Glucose-responsive empagliflozin microneedle delivery system and applications thereof

Abstract

The present application relates to the biomedical field, specifically relates to a glucose-responsive empagliflozin microneedle delivery system and application thereof.The glucose-responsive empagliflozin microneedle delivery system of the present application is characterized in that the microneedle delivery system is a microneedle patch, comprising a substrate and a microneedle array arranged on the substrate; the microneedle in the microneedle array contains glucose-responsive empagliflozin nanoparticles CB@E or PCB@E.The present application has the following technical effects: the empagliflozin nanoparticles of the present application have precise glucose-responsive drug release performance.The microneedle drug delivery system of the present application has the effect of long-term control of blood glucose.In a T2DM rat model, MN@CB@E exhibits long-acting blood glucose control effect, can reduce blood glucose to normal level within 4h and maintain for 24h, and the hypoglycemic duration is significantly better than that of subcutaneous injection of free empagliflozin (only maintains for 6h), and there is no risk of hypoglycemia after administration to healthy rats.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a glucose-responsive empagliflozin microneedle delivery system and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is the most common metabolic disease in clinical practice, characterized primarily by insulin resistance and progressive decline in pancreatic β-cell function, manifesting as chronic hyperglycemia. As a globally prevalent chronic metabolic disease, accounting for over 90% of all diabetes cases, it has become a serious public health problem threatening human health. According to data from the International Diabetes Federation's (IDF) 10th Diabetes Atlas, there were 537 million people aged 20-79 with T2DM globally in 2021, and this number is projected to surge to 784 million by 2045, with case growth rates exceeding 100% in regions such as Africa and the Middle East. T2DM patients are chronically hyperglycemic, easily inducing various serious complications, including chronic kidney disease, cardiovascular disease, diabetic retinopathy, and peripheral neuropathy. These complications not only significantly reduce patients' quality of life but are also leading causes of disability and death. Furthermore, diabetic patients face additional health risks such as increased bone fragility, delayed wound healing, and chronic diabetic ulcers, further exacerbating the disease burden.

[0003] Current clinical treatment for type 2 diabetes mellitus (T2DM) focuses on controlling blood glucose and preventing complications, employing a combination of lifestyle interventions and drug therapy. In drug therapy, insulin requires injection, which, while rapidly lowering blood glucose, can cause local adverse reactions such as swelling, erythema, and itching at the injection site with repeated injections, and may also lead to hypoglycemia, severely impacting patient adherence. There are many types of oral hypoglycemic agents, among which empagliflozin, a representative drug of the sodium-glucose cotransporter 2 (SGLT2) inhibitors, lowers blood glucose by inhibiting the reabsorption of glucose in the proximal tubules of the kidneys and promoting glucose excretion in urine. In addition to its hypoglycemic effect, empagliflozin has been shown to have cardiovascular protective advantages independent of its hypoglycemic effect, significantly reducing the risk of cardiovascular disease in T2DM patients by 38%, especially in preventing heart failure. Its once-daily dosing frequency is also more convenient than other drugs. However, existing oral tablets of empagliflozin have limitations in dosage form. Absorption through the gastrointestinal tract is easily affected by first-pass metabolism, limiting bioavailability and making them unsuitable for patients with gastrointestinal diseases or difficulty swallowing. Long-term use can easily lead to gastrointestinal discomfort such as nausea and diarrhea. Furthermore, the current dosage form lacks glucose responsiveness, failing to achieve on-demand release based on blood glucose levels. This results in insufficient hypoglycemic effect in hyperglycemia, and sustained release in normal blood glucose levels may increase the risk of adverse reactions. It also lacks the ability to self-regulate to avoid hypoglycemia. In addition, current delivery methods lack targeting precision, failing to achieve specific enrichment of empagliflozin at its renal target site, and drug distribution in non-target organs can easily cause toxic side effects. Therefore, there is a need to develop a novel drug delivery system to optimize the glucose responsiveness, targeting, and convenience of small-molecule hypoglycemic drugs such as empagliflozin.

[0004] The development of drug delivery systems (DDS) has provided a new direction for overcoming the limitations of traditional hypoglycemic drugs. An ideal DDS needs to optimize pharmacokinetic and pharmacodynamic properties, achieve targeted delivery, and provide controlled drug release, thereby improving therapeutic efficacy while minimizing the toxic side effects caused by nonspecific distribution. Bioresponsive materials, as a core component of intelligent drug delivery systems, can sense specific biological signals in the body (such as pH, glucose concentration, and enzyme activity) and undergo changes in physical or chemical properties, thereby triggering precise and on-demand drug release. Compared to traditional formulations, they better meet the needs of personalized treatment for type 2 diabetes mellitus (T2DM). Currently, extensive research has been conducted on bioresponsive formulations for large molecule drugs such as insulin, but these biopharmaceuticals suffer from poor storage stability and easy degradation and inactivation, limiting their clinical application. Research on intelligent delivery systems for small molecule hypoglycemic drugs such as empagliflozin is still in its early stages, particularly lacking novel formulations that combine glucose responsiveness, targeted delivery, and convenient delivery characteristics.

