Polyamino acid and preparation method therefor, hydrogel, and chronic wound dressing

Abstract

The present invention relates to the technical field of hydrogels. Disclosed are a polyamino acid and a preparation method therefor, a hydrogel, and a chronic wound dressing. The polyamino acid has a structure represented by formula 1, wherein m, n, x, and y are the degree of polymerization, 30≤m≤60, 10≤n≤40, 1≤x≤100, 1≤y≤100, R1, R2, and R3 are independently selected from C1-C10 alkyl, and R4 is selected from OH, benzyloxy, amino, and C1-C4alkoxy. The polyamino acid is mixed with water to be converted into a temperature-sensitive hydrogel, the temperature-sensitive hydrogel is loaded with linagliptin and curcumin to obtain a drug-loaded temperature-sensitive hydrogel, and the drug-loaded temperature-sensitive hydrogel can be used as a chronic wound dressing for the treatment of a chronic wound and exhibits a good therapeutic effect. In addition, the drug-loaded temperature-sensitive hydrogel of the present invention exhibits few side effects on skin wounds because of the inclusion of fragments and has great application value.

Description

A polyamino acid, its preparation method, hydrogel, and chronic wound dressing.

[0001] This application claims priority to Chinese Patent Application No. 202410919575.7, filed on July 10, 2024, entitled "A Polyamino Acid and its Preparation Method and Hydrogel, Chronic Wound Dressing", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of hydrogel technology, and in particular to a polyamino acid, its preparation method, hydrogel, and chronic wound dressing. Background Technology

[0003] Traditional wound dressings, including gauze, cotton wool, and polymer bandages, often fail to provide a moist environment at the wound interface. Removal of these dressings inevitably damages regenerating tissue. To further promote chronic wound healing, water-rich, three-dimensional hydrogels are widely used to construct platforms for controlling drug delivery. The application of these new dressings has significantly accelerated the regeneration of damaged skin. However, due to the complex microenvironment of the skin wound, chronic wounds often contain substantial exudate and necrotic tissue. This can cause dressing breakage or detachment, hindering local medication application and delaying wound healing. Furthermore, persistent local inflammation is a major cause of chronic wound formation. Therefore, the choice of drug carrier in wound dressings and the selection of the drugs significantly impact treatment efficacy.

[0004] In recent years, hydrogel wound dressings have attracted much attention. Hydrogel wound dressings are made of a mixture of hydrogel, synthetic rubber and adhesives, and have the effects of moisturizing, promoting wound healing, inhibiting scar formation, fighting infection and promoting blood circulation.

[0005] Specifically: 1) The hydrogel and adhesives in hydrogel wound dressings form a protective film on the wound surface, reducing skin moisture evaporation and providing a moisturizing effect; 2) Hydrogel wound dressings provide a moist environment for wound healing, promoting epithelial cell growth and accelerating wound healing; 3) Hydrogel wound dressings have adhesiveness and compliance, which can inhibit connective tissue proliferation and lighten scar formation; 4) The hydrogel in hydrogel wound dressings can also adsorb bacteria on the skin surface and inhibit bacterial growth; 5) The synthetic rubber and hydrogel components in the dressing can also promote subcutaneous vasodilation and maintain normal local blood supply. However, there is currently limited research and development on drug-loadable, safe, and easily degradable hydrogels. Therefore, researching and developing a novel hydrogel as a carrier for preparing hydrogel wound dressings is of great significance.

[0006] Current research indicates that T cells and other specific immune modulators play a crucial role in the treatment of chronic wounds. CD26, also known as dipeptidyl peptidase-4 (DPP-4), is a multifunctional T cell surface glycoprotein. It is an activating antigen and co-stimulatory molecule on the T cell surface. CD26 plays an extremely important role in chronic inflammatory diseases such as rheumatoid arthritis. Therefore, inhibiting CD26 activity is key to addressing chronic inflammatory diseases like rheumatoid arthritis. In vitro cell experiments have shown that CD26 inhibitors effectively interfere with CD26 expression, dimerization, and cell signal transduction, completely inhibiting interleukin-6 expression in peripheral blood mononuclear cells and exhibiting immunosuppressive effects on the differentiation of Th1, Th17, and Th2 lymphocytes. There are various types of CD26 inhibitors, clinically referred to as DPP-4 inhibitors. Compared to other inhibitors, linagliptin (Lin) has a lower dissociation rate from the DPP-4 enzyme, making it more effective than other DPP-4 inhibitors and showing good therapeutic efficacy even at low doses.

