A glucose-responsive insulin delivery system and method of preparation and use thereof

Abstract

The application discloses a glucose-responsive insulin delivery system and a preparation method and application thereof, and belongs to the technical field of biomaterials. The glucose-responsive insulin delivery system provided by the application comprises a delivery carrier and a bioactive component encapsulated in the carrier; the delivery carrier is a metal polyphenol network formed by a coordination reaction between epigallocatechin gallate and metal ions; and the bioactive component comprises glucose oxidase, catalase and insulin. The glucose-responsive insulin delivery system can adjust the release of insulin according to the concentration of glucose in the surrounding environment, so that the blood glucose can be maintained at a normal level.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a glucose-responsive insulin delivery system and a preparation method and application thereof. BACKGROUND

[0002] Diabetes is a chronic metabolic disease, mainly manifested as insufficient insulin secretion or insulin resistance. The blood glucose level of a diabetic patient is often higher than that of a normal person, and the clinical manifestations are polydipsia, polyphagia, polyuria and body wasting. If the blood glucose of a diabetic patient is in an uncontrolled state for a long time, it will cause serious damage to many organs of the body, especially the nerves and blood vessels. According to the data of the International Diabetes Federation in 2024, about 540 million adults worldwide suffer from diabetes, and it is estimated that this number will reach 783 million by 2045. Therefore, it is very urgent to improve the treatment level of diabetes.

[0003] Clinically, diabetes can be treated by directly injecting insulin or insulin-like protein hormones to promote the metabolism of glucose in liver, adipose tissue and skeletal muscle cells, thereby reducing blood glucose level. However, insulin therapy is a heavy burden because it requires multiple subcutaneous injections every day, which reduces the compliance of diabetic patients, and excessive or insufficient injection will cause irreversible serious consequences. Therefore, there is an urgent need for a new insulin administration strategy that can release insulin in response to blood glucose level to effectively regulate blood glucose.

[0004] In healthy individuals, endogenous insulin secreted by pancreatic beta cells has a high response to blood glucose level. Delivering insulin by injecting glucose-responsive carriers can mimic the function of human pancreas to accurately adjust blood glucose level in real time, achieving safe individualized treatment of normal blood glucose. So far, a variety of glucose-responsive carriers have been developed, including glucose-binding protein-based, phenylboronic acid-based and glucose oxidase-based responsive carriers. Among these responsive carriers, glucose oxidase-based responsive carriers are of great concern due to their ultra-high specificity for glucose and high sensitivity to blood glucose level.

[0005] Under physiological conditions, glucose-responsive carriers will face multiple obstacles after entering the body, such as buffering of body fluids, degradation by proteases and immune cells responsible for foreign matter clearance. In addition, as proteins, the secondary structure of glucose oxidase and insulin is crucial for maintaining their biological activity, but the secondary structure of glucose oxidase and insulin is very sensitive to changes in the microenvironment during delivery. Therefore, to be able to release insulin in response to blood glucose level to regulate blood glucose level under such severe conditions, there are still technical obstacles.

[0006] Therefore, in order to solve the above technical problems, a new technical solution is needed to solve this technical problem, especially a glucose-responsive insulin delivery system. SUMMARY

[0007] The purpose of the present application is to solve the technical problem of how to release insulin in response to blood glucose levels to reduce blood glucose levels, and to provide a glucose-responsive insulin delivery system and its preparation method and application to effectively regulate blood glucose.

[0008] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0009] The present application provides a glucose-responsive insulin delivery system, mainly composed of a delivery carrier and a bioactive component encapsulated in the carrier, the delivery carrier and the bioactive component self-assemble to form a nanoparticle structure; the delivery carrier is a metal polyphenol network with pH response characteristics formed by the coordination reaction of epigallocatechin gallate and metal ions; the bioactive component is glucose oxidase, catalase and insulin. The encapsulation in the present application is not a complete physical relationship, but a mixture of bioactive components and delivery carriers through hydrogen bonds, hydrophobic interaction forces, coordination bonds and other forces.

[0010] Preferably, the mass ratio of glucose oxidase, catalase and insulin is (0.1-1.3):(0.1-1.3):(0.1-1.3), to achieve a higher encapsulation rate of the three active components by the metal polyphenol network.

[0011] Preferably, the metal ions and epigallocatechin gallate directly self-assemble into metal polyphenol nanoparticles in phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS), 3-(N-morpholino) propane sulfonic acid buffer (MOPS) or Bis(2-hydroxyethylamino) tris(hydroxymethyl) methane buffer (Bis-Tris).

