Glucose response type insulin delivery system as well as preparation method and application thereof

Abstract

The invention discloses a glucose response type insulin delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological materials. The glucose response type insulin delivery system provided by the invention comprises a delivery carrier and a bioactive component encapsulated in the carrier, the delivery carrier is a metal polyphenol network formed by carrying out coordination reaction on epigallocatechin gallate and metal ions; the bioactive components comprise glucose oxidase, catalase and insulin. The glucose response type insulin delivery system provided by the invention can adjust the release of insulin according to the concentration of glucose in the surrounding environment, so that the blood glucose is maintained at a normal level.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and in particular relates to a glucose-responsive insulin delivery system, a preparation method and applications thereof. Background Art

[0002] Diabetes is a chronic metabolic disease, which is mainly manifested by insufficient insulin secretion or insulin resistance. The blood sugar level of diabetic patients is often higher than that of normal people, and the clinical manifestations are polydipsia, polyphagia, polyuria and weight loss. If the blood sugar of diabetic patients is uncontrolled for a long time, it will cause serious damage to many organs of the body, especially nerves and blood vessels. According to data from the International Diabetes Federation in 2024, about 540 million adults in the world suffer from diabetes, and this number is expected to reach 783 million by 2045. Therefore, it is 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, fat and skeletal muscle cells, thereby lowering blood sugar levels. However, insulin therapy is burdensome because it requires multiple subcutaneous injections per day, which reduces the compliance of diabetic patients, and excessive or insufficient injections can cause irreversible serious consequences. Therefore, there is an urgent need for a new insulin delivery strategy that can responsively release insulin according to blood sugar levels to effectively regulate blood sugar.

[0004] In healthy individuals, endogenous insulin secreted by pancreatic β cells is highly responsive to blood glucose levels. Insulin delivery by injection of glucose-responsive carriers can mimic human pancreatic function to accurately adjust blood glucose levels in real time and achieve 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 have attracted much attention due to their ultra-high specificity for glucose and high sensitivity to blood glucose levels.

[0005] Under physiological conditions, glucose-responsive carriers face multiple obstacles after entering the body, such as buffering of body fluids, degradation of proteases, and immune cells responsible for clearing foreign bodies. In addition, as proteins, the secondary structures of glucose oxidase and insulin are crucial to maintaining their biological activity, but during the delivery process, the secondary structures of glucose oxidase and insulin are very sensitive to changes in the microenvironment. Therefore, there are still technical barriers to being able to responsively release insulin according to blood sugar levels to regulate blood sugar levels under such severe conditions.

[0006] Therefore, in order to solve the above technical problems, it is necessary to provide a new technical solution to solve this technical problem, especially a glucose-responsive insulin delivery system. Summary of the invention

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

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

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

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

[0011] Preferably, the metal ions and epigallocatechin gallate are directly self-assembled 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) to form metal polyphenol nanoparticles.

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

[0013] Preferably, 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.

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

[0015] The present invention also provides a method for preparing a glucose-responsive insulin delivery system, comprising the aforementioned delivery system, specifically comprising the following steps:

[0016] S1. Preparation of 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. Weighing a metal salt, wherein the mass ratio of the metal salt to epigallocatechin gallate is 1:(1-5);

[0019] S13. The glucose oxidase, epigallocatechin gallate, and metal salt weighed above are dissolved in distilled water, respectively; catalase is dissolved in potassium phosphate buffer, and insulin is dissolved in NaHCO3 solution to obtain solutions of the components;

[0020] S2. Preparation of delivery system nanoparticles

[0021] S21. Under stirring conditions, in a reagent bottle containing a phosphate buffer, glucose oxidase, catalase, insulin, epigallocatechin gallate and a metal salt solution are added in order to obtain a mixed solution; the mixed solution is continuously stirred for 30 minutes to prepare metal polyphenol nanoparticles encapsulating glucose oxidase, catalase and insulin;

[0022] S3. Obtaining Nanoparticles for Delivery Systems

[0023] S31. The stirred mixture was centrifuged to obtain a bottom precipitate;