[0005] In the development of glucose-responsive materials, phenylboronic acid (PBA) and its derivatives have attracted widespread attention due to their unique chemical properties. PBA can form reversible borate ester bonds with glucose and other molecules containing diol structures, making it an ideal sensitive group for constructing glucose-responsive drug delivery systems. Compared with other glucose-responsive materials such as glucose oxidase (GOx) and concanavalin A (Con A), PBA has advantages such as high stability, low cytotoxicity, and good biocompatibility, avoiding problems such as tissue damage caused by GOx and immune responses caused by Con A. However, the dissociation constant (pKa) of conventional PBA is approximately 8.8. Under the physiological pH environment of the human body (7.35-7.45), most PBA exists in a non-ionized hydrophobic state, exhibiting weak binding ability to glucose and difficulty in efficiently responding to changes in blood glucose concentration. 4-Carboxy-3-fluorophenylboronic acid (FPBA), as a derivative of PBA, has been shown in studies to have a lower pKa (approximately 3.15 (carboxyl group) and 7.2 (boronic acid group)). It can still maintain good glucose binding activity under physiological pH conditions, significantly improving response sensitivity and providing key material support for the construction of highly efficient glucose-responsive formulations.

[0006] The selection of carrier materials is another crucial step in constructing an efficient drug delivery system. Chitosan (Cs), a natural cationic polysaccharide, has a degradation product, glucosamine, that is non-toxic to cells and exhibits good biocompatibility, biodegradability, and low toxicity, and has been approved by the US FDA for use as a biomaterial. The amino (–NH2) and hydroxyl (–OH) groups in the chitosan molecule provide convenient structural modification, allowing for the introduction of functional groups through chemical modification to impart specific properties. More importantly, chitosan possesses selective renal aggregation characteristics; its amino and glucosamine molecules can bind to macroprotein receptors on the surface of renal tubular epithelial cells, achieving targeted drug delivery to the kidneys. This highly aligns with the renal target characteristics of empagliflozin, providing favorable conditions for increasing drug concentration at the target site and enhancing therapeutic efficacy. Polyvinyl alcohol (PVA), another FDA-approved biocompatible material, has good water solubility and stability. It is often used as a stabilizer in nano-formulations. By enhancing the cross-linking network density of the carrier material, it reduces premature drug leakage and prolongs the drug release cycle. When used in combination with chitosan and FPBA, it can further optimize the physicochemical properties and drug release behavior of the formulation.

[0007] Transdermal drug delivery technology effectively avoids the first-pass metabolism of oral administration and the local irritation of injection, making it a research hotspot in the field of drug delivery. Microneedle (MN) array patches, as an important carrier for transdermal delivery, typically have needle lengths between 600-800 μm. These needles can penetrate the stratum corneum, the main barrier to transdermal drug absorption, without touching the nerves and blood vessels in the dermis, achieving painless and minimally invasive drug delivery. Microneedle patches do not require professional operation and can be used by patients themselves, significantly improving the convenience of drug administration, especially suitable for patients with chronic diseases requiring long-term medication. Currently, microneedle technology has been successfully applied to the transdermal delivery of biological drugs such as insulin and vaccines, and some glucose-responsive insulin microneedle formulations have entered preclinical or clinical research stages. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art, avoid the damage of empagliflozin nanoparticles to the gastrointestinal tract, reduce the accumulation of nanoparticles in the liver and better target the kidneys, and at the same time solve the core problems of insufficient responsiveness, low bioavailability and inconvenient administration of existing empagliflozin dosage forms, and provide a glucose-responsive empagliflozin microneedle delivery system and its application.

[0009] This invention aims to overcome the shortcomings of existing empagliflozin formulations by designing a drug delivery system, enhancing the glucose responsiveness of empagliflozin nanoparticles, achieving on-demand drug release in hyperglycemic states and a slower release rate in normal blood glucose states, thereby effectively avoiding the risk of hypoglycemia. Simultaneously, it addresses the issues of low bioavailability and poor targeting in oral formulations by leveraging the renal targeting properties of the carrier material to improve the enrichment efficiency of empagliflozin nanoparticles at the target site. Furthermore, it provides a minimally invasive and convenient microneedle transdermal drug delivery system to replace traditional oral administration, reducing the incidence of adverse drug reactions and improving long-term patient adherence. This invention develops a novel formulation with excellent stability and good biocompatibility, solving practical problems in the clinical application of empagliflozin and possessing significant potential for widespread application. This invention designs a novel delivery system that combines glucose-responsive empagliflozin nanoparticles with microneedle patches, constructing a novel delivery system that combines targeting, responsiveness, and convenience. The combination of glucose-responsive materials (FPBA), targeting carrier materials (chitosan), and microneedle transdermal delivery technology provides a feasible path for the development of glucose-responsive empagliflozin nanoparticles. This drug delivery system enables on-demand drug release, renal targeting, and minimally invasive and convenient drug administration, which has significant clinical value and application prospects for improving the treatment effect of type 2 diabetes mellitus, enhancing the patient's medication experience, and reducing the risk of complications.