[0012] Preferably, the metal ions and epigallocatechin gallate directly self-assemble into metal polyphenol nanoparticles in the phosphate buffered saline (PBS), and the concentration of the phosphate buffered saline (PBS) used is 10 mM / L. The interaction between the metal ions and the PBS buffer can promote nucleation and form metal polyphenol nanoparticles. The concentration of the phosphate buffered saline (PBS) is 10 mM / L to promote the formation of a larger number of inner cores.

[0013] Preferably, the metal ions are one or more of zinc ions, iron ions or aluminum ions, the compound corresponding to the zinc ions is zinc nitrate, the compound corresponding to the iron ions is ferric chloride, and the compound corresponding to the aluminum ions is aluminum chloride.

[0014] Preferably, the metal ion is iron ion, and the compound corresponding to the iron ion is ferric chloride hexahydrate.

[0015] The application also provides a preparation method of the glucose-responsive insulin delivery system, comprising the delivery system described above, and specifically comprising the following steps:

[0016] S1. Preparation of dissolved solutions of each component

[0017] S11. Weigh epigallocatechin gallate, glucose oxidase, catalase, and insulin, and the mass ratio of each component is 5:(0.1-1.3):(0.1-1.3):(0.1-1.3);

[0018] S12. Weigh the metal salt, and the mass ratio of the metal salt to epigallocatechin gallate is 1:(1-5);

[0019] S13. Dissolve the above-weighed glucose oxidase, epigallocatechin gallate, and metal salt in distilled water, respectively; dissolve the catalase in a potassium phosphate buffer, and dissolve the insulin in a NaHCO3 solution, to obtain each component in the form of a dissolved solution;

[0020] S2. Preparation of delivery system nanoparticles

[0021] S21. Under stirring conditions, sequentially add the dissolved solutions of glucose oxidase, catalase, insulin, epigallocatechin gallate, and metal salt in a reagent bottle containing a phosphate buffer to obtain a mixed solution; continue stirring the mixed solution for 30 min, to prepare metal polyphenol nanoparticles encapsulating glucose oxidase, catalase, and insulin;

[0022] S3. Obtain delivery system nanoparticles

[0023] S31. Centrifuge the mixed solution after stirring to obtain a bottom precipitate;

[0024] S32. Wash the bottom precipitate with distilled water at least three times to obtain nanoparticles;

[0025] S4. Store the delivery system nanoparticles

[0026] Disperse the obtained nanoparticles in a phosphate buffer for subsequent use, to obtain nanoparticles with a desired particle size.

[0027] Preferably, the concentration of the potassium phosphate buffer in step S13 is 50 mM / L, and the pH is 7.0; the concentration of NaHCO3 is 0.1 M / L, and the pH is 8.5.

[0028] The present invention also provides a drug for treating diabetes, comprising the glucose-responsive insulin delivery system nanoparticles prepared above.

[0029] The beneficial effects of this invention are as follows:

[0030] The delivery system provided by this invention mainly consists of a delivery carrier and nanoparticles formed by the self-assembly of bioactive components encapsulated within the carrier. The delivery carrier is a pH-responsive metal polyphenol network formed by the coordination reaction of epigallocatechin gallate and metal ions. The bioactive components are glucose oxidase, catalase, and insulin. This glucose-responsive insulin delivery system, using a metal polyphenol network as a delivery carrier to encapsulate glucose oxidase, catalase, and insulin, not only achieves a high loading of insulin in the bioactive components but also maintains the bioactivity of glucose oxidase, catalase, and insulin during encapsulation and delivery, thereby enabling a rapid response to glucose.

[0031] The delivery carrier and bioactive components are mixed together through hydrogen bonds, hydrophobic interactions, and coordination bonds, rather than being completely encapsulated. Each component can contact glucose but does not immediately dissolve due to these interactions. When the glucose concentration is too high, glucose oxidase oxidizes glucose to gluconic acid. As the gluconic acid content increases, the pH value decreases. Due to the pH-responsive characteristics of the metallopolyphenol network, the network dissociates, gradually opening up the entire delivery system to achieve rapid insulin release at high blood glucose levels.

[0032] At normal blood glucose levels, glucose oxidase oxidizes glucose to produce less gluconic acid, and the metallopolyphenol network can stably encapsulate insulin, allowing for slow insulin release and avoiding the risk of hypoglycemia. Simultaneously, it prolongs the duration of insulin's action in the body, reducing injection frequency and greatly alleviating the discomfort of multiple daily injections for diabetic patients. Data from the embodiments show that the glucose-responsive insulin delivery system provided by this invention can maintain normal blood glucose levels for more than 24 hours after a single subcutaneous injection, and can be applied to the treatment of type 1 diabetes (T1D) with glucose-mediated transdermal insulin delivery, showing promise for widespread application in diabetes treatment.