[0007] Curcumin (CUR) is a natural compound that has good anti-inflammatory and anti-cancer properties as an immunosuppressant. It is often used for lowering blood lipids, anti-tumor, anti-inflammatory, choleretic, and antioxidant purposes. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a polyamino acid, a method for preparing the same, a hydrogel, and a dressing for chronic wounds. The polyamino acid can be converted into a thermosensitive hydrogel, and the drug-loaded hydrogel obtained by loading drugs onto it can be used as a dressing to promote the healing of chronic wounds.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a polyamino acid having the structure shown in Formula 1:

[0011] Where m, n, x, and y represent the degree of polymerization, 30≤m≤60, 10≤n≤40, 1≤x≤100, and 1≤y≤100; preferably, R1, R2, and R3 are independently selected from C1 to C2. 10 Alkyl groups.

[0012] The C1~C 10 The alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.

[0013] Preferably, R4 is selected from OH, benzyloxy, amino, and C1-C4 alkoxy.

[0014] More preferably in this invention, the values ​​of 40≤m≤50, 20≤n≤30, 1≤x≤10, and 1≤y≤10 are specified.

[0015] More preferably, R1, R2, and R3 are independently selected from C1 to C6 alkyl groups.

[0016] The C1 to C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, and hexyl.

[0017] More preferably, R4 is selected from OH or benzyloxy group.

[0018] In a further preferred embodiment of the present invention, m is 45, n is 28 or 30, and x+y is 5 or 9.

[0019] More preferably, R1, R2, and R3 are independently selected from methyl or ethyl.

[0020] More preferably, the R4 is selected from OH.

[0021] Preferably, in this invention, R1, R2, and R3 are all selected from methyl groups, and R4 is selected from OH groups;

[0022] Alternatively, R1, R2, and R3 are all selected from methyl groups, and R4 is selected from benzyloxy groups;

[0023] Alternatively, R1, R2, and R3 may all be selected from methyl groups, and R4 may be selected from amino groups.

[0024] In some specific embodiments of the present invention, the polyamino acid has the following structure:

[0025] In all polyamino acid structural formulas of this invention, those without a degree of polymerization value outside the parentheses indicate that the degree of polymerization of the polymeric segment is 1.

[0026] The present invention also provides a method for preparing the above-mentioned polyamino acid, comprising the following steps:

[0027] 1) The polyamino acid shown in Formula 2 and BLA-NCA are subjected to ring-opening polymerization to obtain the polyamino acid shown in Formula 3;

[0028] 2) After the polyamino acid shown in Formula 3 is reacted with acid to remove the benzyl group, it is then reacted with EDC and NHS to obtain the polyamino acid shown in Formula 1.

[0029] Where m, n, x, and y are the degree of aggregation, 30≤m≤60, 10≤n≤40, 1≤x≤100, and 1≤y≤100;

[0030] Preferably, R1, R2, R3, and R5 are independently selected from C1 to C2. 10 Alkyl groups;

[0031] Preferably, R4 is selected from OH, benzyloxy, amino, and C1-C4 alkoxy.

[0032] The more preferred and further preferred ranges of m, n, R1, R2, R3, R4, and R5 are the same as those described above, and will not be repeated here.

[0033] Preferably, in step 2) of the present invention, the addition of EDC is used to activate the carboxyl group, making it easier to react with NHS to generate N-hydroxysuccinimide (NHS ester) with affinity.

[0034] Preferably, the polyamino acid of Formula 2 is obtained by ring-opening polymerization of the polyamino acid of Formula 4 and the compound of Formula 5.