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

[0025] S4. Storage of Nanoparticles in Delivery Systems

[0026] The obtained nanoparticles are dispersed in 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 the present invention are:

[0030] The delivery system provided by the present invention is mainly composed of nanoparticles formed by self-assembly of a delivery carrier and a bioactive component encapsulated in the carrier; the delivery carrier is a metal polyphenol network with pH response characteristics formed by coordination reaction between epigallocatechin gallate and metal ions; the bioactive components are glucose oxidase, catalase and insulin. The glucose-responsive insulin delivery system provided by the present invention uses the metal polyphenol network as a delivery carrier to encapsulate glucose oxidase, catalase and insulin, which can not only achieve a high loading amount of insulin in the bioactive components, but also maintain the bioactivity of glucose oxidase, catalase and insulin during encapsulation and delivery, thereby achieving a rapid response to glucose.

[0031] The delivery carrier and the bioactive components are mixed together rather than completely encapsulated by hydrogen bonds, hydrophobic interactions, coordination bonds and other forces. Each component can contact glucose but will not dissolve immediately under the forces of hydrogen bonds, hydrophobic interactions, coordination bonds and other forces. When the glucose concentration is too high, glucose oxidase can oxidize glucose to produce gluconic acid. As the content of gluconic acid increases, the pH value decreases. Due to the pH response characteristics of the metal polyphenol network, the metal polyphenol network dissociates, thereby gradually opening the entire delivery system to achieve rapid release of insulin under high blood sugar levels.

[0032] Under normal blood sugar level, glucose oxidase oxidizes glucose to generate less gluconic acid, and the metal polyphenol network can stably encapsulate insulin, so that insulin is slowly released, avoiding the risk of hypoglycemia in patients. At the same time, the in vivo action time of insulin is extended to reduce the injection frequency, greatly alleviating the pain of multiple injections in diabetic patients a day. The data of the embodiment show that the glucose-responsive insulin delivery system provided by the invention can maintain a normal blood sugar state of more than 24h after a subcutaneous injection, can be applied to the treatment of glucose-mediated insulin transdermal administration type 1 diabetes (T1D), and is expected to be widely used in the treatment of diabetes.

[0033] The present invention further adds catalase to the biologically active 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) The present invention innovatively prepares a delivery system with glucose-responsive insulin release characteristics by a simple and rapid one-step co-deposition method. The delivery system is a nanoparticle formed by a coordination reaction between epigallocatechin gallate and metal ions, and co-assembly encapsulates the biologically active components glucose oxidase, catalase and insulin. The preparation method is simple, easy to operate, rapid and gentle, and is suitable for industrial promotion and production applications.

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

[0037] (3) The glucose-responsive insulin delivery system provided by the present invention has significant responsiveness to blood sugar levels. When the glucose concentration is too high, glucose oxidase can oxidize glucose to generate gluconic acid. As the content of gluconic acid increases, the pH value decreases, and the metal polyphenol network with pH-responsive characteristics dissociates, thereby achieving rapid release of insulin under high blood sugar levels; while under normal blood sugar levels, the metal polyphenol network can stably encapsulate insulin, allowing insulin to be released slowly, avoiding the risk of hypoglycemia in patients. It can be seen from the experimental data that the delivery system can maintain a normal blood sugar state for more than 24 hours after a single subcutaneous injection.

[0038] (4) The glucose-responsive insulin delivery system provided by the present invention has a long retention time in the body, thereby prolonging the in vivo action time of insulin, reducing the injection frequency, and greatly alleviating the pain of multiple injections per day for diabetic patients. The present invention can provide new ideas and possibilities for the preparation and application of a simple and efficient glucose-responsive insulin delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a flow chart of preparing a glucose-responsive insulin delivery system and a flow chart of its production effect provided in Example 1 of the present invention.

[0040] Figure 2 It is a particle size distribution diagram of the products prepared in Examples 1 and 4 and Comparative Examples 1 and 2 of the present invention measured by dynamic light scattering method.

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

[0042] Figure 4 This is the pH response curve of 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 high blood sugar and normal blood sugar are used alternately every hour.