[0033] The present invention further adds catalase to the bioactive components, which can further decompose the hydrogen peroxide (H2O2) produced by glucose oxidase into H2O and O2, thereby effectively avoiding inflammatory damage at the injection site.

[0034] In addition, the present invention also has the following technical effects:

[0035] (1) This invention innovatively prepares a glucose-responsive insulin delivery system using a simple and rapid one-step co-deposition method. This delivery system is a nanoparticle formed by the coordination reaction of epigallocatechin gallate and metal ions, simultaneously encapsulating the bioactive components glucose oxidase, catalase, and insulin through co-assembly. The preparation method is simple, easy to operate, rapid, and mild, making it suitable for industrial-scale production and application.

[0036] (2) The glucose-responsive insulin delivery system provided by the present invention does not affect the secondary structure of the encapsulated proteins (insulin, glucose oxidase and catalase), thereby helping to maintain the biological activity of each protein.

[0037] (3) The glucose-responsive insulin delivery system provided by this invention exhibits significant responsiveness to blood glucose levels. Specifically, when glucose concentration is too high, glucose oxidase oxidizes glucose to gluconic acid. As the gluconic acid content increases, the pH value decreases, causing the pH-responsive metal polyphenol network to dissociate, thus enabling rapid insulin release at high blood glucose levels. At normal blood glucose levels, the metal polyphenol network stably encapsulates insulin, resulting in slow insulin release and avoiding the risk of hypoglycemia. Experimental data show that this delivery system can maintain normal blood glucose levels for more than 24 hours after a single subcutaneous injection.

[0038] (4) The glucose-responsive insulin delivery system provided by this invention has a longer retention time in vivo, thereby prolonging the duration of insulin action and reducing the frequency of injections, greatly alleviating the suffering of diabetic patients who have to inject multiple times a day. This invention can provide new ideas and possibilities for the preparation and application of simple and efficient glucose-responsive insulin delivery systems. Attached Figure Description

[0039] Figure 1 This is a flowchart of the preparation of a glucose-responsive insulin delivery system and its effects provided in Embodiment 1 of the present invention.

[0040] Figure 2 These are particle size distribution diagrams of the products prepared in Examples 1 and 4, and Comparative Examples 1 and 2 of this invention, determined by dynamic light scattering method.

[0041] Figure 3 This is to maintain the secondary structure of each bioactive component in the MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase and insulin) nanoparticles prepared in Example 1.

[0042] Figure 4 The pH response curve is shown for the MPN@GCI nanoparticles prepared in Example 1.

[0043] Figure 5 This is the blood glucose response curve of the MPN@GCI nanoparticles prepared in Example 1.

[0044] Figure 6 This is the insulin release curve of the MPN@GCI nanoparticles prepared in Example 1 when alternating between hyperglycemia and normal blood glucose every hour.

[0045] Figure 7 This is a comparison diagram of the in vivo retention of MPN@GCI nanoparticles prepared in Example 1 and free insulin.

[0046] Figure 8 This is a comparison curve of the in vivo retention of MPN@GCI nanoparticles prepared in Example 1 and free insulin.

[0047] Figure 9 This is a graph showing the hypoglycemic effect of the MPN@GCI nanoparticles prepared in Example 1 in a diabetic animal model.

[0048] Figure 10 This is a comparison graph of the blood glucose change curves over time in healthy animal models prepared in Example 1 and Comparative Example 2.

[0049] Figure 11 This is a comparison chart of the hypoglycemia index of the products prepared in Example 1 and Comparative Example 2 in healthy animal models.

[0050] Figure 12 The images show a comparison of the injection sites and tissue sections of mice after subcutaneous injection of MPN@GCI nanoparticles prepared in Example 1 and MPN@GI (metal polyphenol nanoparticles loaded with glucose oxidase and insulin) nanoparticles prepared in Example 4. Detailed Implementation

[0051] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0052] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0053] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0054] This invention provides a glucose-responsive insulin delivery system, mainly composed of a delivery carrier and nanoparticles formed by the self-assembly of bioactive components encapsulated within the carrier, with a preferred particle size of 230–290 nm. The delivery carrier is a pH-responsive metal polyphenol network formed by the coordination reaction of epigallocatechin gallate and metal ions; the bioactive components are glucose oxidase, catalase, and insulin, with the preferred mass ratio of glucose oxidase, catalase, and insulin being (0.1–1.3):(0.1–1.3):(0.1–1.3). In this invention, a more preferred mass ratio of glucose oxidase, catalase, and insulin is (0.5–1.0):(0.5–1.0):(0.5–1.0).

