A blood glucose-responsive, visualized, long-acting microneedle and its preparation method

Abstract

This invention relates to the field of biomedicine, specifically to a glucose-responsive, visualized, long-acting microneedle and its preparation method. The microneedle body comprises a glucose-responsive controlled-release shell, a hypoglycemic drug core, and a colorimetric propellant core. The controlled-release shell is composed of a cross-linked natural or synthetic polymer network framework grafted with materials that assist in achieving the shell's glucose-responsive function. The hypoglycemic drug core contains solid powder of the hypoglycemic drug. The colorimetric propellant core consists of a colorimetric agent and a propellant. The hypoglycemic drug core occupies 50%-95% of the needle body volume. The microneedle obtained by this invention can simultaneously achieve multiple functions such as intelligent responsive drug release, high drug loading, tracking drug release within the needle, and improved drug stability. It also provides a new, universally applicable platform for the development of other intelligent microneedle drug delivery systems.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a blood glucose-responsive, visualized, long-acting microneedle (a triple structure of controlled-release shell, hypoglycemic drug core, and colorimetric propulsion core) and its preparation method. Background Technology

[0002] Diabetes poses a serious threat to human health and is one of the four leading causes of death and disability from non-communicable diseases worldwide. The latest data released by the World Health Organization and the International Diabetes Federation shows that the number of people with diabetes globally is growing at an alarming rate, reaching 643 million in 2021, accounting for 10.5% of the global population. Long-term high blood sugar can lead to amputation, blindness, kidney failure, cardiovascular disease, and even death. Therefore, developing convenient and effective hypoglycemic agents is urgently needed worldwide.

[0003] Currently, the most widely used treatment for diabetes is subcutaneous insulin injection. However, the pain associated with this treatment, as well as problems such as induration and inflammation at the injection site caused by long-term injections, lead to poor patient compliance. More importantly, the dosage of insulin injections is mostly determined based on the patient's continuous blood glucose monitoring results before treatment. However, due to changes in the patient's daily diet and real-time physical condition, coupled with the progression of diabetes, the initially determined dosage may not be entirely suitable for subsequent treatment. The dosage significantly affects the efficacy of hypoglycemic drugs and the patient's health and safety—too low a dosage will not effectively lower blood glucose; while too high a dosage may lead to hypoglycemia, and in severe cases, even shock and death. In addition, insulin injection solutions require strict transportation and storage conditions. Before use, they must be transported under cold chain and stored in a refrigerator. Once opened, they should generally not be stored at room temperature for more than 4 weeks.

[0004] Microneedles, which are virtually painless and easy to use, have been proven to be an advanced method of drug delivery, enabling drug administration in a minimally invasive manner. Studies have shown that by integrating blood glucose-responsive components into insulin microneedles, mimicking the way pancreatic β-cells in healthy individuals sense blood glucose fluctuations and regulate insulin release, blood glucose-responsive insulin release can be achieved, maintaining blood glucose homeostasis with minimal patient involvement. However, existing blood glucose-responsive microneedles still have the following common problems that are difficult to solve: (1) Low drug loading and short duration of action. Most existing microneedles use the method of dissolving drugs in a monomer solution of the polymer material of the needle body to load drugs. Therefore, the drug loading mainly depends on the solubility of the drug in the monomer solution. However, many drugs, including large molecular protein drugs such as insulin, have limited solubility in polymer monomer solutions. In addition, the volume of microneedles is small, and the drug loading is usually no more than 1 μg / needle, which means that patients still need to administer the drug multiple times a day. (2) Incomplete drug release. When the polymer needle body swells upon contact with body fluids, the diffusion rate of the loaded drugs, especially large molecular drugs such as insulin, in the gel needle body is slow, and the drug release is likely to be incomplete. This leads to waste and increases the cost of medication for patients. On the other hand, it may also make it difficult to control the dose-effect relationship of the formulation. (3) There is no obvious indicator to show the degree of drug release. Patients have no way of knowing when the drug has been released, making it difficult to guide the timing of the next administration. Changing the medication in advance will cause waste, while not changing the medication in time will prevent timely regulation of blood sugar and interrupt treatment. (4) If the microneedles are prepared by monomer polymerization, there will be a certain amount of monomer residue. The toxicity of the monomer is not conducive to long-term administration. The above problems limit their use in the treatment of diabetes. In summary, the development of a new hypoglycemic agent with good compliance, convenient for patients to administer medication on their own for a long time, and capable of simulating the "on-demand release" of endogenous insulin according to the patient's real-time blood sugar level and convenient for transportation and storage is of great theoretical significance and extremely high application value for solving the many drawbacks of existing formulations, improving the severe situation of diabetes prevention and control, and guiding the design of new biological macromolecular drug delivery systems. Summary of the Invention

[0005] The purpose of this invention is to solve the problems existing in the prior art and provide a blood glucose-responsive, visualized, long-acting microneedle (a triple structure of controlled-release shell, hypoglycemic drug core, and colorimetric propulsion core) and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A glucose-responsive, visualized, long-acting microneedle comprises a glucose-responsive controlled-release shell, a hypoglycemic drug core, and a colorimetric propellant core. The controlled-release shell is composed of a cross-linked natural or synthetic polymer network framework grafted with materials that assist in achieving the blood glucose-responsive function of the shell. The hypoglycemic drug core contains a solid powder of hypoglycemic drug. The colorimetric propellant core is composed of a colorimetric agent and a propellant. The hypoglycemic drug core occupies 50%-95% of the needle body volume.

