Collagen gel delivery system integrated with polypeptide drug as well as preparation method and application of collagen gel delivery system

Abstract

The invention relates to the technical field of biological medicine, in particular to a collagen gel delivery system integrated with polypeptide medicine and a preparation method and application thereof.The collagen gel delivery system is characterized in that collagen serves as a basic gel matrix, covalent cross-linking is conducted through 4-arm-PEG-NHS, then active polypeptide is connected into a gel network through amido bonds, and the collagen gel delivery system integrated with the polypeptide medicine is obtained. The active polypeptide is an antibacterial peptide and / or an MMP-9 cyclopeptide inhibitor. The collagen gel delivery system is expected to realize the efficient and ordered healing promotion effect on the wound infected by diabetes mellitus through a multi-target synergistic mechanism such as anti-infection, anti-inflammation, collagen degradation inhibition and collagen synthesis promotion, and provides a new idea and a new option for clinical response.

Description

A collagen gel delivery system for integrated peptide drugs, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a collagen gel delivery system for integrated polypeptide drugs, its preparation method, and its application. Background Technology

[0002] The healing of diabetic wounds has long faced severe challenges. The key lies in the complex pathological microenvironment formed by persistent high blood sugar, susceptibility to infection, excessive activation of matrix metalloproteinases (MMPs, especially MMP-9), and chronic inflammation, which seriously hinders the tissue repair process.

[0003] Chinese invention patent CN117180181B discloses a glucose MMP-9 matrix enzyme dual-response layered drug release gel for diabetic wound healing, its preparation method, and its application, belonging to the field of pharmaceutical products. The wound healing gel of this invention is obtained by amidation and boron ester bond reactions of 4-arm-PEG-NHS, 3-aminophenylboronic acid, and PVA, with the addition of anti-inflammatory or repairing drugs. Short-term and long-term drug release gels are prepared by controlling the molecular weight and ratio of the raw materials. The gel product of this invention is designed for the complex and unique environment of diabetic wounds, achieving intelligent glucose MMP-9 matrix enzyme dual response to avoid indiscriminate drug release. Simultaneously, the functional drug release of the gel is controlled according to different stages of wound development, achieving comprehensive wound healing through integrated anti-inflammatory and repair mechanisms.

[0004] Although collagen has excellent biocompatibility and tissue regeneration ability, its inherent mechanical properties are insufficient and it cannot actively regulate the pathological microenvironment, which seriously limits its application value in diabetic wounds. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide an integrated collagen gel delivery system for polypeptide drugs, its preparation method and application.

[0006] The objective of this invention is achieved through the following technical solution: a collagen gel delivery system for integrated peptide drugs, wherein the collagen gel delivery system uses collagen as a base gel matrix, is covalently cross-linked by 4-arm-PEG-NHS, and then an active peptide is linked to the gel network by amide bonds, wherein the active peptide is an antimicrobial peptide and / or an MMP-9 cyclic peptide inhibitor.

[0007] Preferably, the mass ratio of collagen to 4-arm-PEG-NHS is 1:0.5-5.0, and the mass ratio of collagen to active peptides is 1:0.01-0.10.

[0008] Preferably, the active polypeptide is composed of an antimicrobial peptide and an MMP-9 cyclic peptide inhibitor in a mass ratio of 1-10:1.

[0009] Preferably, the structural formula of the antimicrobial peptide (Tet-213) is as follows: .

[0010] Preferably, the structural formula of the MMP-9 cyclic peptide inhibitor (Mi) is: .

[0011] The structure of the 4-arm-PEG-NHS is shown as follows: .

[0012] 4-arm-PEG-NHS (four-arm polyethylene glycol-active ester) is a multi-arm star-shaped polyethylene glycol derivative. Its molecular structure contains a central polyethylene glycol backbone and four terminal N-hydroxysuccinimide (NHS) groups, giving it multiple reaction sites and enabling it to participate efficiently in chemical cross-linking reactions.