[0055] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0056] The glucose-responsive insulin delivery system provided by this invention can intelligently respond to changes in blood glucose levels, releasing insulin rapidly at high blood glucose levels and slowly at normal levels, thus achieving glucose-responsive insulin release. This not only quickly controls blood glucose within the normal range, avoiding diabetic hyperosmolar coma caused by hyperglycemia, but also allows the system to release appropriate amounts of insulin according to blood glucose levels through glucose-dependent release, avoiding hypoglycemia. Specifically, at normal blood glucose levels, glucose oxidase oxidizes glucose to produce less gluconic acid, and the metallopolyphenol network can stably encapsulate insulin, resulting in slow insulin release and avoiding the risk of hypoglycemia in patients.

[0057] This invention provides a method for preparing the glucose-responsive insulin delivery system described above, comprising the following steps:

[0058] S1. Prepare solutions of each component

[0059] S11. Weigh out epigallocatechin gallate, glucose oxidase, catalase and insulin, with a mass ratio of 5:(0.1~1.3):(0.1~1.3):(0.1~1.3);

[0060] S12. Weigh the metal salt, wherein the mass ratio of the metal salt to epigallocatechin gallate is 1:(1-5);

[0061] S13. Dissolve the previously weighed glucose oxidase, epigallocatechin gallate, and metal salt in distilled water; dissolve catalase in potassium phosphate buffer; and dissolve insulin in NaHCO3 solution to obtain the solution form of each component.

[0062] S2. Preparation of nanoparticles for the delivery system

[0063] S21. Under stirring conditions, glucose oxidase, catalase, insulin, epigallocatechin gallate and metal salt solutions were added sequentially to a reagent bottle containing phosphate buffer to obtain a mixed solution; the mixed solution was stirred continuously for 30 min to prepare metal polyphenol nanoparticles encapsulating glucose oxidase, catalase and insulin.

[0064] S3. Obtaining nanoparticles from the delivery system

[0065] S31. Centrifuge the stirred mixture to obtain the bottom precipitate;

[0066] S32. Wash the bottom precipitate with distilled water at least three times to obtain nanoparticles;

[0067] S4. Preservation of nanoparticles in the delivery system

[0068] The obtained nanoparticles were dispersed in phosphate buffer for subsequent use to obtain nanoparticles of the desired particle size.

[0069] Preferably, the mass ratio of epigallocatechin gallate, glucose oxidase, catalase and insulin is 5:(0.5-1.0):(0.5-1.0):(0.5-1.0).

[0070] In this invention, the preferred melting temperature is 0–35°C.

[0071] The present invention does not have any special requirements for the specific implementation process of the dissolution.

[0072] In this invention, the buffer solution is preferably phosphate-buffered saline (PBS), Duchenne phosphate-buffered saline (DPBS), 3-(N-morpholino)propanesulfonic acid buffer (MOPS), or bis(2-hydroxyethylamino)tris(hydroxymethyl)methane buffer (Bis-Tris). The molar concentration of the buffer solution is preferably 5–20 mmol / L. The pH value of the buffer solution is preferably 7.0–12.0.

[0073] In this invention, the metal salt is preferably a water-soluble zinc salt, a water-soluble iron salt, or a water-soluble aluminum salt, more preferably zinc nitrate, ferric chloride, or aluminum chloride. Specifically, the metal salt is preferably ferric chloride hexahydrate, because ferric chloride hexahydrate has a stronger coordination ability.

[0074] In this invention, the mass ratio of epigallocatechin gallate ester to metal salt is (1-5):1, more preferably (1-2.5):1.

[0075] In this invention, the metal salt is preferably mixed in the form of an aqueous solution of the metal salt.

[0076] In this invention, the mass concentration of the metal salt aqueous solution is preferably 10 mg / mL.

[0077] In this invention, the mixing preferably includes the following steps: mixing the mixed solution and the buffer solution to obtain a buffer mixture; and adding the metal salt dropwise to the buffer mixture under continuous stirring. In this invention, the stirring rate is preferably 600 rpm, and the stirring time is preferably 30 min; this invention does not have special requirements for the dropwise addition rate. In this invention, the volume of the metal salt solution is preferably such that all epigallocatechin gallate has undergone a coordination reaction with the metal salt, in order to stabilize the formed nanoparticle structure and reduce side reactions.