[0008] The controlled-release shell is formed by grafting a phenylboronic acid derivative onto a backbone that has been grafted with crosslinkable groups via covalent bonds; wherein the grafting rate of the phenylboronic acid derivative onto the backbone is 2%-56%, and the grafting rate of the crosslinkable groups is 1%-55%.

[0009] The skeleton is one or more of chitosan, alginate, gelatin, silk protein, carboxymethyl cellulose, dextran, polyvinyl alcohol, and polyvinylpyrrolidone; preferably chitosan and / or polyvinyl alcohol.

[0010] The phenylboronic acid derivative is one or more of 3-aminophenylboronic acid, 2-carboxyphenylboronic acid, 3-fluoro-4-hydroxyphenylboronic acid, 4-carboxy-3-fluorophenylboronic acid, 4-carboxyphenylboronic acid, 4-formylphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxy-3-chlorophenylboronic acid, 3-carboxy-4-fluorophenylboronic acid, and 3-acrylamidophenylboronic acid; preferably 4-carboxy-3-chlorophenylboronic acid and / or 3-acrylamidophenylboronic acid.

[0011] The substances providing crosslinkable groups are methacrylate / anhydride compounds such as glyceryl methacrylate and methyl methacrylate; and acrylamide compounds such as 3-acrylamidophenylboronic acid, acrylamide, and N-hydroxymethylacrylamide.

[0012] The hypoglycemic drug solid powder is one or more of the following: sulfonylureas, biguanides, α-glucosidase inhibitors, insulin, thiazolidinediones (TZDs), meglitinides, GLP-1 receptor agonists, DPP-IV inhibitors, and SGLT-2 inhibitors.

[0013] A method for preparing a blood glucose-responsive, visualized, long-acting microneedle.

[0014] (1) Natural or synthetic polymer network framework materials are prepared by physical cross-linking or chemical cross-linking. Benzeneboronic acid derivatives that can help achieve blood glucose response are grafted onto the network framework and then cast into a microneedle mold. After drying, they are used as a controlled-release shell.

[0015] (2) Fill the controlled-release shell with powder containing hypoglycemic drug solids as a drug core;

[0016] (3) After mixing the color developer with the swelling polymer, small solid microneedles (with the same needle spacing as the controlled-release shell) are prepared as the color-developing booster core, and a colorless and transparent material is used to prepare the backing layer of the small microneedles.

[0017] (4) Apply an adhesive to the controlled-release shell after drug loading, and then insert the color-developing booster core microneedles into the controlled-release shell filled with hypoglycemic drugs in a nested manner to secure them, thus completing the preparation of the triple structure microneedles.

[0018] To elaborate further,

[0019] 1) Modification of polymer materials is achieved by grafting physically or chemically crosslinkable groups onto the molecular chains of natural or synthetic polymer materials through covalent bonds (such as the transesterification reaction of -OH with esters), with a grafting rate of 1%-55% for crosslinkable groups; phenylboronic acid derivatives are grafted onto polymer materials through covalent bonds (such as the amide formation reaction of -NH2 with -COOH) (grafting rate of 2%-56%), and then the material solution is cast into a microneedle mold to fill the mold, and dried to obtain the controlled-release shell;

[0020] 2) Fill the dried controlled-release shell with the powder containing the hypoglycemic drug solid, so that the drug powder fills the micropores of the shell;

[0021] 3) Dissolve or disperse the booster and color developer to prepare a color-developing booster solution or suspension. Add the solution to a small microneedle mold with the same needle spacing as in step 1), fill the mold with the liquid, dry it, and then pour a colorless transparent backing on top. Peel the microneedles off the mold to obtain the color-developing booster core.

[0022] 4) Apply an adhesive to the microneedle filled with drug obtained in step 2), and then insert the color-developing booster core obtained in step 3) into it in a nested manner. After bonding, peel the microneedle off the mold to obtain a triple structure microneedle of "controlled release shell - powder core - color-developing booster core".

[0023] The mass ratio of the color developer and the booster is 0.01-0.5; wherein the color developer is one or more of organic or inorganic pigments (such as red iron oxide, reactive brilliant blue KN-R); the booster is one or more of polyoxyethylene, povidone, and acrylic polymer, preferably povidone.

[0024] The controlled-release outer shell needles prepared using this method have a regular and uniform shape, are neatly arranged, and allow the drug to fully fill the interior of the controlled-release shell (e.g., Figure 7 It has a high drug loading capacity, up to 1.1 mg / cm³. 2 (11±0.7μg / needle). The microneedles have good rigidity, enabling them to meet the requirements for skin puncture while maintaining a high drug loading capacity (as shown in Table 1). Figure 8This can mimic the "on-demand release" trend of endogenous insulin, adjusting the release rate of hypoglycemic drugs according to blood glucose concentration (e.g., Figure 9 Furthermore, with the release of the drug, the colorimetric effect significantly promotes the swelling of the inner core (e.g., Figure 10 Less than 0.35cm 2 Microneedles can maintain normal blood glucose levels in diabetic model mice for 16 hours (e.g. Figure 11 Furthermore, during the administration process, diabetic mice injected intraperitoneally with 1.5 g / kg glucose experienced hyperglycemia, but their blood glucose levels returned to normal within 10 minutes (e.g., Figure 12 Furthermore, the microneedles prepared using this method do not cause hypoglycemia in healthy mice (e.g., Figure 13 ).