[0013] Another objective of the present invention is achieved by the following technical solution: a method for preparing a collagen gel delivery system for integrated polypeptide drugs, comprising the following steps: (1) dissolving type I collagen lyophilized sponge extracted from bovine Achilles tendon in pure aqueous solution, stirring magnetically overnight to prepare a collagen solution; dissolving 4-arm-PEG-NHS powder in sterile water to prepare a 4-arm-PEG-NHS solution; dissolving active polypeptide in sterile ultrapure water to prepare an active polypeptide solution, and storing it at -20℃ after dispensing; the active polypeptide is an antimicrobial peptide and / or an MMP-9 cyclic peptide inhibitor; (2) taking the corresponding volumes of collagen solution, active polypeptide solution and 4-arm-PEG-NHS solution according to the mass ratio and mixing them evenly to obtain a mixture, placing the mixture in a constant temperature box at 37℃, and after 4-8 min, the hydrogel crosslinks from the solution to a gel.

[0014] Preferably, in step (1), the mass concentration of the collagen solution is 5-15 mg / mL, the mass concentration of the 4-arm-PEG-NHS solution is 5-50 mg / mL, and the mass concentration of the active polypeptide solution is 0.1-10.0 mg / mL.

[0015] Preferably, in step (1), the active polypeptide is composed of an antimicrobial peptide and an MMP-9 cyclic peptide inhibitor in a mass ratio of 1-10:1, the mass concentration of the antimicrobial peptide solution is 0.1-2.0 mg / mL, and the mass concentration of the MMP-9 cyclic peptide inhibitor solution is 1-10 mg / mL.

[0016] Preferably, in step (2), the mixing step is as follows: take the corresponding volume of collagen solution into a centrifuge tube according to the mass ratio, add the active peptide solution, and gently vortex to mix; then, quickly add 4-arm-PEG-NHS solution, and immediately use a pipette to repeatedly blow and mix 10-30 times to ensure thorough mixing.

[0017] Another object of the present invention is achieved through the following technical solution: the application of a collagen gel delivery system integrating polypeptide drugs in the preparation of biological products that promote the healing of diabetic infected wounds. More specifically, the application of the collagen gel delivery system in the preparation of drugs or tissue repair materials such as dressings that promote the healing of diabetic infected wounds.

[0018] The beneficial effects of this invention are as follows: The collagen gel delivery system of this invention uses collagen as the base gel matrix and covalently cross-links it with four-arm polyethylene glycol (4-arm-PEG-NHS) modified with terminal active esters to enhance the mechanical properties of the gel. In addition, two highly targeted active peptides (broad-spectrum antimicrobial peptide Tet-213 and MMP-9 highly specific inhibitory cyclic peptide Mi) are "chained" in the gel network through amide bonds to achieve dual objectives: 1) Optimize the release behavior of peptide drugs, significantly improving their stability and retention time in the diabetic wound environment; 2) The two peptides precisely intervene in two key pathological links of diabetic wounds, namely, Tet-213 effectively controls infection, while Mi actively regulates MMP9-mediated persistent inflammation and excessive degradation of the extracellular matrix, thereby jointly creating an ideal pathological microenvironment conducive to tissue regeneration for collagen.

[0019] This invention's collagen gel delivery system integrates clinically validated matrix dressings (collagen, PEG) with emerging peptide drugs through rational design, constructing a multifunctional dressing with clearly defined functions and synergistic effects among its components. It achieves a "source-source and cost-saving" approach to tissue regeneration through "collagen replenishment and degradation inhibition," actively and continuously reshaping the pathological microenvironment. This collagen gel delivery system is expected to achieve efficient and orderly healing of diabetic infected wounds through a multi-target synergistic mechanism involving anti-infection, anti-inflammation, inhibition of collagen degradation, and promotion of collagen synthesis, providing new ideas and options for clinical treatment. Attached Figure Description

[0020] Figure 1 shows the HPLC test results of the two active peptides of the present invention; where A is the HPLC test result of Tet-213 and B is the HPLC test result of Mi.