[0078] In this invention, the temperature of the coordination reaction is preferably 0 to 35°C.

[0079] In this invention, the coordination reaction is preferably carried out under continuous stirring conditions, the stirring rate is preferably 600 rpm, and the stirring time is preferably 30 min.

[0080] In this invention, the coordination reaction yields a coordination reaction solution. Preferably, the coordination reaction solution undergoes post-processing to obtain the glucose-responsive insulin delivery system. The post-processing preferably includes sequential centrifugation and washing with water. The centrifugation speed is preferably 10000 r / min, and the centrifugation time is preferably 30 min. The precipitate from the centrifugation is preferably washed with water, specifically with distilled water, and preferably three times. The specific implementation of the washing process is not particularly important in this invention.

[0081] In this invention, the glucose-responsive insulin delivery system is preferably stored at 4°C.

[0082] This invention provides the application of the glucose-responsive insulin delivery system described in the above technical solution or the glucose-responsive insulin delivery system prepared by the preparation method described in the above technical solution in the preparation of drugs for treating diabetes.

[0083] The following description is provided in conjunction with specific embodiments and accompanying drawings:

[0084] Example 1

[0085] according to Figure 1 The flowchart shown indicates that the pH of the phosphate-buffered saline (PBS) buffer used is 7.4 and the molar concentration is 10 mmol / L.

[0086] A mixed solution was prepared by mixing 200 μL of epigallocatechin gallate (EGCG) solution, 200 μL of insulin solution, 200 μL of glucose oxidase solution, and 200 μL of catalase solution. The mass concentrations of epigallocatechin gallate (EGCG) in the mixed solution were 10 mg / mL, insulin (1 mg / mL), glucose oxidase (1 mg / mL), and catalase (1 mg / mL).

[0087] The mixed solution and phosphate-buffered saline (PBS) buffer were mixed at a volume ratio of 1:1.5. Then, 100 μL of ferric chloride hexahydrate solution (10 mg / mL) was added dropwise to the mixed solution while stirring at 600 rpm. The solution color changed from colorless to purple. The mixture was then stirred for another 30 min to complete the coordination reaction. The precipitated particles were collected by centrifugation (10000 rpm, 20 min), washed three times with deionized water, and the glucose-responsive insulin delivery system nanoparticles were obtained, designated MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase, and insulin). MPN@GCI was stored at 4 °C.

[0088] Example 2

[0089] according to Figure 1 The flowchart shown indicates that the pH of the PBS buffer used is 7.4 and the molar concentration is 10 mmol / L.

[0090] A mixed solution was prepared by mixing 100 μL of epigallocatechin gallate (EGCG) solution, 20 μL of insulin solution, 200 μL of glucose oxidase solution, and 200 μL of catalase solution. The mass concentrations of epigallocatechin gallate (EGCG) solution, insulin solution, glucose oxidase solution, and catalase solution in the mixed solution were 10 mg / mL, 1 mg / mL, 1 mg / mL, and 1 mg / mL, respectively.

[0091] The mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5. Then, 100 μL of ferric chloride hexahydrate solution (10 mg / mL) was added dropwise to the mixed solution while stirring at 600 rpm. The solution color subsequently changed from colorless to purple. The mixture was then stirred for another 30 min to complete the coordination reaction. The precipitated particles were collected by centrifugation (10000 rpm, 20 min), washed three times with deionized water, and the glucose-responsive insulin delivery system nanoparticles were obtained, denoted as MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase, and insulin). MPN@GCI was stored at 4 °C.

[0092] Example 3

[0093] according to Figure 1 The flowchart shown indicates that the pH of the PBS buffer used is 7.4 and the molar concentration is 10 mmol / L.

[0094] A mixed solution was prepared by mixing 500 μL of epigallocatechin gallate (EGCG) solution, 260 μL of insulin solution, 200 μL of glucose oxidase solution, and 200 μL of catalase solution. The mass concentrations of epigallocatechin gallate (EGCG) and insulin (EGCG) in the mixed solution were 10 mg / mL, 1 mg / mL, 1 mg / mL, and 1 mg / mL, respectively.

[0095] The mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5. Then, 100 μL of ferric chloride hexahydrate solution (10 mg / mL) was added dropwise to the mixed solution while stirring at 600 rpm. The solution color subsequently changed from colorless to purple. The mixture was then stirred for another 30 min to complete the coordination reaction. The precipitated particles were collected by centrifugation (10000 rpm, 20 min), washed three times with deionized water, and the glucose-responsive insulin delivery system nanoparticles were obtained, denoted as MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase, and insulin). MPN@GCI was stored at 4 °C.