[0035] The more preferred and further preferred ranges of m, R1, and R2 are the same as those described above, and will not be repeated here.

[0036] Preferably, in step 1), the molar ratio of the polyamino acid and BLA-NCA shown in Formula 2 is 1:(5-15); more preferably, it is 1:(9-10). In some specific embodiments of the present invention, the molar ratio is 1:9.10.

[0037] Preferably, in step 2), the molar ratio of the polyamino acid and acid shown in Formula 3 is 1:(500-2000); more preferably, 1:(1000-1500). In some specific embodiments of the present invention, the molar ratio is 1:1210.

[0038] Preferably, in step 2), the molar ratio of the polyamino acid and NHS shown in Formula 3 is 1:(10-15); more preferably, 1:(12-13). In some specific embodiments of the present invention, the molar ratio is 1:12.3.

[0039] Preferably, the acid in step 2) is selected from one or more of hydrobromic acid, acetic acid, hydrochloric acid, and anhydrous hydrofluoric acid.

[0040] The present invention also provides a thermosensitive hydrogel comprising the above-mentioned polyamino acid or the polyamino acid prepared by the above-mentioned preparation method, and water.

[0041] The conversion temperature of the thermosensitive hydrogel is related to the concentration of the polyamino acid. In some specific embodiments of the present invention, the sol-hydrogel phase diagram and rheological diagram of the thermosensitive hydrogel are provided to prove the above conclusion.

[0042] The present invention also provides a drug-loaded thermosensitive hydrogel, comprising the above-described thermosensitive hydrogel and a drug loaded in the thermosensitive hydrogel.

[0043] The present invention also provides a chronic wound dressing comprising the above-described thermosensitive hydrogel and linagliptin and curcumin loaded in the thermosensitive hydrogel.

[0044] Preferably, the linagliptin content in the chronic wound dressing of this invention is 1 wt% to 8 wt%; more preferably, it is 2.5 to 5.5 wt%. In some specific embodiments of this invention, it is preferably 4 wt%.

[0045] Preferably, the curcumin content in the chronic wound dressing is 1 wt% to 8 wt%; more preferably, it is 2.5 to 5.5 wt%. In some specific embodiments of the present invention, it is preferably 4 wt%.

[0046] This invention uses the aforementioned thermosensitive hydrogel as a drug carrier to load the drugs linagliptin and curcumin, thus preparing a chronic wound dressing (Gel-NHS). CUR+LIN ).

[0047] Preferably, the chronic wound dressing (Gel-NHS) CUR+LIN In application, it is obtained by mixing and converting the aqueous solution of the polyamino acid described in this invention, linagliptin, and curcumin. The chronic wound dressing (Gel-NHS) CUR+LIN The synergistic effect of linagliptin and curcumin in promoting the healing of chronic wounds is superior to that of the thermosensitive hydrogel described in this invention, which carries linagliptin alone or curcumin alone.

[0048] Compared with the prior art, the polyamino acid provided by the present invention has the structure shown in Formula 1, wherein m, n, x, and y are the degrees of polymerization, 30≤m≤60, 10≤n≤40, 1≤x≤100, and 1≤y≤100. R1, R2, and R3 are independently selected from C1 to C2. 10 The alkyl group, R4, is selected from OH, benzyloxy, amino, and C1-C4 alkoxy groups. The polyamino acid is mixed with water to form a thermosensitive hydrogel. The thermosensitive hydrogel is loaded with linagliptin and curcumin to obtain a drug-loaded thermosensitive hydrogel. This drug-loaded thermosensitive hydrogel can be used as a dressing for chronic wounds, exhibiting good therapeutic effects. Furthermore, the drug-loaded thermosensitive hydrogel of this invention contains fragments, resulting in fewer side effects on skin wounds and greater application value. Attached Figure Description

[0049] Figure 1 shows mPEG. 45 -b-PAla 28SEM image of -bP(Asp-co-(NHS-Asp)4), scale bar 20 μm;