[0010] The technical solution of this invention to solve the technical problem is as follows: This invention provides a glucose-responsive empagliflozin microneedle delivery system. The microneedle delivery system is a microneedle patch, comprising a substrate and a microneedle array disposed on the substrate; the microneedles in the microneedle array contain glucose-responsive empagliflozin nanoparticles CB@E or PCB@E. The CB@E is made by first condensing 4-carboxy-3-fluorophenylboronic acid (FPBA) and chitosan (Cs) into a glucose-responsive polymer matrix CB, and then encapsulating empagliflozin using an iontophoresis method. The PCB@E is prepared by first reacting polyvinyl alcohol (PVA) with succinic anhydride (SA) to prepare carboxylated PVA (cPVA), then crosslinking cPVA with CB through amide bonds to obtain PCB, and finally encapsulating empagliflozin using an ion gel method.

[0011] Furthermore, the preparation method of the glucose-responsive empagliflozin nanoparticles CB@E specifically includes the following steps: 1) Chitosan (Cs) was dissolved in 0.1M HCl and stirred at room temperature until completely dissolved. FPBA, EDC, and NHS were dissolved in a molar ratio of 1:1:1 and stirred continuously at room temperature for 4 hours to prepare an FPBA-NHS solution. Subsequently, the FPBA-NHS solution was added to the chitosan (Cs) solution, the pH was adjusted to 4.5, and the reaction was carried out at room temperature for 24 hours. The molar ratio of Cs(-NH2):FPBA was 1:1. The reaction solution was collected, dialyzed, and the molecular weight cutoff was 3.5 kDa. The solution was then lyophilized to obtain CB. 2) Dissolve CB in 2% acetic acid solution, adjust pH to 4.5, add empagliflozin ethanol solution and stir for 10 min. The mass ratio of empagliflozin to CB is 20 μg: 60 mg. Then add sodium tripolyphosphate (STPP) solution dropwise, stir for 10 min and centrifuge at 12,000 rpm, 4 ℃ for 15 min. Wash the precipitate and freeze dry to obtain glucose-responsive empagliflozin nanoparticles CB@E.

[0012] In the above-described reaction for preparing CB, under the synergistic catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), the carboxyl group (–COOH) of 4-carboxy-3-fluorophenylboronic acid (FPBA) undergoes a condensation reaction with the amino group (–NH2) on the chitosan molecule to form an amide bond. The core role of the EDC / NHS system is to activate the carboxyl group to promote the efficient formation of the amide bond. After dialysis and lyophilization, CB was obtained with an FPBA grafting rate of 46%.

[0013] Furthermore, the preparation method of the glucose-responsive empagliflozin nanoparticles PCB@E is as follows: 1) Polyvinyl alcohol (PVA) powder was added to water, heated to dissolve, cooled and then DMAP (10% w / w succinic anhydride (SA)) was added and stirred for 24 h. The molar ratio of PVA (-OH):SA was 1:1. The reaction solution was collected, dialyzed to a molecular weight cutoff of 3.5 kDa, and freeze-dried after five days to obtain cPVA. 2) Dissolve CB in 0.1M HCl, dissolve cPVA, EDC and NHS, and activate at room temperature for 4 hours. The molar ratio of cPVA(-COOH):EDC:NHS is 1:1:1. Add this solution to the CB solution and react at room temperature for 24 hours. Then collect the reaction solution and dialyze it. The molecular weight cutoff is 3.5kDa. After five days, freeze-dry to obtain PCB. The CB preparation method is the same as the CB preparation method in the CB@E preparation method. 3) Dissolve PCB in 2% acetic acid solution, adjust pH to 4.5, add empagliflozin ethanol solution and stir for 10 min. The mass ratio of empagliflozin to PCB is 20 μg: 60 mg. Then add sodium tripolyphosphate (STPP) solution dropwise, stir for 10 min and centrifuge at 12,000 rpm, 4 ℃ for 15 min. Wash the precipitate and freeze dry to obtain glucose-responsive empagliflozin nanoparticles PCB@E.

[0014] Furthermore, the CB@E has a particle size of 126 nm, a zeta potential of +25 mV, an encapsulation efficiency (EE%) of 38%, and a loading efficiency (LE%) of 17%. All nanoparticles have a spherical structure and exhibit good stability after 30 days of storage at 4°C.

[0015] Furthermore, the PCB@E has the following characteristics: particle size 122nm, zeta potential +21mV, EE% 21%, LE% 9%. All nanoparticles are spherical and exhibit good stability after 30 days of storage at 4°C.

[0016] Furthermore, the microneedle patch is prepared using photocuring technology.