[0096] Example 4

[0097] The PBS buffer used had a pH of 7.4 and a molar concentration of 10 mmol / L.

[0098] A mixed solution was prepared by mixing epigallocatechin gallate (EGCG) solution, insulin solution, and glucose oxidase solution. The mass concentrations of epigallocatechin gallate (EGCG) and insulin (EGCG) in the mixed solution were 10 mg / mL, 1 mg / mL, and 1 mg / mL, respectively.

[0099] The mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5. Then, a 10 mg / mL ferric chloride hexahydrate solution was added dropwise to the mixed solution while stirring at 600 rpm. The solution subsequently changed from colorless to purple. The mixture was stirred for another 30 min to complete the coordination reaction. The precipitated particles were collected by centrifugation (10000 rpm, 20 min), washed three times with deionized water, and the glucose-responsive insulin delivery system nanoparticles were obtained, denoted as MPN@GI (metal polyphenol nanoparticles loaded with glucose oxidase and insulin). MPN@GI was stored at 4 °C.

[0100] Comparative Example 1

[0101] The PBS buffer used had a pH of 7.4 and a molar concentration of 10 mmol / L.

[0102] A 10 mg / mL epigallocatechin gallate (EGCG) solution and PBS buffer solution were mixed at a volume ratio of 1:1.5. Then, a 10 mg / mL ferric chloride hexahydrate solution was added dropwise to the mixture while stirring at 600 rpm. The solution changed color from colorless to purple. The mixture was then stirred for another 30 min to complete the coordination reaction. The precipitated particles were collected by centrifugation (10000 rpm, 20 min), washed three times with deionized water, and the glucose-responsive insulin delivery system nanoparticles, denoted as MPN (metal polyphenol nanoparticles), were obtained. The MPN was stored at 4 °C.

[0103] Comparative Example 2

[0104] The PBS buffer used had a pH of 7.4 and a molar concentration of 10 mmol / L.

[0105] Epigallocatechin gallate (EGCG) solution and insulin solution were mixed to obtain a mixed solution. The mass concentration of epigallocatechin gallate in the mixed solution was 10 mg / mL and the mass concentration of insulin was 1 mg / mL.

[0106] The mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5. Then, a 10 mg / mL ferric chloride hexahydrate solution was added dropwise to the mixed solution while stirring at 600 rpm. The solution subsequently changed from colorless to purple. The mixture was stirred for another 30 min to complete the coordination reaction. The precipitated particles were collected by centrifugation (10000 rpm, 20 min), washed three times with deionized water, and the glucose-responsive insulin delivery system nanoparticles were obtained, designated MPN@Insulin (insulin-loaded metal polyphenol nanoparticles). MPN@Insulin was stored at 4 °C.

[0107] Test Example 1

[0108] The nanoparticles synthesized in Examples 1 and 4, and Comparative Examples 1 and 2 were tested using dynamic light scattering method, and the particle size distribution curves are shown below. Figure 2 As shown, the sizes of MPN (metal polyphenol nanoparticles), MPN@Insulin (metal polyphenol nanoparticles loaded with insulin), MPN@GOx&Insulin (metal polyphenol nanoparticles loaded with glucose oxidase and insulin), and MPN@GOx&CAT&Insulin (metal polyphenol nanoparticles loaded with glucose oxidase, catalase, and insulin) are 214.2 nm, 233.9 nm, 255.7 nm, and 280.3 nm, respectively. Their polymer dispersibility index (PDI value) is less than 0.4, indicating that the particle products prepared in Examples 1 and 4 and Comparative Examples 1 and 2 of this invention are nanoscale particles with a narrow particle size distribution.

[0109] Test Example 2

[0110] The secondary structure of proteins is crucial for maintaining their biological activity. Therefore, we investigated the secondary structures of insulin, GOx (glucose oxidase), and CAT (catalase) before encapsulation (native) and after release. Figure 3 As shown in Figure ac, the circular dichroism curves of the MPN-released proteins and the native proteins (Insulin, GOx, and CAT) showed no significant difference. Analysis of the secondary structure ratios of each protein further confirmed that encapsulation does not affect the secondary structure of these proteins. Figure 3 This demonstrates that encapsulation does not affect the biological activity of these proteins.

[0111] Test Example 3

[0112] Insulin's glucose-responsive release is based on the pH-responsive disintegration of MPN and the glucose specificity of GOx. Therefore, to confirm the glucose response of MPN@GCI, MPN@GCI particles were incubated at different glucose concentrations (0 mg / mL, 1 mg / mL, 4 mg / mL) and the pH value was monitored in real time.

