Sustained-release gel preparation capable of responding to glucose to release growth factors as well as preparation method and application of sustained-release gel preparation

Abstract

The invention relates to the technical field of sustained-release gel preparations, and discloses a sustained-release gel preparation capable of responding to glucose to release growth factors as well as a preparation method and application of the sustained-release gel preparation. According to the invention, a gel preparation containing a'growth factor-FPBA-polylysine-hyaluronic acid 'quaternary composite system is constructed, hyaluronic acid is used as a gel matrix, and FPBA-grafted polylysine and the hyaluronic acid matrix are compounded to form a gel carrier framework; the gel carrier framework is used for realizing mechanical protection and specific condition response type delivery on the growth factors, and the utilization of organisms on the growth factors is realized through a synergistic effect. Wherein the hyaluronic acid can promote the contact between the gel and a wound, the existence of the FPBA-grafted polylysine makes the active component of the growth factor difficult to degrade, and when the gel is in contact with the wound, the glucose responsive release effect of the growth factor is realized through the reversible combination of the FPBA and the glucose; therefore, the gel is suitable for dressing application of diabetes wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sustained-release gel preparation, and particularly relates to a sustained-release gel preparation for releasing growth factors in response to glucose and a preparation method thereof. BACKGROUND

[0002] Diabetes is a chronic metabolic disease that affects millions of people worldwide. Diabetic chronic wounds are one of the important complications of diabetes. Factors such as excessive inflammation, oxidative stress, peripheral neuropathy and vascularization disorders in the diabetic chronic wound area lead to difficulty in healing, and even failure to heal. In severe cases, amputation may be required. Therefore, choosing appropriate and effective treatment methods to promote diabetic wound healing has become a major challenge for global healthcare systems.

[0003] Growth factors are biologically active polypeptides, including insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived proliferation factor (PDGF), and keratinocyte growth factor (KGF), etc. Therefore, growth factors are particularly suitable for the treatment of diabetic chronic wounds. In addition, growth factors play a key role in all stages of the healing process and can accelerate the wound healing process.

[0004] However, during the application of growth factors in wound treatment, the released growth factors are easily affected by highly active proteases (such as peptidases) in the wound environment, resulting in a decrease in effective dose and poor biological activity. Therefore, it is necessary to develop an effective growth factor delivery system to improve the bioavailability and therapeutic effect of growth factors.

[0005] At present, there is no suitable growth factor delivery system that can be effectively applied to the treatment of diabetic chronic wounds. SUMMARY

[0006] In order to solve the problems of low bioavailability and poor therapeutic effect in the growth factor delivery system, the present application provides a sustained-release gel preparation for releasing growth factors in response to glucose and a preparation method thereof.

[0007] The specific technical scheme of the present application is as follows: The present application provides a sustained-release gel preparation for releasing growth factors in response to glucose, which comprises the following components: growth factors, polylysine grafted with 4-carboxy-3-fluorophenylboronic acid (FPBA), and hyaluronic acid. The molar ratio of the amino group of the polylysine grafted with 4-carboxy-3-fluorophenylboronic acid to the carboxyl group of the hyaluronic acid is (0.1-1):1.

[0008] In order to solve the problem that growth factors cannot be effectively delivered to organisms due to the influence of proteases in the wound environment, the present application constructs a sustained-release gel preparation containing a "growth factor-FPBA-polylysine-hyaluronic acid" four-component complex system. The sustained-release gel preparation uses hyaluronic acid as a gel matrix, and forms a gel carrier framework by compounding polylysine grafted with FPBA with the hyaluronic acid matrix. The gel carrier framework realizes mechanical protection and specific condition-responsive delivery of growth factors, and the two functions work together to realize the utilization of growth factors by organisms.

[0009] Hyaluronic acid can promote the contact between the sustained-release gel preparation and the wound, which is conducive to the transdermal delivery of the growth factor active ingredient, and in addition, the presence of polylysine grafted with FPBA makes the growth factor active ingredient less likely to be degraded. As the core effect of the sustained-release gel preparation of the present application, the gel of the "growth factor-FPBA-polylysine-hyaluronic acid" four-component complex system can protect the growth factor from being degraded by proteases in the wound environment through the carrier framework formed by polylysine grafted with FPBA, and when contacting the wound, the glucose-responsive release effect of the growth factor is realized through the reversible binding of FPBA and glucose, so that the gel is suitable for application in the dressing of diabetic wound healing. In summary, the sustained-release gel preparation of the present application can quickly respond and release after contacting the wound, and the bioavailability of the growth factor is high.