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

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

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

[0048] Fig.10 It is a comparison chart of the blood sugar change curves of healthy animal models of the products prepared in Example 1 and Comparative Example 2 over time.

[0049] Fig.11 It is a comparison chart of the low blood sugar index of healthy animal models of the products prepared in Example 1 and Comparative Example 2.

[0050] Fig.12 It is a comparison picture of the injection site of mouse skin and tissue sections 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 DESCRIPTION

[0051] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0052] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

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

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

[0056] The glucose-responsive insulin delivery system provided by the present invention can intelligently respond to changes in blood sugar levels, quickly release at high blood sugar levels, and slowly release at normal levels, thereby achieving glucose-responsive release of insulin. This can not only quickly control blood sugar within a normal range, avoiding diabetic hyperosmotic coma caused by high blood sugar; it can also enable the system to release an appropriate amount of insulin according to blood sugar levels through glucose-dependent release, thereby avoiding the occurrence of hypoglycemia, that is, at normal blood sugar levels, glucose oxidase oxidizes glucose to generate less gluconic acid, and the metal polyphenol network can stably encapsulate insulin, allowing insulin to be slowly released, thereby avoiding the risk of hypoglycemia in patients.

[0057] The present invention provides a method for preparing the glucose-responsive insulin delivery system described in the above technical solution, comprising the following steps:

[0058] S1. Preparation of solutions of each component

[0059] 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);

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

[0061] S13. The glucose oxidase, epigallocatechin gallate, and metal salt weighed above are dissolved in distilled water, respectively; catalase is dissolved in potassium phosphate buffer, and insulin is dissolved in NaHCO3 solution to obtain solutions of the components;

[0062] S2. Preparation of delivery system nanoparticles

[0063] S21. Under stirring conditions, in a reagent bottle containing a phosphate buffer, glucose oxidase, catalase, insulin, epigallocatechin gallate and a metal salt solution are added in order to obtain a mixed solution; the mixed solution is continuously stirred for 30 minutes to prepare metal polyphenol nanoparticles encapsulating glucose oxidase, catalase and insulin;

[0064] S3. Obtaining Nanoparticles for Delivery Systems

[0065] S31. The stirred mixture was centrifuged to obtain a bottom precipitate;

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

[0067] S4. Storage and delivery system nanoparticles

[0068] The obtained nanoparticles are dispersed in phosphate buffer for subsequent use to obtain nanoparticles with a 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 the present invention, the dissolving temperature is preferably 0 to 35°C.

[0071] The present invention has no special requirements on the specific implementation process of the dissolution.

[0072] In the present invention, the buffer solution is preferably 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). The molar concentration of the buffer solution is preferably 5 to 20 mmol / L. The pH value of the buffer solution is preferably 7.0 to 12.0.

[0073] In the present 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. The metal salt is preferably ferric chloride hexahydrate because ferric chloride hexahydrate has a stronger coordination ability.

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

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

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

[0077] In the present invention, the mixing preferably includes the following steps: mixing the mixed solution and a buffer solution to obtain a buffered mixed solution; and dropping the metal salt into the buffered mixed solution under continuous stirring. In the present invention, the stirring rate is preferably 600 rpm, and the stirring time is preferably 30 min; the present invention has no special requirements for the speed of the dropping. In the present invention, the volume of the metal salt solution is preferably based on the coordination reaction between all epigallocatechin gallate and the metal salt, so as to stabilize the formed nanoparticle structure and reduce side reactions.

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

[0079] In the present 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 the present invention, the coordination reaction obtains a coordination reaction liquid. The present invention preferably performs post-treatment on the coordination reaction liquid to obtain the glucose-responsive insulin delivery system. In the present invention, the post-treatment preferably includes: centrifugal separation and water washing in sequence. In the present invention, the rotation speed of the centrifugal separation is preferably 10000r / min, and the time of the centrifugal separation is preferably 30min. The present invention preferably washes the precipitate obtained by the centrifugal separation with water. In the present invention, the water washing is specifically preferably distilled water washing. In the present invention, the number of times of the water washing is preferably 3 times. The present invention has no special requirements for the specific implementation process of the water washing.

