydimethylsiloxane PDMS mold microneedle array model in a vacuum negative pressure device, and maintaining the temperature at 30-50° C. The polydimethylsiloxane PDMS mold microneedle array model is an n*m rectangular array of microneedles, the center spacing between adjacent microneedles is 500-1200 μm, the height of each microneedle is 200-3000 μm, m and n are both integers ≥2, the needle diameter of each microneedle is 5-10 μm, and the bottom width of each microneedle is 250-400 μm; Step 5: After the vacuum negative pressure device is deflated, quickly remove the bubbles on the surface of the hydrogel solution in the PDMS mold microneedle array model, and repeat this step 3-5 times; Step 6: Drying, blue light cross-linking for 1-5 minutes, and finally demolding to obtain hydrogel microneedles; the wavelength of the blue light source is 405nm.

2. The method for preparing a pH-responsive ultralong double-network hydrogel composite microneedle according to claim 1, characterized in that: The hydrogel composition in the second step is a double cross-linked hydrogel composition, which is obtained by batch mixing or direct mixing. The batch mixing method is to first mix the photoinitiator and the photocurable hydrogel monomer evenly to obtain a hydrogel matrix, and then mix the hydrogel matrix with the modified oxidized hyaluronic acid evenly to obtain a double cross-linked hydrogel composition; the direct mixing method is to mix the photoinitiator, the photocurable hydrogel monomer and the modified oxidized hyaluronic acid at the same time to obtain a double cross-linked hydrogel composition.

3. The method for preparing a pH-responsive ultralong double-network hydrogel composite microneedle according to claim 1, characterized in that: In the fourth step, the dimethylsiloxane PDMS mold microneedle array model is a 20*20 microneedle rectangular array, the center spacing between adjacent microneedles is 550 μm, the needle diameter of each microneedle is 5 μm, and the bottom width of each microneedle is 250 μm.

4. The method for preparing a pH-responsive ultralong double-network hydrogel composite microneedle according to claim 1, characterized in that: In the fourth step, the method for preparing the polydimethylsiloxane PDMS mold microneedle array model is to pour the polydimethylsiloxane solution into the microneedle array mold and perform a curing treatment to obtain the polydimethylsiloxane PDMS mold microneedle array model, and the curing treatment time is 40-80 minutes.

5. The method for preparing a pH-responsive ultralong double-network hydrogel composite microneedle according to claim 1, characterized in that: The drying time in the sixth step is 6-12 hours.

6. A pH-responsive ultra-long double-network hydrogel composite microneedle prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The raw materials include a photo-crosslinking part and a self-crosslinking part. The photo-crosslinking part is a photoinitiator and a photocurable hydrogel monomer. The photocurable hydrogel monomer is methacrylated gelatin. The self-crosslinking part is modified oxidized hyaluronic acid. The CHO bond of the modified oxidized hyaluronic acid undergoes a Schiff base reaction with the amino group of the methacrylated gelatin.

7. The pH-responsive ultralong double-network hydrogel composite microneedle according to claim 6, characterized in that: According to different ratios of oxidized hyaluronic acid and methacryloyl gelatin and different polydimethylsiloxane (PDMS) mold microneedle array models, a pH-responsive ultra-long double-network hydrogel microneedle with adjustable mechanical strength and length in the range of 200-3000 μm was prepared. The pH-responsive ultra-long double-network hydrogel microneedle has variable shapes such as conical, pyramidal, cylindrical, and hollow, and can respond to pH intelligently and release loaded drugs.

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