[0010] As a preferred embodiment of the above sustained-release gel preparation, the molar ratio of the amino group of the polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid to the carboxyl group of hyaluronic acid is 0.5:1.

[0011] The ratio of the amino group of the polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid to the carboxyl group of hyaluronic acid affects the bioadhesion performance of the sustained-release gel preparation. When the molar ratio of the amino group of the polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid to the carboxyl group of hyaluronic acid is 0.5:1, the bioadhesion performance of the sustained-release gel preparation is the best.

[0012] As a preferred embodiment of the above sustained-release gel preparation, the grafting rate of the polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid is 13% to 40%.

[0013] The grafting rate of the polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid affects the mechanical properties of the sustained-release gel preparation. When the grafting rate of the polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid is 13% to 40%, the sustained-release gel preparation has a larger Young's modulus and the best mechanical properties.

[0014] As the preferred embodiment of the above sustained-release gel preparation, the growth factor is one or more of insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived proliferation factor (PDGF), and keratinocyte growth factor (KGF).

[0015] As the preferred embodiment of the above sustained-release gel preparation, the mass ratio of the growth factor to hyaluronic acid is 0.01-0.1:1. The amount of the growth factor added can be adjusted as needed.

[0016] In addition, the present application provides a method for preparing a sustained-release gel preparation for releasing growth factors in response to glucose, comprising the following steps: (1) mixing 4-carboxy-3-fluorophenylboronic acid (FPBA) with 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide (EDC), activating with N-hydroxysuccinimide (NHS), and reacting for 8-12 hours to obtain FPBA-NHS; (2) adding FPBA-NHS to a polylysine solution, adjusting the pH to 6-7, and reacting for 12-24 hours to obtain polylysine grafted with FPBA; (3) mixing the growth factor with the polylysine grafted with FPBA, adding hyaluronic acid, adjusting the pH to 7-8, and forming a gel.

[0017] In the present application, FPBA is activated in step (1) to prepare pre-reacted FPBA-NHS, which is then successfully grafted to polylysine. The polylysine grafted with FPBA is reacted with hyaluronic acid to construct a gel carrier framework. In step (3), the growth factor is loaded into the gel carrier framework to achieve the construction of a delivery system for the growth factor. In the delivery system for the growth factor in the present application, the contact of the gel with the wound is facilitated by hyaluronic acid, which is conducive to the transdermal delivery of the active ingredient of the growth factor; when contacting the wound, the carrier framework formed by the polylysine grafted with FPBA protects the growth factor from being easily degraded by wound proteases; when contacting the wound, the glucose-responsive release effect of the growth factor is achieved through the reversible binding of FPBA to glucose, so that the gel is suitable for application in the dressing of diabetic wound healing.

[0018] As the preferred embodiment of the above preparation method, in step (1), the molar ratio of 4-carboxy-3-fluorophenylboronic acid to N-hydroxysuccinimide is 1:0.8-1.2.

[0019] As the preferred embodiment of the above preparation method, in step (2), the mass ratio of the FPBA-NHS to the polylysine is 1:5-10.

[0020] Based on the above, the application provides an application of the sustained-release gel preparation releasing growth factors in response to glucose in preparation of a medical dressing.

[0021] Based on the above, the application provides an application of the sustained-release gel preparation releasing growth factors in response to glucose in preparation of a medicine for promoting healing of a diabetic wound.

[0022] Compared with the prior art, the application has the following technical effects: The application constructs a sustained-release gel preparation containing a four-component complex system of "growth factor-FPBA-polylysine-hyaluronic acid". The sustained-release gel preparation of the application uses hyaluronic acid as a gel matrix, and forms a gel carrier framework by compounding polylysine grafted with FPBA with the hyaluronic acid matrix. The gel carrier framework is used to realize mechanical protection and specific condition-responsive delivery of growth factors, and the biological utilization of growth factors is realized by synergistic action. The hyaluronic acid can promote the contact between the gel preparation and a wound, and is conducive to the transdermal delivery of the active ingredient of the growth factor. Meanwhile, due to the presence of the polylysine grafted with FPBA, the active ingredient of the growth factor is not prone to degradation. When contacting the wound, the glucose-responsive release effect of the growth factor is realized by the reversible combination of FPBA and glucose, so that the gel preparation is suitable for application in a dressing for diabetic wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The nuclear magnetic resonance spectrum of polylysine grafted with 4-carboxyl-3-fluorophenylboronic acid prepared for an example embodiment of the application.