[0021] Figure 2 shows the MS results of the two active peptides of the present invention; where A is the MS result of Tet-213 and B is the MS result of Mi.

[0022] Figure 3 shows the MALDI-MS results of the 4-arm-PEG-NHS of this invention.

[0023] Figure 4 shows the 4-arm-PEG-NHS of the present invention. 1 HNMR results.

[0024] Figure 5 is a comparison of the T&Mi@CP hydrogel of the present invention before and after gelation; where A is before hydrogelation and B is after hydrogelation.

[0025] Figure 6 is a schematic diagram of the adhesion and deformation ability of the T&Mi@CP hydrogel of the present invention to pigskin.

[0026] Figure 7 shows the antibacterial experiment of the hydrogels of different components of the present invention; where A is a representative colony image of E. coli from different groups, and B is a representative colony image of S. aureus from different groups; where Control is the control group without hydrogel treatment, CP gel is a hydrogel cross-linked with collagen and 4-arm-PEG-NHS, Mi@CP gel is a hydrogel cross-linked with collagen, 4-arm-PEG-NHS and Mi, T@CP gel is a hydrogel cross-linked with collagen, 4-arm-PEG-NHS and Tet 213, and T&Mi@CP gel is a hydrogel cross-linked with collagen, 4-arm-PEG-NHS, Mi and Tet 213.

[0027] Figure 8 shows cell experiments of hydrogels with different components of the present invention; where A is the CCK8 experiment of hydrogels with different components, and B is the hemolysis experiment of fresh red blood cells by hydrogels with different components; where Control is the control group without hydrogel treatment, CPgel is a hydrogel cross-linked with collagen and 4-arm-PEG-NHS, Mi@CP gel is a hydrogel cross-linked with collagen, 4-arm-PEG-NHS and Mi, T@CP gel is a hydrogel cross-linked with collagen, 4-arm-PEG-NHS and Tet 213, and T&Mi@CP gel is a hydrogel cross-linked with collagen, 4-arm-PEG-NHS, Mi and Tet 213. Detailed Implementation

[0028] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the embodiments and figures 1-8. The content mentioned in the embodiments is not intended to limit the present invention.

[0029] Example 1 Preparation of Collagen Gel Delivery System (1) Type I collagen lyophilized sponge extracted from bovine Achilles tendon was dissolved in pure water and magnetically stirred overnight to prepare a stock solution with a concentration of 10 mg / mL. 4-arm-PEG-NHS powder was dissolved in sterile water to prepare a stock solution with a concentration of 27.5 mg / mL. Antimicrobial peptide (Tet-213) and MMP-9 inhibitor (Mi) were dissolved in sterile ultrapure water to prepare Tet-213 stock solution with a concentration of 1 mg / mL and Mi stock solution with a concentration of 5 mg / mL, respectively. After aliquoting, the solutions were stored at -20°C.

[0030] (2) Take 1 mL of collagen solution into a centrifuge tube, add 50 µL of Tet-213 stock solution and 2 µL of Mi stock solution in sequence, and vortex gently to mix. Then, quickly add 1 mL of 4-arm-PEG-NHS solution, and immediately use a pipette to mix repeatedly at least 10 times to ensure thorough mixing. Place the mixture in a 37°C incubator. After 5 min, the hydrogel will crosslink from the solution to a gel.

[0031] Example 2 Preparation of Collagen Gel Delivery System (1) Type I collagen lyophilized sponge extracted from bovine Achilles tendon was dissolved in pure water and magnetically stirred overnight to prepare a stock solution with a concentration of 10 mg / mL. 4-arm-PEG-NHS powder was dissolved in sterile water to prepare a stock solution with a concentration of 5 mg / mL. Antimicrobial peptide (Tet-213) and MMP-9 inhibitor (Mi) were dissolved in sterile ultrapure water to prepare Tet-213 stock solution with a concentration of 1 mg / mL and Mi stock solution with a concentration of 5 mg / mL, respectively. After aliquoting, the solutions were stored at -20°C.