[0025] While there are existing inventions related to core-shell microneedles, their preparation requires high temperatures or organic solvents, resulting in complex processes and high costs. This invention, however, uses an aqueous solution, eliminating the need for high temperatures and simplifying the process. Currently, glucose-responsive microneedles are also prepared using phenylboronic acid derivatives. However, firstly, existing inventions produce uniform microneedles with low drug loading; the drug loading of uniform insulin microneedles is generally no more than 1 μg / needle, while the core-shell microneedles of this patent have a drug loading as high as 11 ± 0.7 μg / needle. Furthermore, the preparation of core-shell glucose-responsive microneedles is challenging, requiring high standards for the thickness of the microneedle shell, the degree of cross-linking, and the grafting rate of the phenylboronic acid derivative. Secondly, compared to existing inventions, the chitosan used in this invention is rich in amino groups. As Lewis bases, amino groups can stabilize the phenylboronic acid-glucose complex, contributing to improved glucose responsiveness of the microneedles. Thirdly, the core-shell glucose-responsive microneedles prepared according to this patent exhibit better glucose-responsive drug release effects, whereas many similar inventions do not specify glucose-sensitive drug release data.

[0026] The beneficial effects of this invention are:

[0027] The present invention features a microneedle with a triple structure of "controlled-release outer shell - hypoglycemic drug core - color-developing propulsion inner core". This microneedle can simulate the "on-demand release" of endogenous insulin according to the patient's real-time blood glucose level, maintain the patient's blood glucose homeostasis, effectively improve the drug loading and stability of hypoglycemic drugs, prolong the duration of drug effect and facilitate formulation storage; it can also instruct the patient to replace the microneedle in a timely manner, achieving seamless drug delivery.

[0028] 1) This invention is inexpensive, suitable for mass production, and can be widely used in the field of transdermal drug delivery for hypoglycemic agents. Furthermore, the drug release behavior of blood glucose-responsive microneedles can be precisely controlled by altering the degree of cross-linking and the type and amount of blood glucose-responsive compounds used for grafting. For example, by increasing the amount of blood glucose-responsive compounds used in the preparation process, the swelling degree of the controlled-release shell at high sugar concentrations can be increased, thereby improving the blood glucose responsiveness of the microneedles.

[0029] 2) The blood glucose-responsive microneedles of the present invention have a high drug loading capacity and can adjust the release rate and dosage of hypoglycemic drugs according to the patient's real-time blood glucose concentration, thereby prolonging the duration of the drug's effect in maintaining stable blood glucose levels, while reducing the risk of hypoglycemia and improving patient compliance with long-term medication.

[0030] 3) When using the blood glucose-responsive microneedles of this invention, patients can observe the diffusion of the inner core color through the backing of the needle patch to indicate the release of the drug inside the needle, which makes it convenient for patients to replace the needle patch in a timely manner and achieve uninterrupted "closed-loop" treatment. Attached Figure Description

[0031] Figure 1 The hydrogen nuclear magnetic resonance spectra of CTS, GMA-CTS, and GMA-CTS-FCPBA provided in the embodiments of the present invention.

[0032] Figure 2 Infrared spectra of CTS, GMA-CTS, and GMA-CTS-FCPBA provided for embodiments of the present invention.

[0033] Figure 3 The hydrogen nuclear magnetic resonance spectra of PVA and PVA-MA provided in the embodiments of the present invention.

[0034] Figure 4 The infrared spectra of PVA and PVA-MA provided for embodiments of the present invention.

[0035] Figure 5 This is a schematic diagram illustrating the preparation process of a blood glucose-responsive, visualized, long-acting microneedle for drug delivery, as provided in an embodiment of the present invention.

[0036] Figure 6 Hollow microneedle shells prepared with different concentrations of GMA-CTS-FCPBA are provided in the embodiments of the present invention, wherein the concentration of A is 30 mg / mL, the concentration of B is 60 mg / mL, and the concentration of C is 120 mg / mL.

[0037] Figure 7 Micrographs of long-acting microneedles with blood glucose-responsive drug delivery provided in embodiments of the present invention.

[0038] Figure 8 The image shows hematoxylin-eosin staining of mouse skin punctures using a micro-target provided in an embodiment of the present invention.

[0039] Figure 9 This is a diagram illustrating the in vitro blood glucose response drug release effect of microneedles provided in an embodiment of the present invention.

[0040] Figure 10 The image shows the change in the size of the colored spots before and after drug release, observed from above the microneedle patch, as provided in an embodiment of the present invention (left: before drug release; right: after drug release).

[0041] Figure 11 The following is a diagram showing the effect of blood glucose changes after microneedle administration to diabetic mice, provided for embodiments of the present invention (A is the blank microneedle group without insulin; B is the subcutaneous injection group; C is the microneedle of sample 2 in Example 4; D is the microneedle group prepared by using the controlled-release shell prepared with the material of sample 4 in Example 1 and following the microneedle preparation method of sample 2 in Example 4).