[0017] Further, the microneedle patch preparation method is as follows: CB@E or PCB@E nanoparticles are dispersed in an N-vinylpyrrolidone (NVP) solution containing 0.5 mol% ethylene glycol dimethacrylate (EGDMA) and 1 mol% photoinitiator Irgacure 2959. The solution is then cast into a PDMS mold. After centrifugation at 3500 rpm for 10 min to remove air bubbles, the mold is cured with 365 nm UV light for 20 min. NOA-86H is added as a substrate and UV cured for another 10 min. The microneedle patch is then demolded. Each patch has a drug loading capacity of approximately 346 ± 26 μg, enabling minimally invasive drug delivery by penetrating the stratum corneum.

[0018] Furthermore, the PDMS mold is a 20×20 array with a needle length of 1000μm, a needle base of 410μm×410μm, and a needle spacing of 750μm.

[0019] In this invention, a microneedle punctures the skin and rapidly swells upon contact with tissue fluid, releasing glucose-responsive empagliflozin nanoparticles. These nanoparticles then enter subcutaneous blood vessels, where, under hyperglycemic conditions (400 mg / dL), glucose and empagliflozin compete for FPBA binding, promoting empagliflozin release. At normal blood glucose levels (100 mg / dL), this competition weakens, and the release rate slows, achieving on-demand drug release.

[0020] The present invention also provides the application of the glucose-responsive empagliflozin microneedle delivery system described herein in the preparation of medicaments for treating type 2 diabetes.

[0021] The improvements of this invention are as follows: 1. The glucose-responsive nanoparticles of the present invention have a relatively regular spherical appearance, effectively load empagliflozin in a reversible covalent manner, and have good stability during storage.

[0022] 2. The glucose-responsive empagliflozin nanoparticles of this invention exhibit excellent glucose responsiveness, enabling differential drug release in high-glucose and low-glucose solutions. The glucose responsiveness is based on a glucose-responsive polymer matrix. During nanoparticle formation, the functional group FPBA of this polymer binds to empagliflozin via reversible borate bonds. In a hyperglycemic state (400 mg / dL), glucose and empagliflozin competitively bind to FPBA, promoting empagliflozin release. In a normal blood glucose state (100 mg / dL), the competition weakens, the release rate slows, and on-demand drug release is achieved.

[0023] 3. The intelligent transdermal drug delivery system of this invention was constructed using microneedles encapsulated with glucose-responsive empagliflozin nanoparticles. The drug-loaded microneedles exhibit good puncture resistance and drug release behavior, with a single patch loading of approximately 346±26 μg.

[0024] 4. The intelligent transdermal drug delivery system described in this invention can achieve renal targeting and good glycemic control in vivo. Microneedles puncture the skin and rapidly swell upon contact with tissue fluid, releasing glucose-responsive empagliflozin nanoparticles. These nanoparticles then enter subcutaneous blood vessels and, due to the renal targeting properties of chitosan, accumulate in the kidney region. Under hyperglycemic conditions, empagliflozin is released in a responsive manner, achieving long-term regulation of blood glucose.

[0025] 5. The intelligent transdermal drug delivery system described in this invention exhibits good biocompatibility. The pharmaceutical excipients used to prepare this invention have been approved by the FDA and have demonstrated good safety in in vivo experiments.

[0026] The present invention has the following technical effects: 1. The empagliflozin nanoparticles described in this invention possess precise glucose-responsive drug release performance. In vitro experiments have demonstrated that under a hyperglycemic environment of 400 mg / dL, the drug release of CB@E and PCB@E is significantly higher than that under normal blood glucose and glucose-free environments. Among them, CB@E has a greater cumulative drug release within 24 hours and exhibits pulsatile drug release characteristics, which can simulate the glycemic regulation function of pancreatic β cells; 2. The microneedle drug delivery system described in this invention has a long-term blood glucose control effect. In a T2DM rat model, MN@CB@E exhibits a long-acting blood glucose control effect, reducing blood glucose to normal levels within 4 hours and maintaining it for 24 hours. The duration of glucose reduction is significantly better than that of subcutaneous injection of free empagliflozin (which only lasts for 6 hours), and there is no risk of hypoglycemia after administration to healthy rats.

[0027] 3. The empagliflozin nanoparticles described in this invention can be effectively concentrated in the kidneys, improving drug utilization efficiency.

[0028] 4. The glucose-responsive empagliflozin nanoparticle microneedle drug delivery system described in this invention exhibits good biocompatibility and supports long-term administration. H&E staining results showed no tissue damage in the heart, liver, spleen, lungs, and kidneys of rats after administration, demonstrating good biocompatibility. Attached Figure Description

[0029] Figure 1 Infrared spectra of glucose-responsive polymer matrices CB and PCB, and particle sizes of empagliflozin nanoparticles CB@E and PCB@E prepared based thereon. Figure 1(a) is the Fourier transform infrared (FTIR) characterization of FPBA-functionalized chitosan (CB). Figure 1 (b) is the Fourier transform infrared (FTIR) spectrum characterization of the crosslinked product (PCB) of CB and carboxylated PVA. Figure 1 (c) shows the particle size distribution and transmission electron microscopy (TEM) morphology characterization of CB@E nanoparticles. Figure 1 (d) shows the particle size distribution and transmission electron microscopy (TEM) morphology characterization of PCB@E nanoparticles.