[0113] The specific procedure is as follows: MPN@GCI particles were redispersed in 1 mL of glucose aqueous solution (mass concentrations of 0 mg / mL, 1 mg / mL, and 4 mg / mL, respectively), and incubated in a shaker at 37°C and 200 rpm. At pre-set time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 5 h, and 8 h), the pH value of the solution containing MPN@GCI particles was recorded accurately using pH test paper. The pH of MPN@GCI showed a strong dependence on glucose concentration and incubation time (e.g., ...). Figure 4 As the culture time increased, the pH of the 0 mg / mL glucose aqueous solution remained unchanged at 7, while the pH of the 1 mg / mL glucose solution gradually decreased to 5.5, and the pH of the 4 mg / mL glucose solution gradually decreased to 4.0. This indicates that the glucose in the solution containing MPN@GCI particles was oxidized to gluconic acid under the catalysis of glucose oxidase, thereby lowering the pH.

[0114] Test Example 4

[0115] To further confirm the glucose-responsive release of MPN@GCI, release curves of MPN@GCI at different glucose concentrations were determined. Figure 5 As shown, the insulin release curve resembles the trend of pH changes, depending on glucose concentration and incubation time. Overall, insulin is released continuously with increasing incubation time, with faster release at high glucose concentrations. At the end of the experiment, the cumulative release of insulin was 202 μg / mL at a glucose concentration of 4 mg / mL, compared to only 73 μg / mL at 1 mg / mL and nearly 46 μg / mL at 0 mg / mL. These results indicate that MPN@GCI possesses the characteristic of releasing insulin in response to glucose concentration.

[0116] Test Example 5

[0117] To simulate the fluctuations in blood sugar levels experienced by diabetic patients in their daily lives, we alternate between high-glycemic and normal-glycemic levels every hour. Figure 6 As shown, the insulin release curve in MPN@GCI exhibits a fluctuating release pattern, meaning that the release amount increases with increasing glucose concentration, but decreases when glucose concentration decreases. These results further confirm the glucose-responsive insulin release characteristic of MPN@GCI.

[0118] Test Example 6

[0119] To assess the retention of insulin in vivo, free FITC-labeled insulin (F-Insulin) and FITC-labeled MPN@GCI (MPN@GCI prepared in Example 1) were injected subcutaneously into STZ-induced T1D mice, and in vivo fluorescence imaging was performed. Figure 7 As shown. Compared to MPN@GCI, the brightness of free F-Insulin fades much faster, becoming almost invisible after 4 hours. Figure 8 As shown, slow retention was observed for MPN@GCI, with 10% of the total fluorescence retained at 12h, which is beneficial for prolonging the in vivo hypoglycemic effect of MPN@GCI.

[0120] Example 5

[0121] To evaluate the efficacy of MPN@GCI in treating diabetes, streptozotocin-induced T1D mice were used. The T1D mice were then divided into four groups, receiving subcutaneous injections of saline (NS group), free insulin, MPN@Insulin, and MPN@GCI, respectively. Blood glucose levels in each group were closely monitored at pre-set time intervals. Figure 9 As shown, blood glucose levels in the free insulin group rapidly decreased to normal levels after approximately half an hour, but returned to hyperglycemia (>200 mg / dL) after 2 hours. Blood glucose levels in the MPN@Insulin group returned to normal after approximately 1 hour and remained normal for approximately 3 hours. MPN@GCI injection also rapidly reduced blood glucose levels to normal within 1 hour and maintained them for over 24 hours. Importantly, no hypoglycemia was observed during this period. Furthermore, to clarify the risk of hypoglycemia induced by MPN@GCI, healthy mice were injected with saline (NS group), free insulin, MPN@Insulin, and MPN@GCI. Blood glucose levels were monitored at the desired time points (e.g., ) Figure 10 As shown in the figure, a hypoglycemic index is calculated based on this (a low value indicates a lower risk of hypoglycemia), and displayed on the chart. Figure 11 In mice treated with free insulin, the hypoglycemic index was significantly higher than that of mice treated with MPN@GCI, indicating that the hypoglycemic risk of MPN@GCI is very low. Clearly, although free insulin has a rapid hypoglycemic effect, its hypoglycemic activity carries the risk of hypoglycemia. Encapsulating insulin in MPN reduces the risk of hypoglycemia (e.g., ...). Figure 10 As shown in the figure, it is clearly demonstrated that MPN has the ability to control the release of insulin. These results indicate that MPN@GCI exhibits a good in vivo hypoglycemic effect and can reduce the risk of hypoglycemia.