[0042] Figure 12 The image provided in this embodiment of the invention shows the effect of obtaining blood glucose changes in diabetic mice through microneedle administration in a glucose tolerance test.

[0043] Figure 13 The image provided in this embodiment of the invention shows the effect of blood glucose changes in healthy mice after microneedle administration. Detailed Implementation

[0044] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0045] The microneedle with a triple structure of "controlled-release outer shell - hypoglycemic drug core - color-developing propulsion inner core" obtained by this invention can simultaneously achieve multiple functions such as intelligent responsive drug release, high drug loading, tracking drug release within the needle, and improving drug stability. It also provides a new platform with universal applicability for the development of other intelligent microneedle drug delivery systems.

[0046] Example 1: Preparation of Blood Glucose-Response Controlled-Release Materials of Different Materials

[0047] Sample 1

[0048] Preparation of 4-carboxy-3-fluorophenylboronic acid-grafted crosslinkable chitosan (GMA-CTS-FCPBA): 2 g of chitosan (CTS) (weight-average molecular weight 3000 kD, degree of deacetylation 95%) was dissolved in 200 mL of 0.4 mol / L acetic acid, and 1.76 g of glycidyl methacrylate (GMA) was added. The reaction solution was heated to 60 °C and reacted for 4 h, then cooled in an ice-water bath to stop the reaction. Unreacted raw materials were removed by dialyzing with 30% ethanol and distilled water for 3 days respectively, and then freeze-dried to obtain GMA-CTS. Weigh 1g of the prepared GMA-CTS and dissolve it in 100mL of 0.4mol / L acetic acid. Add 1g of 4-carboxy-3-fluorophenylboronic acid (FCPBA), 1g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g of N-hydroxysuccinic acid imine to the solution in sequence. Stir to dissolve and adjust the pH of the solution to 4.7 with 0.01mol / L acetic acid. Stir at 25℃ for 48h, remove unreacted raw materials by ultrafiltration and centrifugation, and freeze-dry to obtain the blood glucose-responsive controlled-release material GMA-CTS-FCPBA.

[0049] The above-mentioned characterization results of the proton NMR and infrared spectra of CTS, GMA-CTS, and GMA-CTS-FCPBA are as follows: Figure 1 , 2 . Figure 1 The peaks with chemical shifts at 5.41 ppm and 5.90 ppm represent proton peaks of the olefin in GMA, while the peaks with chemical shifts between 7.31 and 7.52 ppm represent proton peaks of the benzene ring in FCPBA. (In the CTS infrared spectrum...) Figure 2 ), 3437cm -1 The peaks are stretching vibration peaks of OH and NH; compared with CTS, CTS-GMA has a peak at 1605 cm⁻¹. -1 The peak intensities of the C=O and C=C stretching vibrations of GMA at this location are significantly increased, and at 1085 cm⁻¹ -1 A new stretching vibration peak of COC appeared at 1567 cm⁻¹; compared with CTS-GMA, GMA-CTS-FCPBA showed a peak at 1567 cm⁻¹. -1 1639cm -1 and 1711cm -1 The peaks that appear are stretching vibration peaks of the FCPBA benzene ring skeleton. The above results prove that GMA and FCPBA have been successfully grafted onto CTS.

[0050] Sample 2

[0051] Preparation of 4-formylphenylboronic acid-grafted crosslinkable chitosan (GMA-CTS-FPBA): 2 g of CTS (weight-average molecular weight 3000 kD, degree of deacetylation 95%) was dissolved in 200 mL of 0.4 mol / L acetic acid, and 1.76 g of glycidyl methacrylate (GMA) was added. The reaction solution was heated to 60 °C and reacted for 4 h, then cooled in an ice-water bath to stop the reaction. Unreacted raw materials were removed by dialyzing with 30% ethanol and distilled water, and the mixture was freeze-dried to obtain GMA-CTS. 1 g of the prepared GMA-CTS was weighed and dissolved in 100 mL of 0.4 mol / L acetic acid, and 0.5 g of FPBA was weighed and dissolved in 50 mL of methanol. The mixture was stirred at 25 °C for 24 h, then centrifuged at 10000 rpm for 5 min to collect the precipitate. The precipitate was washed successively with methanol, anhydrous ethanol, and water, and then freeze-dried to obtain GMA-CTS-FPBA.

[0052] Sample 3

[0053] 5 g of polyvinyl alcohol (PVA) was dissolved in 100 mL of dimethyl sulfoxide, and 0.42 g of 4-dimethylaminopyridine and 4.86 g of GMA were added. The reaction solution was heated to 60 °C and reacted for 6 h. Then, 5 times the volume of acetone was added to the reaction system, and a precipitate was formed. The precipitate was filtered, washed three times with anhydrous ethanol, and dried at room temperature to obtain PVA-MA. The results of the 1H NMR and IR characterization of PVA and PVA-MA are as follows: Figure 3 , 4 . Figure 3 The peaks with chemical shifts at 5.61 ppm and 5.98 ppm are the proton peaks of the olefins in GMA; Figure 4 In the middle, PVA-MA compared to PVA at 1670cm -1 The newly appearing peaks nearby are the stretching vibration peaks of C=O and C=C on GMA. All of the above results prove that GMA has been successfully grafted onto PVA.