[0030] Figure 2 Characterization of the skin penetration properties of MN array patches. Figure 2 The image shows an in vivo drug delivery process using microneedle patches applied to the skin on the back of SD rats. Figure 2 Images A-II are bright-field micrographs of trypan blue stained skin surface on rats after microneedle patches were applied. Figure 2 Images A-III are scanning electron microscope (SEM) characterization images of the microneedle patch. Figure 2 Figure A-IV is a high-magnification scanning electron microscope (SEM) image of a single microneedle marked with a yellow box in Figure A-III. Figure 2 BI refers to hematoxylin-eosin (H&E) stained sections of normal rat skin (blank control group) without microneedle application. Figure 2 B-II is an H&E stained section of rat skin after the microneedle patch of the present invention was applied.

[0031] Figure 3 The net amount of engorgement effectively loaded onto the MN array patch.

[0032] Figure 4 pKa is for FPBA.

[0033] Figure 5 Comparison of cumulative net empagliflozin release between CB@E and PCB@E, and comparison of cumulative net empagliflozin release between MN@CB@E and MN@PCB@E. Figure 5 (a) shows the cumulative drug release curves of free CB@E and PCB@E nanoparticles in a high-sugar environment. Figure 5 (b) shows the cumulative drug release curves of microneedle-loaded CB@E and PCB@E nanoparticles in a high-sugar environment.

[0034] Figure 6 In vivo study of MN array patches for the treatment of diabetic rat models.

[0035] Figure 7 Histological evaluation of major organs stained with H&E. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0037] The sources of the experimental materials involved in this invention are described below:

[0038]

[0039]

[0040]

[0041] Example 1: Synthesis of glucose-responsive polymer matrix and preparation of empagliflozin nanoparticles I. Synthesis of FPBA-functionalized chitosan (CB) 1 g of chitosan (Cs) was dissolved in 20 ml of 0.1 M HCl and stirred at room temperature until completely dissolved. FPBA, EDC, and NHS were dissolved in 10 ml of DSMO at a molar ratio of 1:1:1, and then stirred continuously at room temperature for 4 hours to prepare an FPBA-NHS solution. Subsequently, the FPBA-NHS ester solution was added to the Cs solution, and the pH was adjusted to 4.5 with 0.1 M NaOH. The reaction was carried out at room temperature for 24 hours, with a Cs(-NH2):FPBA molar ratio of 1:1. The reaction solution was then collected, dialyzed (molecular weight cutoff 3.5 kDa), and freeze-dried for five days to obtain CB.

[0042] II. Synthesis of PVA-Chitosan FPBA (PCB) 1 g of PVA powder was added to 20 mL of deionized water and heated to 90°C with constant stirring until a clear solution was obtained. The solution was cooled to 65°C, DMAP (10% w / w succinic anhydride (SA)) was added, and the mixture was stirred for 24 h. The molar ratio of PVA(-OH):SA was 1:1. The reaction solution was then collected, dialyzed (molecular weight cutoff 3.5 kDa), and freeze-dried for five days to obtain cPVA.

[0043] 200 mg of CB was dissolved in 5 ml of 0.1 M HCl and stirred at room temperature until completely dissolved. cPVA, EDC, and NHS were dissolved in 15 ml of DMSO and activated at room temperature for 4 hours, with a cPVA(-COOH):EDC:NHS molar ratio of 1:1:1. This solution was added to the CB solution and reacted at room temperature for 24 hours. The reaction solution was then collected, dialyzed (molecular weight cutoff 3.5 kDa), and freeze-dried after five days to obtain PCB.

[0044] III. Preparation of glucose-responsive empagliflozin nanoparticles 60 mg of CB was dissolved in 10 ml of 2% acetic acid, and the pH was adjusted to 4.5. Distilled water was added to bring the final volume to 3 mg / ml. Then, 1 ml of empagliflozin in ethanol (20 μg / ml) was added and stirred for 10 minutes. Following this, sodium tripolyphosphate (STPP) solution (0.5 mg / ml) was added dropwise while stirring. The CB:STPP volume ratio was approximately 1:0.5. The reaction mixture was then collected and centrifuged at 12,000 rpm at 4°C for 15 minutes. The solution was washed with ethanol and water, and then with water, respectively. The final CB@E was stored at -20°C. PCB@E was prepared using the same method and stored at -20°C.