[0050] Figure 2 shows the synthetic route and structural characterization, where 2A is the polyamino acid mPEG. 45 -b-PAla 28 The synthetic route for -bP(Asp-co-(NHS-Asp)4), where 2B is mPEG. 45 -b-Pala 28 -b-PBLA5, mPEG45-b-PAla28-b-PAsp5 and mPEG 45 -b-PAla 28 -bP(Asp-co-(NHS-Asp)4) 1 1H NMR spectrum, 2C is mPEG 45 -b-PAla 28 -b-PAsp5 and mPEG 45 -b-PAla 28 FT-IR spectrum of -bP(Asp-co-(NHS-Asp)4);

[0051] Figure 3 shows the phase diagram (A) and rheological diagram (B) of the drug-loaded sol and drug-loaded hydrogel in Example 1;

[0052] Figure 4 shows the adhesion performance diagram, where A is the adhesion test diagram of Gel-NHS prepared in Example 1 on the skin, and B is a comparison diagram of the adhesion performance of Gel-NHS and Gel.

[0053] Figure 5 shows the therapeutic effect verification diagram of the chronic wound dressing prepared in Example 1. In Figure 5, A is the local wound image of each specimen in each experimental group at different time points, and the inner diameter of the silicone ring is 10 mm. In Figure 5, B is the change of the wound area ratio of each experimental group at different days. Detailed Implementation

[0054] To further illustrate the present invention, the following detailed description of the polyamino acid, its preparation method, hydrogel, and chronic wound dressing provided by the present invention is provided in conjunction with embodiments.

[0055] Example 1

[0056] 1) mPEG 45 -b-PAla 28 Synthesis of -bP(Asp-co-(NHS-Asp)4)

[0057] The synthesis of this material is divided into three parts. First, polyethylene glycol monomethyl ether-b-polyalanine-b-polyaspartic acid benzyl ester block copolymer (mPEG) 45 -b-Pala28 Ala-NCA (-b-PBLA5) was synthesized via ring-opening polymerization of NCA initiated by mPEG-NH2 (Mn = 2000 Da). 4 g of mPEG-NH2 was weighed and placed in an ampoule, and 200 mL of toluene was added. The mixture was magnetically stirred in an oil bath at 130°C and refluxed for 4 hours. The toluene flowing out of the separator was collected, and the ampoule was then connected to a cold trap to remove the remaining toluene. The solid in the ampoule was completely dissolved in DMF, and 6.90 g of Ala-NCA was added. While stirring, a vacuum pump was connected to remove the CO2 generated by the reaction. After purging with nitrogen, the reaction was carried out at room temperature for 72 hours to obtain mPEG. 45 -PA was directly added to 4.50 g of BLA-NCA without post-treatment, and the reaction was continued for 72 hours under nitrogen purging. After the reaction, the product was precipitated with 10 times its volume of anhydrous diethyl ether, and the resulting solid was connected to a cold trap to remove the remaining diethyl ether under vacuum. The product was dissolved in as little DMF as possible and poured into a dialysis bag with a molecular weight cutoff of 3500 Da, then placed in a beaker containing primary water for dialysis. After dialysis, the product was freeze-dried to obtain mPEG. 45 -b-Pala 28 -b-PBLA5.

[0058] Secondly, polyethylene glycol monomethyl ether-b-polyalanine-b-polyaspartic acid block copolymer (mPEG) 45 -b-PAla 28 -b-PAsp5) is derived from mPEG 45 -b-Pala 28 -b-PBLA5 was obtained by deprotection with hydrobromic acid. 6.0 g of mPEG was weighed. 45 -b-PAla 28 -b-PBLA5 was placed in a round-bottom flask, and 60.0 mL of acetic acid was slowly added along the wall while magnetically stirring until homogeneous. Then, 18.0 mL of hydrogen bromide solution was added, and the mixture was stirred at room temperature for 1.5 hours. The mixture was then settled with anhydrous diethyl ether. Finally, the remaining diethyl ether was removed by vacuum pumping. The product was dissolved in as little DMF as possible and poured into a dialysis bag with a molecular weight cutoff of 3500 Da. The bag was then placed in a beaker containing pre-distilled water for dialysis. After dialysis, the product was freeze-dried to obtain mPEG. 45 -b-PAla 28 -b-PAsp5 (also known as Gel).