[0032] (2) Take 1 mL of collagen solution into a centrifuge tube, add 50 µL of Tet-213 stock solution and 2 µL of Mi stock solution in sequence, and vortex gently to mix. Then, quickly add 1 mL of 4-arm-PEG-NHS solution, and immediately use a pipette to mix repeatedly at least 10 times to ensure thorough mixing. Place the mixture in a 37°C incubator. After 5 min, the hydrogel will crosslink from the solution to a gel.

[0033] Example 3 Preparation of Collagen Gel Delivery System (1) Type I collagen lyophilized sponge extracted from bovine Achilles tendon was dissolved in pure water and magnetically stirred overnight to prepare a stock solution with a concentration of 10 mg / mL. 4-arm-PEG-NHS powder was dissolved in sterile water to prepare a stock solution with a concentration of 50 mg / mL. Antimicrobial peptide (Tet-213) and MMP-9 inhibitor (Mi) were dissolved in sterile ultrapure water to prepare Tet-213 stock solution with a concentration of 1 mg / mL and Mi stock solution with a concentration of 5 mg / mL, respectively. After aliquoting, the solutions were stored at -20°C.

[0034] (2) Take 1 mL of collagen solution into a centrifuge tube, add 50 µL of Tet-213 stock solution and 2 µL of Mi stock solution in sequence, and vortex gently to mix. Then, quickly add 1 mL of 4-arm-PEG-NHS solution, and immediately use a pipette to mix repeatedly at least 10 times to ensure thorough mixing. Place the mixture in a 37°C incubator. After 5 min, the hydrogel will crosslink from the solution to a gel.

[0035] Example 4 Preparation of Collagen Gel Delivery System (1) Type I collagen lyophilized sponge extracted from bovine Achilles tendon was dissolved in pure water and magnetically stirred overnight to prepare a stock solution with a concentration of 10 mg / mL. 4-arm-PEG-NHS powder was dissolved in sterile water to prepare a stock solution with a concentration of 27.5 mg / mL. Antimicrobial peptide (Tet-213) was dissolved in sterile ultrapure water to prepare a Tet-213 stock solution with a concentration of 1 mg / mL, which was then aliquoted and stored at -20°C.

[0036] (2) Take 1 mL of collagen solution into a centrifuge tube, add 50 µL of Tet-213 stock solution, and vortex gently to mix. Then, quickly add 1 mL of 4-arm-PEG-NHS solution, and immediately use a pipette to mix repeatedly at least 10 times to ensure thorough mixing. Place the mixture in a 37°C incubator. After 5 min, the hydrogel will crosslink from solution to gel.

[0037] Example 5 Preparation of Collagen Gel Delivery System (1) Type I collagen lyophilized sponge extracted from bovine Achilles tendon was dissolved in pure water and magnetically stirred overnight to prepare a stock solution with a concentration of 10 mg / mL. 4-arm-PEG-NHS powder was dissolved in sterile water to prepare a stock solution with a concentration of 27.5 mg / mL. MMP-9 inhibitor (Mi) was dissolved in sterile ultrapure water to prepare a Mi stock solution with a concentration of 5 mg / mL, aliquoted, and stored at -20°C.

[0038] (2) Take 1 mL of collagen solution into a centrifuge tube, add 2 µL of Mi stock solution, and vortex gently to mix. Then, quickly add 1 mL of 4-arm-PEG-NHS solution, and immediately use a pipette to mix repeatedly at least 10 times to ensure thorough mixing. Place the mixture in a 37°C incubator. After 5 min, the hydrogel will crosslink from the solution to a gel.