[0045] Infrared results showed characteristic peaks of amide bonds, indicating successful synthesis of CB, with an FPBA grafting rate of 46%. The infrared results also showed a shift in the characteristic peaks of amide bonds, with the appearance of carbonyl peaks characteristic of PVA, suggesting successful synthesis of PCB. Figure 1 The prepared nanoparticles have a regular appearance; the CB@E particle size is approximately 126 nm; the PCB@E particle size is approximately 122 nm. Figure 1 ). Figure 1 (a) is the Fourier transform infrared spectrum, verifying that FPBA was successfully grafted onto chitosan via amide bonds, thus synthesizing the target polymer CB; 1578 cm⁻¹ -1 1677cm -1 The novel characteristic peak at 1642 cm⁻¹ is a characteristic absorption peak of the amide bond, providing direct evidence of the successful synthesis of CB. Figure 1(b) shows the Fourier transform infrared spectrum, verifying the successful crosslinking of CB and carboxylated PVA through amide bonds, thus synthesizing the target polymer PCB; -1 The novel amide bond characteristic peak at the TEM image is direct evidence of the successful synthesis of PCB. Figure 1(c) shows the particle size distribution and TEM morphology of CB@E nanoparticles. It can be seen that the particle size of CB@E nanoparticles is concentrated in the range of 100-150 nm, with uniform size, good dispersion, and regular spherical shape without obvious agglomeration. Figure 1(d) shows the particle size distribution and TEM morphology of PCB@E nanoparticles. It can be seen that the particle size of PCB@E nanoparticles is concentrated in the range of 100-200 nm, with uniform size, spherical shape, and uniform dispersion without obvious agglomeration.

[0046] Example 2: Microneedle Patch Preparation and Skin Penetration Experiment I. Preparation of microneedle array patches loaded with empagliflozin nanoparticles CB@E or PCB@E nanoparticles were dispersed in an N-vinylpyrrolidone solution containing EGDMA (0.5 mol%) and Irgacure 2959 (1 mol%). After uniform ultrasonic dispersion, the nanoparticles were cast into a PDMS mold (20×20 array, needle length 1000 μm, needle base 410 μm × 410 μm, needle spacing 750 μm). The mold was centrifuged at 3500 rpm for 10 minutes to remove air bubbles, and after removing excess solution, it was cured with 365 nm UV light for 20 minutes. NOA-86H was added as a backing, and the mold was cured again with UV light for 10 minutes. After demolding, the nanoparticles were stored in a cool, dry place.

[0047] The microneedle patch loaded with CB@E nanoparticles is MN@CB@E.

[0048] The microneedle patch loaded with PCB@E nanoparticles is MN@PCB@E.

[0049] II. Verification of the skin penetration ability of microneedle patches Skin from the back of euthanized rats was shaved, cleaned with saline, dried, and fixed onto a paraffin block. A microneedle patch was pressed vertically at 90° to the skin for 3 minutes, then removed. Immediately afterward, the skin was stained with 1 mg / ml trypan blue for 10 minutes, and residual dye was washed away with ethanol. The morphology of the skin micropores was observed. Simultaneously, H&E-stained sections of the skin were prepared, and penetration traces were observed under a microscope. Results showed clear micropores on the skin surface, and microneedle penetration channels were visible in the sections, confirming good penetration ability. SEM showed that the microneedles remained structurally intact and non-soluble 72 hours after application (Figure 2). Figure 2 The AI ​​analysis shows that microneedle patches can be conveniently applied to rat skin for non-invasive transdermal drug delivery, demonstrating feasibility for in vivo drug delivery and providing support for clinical home-based self-administration. Figure 2 As can be seen from A-II, a regular and uniform array of micropores was formed on the skin surface, directly proving that the microneedles can effectively penetrate the stratum corneum of the skin, forming a stable transdermal drug delivery channel and possessing excellent skin penetration ability. Figure 2 As can be seen from A-III and A-IV, the microneedles are arranged in a regular pyramidal array, with a uniform structure, sharp tips, good formability, and no defects or deformation, meeting the structural design requirements for transdermal drug delivery and ensuring stable skin penetration. Figure 2 The BI results show that the stratum corneum, epidermis, and dermis of the rat skin are intact and continuous, with clear layers, no damage, and no inflammatory cell infiltration, providing a negative control benchmark for the drug-treated group. Figure 2As can be seen from B-II, the microneedles successfully penetrated the stratum corneum and epidermis of the skin, reaching the dermis to form a drug delivery channel; at the same time, the overall skin structure showed no serious tearing, obvious inflammation or necrosis, which not only verified the effective penetration depth of the microneedles, but also proved their good skin safety and biocompatibility.

[0050] III. Determination of Drug Loading Capacity of Microneedle Patches Five MN@CB@E patches were immersed in 1 ml of 0.5% Tween 80:ethanol (1:1) solution and shaken at 37℃ and 100 rpm for 72 h until the microneedle tips disintegrated. The solution was filtered through a 0.45 μm filter membrane, and the drug concentration was determined by HPLC to calculate the drug loading. The results showed that the drug loading per patch was 346±26 μg, meeting the therapeutic dose requirement (Figure 3).