[0059] Finally, mPEG45-b-PAla 28 -bP(Asp-co-(NHS-Asp)4) is derived from mPEG 45 -b-PAla 28 -b-PAsp5 was obtained via an EDC / NHS reaction. 2.0 g mPEG was weighed.45 -b-PAla 28 -b-PAsp5 was placed in a round-bottom flask, dissolved in DMF, and then mixed with 2.24 g EDC and 0.54 g NHS. The mixture was stirred magnetically at room temperature for 3 days. The product was then dissolved in as little DMF as possible and poured into a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed in a beaker containing pre-existing water. After dialysis, the product was freeze-dried to obtain polyamino acid mPEG. 45 -b-PAla 28 -bP(Asp-co-(NHS-Asp)4) (also known as Gel-NHS).

[0060] Figure 1 shows mPEG. 45 -b-PAla 28 SEM image of -bP(Asp-co-(NHS-Asp)4), scale bar 20 μm.

[0061] Figure 2A shows polyamino acid mPEG. 45 -b-PAla 28 Synthetic route of -bP(Asp-co-(NHS-Asp)4).

[0062] Figure 2B shows mPEG. 45 -b-Pala 28 -b-PBLA5, mPEG 45 -b-PAla 28 -b-PAsp5 and mPEG 45 -b-PAla 28 -bP(Asp-co-(NHS-Asp)4) 1 The 1H NMR spectrum indicates that the polyamino acid mPEG was successfully synthesized in this invention. 45 -b-PAla 28 -bP(Asp-co-(NHS-Asp)4).

[0063] Figure 2C shows mPEG. 45 -b-PAla 28 -b-PAsp5 and mPEG 45 -b-PAla 28 The FT-IR spectrum of -bP(Asp-co-(NHS-Asp)4) indicates that the NHS group has been successfully grafted onto the hydrogel.

[0064] 2) Preparation of drug-loaded hydrogel solution

[0065] The polyamino acid mPEG obtained in step 1) 45 -b-PAla 28-bP(Asp-co-(NHS-Asp)4) was prepared into a 6wt% hydrogel solution. 60 mg of the polyamino acid material was weighed and pre-cooled in ice for 20 min. 1 mL of PBS buffer solution was added to the 6wt% solution. To prevent water seepage after the ice melted, the bottle cap was sealed with a sealing film. After standing for 20 min to allow the polyamino acid to fully absorb water, 4.5 μg of curcumin (CUR) and 4.5 μg of linagliptin (Lin) were added. Finally, the mixture was placed in a 4°C refrigerator and stirred thoroughly to dissolve overnight, yielding a drug-loaded hydrogel solution (i.e., a drug-loaded sol) with 4wt% linagliptin and 4wt% curcumin.

[0066] 3) Preparation of dressings for chronic wounds

[0067] The drug-loaded hydrogel solution obtained in step 2) is converted into a drug-loaded hydrogel and used directly as a wound dressing for the treatment of chronic wounds.

[0068] This invention uses rheological screening to select hydrogels that meet the gelation conditions of human body surface temperature. Figure 3 shows the phase diagram (A) and rheological diagram (B) of the drug-loaded sol and drug-loaded hydrogel.

[0069] Furthermore, the adhesion performance of the drug-loaded hydrogel was verified by adhesion experiments, as shown in Figure 4. Figure 4 is an adhesion performance diagram, where A is the adhesion test diagram of Gel-NHS prepared in Example 1 on the skin, and B is a comparison diagram of the adhesion performance of Gel-NHS and Gel. Figure 4 shows that the polyamino acid Gel-NHS (mPEG) 45 -b-PAla 28 -bP(Asp-co-(NHS-Asp)4) exhibits better adhesion properties than Gel(mPEG). 45 -b-PAla 28 -b-PAsp5).