[0054] Then, 0.8 g of the obtained blood glucose-responsive controlled-release material (PVA-MA) was dissolved in 4 mL of distilled water, and 0.1 g of 3-acrylamidophenylboronic acid (APBA) and 0.01 g of N,N′-methylenebisacrylamide were added as crosslinking agents, and 0.02 g of Irgacure 2959 was added as a photoinitiator. The mixture was stirred and dissolved, and then a hollow controlled-release shell was prepared according to the further description in Example 2.

[0055] Sample 4

[0056] Preparation of 4-carboxy-3-fluorophenylboronic acid-grafted crosslinkable chitosan (GMA-CTS-FCPBA): 2 g of chitosan (CTS) (weight-average molecular weight 3000 kD, degree of deacetylation 95%) was dissolved in 200 mL of 0.4 mol / L acetic acid, and 1.76 g of glycidyl methacrylate (GMA) was added. The reaction solution was heated to 60 °C and reacted for 4 h, then cooled in an ice-water bath to stop the reaction. Unreacted raw materials were removed by dialyzing with 30% ethanol and distilled water, and the product was freeze-dried to obtain GMA-CTS. Weigh 1g of the prepared GMA-CTS and dissolve it in 200mL of 0.4mol / L acetic acid. Add 2g of 4-carboxy-3-fluorophenylboronic acid (FCPBA), 2g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2g of N-hydroxysuccinic acid imine to the solution in sequence. Stir to dissolve and adjust the pH of the solution to 4.7 with 0.01mol / L acetic acid. Stir at 25℃ for 48h. Remove unreacted raw materials by ultrafiltration and centrifugation. Freeze-dry to obtain the blood glucose-responsive controlled-release material GMA-CTS-FCPBA.

[0057] Example 2: Preparation of a blood glucose-responsive controlled-release shell

[0058] Sample 1

[0059] 0.3 g of sample 1GMA-CTS-FCPBA from Example 1 was dissolved in 5 mL of 0.05 mol / L acetic acid. 6 mg of N,N′-methylenebisacrylamide was added as a crosslinking agent, and 6 mg of Irgacure 2959 was added as a photoinitiator. After stirring and dissolving, a blood glucose-responsive controlled-release material solution was obtained. The blood glucose-responsive controlled-release material solution was cast into a polydimethylsiloxane (PDMS) microneedle mold (needle length 1000 μm, needle bottom diameter 440 μm, needle tip distance 1000 μm, 10×10 array). Air was extracted from the mold needle holes under a vacuum of 0.09 MPa, allowing the solution to enter the mold needle holes. After scraping away the solution containing air bubbles in the mold grooves, the blood glucose-responsive controlled-release material solution was added again. This process was repeated until no air bubbles were generated in the grooves. The mold was dried overnight at 40°C and then subjected to UV light (365 nm, 60 mW / cm²). 2 Hollow controlled-release shell was obtained by irradiation for 10 minutes.

[0060] Sample 2

[0061] 0.15 g of sample 1GMA-CTS-FCPBA from Example 1 was dissolved in 5 mL of 0.05 mol / L acetic acid. 3 mg of N,N′-methylenebisacrylamide was added as a crosslinking agent, and 3 mg of Irgacure 2959 was added as a photoinitiator. After stirring and dissolving, a blood glucose-responsive controlled-release material solution was obtained. The blood glucose-responsive controlled-release material solution was placed in a PDMS mold and centrifuged horizontally at 5000 rpm for 5 min at 20°C. The mold was then rotated 180 degrees and centrifuged horizontally at 5000 rpm for 5 min to ensure the controlled-release material solution fully entered the microneedle mold to form needle tips. The mold was dried overnight at 40°C and then subjected to UV light (365 nm, 60 mW / cm²). 2 Hollow controlled-release shells were obtained by irradiation for 10 minutes. Figure 6 A.

[0062] Sample 3

[0063] 0.3 g of sample 1GMA-CTS-FCPBA from Example 1 was dissolved in 5 mL of 0.05 mol / L acetic acid. 6 mg of N,N′-methylenebisacrylamide was added as a crosslinking agent, and 6 mg of Irgacure 2959 was added as a photoinitiator. After stirring and dissolving, a blood glucose-responsive controlled-release material solution was obtained. The blood glucose-responsive controlled-release material solution was placed in a PDMS mold and centrifuged horizontally at 5000 rpm for 5 min at 20°C. The mold was then rotated 180 degrees and centrifuged horizontally at 5000 rpm for 5 min to ensure the controlled-release material solution fully entered the microneedle mold to form needle tips. The mold was dried overnight at 40°C and then subjected to UV light (365 nm, 60 mW / cm²). 2 Hollow controlled-release shells were obtained by irradiation for 10 minutes. Figure 6 B.