[0051] Example 3: In vitro glucose-responsive drug release experiment I. FPBA pKa Determination A DSMO stock solution of 2 mg / ml FPBA was prepared and diluted to a working solution of 0.02 mg / ml. The solutions were then added to buffers of different pH values ​​(pH 2-12). The spectrum from 200 to 400 nm was scanned using a UV spectrophotometer, and 286 nm was determined to be the wavelength of maximum absorption. A plot was drawn with absorbance on the ordinate and pH on the abscissa. The pKa of FPBA was determined to be 5.0 and 8.0 using the inflection point method (Figure 4).

[0052] II. Glucose-responsive drug release from nanoparticles Equal amounts of CB@E and PCB@E (converted to LE%) prepared in Example 1 were weighed and dispersed in 1 ml of 0.5% Tween 80 release medium. These were then placed in dialysis bags (molecular weight cutoff 3.5 kDa) and in 20 ml of release medium containing 0, 100, and 400 mg / dL glucose, and incubated at 37°C with constant shaking. 1 ml samples were taken at 0, 0.5, 2, 4, 6, and 24 hours, and an equal volume of fresh medium was added. After centrifugation, the drug concentration was determined by HPLC. The results showed that under a hyperglycemic environment of 400 mg / dL, the release amounts of CB@E and PCB@E were significantly higher than those in the normal blood glucose group, and CB@E exhibited superior glucose responsiveness (Figure 5). Figure 5 As shown in (a), both nanoparticles exhibit sustained drug release capabilities under high glucose conditions, with a cumulative drug release of approximately 83 μg for CB@E and approximately 50 μg for PCB@E over 24 hours, perfectly matching the difference in their drug loading efficiency. The high glucose-responsive drug release efficiency of CB@E is significantly superior to that of PCB@E, validating the glucose-responsive drug release function of both carriers. Figure 5As shown in (b), after the nanoparticles are embedded in the microneedles, they still maintain excellent high glucose response drug release capabilities, and the drug release trend is completely consistent with that of the free nanoparticles. The cumulative drug release amount of MN@CB@E in 24 hours is about 38 μg and that of MN@PCB@E is about 21 μg. The difference in drug release between the two is statistically significant. This proves that the microneedle matrix does not interfere with the glucose response characteristics of the nanoparticles and verifies the feasibility of the microneedle delivery system.

[0053] III. Glucose-responsive drug release via microneedle-loaded nanoparticles MN@CB@E and MN@PCB@E patches were placed in dialysis bags (molecular weight cutoff 3.5 kDa) and then placed in 10 ml of release medium containing different glucose concentrations, and incubated at 37°C with constant temperature shaking. 0.5 ml samples were taken at the above time points, and fresh medium was added. Drug concentration was determined by HPLC. The results were consistent with the trend of direct drug release from nanoparticles, confirming that microneedle delivery does not affect glucose responsiveness (Figure 5).

[0054] Example 4: In vivo blood glucose control experiment I. Establishment of a T2DM rat model Male SD rats aged 6-8 weeks (weighing 150-200g) were selected and, after 3 days of acclimatization, randomly divided into a healthy group, a T2DM control group, and a T2DM experimental group. After fasting for 8 hours, rats in the experimental group were injected intraperitoneally with nicotinamide (230mg / kg), followed by an injection of STZ (65mg / kg) 15 minutes later. Blood glucose levels were measured in the tail vein on days 5 and 10 after modeling; a blood glucose level >200mg / dl was considered a successful model establishment.

[0055] II. In vivo blood glucose control experiment T2DM rats that successfully developed the model were divided into three groups (n=5): a blank microneedle group, an MN@CB@E group, and a subcutaneous injection group of free empagliflozin (Sc.Emp); an additional healthy rat + MN@CB@E group (n=5) was also included. After fasting overnight, the blank group received microneedles without the drug, the MN@CB@E group received microneedles loaded with nanoparticles, and the Sc.Emp group received a subcutaneous injection of 0.35 mg / rat. Blood glucose levels were measured in the tail vein at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 18, 24, and 72 hours after drug administration. The results showed that blood glucose levels in the MN@CB@E group decreased to normal within 4 hours and remained at that level for 24 hours, with no risk of hypoglycemia; while in the Sc.Emp group, the level only remained normal for 6 hours (Figure 6).

[0056] Example 5: Biocompatibility Evaluation I. Grouping and Administration of Experimental Animals Healthy rats, T2DM control rats, and T2DM+MN@CB@E treatment group rats were selected. After administration, they were continuously observed for 72 hours, and their body weight changes and behavioral status were recorded. Thirty days after administration, the rats were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were quickly removed.

[0057] II. Pathological examination of major organs Organs were fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with H&E. The tissue structure, cell morphology, and distribution were then observed under a light microscope. The results showed that the organ tissue structure of the three groups of rats was intact, the cell morphology was normal, and there were no abnormalities such as inflammatory infiltration or cell necrosis, confirming the good biocompatibility of the formulation (Figure 7). The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A glucose-responsive empagliflozin microneedle delivery system, characterized in that, The microneedle delivery system is a microneedle patch, comprising a substrate and a microneedle array disposed on the substrate; the microneedles in the microneedle array contain glucose-responsive empagliflozin nanoparticles CB@E or PCB@E; The CB@E is made by first condensing 4-carboxy-3-fluorophenylboronic acid FPBA with chitosan Cs into a glucose-responsive polymer matrix CB, and then encapsulating empagliflozin using an iontophoresis method. The PCB@E is prepared by first reacting polyvinyl alcohol (PVA) with succinic anhydride (SA) to prepare carboxylated PVA, i.e., cPVA, then crosslinking cPVA with CB via amide bonds to obtain PCB, and finally encapsulating empagliflozin using an iontophoresis method.