[0024] Figure 2 The actual photograph of the gel prepared for example 2 of the application promoting wound healing of a diabetic model rat. DETAILED DESCRIPTION

[0025] The application will be further described below in conjunction with examples. Those skilled in the art will be able to implement the application based on the description. In addition, the examples of the application involved in the following description are generally only examples of a part of the application, rather than all examples. Therefore, based on the examples in the application, all other examples obtained by those skilled in the art without making creative efforts shall fall within the protection scope of the application.

[0026] Example 1 (1) Synthesis of FPBA-NHS Take 2 g of 4-carboxyl-3-fluorophenylboronic acid (FPBA) and 2 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) into 50 mL of dimethylformamide and dissolve thoroughly on ice bath, then add 3 g of N-hydroxysuccinimide (NHS) for activation, react for 10 hours at room temperature, extract, spin dry to obtain FPBA-NHS solid.

[0027] (2) Grafting 4-carboxyl-3-fluorophenylboronic acid Take 100 mg of polylysine hydrochloride (MV is 2000-5000) and dissolve in 10 mL of PBS to obtain a polylysine solution; take 22 mg of the synthetic product FPBA-NHS of step (1) and dissolve in 15 mL of dimethyl sulfoxide, then add dropwise into the polylysine solution, control the pH to be 6.5, react for 20 hours at room temperature, after dialysis treatment with a molecular weight cutoff of 1 KDa, freeze-dry to obtain polylysine grafted with FPBA. The result is shown in Figure 1 . The grafting rate of FPBA is 13% calculated by nuclear magnetic peak area integration.

[0028] (3) Preparation of growth factor-loaded gel Take 10 mg of polylysine grafted with FPBA prepared in step (2), 1 mL of epidermal growth factor with a concentration of 100 ng / mL, and add to 5 mL of deionized water, adjust the pH to 7.4 with sodium hydroxide solution, place in a 4°C refrigerator for 10 min, then add hyaluronic acid, and add 2 g of EDC and 2 g of NHS for catalytic reaction, mix well, and crosslink at room temperature to obtain a gel.

[0029] Among them, the amount of hyaluronic acid added is according to the molar ratio of carboxyl in hyaluronic acid:amino group of polylysine grafted with FPBA = 1:0.5. The molar amount of amino group of polylysine grafted with FPBA is: 10 mg x ungrafted rate / polylysine monomer relative molecular weight, i.e. (10 mg x 87%) / (128 g / mol).

[0030] Example 2 The difference from Example 1 is only that the amount of FPBA-NHS taken in step (2) is 80 mg. The other steps are the same as Example 1. The specific operation is as follows: (1) Synthesis of FPBA-NHS The same as Example 1.

[0031] (2) Grafting 4-carboxyl-3-fluorophenylboronic acid Take 100 mg of polylysine hydrochloride (MV is 2000~5000) to be dissolved in 10 mL of PBS to obtain a polylysine solution; take 80 mg of the synthetic product FPBA-NHS of step (1) to be dissolved in 15 mL of dimethyl sulfoxide, then add the polylysine solution drop by drop, control the pH to be 6.5, react at room temperature for 20 hours, after dialysis treatment with a molecular weight cutoff of 1KDa, freeze-drying to obtain polylysine grafted with FPBA. According to the calculation of nuclear magnetic peak area integration, the grafting rate of FPBA is 29%.

[0032] (3) Preparation of growth factor-loaded gel Take the polylysine grafted with FPBA (10 mg, grafting rate is 29%) prepared in step (2) and epidermal growth factor (1 mL) with a concentration of 100 ng / mL to be added to 5 mL of deionized water, adjust the pH to be 7.4 by using sodium hydroxide solution, put it into a 4°C refrigerator for 10 min, then add hyaluronic acid, and add 2 g of EDC and 2 g of NHS to catalyze the reaction, mix well, and cross-link at room temperature to obtain a gel. Among them, Example 3 The difference from example 1 is only that the amount of FPBA-NHS taken in step (2) is 110 mg. The other steps are the same as example 1. The specific operation is as follows: (1) Synthesis of FPBA-NHS The same as example 1.