[0122] Example 6

[0123] To evaluate the biosafety of MPN@GCI, mice were subcutaneously injected with normal saline (NS), MPN@GI, and MPN@GCI, respectively. Photographs of the injection sites were taken on days 1, 2, and 7. Mice were sacrificed after day 7, and skin samples were collected for HE staining. Figure 12 As shown, mice injected with MPN@GI developed obvious skin ulcers on the first day, while those injected with MPN@GCI... Figure 12 As shown in the skin tissue sections, significant skin defects were observed in the MPN@GI group, while no significant skin defects were observed in the control group and the MPN@GCI group. This demonstrates that CAT encapsulated in MPN can effectively remove hydrogen peroxide and prevent inflammatory damage, exhibiting good biocompatibility.

[0124] This invention provides an insulin delivery system capable of rapidly or slowly releasing insulin based on blood glucose concentration, its preparation method, and its application in the management of diabetes. This delivery system not only releases insulin in response to blood glucose levels but also does not destroy bioactive components, thus reducing the number of insulin injections required for diabetic patients and extending the time required to maintain normal blood glucose levels after a single administration.

Claims

1. A glucose-responsive insulin delivery system, characterized in that: The invention comprises a delivery carrier and bioactive components encapsulated within the carrier, wherein the delivery carrier and bioactive components self-assemble to form a nanoparticle structure; the delivery carrier is a pH-responsive metal polyphenol network formed by the coordination reaction of epigallocatechin gallate and metal ions; the bioactive components include glucose oxidase, catalase, and insulin. The mass ratio of glucose oxidase, catalase and insulin is (0.1-1.3):(0.1-1.3):(0.1-1.3), and the mass ratio of the metal salt corresponding to the metal ion to epigallocatechin gallate is 1:(1-5). The metal ions and epigallocatechin gallate directly self-assembled in phosphate-buffered saline (PBS) to form metal polyphenol nanoparticles, with the concentration of PBS being 10 mM / L. The glucose-responsive insulin delivery system has a particle size of 230–290 nm.

2. The glucose-responsive insulin delivery system as described in claim 1, characterized in that: The metal ion is one or more of zinc ion, iron ion or aluminum ion, the compound corresponding to the zinc ion is zinc nitrate, the compound corresponding to the iron ion is ferric chloride, and the compound corresponding to the aluminum ion is aluminum chloride.

3. The glucose-responsive insulin delivery system as described in claim 2, characterized in that: The metal ion is an iron ion, and the compound corresponding to the iron ion is ferric chloride hexahydrate.

4. A method for preparing a glucose-responsive insulin delivery system as described in any one of claims 1-3, characterized in that, Specifically, the following steps are included: S1. Prepare solutions of each component S11. Weigh out epigallocatechin gallate, glucose oxidase, catalase and insulin, with a mass ratio of 5:(0.1~1.3):(0.1~1.3):(0.1~1.3); S12. Weigh the metal salt, wherein the mass ratio of the metal salt to epigallocatechin gallate is 1:(1-5); S13. Dissolve the previously weighed glucose oxidase, epigallocatechin gallate, and metal salt in distilled water; dissolve catalase in potassium phosphate buffer; and dissolve insulin in NaHCO3 solution to obtain the solution form of each component. S2. Preparation of nanoparticles for the delivery system S21. Under stirring conditions, glucose oxidase, catalase, insulin, epigallocatechin gallate and metal salt solutions were added sequentially to a reagent bottle containing phosphate buffer to obtain a mixed solution. The mixed solution was stirred continuously for 30 min to prepare metal polyphenol nanoparticles encapsulating glucose oxidase, catalase and insulin. S3. Obtaining nanoparticles from the delivery system S31. Centrifuge the stirred mixture to obtain the bottom precipitate; S32. Wash the bottom precipitate with distilled water at least three times to obtain nanoparticles; S4. Preserve the nanoparticles in the delivery system: Disperse the obtained nanoparticles in phosphate buffer for subsequent use to obtain nanoparticles of the desired particle size.

5. The method for preparing a glucose-responsive insulin delivery system as described in claim 4, characterized in that: In step S13, the concentration of the potassium phosphate buffer solution is 50 mM / L and the pH is 7.0; the concentration of NaHCO3 is 0.1 M / L and the pH is 8.

5.

6. A drug for treating diabetes, characterized in that: The nanoparticles comprising the glucose-responsive insulin delivery system prepared according to claim 5.

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