[0064] Sample 4

[0065] 0.6 g of sample 1GMA-CTS-FCPBA from Example 1 was dissolved in 5 mL of 0.05 mol / L acetic acid. 12 mg of N,N′-methylenebisacrylamide was added as a crosslinking agent, and 12 mg of Irgacure 2959 was added as a photoinitiator. After stirring and dissolving, a blood glucose-responsive controlled-release material solution was obtained. The blood glucose-responsive controlled-release material solution was placed in a PDMS mold and centrifuged horizontally at 5000 rpm for 5 min at 20°C. The mold was then rotated 180 degrees and centrifuged horizontally at 5000 rpm for 5 min to ensure the controlled-release material solution fully entered the microneedle mold to form needle tips. The mold was dried overnight at 40°C and then subjected to UV light (365 nm, 60 mW / cm²). 2 Hollow controlled-release shells were obtained by irradiation for 10 minutes. Figure 6 C.

[0066] Depend on Figure 6 It can be seen that as the concentration of the controlled-release material solution increases, the thickness of the microneedle controlled-release shell gradually increases, and the hollow volume gradually decreases.

[0067] Example 3: Preparation of the Color-Developing Boosting Core

[0068] Sample 1

[0069] 50 mg of red iron oxide was dispersed in 5 mL of 30% PVP (K90) aqueous solution. Small-sized microneedles (300 μm length, 150 μm base diameter, 1000 μm tip-to-tip distance, 10 × 10 array) with the same needle spacing as the controlled-release shell were prepared using a centrifugal loading method. After three cycles of loading and drying, Nolan resin was cast into a mold groove and subjected to UV light at 365 nm and 60 mW / cm². 2 Irradiate for 10 minutes, demold, and obtain small-sized microneedles with red tips containing a colorless and transparent base layer, i.e., color-developing booster core.

[0070] Sample 2

[0071] Dissolve 4g of PVA-MA in 50mL of water, add 0.08g of Reactive Brilliant Blue KN-R, react at 25℃ for 24h, and dialyze against distilled water to remove unreacted Reactive Blue KN-R. Dissolve 0.8g of PVA-MA in 4mL of distilled water, add 10mg of N,N′-methylenebisacrylamide as a crosslinking agent, and add 20mg of Irgacure 2959 as a photoinitiator. After complete dissolution, prepare microneedles with the same needle spacing as the controlled-release shell using a centrifugal loading method. Repeat the loading-drying process three times, using a UV lamp (365nm, 60mW / cm²). 2Irradiate for 10 minutes to obtain a solid needle tip. Pour Nolan resin into the mold groove and heat under a UV lamp (365nm, 60mW / cm²). 2 Irradiate for 10 minutes, demold, and obtain small-sized microneedles with a colorless and transparent base layer and blue needle tips, i.e., color-developing booster core.

[0072] Example 4: Preparation of blood glucose-responsive, visualized, long-acting microneedles

[0073] Sample 1

[0074] Insulin was loaded into the controlled-release shell prepared as Sample 2 in Example 2, and centrifuged horizontally at 5000 rpm for 5 min at 4°C. The mold was then rotated 180 degrees and centrifuged horizontally at 5000 rpm for 5 min to recover excess insulin powder from the grooves. An adhesive was then applied to the grooves, and the colorimetric booster core of Sample 1 from Example 3 was inserted in a nested manner. After secure adhesion and demolding, a glucose-responsive, visualized, long-acting microneedle was obtained. The drug loading was calculated to be 1.9 ± 0.08 mg / cm³ by weighing the difference in mass before and after loading the controlled-release shell with insulin. 2 (19±0.8μg / needle) The pressure value when the microneedle tip bends, as tested by a texture analyzer, is 0.048N, which does not meet the minimum hardness required for the microneedle to pierce the skin (0.06N), and may cause the microneedle to break when inserted into the skin (see Table 1).

[0075] Sample 2

[0076] Insulin was loaded into the controlled-release shell prepared in Sample 3 of Example 2. After horizontal centrifugation at 5000 rpm for 5 min at 20°C, the mold was rotated 180 degrees and centrifuged horizontally at 5000 rpm for 5 min to recover excess insulin powder from the grooves. An adhesive was then applied to the grooves. The colorimetric pusher core prepared in Sample 1 of Example 3 was inserted in a nested manner. After demolding, a glucose-responsive, visualized, long-acting microneedle was obtained. The drug loading was calculated to be 1.1 ± 0.07 mg / cm³ by weighing the difference in mass before and after loading the controlled-release shell with insulin. 2 (11±0.7μg / needle), the overall morphology and integrity of the microneedles were characterized using optical microscopy. Figure 7 The pressure value when the microneedle tip bends, as measured by a texture analyzer, is 0.11 N, which meets the minimum hardness (0.06 N) required for the microneedle to pierce the skin (see Table 1).

[0077] Sample 3

[0078] Insulin was loaded into the controlled-release shell prepared in Sample 4 of Example 2. After centrifugation at 5000 rpm for 5 min at 20°C, the mold was rotated 180 degrees and centrifuged at 5000 rpm for 5 min to recover excess insulin powder from the grooves. An adhesive was then applied to the grooves. The colorimetric pusher core prepared in Sample 1 of Example 3 was then inserted in a nested manner. After demolding, a glucose-responsive, visualized, long-acting microneedle was obtained. The drug loading was calculated to be 0.5 ± 0.03 mg / cm³ by weighing the difference in mass before and after loading the controlled-release shell with insulin. 2 (5±0.3μg / needle), the pressure value when the microneedle tip bends, as tested by a texture analyzer, is 0.15N, which meets the minimum hardness required for the microneedle to pierce the skin (see Table 1).