[0033] (2) Grafting 4-carboxyl-3-fluorophenylboronic acid Take 100 mg of polylysine hydrochloride (MV is 2000~5000) to be dissolved in 10 mL of PBS to obtain a polylysine solution; take 100 mg of the synthetic product FPBA-NHS of step (1) to be dissolved in 15 mL of dimethyl sulfoxide, then add the polylysine solution drop by drop, control the pH to be 6.5, react at room temperature for 20 hours, after dialysis treatment with a molecular weight cutoff of 1KDa, freeze-drying to obtain polylysine grafted with FPBA. According to the calculation of nuclear magnetic peak area integration, the grafting rate of FPBA is 40%.

[0034] (3) Preparation of growth factor-loaded gel Grafting FPBA polylysine (10 mg, grafting rate 40%) prepared in step (2) and epidermal growth factor (1 mL) with a concentration of 100 ng / mL were added to 5 mL of deionized water, and sodium hydroxide solution was used to adjust pH to 7.4, and then placed in a 4°C refrigerator for 10 min, then hyaluronic acid was added, and 2 g of EDC and 2 g of NHS were added to catalyze the reaction, and then mixed, and then crosslinked at room temperature to obtain a gel. The amount of hyaluronic acid added was weighed according to the molar ratio of carboxyl in hyaluronic acid:amino in grafting FPBA polylysine = 1:0.5.

[0035] Example 4 The difference from Example 1 is only that the amount of hyaluronic acid weighed in step (3) is according to the molar ratio of carboxyl in hyaluronic acid:amino in grafting FPBA polylysine = 1:0.1. The other steps are the same as Example 1.

[0036] Example 5 The difference from Example 1 is only that the amount of hyaluronic acid weighed in step (3) is according to the molar ratio of carboxyl in hyaluronic acid:amino in grafting FPBA polylysine = 1:1. The other steps are the same as Example 1.

[0037] Comparative Example 1 The difference from Example 1 is only that the amount of FPBA-NHS weighed in step (2) is 15 mg, and grafting FPBA polylysine is obtained, and the grafting rate is 8%. The other steps are the same as Example 1.

[0038] Comparative Example 2 The difference from Example 1 is only that the amount of FPBA-NHS weighed in step (2) is 180 mg, and grafting FPBA polylysine is obtained, and the grafting rate is 45%. The other steps are the same as Example 1.

[0039] Comparative Example 4 The difference from Example 1 is only that the amount of hyaluronic acid weighed in step (3) is according to the molar ratio of carboxyl in hyaluronic acid:amino in grafting FPBA polylysine = 1:0.08. The other steps are the same as Example 1.

[0040] Comparative Example 5 The difference from Example 1 is only that the amount of hyaluronic acid weighed in step (3) is according to the molar ratio of carboxyl in hyaluronic acid:amino in grafting FPBA polylysine = 1:1.1. The other steps are the same as Example 1.

[0041] Performance test 1. The gels obtained in Examples 1 to 5 and Comparative Examples 1 to 4 were subjected to bioadhesion strength test and Young's modulus test, and the results are shown in Table 1.

[0042] Table 1 Bioadhesion strength (kPa) Young's modulus (kPa) Example 1 9.3 7.7 Example 2 15.1 9.2 Example 3 13.4 8.0 Example 4 9.1 5.8 Example 5 7.8 7.9 Comparative Example 1 6.9 2.1 Comparative Example 2 5.7 3.6 Comparative Example 3 5.1 2.9 Comparative Example 4 6.1 2.6 As shown in the data in Table 1, the present application realizes the construction of a delivery system for growth factors by grafting FPBA onto polylysine, then cross-linking the grafted FPBA-polylysine with hyaluronic acid to construct a gel carrier framework, and loading growth factors in the gel carrier framework. The gel thus obtained has good mechanical properties and good bioadhesion.

[0043] As shown by the comparative analysis of Comparative Examples 1 and 2 and Examples 1 to 3, the ratio of the amino groups of the FPBA-grafted polylysine to the carboxyl groups of hyaluronic acid has an effect on the bioadhesion and mechanical properties of the gel. When the molar ratio of the amino groups of the FPBA-grafted polylysine to the carboxyl groups of hyaluronic acid is 0.5:1, the gel has the best bioadhesion.