[0039] The collagen gel delivery system of this invention uses collagen as the base gel matrix and covalently cross-links it with four-arm polyethylene glycol (4-arm-PEG-NHS) modified with terminal active esters to enhance the mechanical properties of the gel. Furthermore, two highly targeted active peptides (broad-spectrum antimicrobial peptide Tet-213 and MMP-9 highly specific inhibitory cyclic peptide Mi) are "chained" in the gel network via amide bonds to achieve dual objectives: 1) optimizing the release behavior of the peptide drugs, significantly improving their stability and retention time in the diabetic wound environment; 2) the two peptides precisely intervene in two key pathological aspects of diabetic wounds, namely, Tet-213 effectively controls infection, while Mi actively regulates MMP9-mediated persistent inflammation and excessive degradation of the extracellular matrix, thereby jointly creating an ideal pathological microenvironment conducive to tissue regeneration for collagen.

[0040] Comparative Example 1: Type I collagen lyophilized sponge extracted from bovine Achilles tendon was dissolved in pure aqueous solution and magnetically stirred overnight to prepare a stock solution with a concentration of 10 mg / mL. 4-arm-PEG-NHS powder was dissolved in sterile water to prepare a stock solution with a concentration of 27.5 mg / mL.

[0041] Take 1 mL of collagen solution into a centrifuge tube, then quickly add 1 mL of 4-arm-PEG-NHS solution. Immediately use a pipette to mix repeatedly at least 10 times to ensure thorough mixing. Place the mixture in a 37°C incubator. After 5 minutes, the hydrogel will crosslink from the solution to a gel.

[0042] The hydrogels prepared in Examples 1-5 and Comparative Example 1 were tested, and the results are as follows: Figures 1A-1B show the HPLC test results for Tet-213 and Mi, respectively. These data indicate that the peptides have high purity and are suitable for in vitro and in vivo experiments.

[0043] Figure 2A shows the MS results of Tet-213 (Theoretical: 1590.92; Observed: 1590.80), and Figure 2B shows the MS results of Mi (Theoretical: 1166.36; Observed: 1166.40). The above results basically indicate that the peptides Tet-213 and Mi have been successfully synthesized.

[0044] Figure 3 shows the MALDI-MS results for 4-arm-PEG-NHS, indicating a molecular weight of approximately 10 kDa. Figure 4 shows the... 1 The HNMR results indicate that 4-arm-PEG-NHS has been successfully synthesized.

[0045] Figures 5A-5B show a comparison of the T&Mi@CP hydrogel prepared in Example 1 before and after gelation. The results show that the hydrogel was viscous and fluid before gelation, providing conditions for injectability. After gelation, the hydrogel adhered to the bottom of the vial and did not detach or change shape with the tilt angle, indicating that the hydrogel has in-situ molding properties and good adhesion.

[0046] Figure 6 shows a schematic diagram of the adhesion and deformation ability of the T&Mi@CP hydrogel prepared in Example 1 on pigskin. The results show that the hydrogel has good adhesion to the surface of pigskin and can conform to the deformation of the pigskin, indicating its adaptability to skin wounds.

[0047] Figures 7A-7B show representative colony images of E. coli and S. aureus obtained from the hydrogels prepared in Examples 1, 4-5, and Comparative Example 1, respectively. The experiments demonstrate that Tet-213 exhibits good antibacterial activity against both Gram-positive and Gram-negative bacteria.

[0048] Figure 8A shows the CCK8 experiments using hydrogels prepared in Examples 1, 4-5, and Comparative Example 1. The results show the cytotoxic effects of different hydrogel components on L929 cells. After co-culturing the cells with the hydrogel extract for 24 hours, CCK8 reagent was added, and the OD value was detected at 450 nm using a microplate reader. The calculation formula is: Cell viability = OD value of the drug group / OD value of the control group * 100%. Compared with the control group, there was no significant difference in cell viability, indicating that the hydrogels of different components were not toxic to cells.

[0049] Figure 8B shows the hemolysis experiment of fresh red blood cells on the hydrogels prepared in Examples 1, 4-5, and Comparative Example 1. 1% TX-100 was used as a positive control, and PBS solution as a negative control. After co-incubating the hydrogel extract with red blood cells, the cells were centrifuged, and the OD value of the supernatant was measured at 540 nm using a microplate reader. The hemolysis rate was calculated as (OD value of experimental group - OD value of negative group) / (OD value of experimental group - OD value of positive group) * 100%. The hemolysis rates of the hydrogels of different components were all below 5%, indicating good biocompatibility.