[0079] Table 1 shows the drug loading and hardness of hollow microneedle shells prepared with different concentrations of GMA-CTS-FCPBA.

[0080]

[0081] As shown in Table 1, when the concentration of GMA-CTS-FCPBA is low (30 mg / mL), the microneedles have a high drug loading capacity of 1.9 ± 0.08 mg / mL. 2 (19±0.8μg / needle), but the microneedles lacked sufficient mechanical strength, potentially leading to breakage during skin insertion and hindering effective drug delivery to the subcutaneous tissue. With increasing polymer concentration (60mg / mL), although the drug loading decreased by 1.1±0.07mg / cm³ (11±0.7μg / needle), the mechanical strength of the microneedles significantly increased, meeting the required hardness for skin penetration. When the polymer concentration continued to increase (120mg / mL), the drug loading of the microneedles decreased sharply by 0.5mg / cm³. 2 (5±0.3μg / root).

[0082] Sample 4

[0083] Semaglutide was filled into the controlled-release shell of Sample 3 from Example 2. After centrifugation at 5000 rpm for 5 min at 4°C, the mold was rotated 180 degrees and centrifuged at 5000 rpm for 5 min. The colorimetric booster core of Sample 1 from Example 3 was then inserted in a nested manner and subjected to ultraviolet light (365 nm, 60 mW / cm²). 2 Irradiate for 10 minutes to crosslink the base layer and the controlled-release shell.

[0084] Example 5: Mechanical property testing of microneedles

[0085] Mice (male Kunming mice, 5-7 weeks old) were limbs fixed and their backs shaved. Sample 2 microneedles from Example 4 were vertically inserted into the mouse skin and left in place for 10 minutes. The microneedles were then removed, the mice were euthanized, and the skin at the microneedle puncture site was dissected and fixed in 4% paraformaldehyde. Paraffin-embedded sections were prepared, stained with hematoxylin and eosin, and observed under a microscope to determine the microneedle insertion. The results are as follows: Figure 8 .

[0086] Depend on Figure 8 It is evident that the mouse's stratum corneum was ruptured, and the microneedles formed sharp, cone-shaped puncture holes within the skin, proving that the microneedles can pierce the mouse's stratum corneum and deliver the drug to the dermis.

[0087] Example 6: In vitro glucose-responsive drug release via microneedles

[0088] Sample 2 of Example 4 was incubated in 8 mL of glucose phosphate buffer solution (pH 7.4) at 37°C with different concentrations (0, 100, and 400 mg / dL). Samples were taken at specific time points, and the insulin content in the suspension was determined by high-performance liquid chromatography (HPLC) to investigate the in vitro blood glucose-responsive drug release of the microneedles. The results are as follows: Figure 9 .

[0089] Depend on Figure 9 It can be seen that when the microneedles were incubated in glucose phosphate buffer solutions of different concentrations for 24 hours, the cumulative insulin release in the 100 mg / dL glucose phosphate buffer solution was 2.1 times that in the glucose-free solution. When the glucose concentration was increased to 400 mg / dL, the cumulative insulin release increased to 2.8 times. The above experimental results prove that the drug release of the microneedles has a blood glucose response.

[0090] Example 7: Changes in the color-promoting inner core before and after microneedle drug release

[0091] The microneedles from Example 6, which were released for 24 hours in a phosphate buffer solution (pH 7.4) containing 400 mg / dL glucose, showed a clear increase in the size and diffusion of the colored spots observed above the microneedle patch. The results are as follows: Figure 10 .

[0092] Depend on Figure 10 As the medication is released, the color-developing core gradually swells, making it easy to remind patients to change the patch in a timely manner.

[0093] Example 8: Blood glucose lowering effect in diabetic mice

[0094] A diabetic mouse model was established by intraperitoneal injection of streptozotocin (150 mg / kg) into mice (Kunming mice, male, 5-7 weeks old). After hair removal from the back, microneedles of Sample 2 from Example 4 and controlled-release shells prepared using the material of Sample 4 from Example 1 were respectively attached. The microneedles prepared according to the method of Microneedle 2 from Example 4 were used as controls, with subcutaneous injection of insulin and blank microneedles without insulin as controls. Blood glucose concentrations of mice were measured using a glucometer at specific time points. The results are as follows. Figure 11 .

[0095] Depend on Figure 11 It was observed that after subcutaneous insulin injection in diabetic mice, blood glucose levels dropped to normal within 1 hour, but returned to a hyperglycemic state in less than 3 hours. In the microneedle administration group of Sample 2 in Example 4, blood glucose levels dropped to normal within 6 hours of administration and remained at normal for more than 16 hours without hypoglycemia. This demonstrates that the microneedles can significantly prolong the duration of normal blood glucose levels in hyperglycemic animals, thereby reducing the frequency of administration and improving patient compliance. However, the microneedles prepared using the controlled-release shell material of Sample 4 in Example 1, and prepared according to the microneedle preparation method of Sample 2 in Example 4, resulted in hypoglycemia within 8-15 hours.