2. The glucose-responsive empagliflozin microneedle delivery system according to claim 1, characterized in that, The preparation method of the glucose-responsive empagliflozin nanoparticles CB@E specifically includes the following steps: 1) Chitosan (Cs) was dissolved in 0.1M HCl and stirred at room temperature until completely dissolved. FPBA, EDC, and NHS were dissolved in a molar ratio of 1:1:1 and stirred continuously at room temperature for 4 hours to prepare an FPBA-NHS solution. Subsequently, the FPBA-NHS solution was added to the chitosan (Cs) solution, the pH was adjusted to 4.5, and the reaction was carried out at room temperature for 24 hours. The molar ratio of Cs(-NH2):FPBA was 1:

1. The reaction solution was collected, dialyzed, and the molecular weight cutoff was 3.5 kDa. The solution was then lyophilized to obtain CB. 2) Dissolve CB in 2% acetic acid solution, adjust pH to 4.5, add empagliflozin ethanol solution and stir for 10 min. The mass ratio of empagliflozin to CB is 20 μg: 60 mg. Then add sodium tripolyphosphate (STPP) solution dropwise, stir for 10 min and centrifuge at 12,000 rpm, 4 ℃ for 15 min. Wash the precipitate and freeze dry to obtain glucose-responsive empagliflozin nanoparticles CB@E.

3. The glucose-responsive empagliflozin microneedle delivery system according to claim 1, characterized in that, The preparation method of the glucose-responsive empagliflozin nanoparticles PCB@E is as follows: 1) Polyvinyl alcohol (PVA) powder was added to water, heated to dissolve, cooled and then DMAP (10% w / w succinic anhydride (SA)) was added and stirred for 24 h. The molar ratio of PVA (-OH):SA was 1:

1. The reaction solution was collected, dialyzed to a molecular weight cutoff of 3.5 kDa, and freeze-dried after five days to obtain cPVA. 2) Dissolve CB in 0.1M HCl, dissolve cPVA, EDC and NHS, and activate at room temperature for 4 hours. The molar ratio of cPVA(-COOH):EDC:NHS is 1:1:

1. Add this solution to the CB solution and react at room temperature for 24 hours. Then collect the reaction solution and dialyze it. The molecular weight cutoff is 3.5kDa. After five days, freeze-dry to obtain PCB. The CB preparation method is the same as the CB preparation method described in claim 2. 3) Dissolve PCB in 2% acetic acid solution, adjust pH to 4.5, add empagliflozin ethanol solution and stir for 10 min. The mass ratio of empagliflozin to PCB is 20 μg: 60 mg. Then add sodium tripolyphosphate (STPP) solution dropwise, stir for 10 min and centrifuge at 12,000 rpm, 4 ℃ for 15 min. Wash the precipitate and freeze dry to obtain glucose-responsive empagliflozin nanoparticles PCB@E.

4. The glucose-responsive empagliflozin microneedle delivery system according to claim 2, characterized in that, The CB@E has a particle size of 126nm, a zeta potential of +25mV, a packaging efficiency (EE%) of 38%, and a loading efficiency (LE%) of 17%.

5. The glucose-responsive empagliflozin microneedle delivery system according to claim 3, characterized in that, The PCB@E has a particle size of 122nm, a zeta potential of +21mV, an EE% of 21%, and an LE% of 9%.

6. The glucose-responsive empagliflozin microneedle delivery system according to claim 1, characterized in that, The microneedle patch is prepared using photocuring technology.

7. The glucose-responsive empagliflozin microneedle delivery system according to claim 6, characterized in that, The microneedle patch preparation method is as follows: CB@E or PCB@E nanoparticles are dispersed in an N-vinylpyrrolidone (NVP) solution containing 0.5 mol% ethylene glycol dimethacrylate (EGDMA) and 1 mol% photoinitiator Irgacure 2959. The solution is then cast into a PDMS mold. After centrifugation at 3500 rpm for 10 min to remove air bubbles, the mold is cured with 365 nm UV light for 20 min. NOA-86H is added as a substrate and UV cured for 10 min. The microneedle patch is then demolded.

8. The glucose-responsive empagliflozin microneedle delivery system according to claim 7, characterized in that, The PDMS mold is a 20×20 array with a needle length of 1000μm, a needle base of 410μm×410μm, and a needle spacing of 750μm.

9. The use of the glucose-responsive empagliflozin microneedle delivery system as described in any one of claims 1-8 in the preparation of a medicament for treating type 2 diabetes.

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