[0050] The collagen gel delivery system of this invention is expected to achieve efficient and orderly healing of diabetic infected wounds through a multi-target synergistic mechanism that includes anti-infection, anti-inflammation, inhibition of collagen degradation, and promotion of collagen synthesis, providing new ideas and options for clinical treatment.

[0051] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A collagen gel delivery system for integrating peptide drugs, characterized in that: This collagen gel delivery system uses collagen as the base gel matrix, covalently crosslinked with 4-arm-PEG-NHS, and then connects active peptides to the gel network via amide bonds. The active peptides are antimicrobial peptides and / or MMP-9 cyclic peptide inhibitors.

2. The collagen gel delivery system for an integrated polypeptide drug according to claim 1, characterized in that: The mass ratio of collagen to 4-arm-PEG-NHS is 1:0.5-5.0, and the mass ratio of collagen to active peptides is 1:0.01-0.

10.

3. The collagen gel delivery system for an integrated polypeptide drug according to claim 1, characterized in that: The active polypeptide is composed of an antimicrobial peptide and an MMP-9 cyclic peptide inhibitor in a mass ratio of 1-10:

1.

4. The collagen gel delivery system for an integrated polypeptide drug according to claim 1, characterized in that: The structural formula of the antimicrobial peptide is: 。 5. The collagen gel delivery system for an integrated polypeptide drug according to claim 1, characterized in that: The structural formula of the MMP-9 cyclic peptide inhibitor is as follows: 。 6. A method for preparing an integrated peptide drug collagen gel delivery system according to any one of claims 1-5, characterized in that: The steps include: (1) Dissolving type I collagen lyophilized sponge extracted from bovine Achilles tendon in pure water, stirring magnetically overnight to prepare collagen solution; dissolving 4-arm-PEG-NHS powder in sterile water to prepare 4-arm-PEG-NHS solution; dissolving active peptide in sterile ultrapure water to prepare active peptide solution, and storing it at -20℃ after dispensing; the active peptide is an antimicrobial peptide and / or MMP-9 cyclic peptide inhibitor; (2) taking the corresponding volume of collagen solution, active peptide solution and 4-arm-PEG-NHS solution according to the mass ratio and mixing them evenly to obtain a mixture. The mixture is placed in a constant temperature box at 37℃. After 4-8 minutes, the hydrogel crosslinks from the solution to a gel.

7. The method for preparing an integrated peptide drug collagen gel delivery system according to claim 6, characterized in that: In step (1), the mass concentration of the collagen solution is 5-15 mg / mL, the mass concentration of the 4-arm-PEG-NHS solution is 5-50 mg / mL, and the mass concentration of the active polypeptide solution is 0.1-10.0 mg / mL.

8. The method for preparing an integrated peptide drug collagen gel delivery system according to claim 6, characterized in that: In step (1), the active polypeptide is composed of an antimicrobial peptide and an MMP-9 cyclic peptide inhibitor in a mass ratio of 1-10:1, the mass concentration of the antimicrobial peptide solution is 0.1-2.0 mg / mL, and the mass concentration of the MMP-9 cyclic peptide inhibitor solution is 1-10 mg / mL.

9. The method for preparing an integrated peptide drug collagen gel delivery system according to claim 6, characterized in that: In step (2), the mixing step is as follows: take the corresponding volume of collagen solution into a centrifuge tube according to the mass ratio, add the active peptide solution, and vortex gently to mix; then, quickly add 4-arm-PEG-NHS solution, and immediately use a pipette to repeatedly blow and mix 10-30 times to ensure thorough mixing.

10. The application of the collagen gel delivery system for an integrated polypeptide drug as described in any one of claims 1-5 in the preparation of a biological product that promotes the healing of diabetic infected wounds.

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