[0096] Diabetic mice (male Kunming mice, 5-7 weeks old) were implanted with Sample 2 microneedles (Example 4) for 8 hours. They were then intraperitoneally injected with glucose-phosphate buffer (pH 7.4, 0.4 g / mL) at a dose of 1.5 g / kg. Healthy mice and mice injected subcutaneously with insulin served as controls. Blood glucose concentrations in the rats were measured using a glucometer at specific time points. The results are as follows: Figure 12 .

[0097] Depend on Figure 12 It was observed that blood glucose levels rose rapidly in mice after intraperitoneal injection of glucose. Specifically, in diabetic mice injected subcutaneously with insulin, blood glucose levels remained elevated without decreasing; in healthy mice, blood glucose levels returned to normal within 20 minutes; while in the microneedle administration group, blood glucose levels in diabetic mice returned to normal within 10 minutes. This demonstrates that the microneedle exhibits blood glucose responsiveness, rapidly responding to hyperglycemia, quickly releasing the drug, and regulating blood glucose to normal levels.

[0098] Healthy male Kunming mice (5-7 weeks old) were shaved from their backs and then fitted with the microneedle patch of Sample 2 from Example 4. Subcutaneous insulin injection served as a control. Blood glucose concentrations in the mice were measured using a glucometer at specific time points. Results are as follows: Figure 13 .

[0099] Depend on Figure 13 It can be seen that after subcutaneous injection of insulin, the blood glucose of mice decreased significantly, and even hypoglycemia occurred. However, the blood glucose of mice treated with microneedles decreased only slightly and hypoglycemia did not occur. This proves that the microneedles have blood glucose responsiveness and can simulate the release of endogenous insulin. Under normal blood glucose conditions, almost no drug is released, which can avoid hypoglycemia.

Claims

1. A blood glucose responsive drug release visualizing long-acting microneedle, characterized by, The microneedle body is composed of a blood glucose response controlled release shell, a hypoglycemic drug core and a color developing booster inner core; the blood glucose response controlled release shell is composed of a network framework of grafted cross-linked natural or synthetic high molecular materials capable of assisting the realization of the blood glucose response function of the shell, the hypoglycemic drug core contains hypoglycemic drug solid powder, and the color developing booster inner core is composed of a color developing agent and a booster; wherein the hypoglycemic drug core accounts for 50%-95% of the volume of the needle body; The blood glucose response controlled release shell is obtained by grafting a phenylboronic acid derivative onto a framework that has been grafted with a cross-linkable group through a covalent bond; wherein the grafting rate of the phenylboronic acid derivative grafted on the framework is 2%-56%, and the grafting rate of the cross-linkable group is 1%-55%; specifically, 4-carboxyl-3-fluorophenylboronic acid is grafted onto cross-linkable chitosan; The hypoglycemic drug is insulin.

2. A preparation method of the blood glucose response type drug release visualized long-acting microneedle according to claim 1, characterized in that, (1) a cross-linked natural or synthetic high molecular network framework material is prepared by physical cross-linking or chemical cross-linking, a phenylboronic acid derivative capable of assisting the realization of the blood glucose response function of the shell is grafted onto the network framework, and then cast into a microneedle mold, and dried to serve as a controlled release shell; (2) hypoglycemic drug solid powder is filled into the controlled release shell as a drug core; (3) a small-size solid microneedle is prepared by mixing a color developing agent and a swelling polymer as a color developing booster inner core, and a small-size microneedle made of colorless transparent material is prepared as a backing layer; (4) an adhesive is coated on the drug-loaded controlled release shell, and then the color developing booster inner core microneedle is inserted into the controlled release shell filled with hypoglycemic drug in a nested manner to stick firmly, thereby completing the preparation of the triple-structure microneedle.

3. The preparation method of the blood glucose response type drug release visualized long-acting microneedle according to claim 2, characterized in that, 1) a group capable of physical cross-linking or chemical cross-linking is grafted onto the molecular chain of a natural or synthetic high molecular material through a covalent bond to modify the high molecular material, and the grafting rate of the cross-linkable group is 1%-55%; a phenylboronic acid derivative is grafted onto the high molecular material through a covalent bond, and then the material solution is cast into a microneedle mold to fill the mold, and dried to obtain a controlled release shell; 2) hypoglycemic drug solid powder is filled into the dried controlled release shell to fill the micropore channels of the shell with drug powder; 3) a color developing booster solution or suspension is prepared by dissolving or dispersing a booster and a color developing agent, and then added to a small microneedle mold with the same needle spacing as step 1) to fill the mold with liquid, and dried to cast a colorless transparent backing on top, and then the microneedle is peeled off from the mold to obtain a color developing booster inner core; 4) an adhesive is coated on the microneedle filled with drug obtained in step 2), and then the color developing booster inner core obtained in step 3) is inserted into it in a nested manner, and after bonding, the microneedle is peeled off from the mold to obtain a "controlled release shell-powder drug core-color developing booster inner core" triple-structure microneedle.

4. The method for preparing the blood glucose-responsive, visualized, long-acting microneedles according to claim 3, characterized in that, The mass ratio of the color developing agent and the booster is 0.01-0.5; wherein the color developing agent is one or more of organic or inorganic pigments; and the booster is one or more of polyoxyethylene, povidone and acrylic polymer.

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