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

[0082] The present invention provides the use 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 is a description of the specific embodiments and drawings:

[0084] Embodiment 1

[0085] according to Figure 1 Flow chart shown: The pH value of the phosphate buffered saline (PBS) buffer used was 7.4 and the molar concentration was 10 mmol / L.

[0086] 200 μL of epigallocatechin gallate (EGCG) solution, 200 μL of insulin solution, 200 μL of glucose oxidase solution and 200 μL of catalase solution were mixed to obtain a mixed solution. In the mixed solution, the mass concentration of epigallocatechin gallate was 10 mg / mL, the mass concentration of insulin was 1 mg / mL, the concentration of glucose oxidase was 1 mg / mL, and the mass concentration of catalase was 1 mg / mL.

[0087] After the mixed solution and phosphate buffered saline (PBS) buffer solution were mixed at a volume ratio of 1:1.5, 100 μL of ferric chloride hexahydrate solution (10 mg / mL) was added dropwise to the mixed solution at a stirring rate of 600 rpm. The color of the solution then changed from colorless to purple, and then the coordination reaction was completed by further stirring for 30 min. The precipitated particles were collected by centrifugation (10000 r / min, 20 min), and washed 3 times with deionized water to obtain glucose-responsive insulin delivery system nanoparticles, which were recorded as MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase and insulin), and MPN@GCI was stored at 4 °C.

[0088] Embodiment 2

[0089] according to Figure 1 Flow chart shown: The pH value of the PBS buffer used was 7.4 and the molar concentration was 10 mmol / L.

[0090] 100 μL of epigallocatechin gallate (EGCG) solution, 20 μL of insulin solution, 200 μL of glucose oxidase solution and 200 μL of catalase solution were mixed to obtain a mixed solution, wherein the mass concentration of epigallocatechin gallate was 10 mg / mL, the mass concentration of insulin was 1 mg / mL, the concentration of glucose oxidase was 1 mg / mL, and the mass concentration of catalase was 1 mg / mL;

[0091] After the mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5, 100 μL of ferric chloride hexahydrate solution (10 mg / mL) was added dropwise to the mixed solution at a stirring rate of 600 rpm. The color of the solution then changed from colorless to purple, and then the coordination reaction was completed by further stirring for 30 min. The precipitated particles were collected by centrifugation (10000 r / min, 20 min), and washed 3 times with deionized water to obtain glucose-responsive insulin delivery system nanoparticles, denoted as MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase and insulin), and MPN@GCI was stored at 4 °C.

[0092] Embodiment 3

[0093] according to Figure 1 Flow chart shown: The pH value of the PBS buffer used was 7.4 and the molar concentration was 10 mmol / L.

[0094] 500 μL of epigallocatechin gallate (EGCG) solution, 260 μL of insulin solution, 200 μL of glucose oxidase solution and 200 μL of catalase solution were mixed to obtain a mixed solution, wherein the mass concentration of epigallocatechin gallate was 10 mg / mL, the mass concentration of insulin was 1 mg / mL, the concentration of glucose oxidase was 1 mg / mL, and the mass concentration of catalase was 1 mg / mL;

[0095] After the mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5, 100 μL of ferric chloride hexahydrate solution (10 mg / mL) was added dropwise to the mixed solution at a stirring rate of 600 rpm. The color of the solution then changed from colorless to purple, and then the coordination reaction was completed by further stirring for 30 min. The precipitated particles were collected by centrifugation (10000 r / min, 20 min), and washed 3 times with deionized water to obtain glucose-responsive insulin delivery system nanoparticles, denoted as MPN@GCI (metal polyphenol nanoparticles loaded with glucose oxidase, catalase and insulin), and MPN@GCI was stored at 4 °C.

[0096] Embodiment 4

[0097] The pH value of the PBS buffer used was 7.4 and the molar concentration was 10 mmol / L.