[0044] As shown by the comparative analysis of Comparative Examples 3 and 4 and Examples 1, 4 and 5, the grafting rate of the FPBA-grafted polylysine has an effect on the bioadhesion and mechanical properties of the gel. When the grafting rate of the FPBA-grafted polylysine is 13% to 40%, the gel has a larger Young's modulus and better mechanical properties. The reason is that when the grafting rate is high, the molecular chains of the FPBA affect the cross-linking of the hyaluronic acid and polylysine, thereby affecting the modulus of the gel; when the grafting rate is high, the excessive molecular chains of the FPBA in the cross-linking product of the gel affect the function of the polylysine in the gel, thereby affecting the adhesion of the gel. When the grafting rate is low, it directly leads to insufficient strength of the gel, because the cross-linking of the molecular chains of the FPBA in the gel framework is beneficial to the improvement of the mechanical properties.

[0045] 2. In order to verify the treatment effect of the gel prepared in the present application on wounds, a wound treatment experiment was conducted on diabetic rats, and the treatment effect is shown in Table 2. Figure 2The method was as follows: A diabetic rat model was established by injecting streptozotocin (STZ) into rats. A 10mm biopsy needle was then used to create a wound on the rat's back. The rats were divided into three groups: a blank control group, a blank gel group, and an example group. At the start of the experiment (day 0), 180mg of the gel prepared in Example 2 was applied to the wound of rats in the example group. The wound condition was photographed and recorded on days 0, 2, 6, and 8, and this group was designated as the Hydrogel+EGF group. 180mg of the gel without EGF, prepared according to Example 2, was applied to the wound of rats in the blank gel group. The wound condition was photographed and recorded on days 0, 2, 6, and 8, and this group was designated as the Hydrogel group. The wound of rats in the blank control group was left untreated and was designated as the Control group.

[0046] Depend on Figure 2 It was observed that the rat wounds treated with the EGF-loaded gel prepared in Example 2 scabbed over on day 8, indicating good healing. This demonstrates that the gel prepared in this invention has a good effect on promoting wound healing.

[0047] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A sustained-release gel formulation that responds to glucose-releasing growth factor, characterized in that: Its raw materials include the following components: growth factors, polylysine grafted with 4-carboxy-3-fluorophenylboronic acid, and hyaluronic acid; The molar ratio of the amino group of the polylysine grafted with 4-carboxy-3-fluorophenylboronic acid to the carboxyl group of hyaluronic acid is (0.1~1):

1.

2. The sustained-release gel formulation of glucose-releasing growth factor as described in claim 1, characterized in that: The molar ratio of the amino group of the polylysine grafted with 4-carboxy-3-fluorophenylboronic acid to the carboxyl group of hyaluronic acid is 0.5:

1.

3. The sustained-release gel formulation of glucose-releasing growth factor as described in claim 1, characterized in that: The grafting rate of polylysine grafted with 4-carboxy-3-fluorophenylboronic acid is 13%~40%.

4. The sustained-release gel formulation of glucose-releasing growth factor as described in claim 1, characterized in that: The growth factor is one or more of insulin, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, and keratinocyte growth factor.

5. The sustained-release gel formulation of glucose-releasing growth factor as described in claim 1, characterized in that: The mass ratio of growth factor to hyaluronic acid is 0.01~0.1:

1.

6. A method for preparing a sustained-release gel formulation that responds to glucose-releasing growth factor, characterized in that: Includes the following steps: (1) Mix 4-carboxy-3-fluorophenylboronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide, add N-hydroxysuccinimide, and react for 8-12 hours to obtain FPBA-NHS; (2) Add FPBA-NHS to polylysine solution, adjust pH to 6-7, react for 12-24 hours to obtain FPBA-grafted polylysine; (3) Mix the growth factor with the grafted FPBA polylysine, add hyaluronic acid, adjust the pH to 7-8, and form a gel.

7. The preparation method according to claim 6, characterized in that: In step (1), the molar ratio of 4-carboxy-3-fluorophenylboronic acid to N-hydroxysuccinimide is 1:0.8~1.

2.

8. The preparation method according to claim 6, characterized in that: In step (2), the mass ratio of FPBA-NHS to polylysine is 1:5~10.

9. The use of the sustained-release gel formulation according to any one of claims 1 to 5, or the sustained-release gel formulation prepared by the preparation method according to any one of claims 6 to 8, in the preparation of medical dressings.

10. The use of the sustained-release gel formulation according to any one of claims 1 to 5, or the sustained-release gel formulation prepared by the preparation method according to any one of claims 6 to 8, in the preparation of a medicament for promoting the healing of diabetic wounds.

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