[0098] An epigallocatechin gallate (EGCG) solution, an insulin solution and a glucose oxidase solution are mixed to obtain a mixed solution. In the mixed solution, the mass concentration of epigallocatechin gallate is 10 mg / mL, the mass concentration of insulin is 1 mg / mL, and the concentration of glucose oxidase is 1 mg / mL.

[0099] After the mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5, a 10 mg / mL ferric chloride hexahydrate solution was added dropwise to the mixed solution at a stirring rate of 600 rpm. The color of the solution then changed from colorless to purple, and then the coordination reaction was completed by further stirring for 30 min. The precipitated particles were collected by centrifugation (10000 r / min, 20 min), and washed 3 times with deionized water to obtain glucose-responsive insulin delivery system nanoparticles, denoted as MPN@GI (metal polyphenol nanoparticles loaded with glucose oxidase and insulin), and MPN@GI was stored at 4 °C.

[0100] Comparative Example 1

[0101] The pH value of the PBS buffer used was 7.4 and the molar concentration was 10 mmol / L.

[0102] After mixing a 10 mg / mL epigallocatechin gallate (EGCG) solution and a PBS buffer solution at a volume ratio of 1:1.5, a 10 mg / mL ferric chloride hexahydrate solution was added dropwise to the mixed solution at a stirring rate of 600 rpm. The color of the solution then changed from colorless to purple, and then the coordination reaction was completed by further stirring for 30 min. The precipitated particles were collected by centrifugation (10000 r / min, 20 min), and washed 3 times with deionized water to obtain glucose-responsive insulin delivery system nanoparticles, recorded as MPN (metal polyphenol nanoparticles), and the MPN was stored at 4 ° C.

[0103] Comparative Example 2

[0104] The pH value of the PBS buffer used was 7.4 and the molar concentration was 10 mmol / L.

[0105] Mixing an epigallocatechin gallate (EGCG) solution and an insulin solution to obtain a mixed solution, wherein the mass concentration of epigallocatechin gallate is 10 mg / mL and the mass concentration of insulin is 1 mg / mL;

[0106] After the mixed solution and PBS buffer solution were mixed at a volume ratio of 1:1.5, a 10 mg / mL ferric chloride hexahydrate solution was added dropwise to the mixed solution at a stirring rate of 600 rpm. The color of the solution then changed from colorless to purple, and then the coordination reaction was completed by further stirring for 30 min. The precipitated particles were collected by centrifugation (10000 r / min, 20 min), and washed 3 times with deionized water to obtain glucose-responsive insulin delivery system nanoparticles, which were recorded as MPN@Insulin (metal polyphenol nanoparticles loaded with insulin). 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 by dynamic light scattering. The particle size distribution curves are shown in FIG. Figure 2 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, and their polymer dispersibility index (PDI value) is less than 0.4, indicating that the particle products prepared in Examples 1 and 4 of the present invention and Comparative Examples 1 and 2 are nano-scale particles with a narrow particle size distribution.

[0109] Test Example 2

[0110] The secondary structure of proteins is key to 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 Figures ac, there is no significant difference in the circular dichroism curves of MPN-released proteins and natural proteins (Insulin, GOx, and CAT). The analysis of the secondary structure ratio of each protein further confirmed that encapsulation did not affect the secondary structure of these proteins ( Figure 3 df), thus demonstrating that encapsulation does not affect the biological activities of these proteins.

[0111] Test Example 3

[0112] The glucose-responsive release of insulin is based on the pH-responsive disassembly of MPN and the glucose specificity of GOx. Therefore, to confirm the response of MPN@GCI to glucose, 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 method is: MPN@GCI particles are redispersed into 1mL glucose aqueous solution (mass concentration is 0mg / mL, 1mg / mL and 4mg / mL respectively), and incubated at 37℃, 200rpm in a shaker. At the pre-set time points (0h, 0.5h, 1h, 2h, 4h, 5h and 8h), the pH value of the solution containing MPN@GCI particles is recorded with an accurate pH test paper. The pH of MPN@GCI shows a strong dependence on glucose concentration and incubation time (such as Figure 4 ). As the culture time increases, the pH value of the 0 mg / mL glucose aqueous solution remains unchanged at 7, the pH value of the 1 mg / mL glucose gradually decreases to 5.5, and the pH value of the 4 mg / mL glucose gradually decreases to 4.0, indicating that the glucose in the solution containing MPN@GCI particles is oxidized to gluconic acid under the catalysis of glucose oxidase, thereby reducing the pH.

[0114] Test Example 4

[0115] In order to further confirm the release of MPN@GCI in response to glucose, the release curves of MPN@GCI at different glucose concentrations were measured. Figure 5 As shown in the figure, the release curve of Insulin is similar to the trend of pH change, which depends on the glucose concentration and incubation time. In general, Insulin is continuously released with the extension of incubation time, and the release is faster at high glucose concentration. At the end of the experiment, the cumulative release of Insulin was 202μg / mL when the glucose concentration was 4mg / mL, while it was only 73μg / mL when the glucose concentration was 1mg / mL, and it was close to 46μg / mL when the glucose concentration was 0mg / mL. The above results show that MPN@GCI has the characteristics of releasing insulin in response to glucose concentration.

[0116] Test Example 5

[0117] To simulate the fluctuations in blood sugar levels in the daily life of diabetic patients, we used high blood sugar and normal blood sugar alternately every hour. Figure 6 As shown in the figure, the release curve of insulin in MPN@GCI is a fluctuating release, that is, the release amount increases with the increase of glucose concentration, but when the glucose concentration decreases, the release of insulin decreases. The obtained results further confirm the glucose-responsive insulin release characteristics of MPN@GCI.

[0118] Test Example 6

[0119] In order to evaluate the retention of insulin in vivo, free FITC (fluorescein isothiocyanate)-labeled insulin (F-Insulin) and FITC-labeled MPN@GCI (MPN@GCI prepared in Example 1) were injected subcutaneously into STZ-induced T1D mice, and animal fluorescence in vivo imaging was performed, as shown in Figure 2. Figure 7 Compared with MPN@GCI, the brightness of free F-Insulin disappears faster and is almost invisible after 4 hours. Figure 8 As shown, for MPN@GCI, a slow retention was observed, with 10% of the total fluorescence retained at 12 h, thus favoring the prolonged in vivo hypoglycemic effect of MPN@GCI.

[0120] Embodiment 5

[0121] To evaluate the efficacy of MPN@GCI in treating diabetes, streptozotocin was used to induce T1D mice. The T1D mice were divided into 4 groups and subcutaneously injected with normal saline (NS group), free Insulin (Free Insulin), MPN@Insulin and MPN@GCI, respectively. The blood glucose levels of each group were closely monitored within a pre-set time. Fig. 9 As shown, the blood glucose level of the group injected with free Insulin quickly dropped to normal blood glucose levels after about half an hour, but returned to hyperglycemia (>200 mg / dL) after 2 hours. The blood glucose level of the MPN@Insulin group returned to normal after about 1 hour and was maintained for about 3 hours. Injection of MPN@GCI can also quickly reduce blood glucose levels to normal within 1 hour and maintain it for more than 24 hours. Importantly, no hypoglycemia was found during this period. In addition, in order to clarify the risk of MPN@GCI-induced hypoglycemia, healthy mice were injected with normal saline (NS group), free Insulin (Free Insulin), MPN@Insulin and MPN@GCI. Blood glucose levels at the desired time points were monitored (such as Fig.10 ), based on which the hypoglycemia index is calculated (low values ​​indicate a lower risk of hypoglycemia) and displayed on Fig.11 The hypoglycemic index of mice treated with free insulin was significantly higher than that of mice treated with MPN@GCI, indicating that the hypoglycemic risk of MPN@GCI is very low. Obviously, although free insulin has a rapid hypoglycemic effect, its hypoglycemic activity has the risk of causing hypoglycemia. Encapsulating insulin into MPN reduces the risk of hypoglycemia (such as Fig.10 The above results show that MPN@GCI exhibits a good hypoglycemic effect in vivo and can reduce the risk of hypoglycemia.

[0122] Embodiment 6

[0123] To evaluate the biosafety of MPN@GCI, normal saline (NS), MPN@GI, and MPN@GCI were subcutaneously injected into the mice. Photos of the injection sites of the mice were taken on days 1, 2, and 7. The mice were killed 7 days later, and the skin was taken for HE staining. Fig.12 As shown in Figure 2, the skin of mice injected with MPN@GI showed obvious skin ulcers on the first day, while that of mice injected with MPN@GCI did not. Fig.12 As shown in the figure, the skin tissue sections showed that the MPN@GI group had obvious skin defects, while the control group and the MPN@GCI group had no obvious skin defects. This shows that the MPN-encapsulated CAT can remove hydrogen peroxide in time, avoid inflammatory damage, and show good biosafety.

[0124] The present invention provides an insulin delivery system capable of releasing insulin quickly or slowly according to the blood sugar concentration, a preparation method thereof, and application in the treatment of diabetes. The delivery system can not only release insulin in response to the blood sugar level, but also does not destroy the bioactive components, and can reduce the number of insulin injections for diabetic patients and delay the time to maintain normal blood sugar levels after a single use.

Claims

1. A glucose-responsive insulin delivery system, characterized in that: The invention comprises a delivery carrier and a bioactive component encapsulated in the carrier, wherein 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 coordination reaction between epigallocatechin gallate and metal ions; the bioactive components include glucose oxidase, catalase and insulin.

2. The glucose-responsive insulin delivery system according to claim 1, characterized in that: The mass ratios of the glucose oxidase, catalase and insulin are respectively (0.1-1.3):(0.1-1.3):(0.1-1.3).

3. The glucose-responsive insulin delivery system according to claim 1, wherein: The metal ions and epigallocatechin gallate are directly self-assembled 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) to form metal polyphenol nanoparticles.

4. The glucose-responsive insulin delivery system according to claim 3, characterized in that: The metal ions and epigallocatechin gallate are directly self-assembled in the phosphate buffer (PBS) to form metal polyphenol nanoparticles, and the concentration of the used phosphate buffer (PBS) is 10 mM / L.

5. The glucose-responsive insulin delivery system according to claim 1, wherein: 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.

6. The glucose-responsive insulin delivery system according to claim 5, characterized in that: The metal ion is an iron ion, and the compound corresponding to the iron ion is ferric chloride hexahydrate.

7. The glucose-responsive insulin delivery system according to any one of claims 1 to 6, characterized in that: The particle size of the glucose-responsive insulin delivery system is 230-290 nm.

8. A method for preparing a glucose-responsive insulin delivery system, characterized in that: The delivery system according to claim 7, comprising the following steps: S1. Preparation of solutions of each component 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); S12. Weighing a metal salt, wherein the mass ratio of the metal salt to epigallocatechin gallate is 1:(1-5); S13. The glucose oxidase, epigallocatechin gallate, and metal salt weighed above are dissolved in distilled water, respectively; catalase is dissolved in potassium phosphate buffer, and insulin is dissolved in NaHCO3 solution to obtain solutions of the components; S2. Preparation of delivery system nanoparticles S21. Under stirring conditions, in a reagent bottle containing a phosphate buffer, glucose oxidase, catalase, insulin, epigallocatechin gallate and a metal salt solution are added in order to obtain a mixed solution; the mixed solution is continuously stirred for 30 minutes to prepare metal polyphenol nanoparticles encapsulating glucose oxidase, catalase and insulin; S3. Obtaining Nanoparticles for Delivery Systems S31. The stirred mixture was centrifuged to obtain a bottom precipitate; S32. washing the bottom precipitate with distilled water at least three times to obtain nanoparticles; S4. Storage of Nanoparticles in Delivery Systems The obtained nanoparticles are dispersed in phosphate buffer for subsequent use to obtain nanoparticles with a desired particle size.

9. The method for preparing a glucose-responsive insulin delivery system according to claim 8, characterized in that: 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.

10. A drug for treating diabetes, characterized in that: The invention comprises the glucose-responsive insulin delivery system nanoparticles prepared according to claim 9.

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