[0070] 4) Verification of the therapeutic effect of dressings for chronic wounds

[0071] A T1D model was established using 6-8 week old male BALB / c mice weighing 17-22g. Based on the successful establishment of the T1D mouse model, a circular full-thickness skin defect was created on the back of the mouse using a 5mm diameter skin sampler. A 5mm inner diameter silicone pad was fixed to the wound with 3M tissue adhesive to prevent wound contraction. The drug-loaded hydrogel was replaced on days 0, 3, 7, 10, and 14 of treatment. The treatment effect is shown in Figure 5. Figure 5A shows local wound images of each specimen in each experimental group at different time points, with the silicone pad having an inner diameter of 10mm. Figure 5B shows the changes in the wound area ratio of each experimental group on different days. The results indicate that, compared with the control group without any treatment and the single-drug group, the drug-loaded hydrogel material (Gel-NHS) described in this invention... CUR+LIN It has a better effect on accelerating the healing of chronic wounds.

[0072] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A polyamino acid, characterized in that, It has the structure shown in Equation 1: Where m, n, x, and y are the degree of aggregation, 30≤m≤60, 10≤n≤40, 1≤x≤100, and 1≤y≤100; R1, R2, and R3 are independently selected from C1 to C2. 10 Alkyl groups; R4 is selected from OH, benzyloxy, amino, and C1-C4 alkoxy.

2. The polyamino acid according to claim 1, characterized in that, The following conditions apply: 40≤m≤50, 20≤n≤30, 1≤x≤10, 1≤y≤10; R1, R2, and R3 are independently selected from C1 to C6 alkyl groups; R4 is selected from OH or benzyloxy group.

3. The polyamino acid according to claim 1, characterized in that, R1, R2, and R3 are all selected from methyl groups, and R4 is selected from OH groups; Alternatively, R1, R2, and R3 are all selected from methyl groups, and R4 is selected from benzyloxy groups; Alternatively, R1, R2, and R3 may all be selected from methyl groups, and R4 may be selected from amino groups.

4. The method for preparing polyamino acids according to claim 1, characterized in that, Includes the following steps: 1) The polyamino acid shown in Formula 2 and BLA-NCA are subjected to ring-opening polymerization to obtain the polyamino acid shown in Formula 3; 2) After the polyamino acid shown in Formula 3 is reacted with acid to remove the benzyl group, it is then reacted with EDC and NHS to obtain the polyamino acid shown in Formula 1. Where m, n, x, and y are the degree of aggregation, 30≤m≤60, 10≤n≤40, 1≤x≤100, and 1≤y≤100; R1, R2, R3, and R5 are independently selected from C1 to C2. 10 Alkyl groups; R4 is selected from OH, benzyloxy, amino, and C1-C4 alkoxy.

5. The preparation method according to claim 4, characterized in that, The polyamino acid shown in Formula 2 is obtained by ring-opening polymerization of the polyamino acid shown in Formula 4 and the compound shown in Formula 5.

6. The preparation method according to claim 4, characterized in that, In step 1), the molar ratio of polyamino acid and BLA-NCA shown in Formula 2 is 1:(5-15). In step 2), the molar ratio of polyamino acid to acid shown in formula 3 is 1:(500-2000). In step 2), the molar ratio of polyamino acid and NHS shown in Formula 3 is 1:(10-15).

7. The preparation method according to claim 4, characterized in that, The acid in step 2) is selected from one or more of hydrobromic acid, acetic acid, hydrochloric acid, and anhydrous hydrofluoric acid.

8. A thermosensitive hydrogel, characterized in that, It includes the polyamino acid according to any one of claims 1 to 3 or the polyamino acid prepared by the preparation method according to any one of claims 4 to 7, and water.

9. A drug-loaded thermosensitive hydrogel, characterized in that, Includes the thermosensitive hydrogel of claim 8 and the drug loaded in the thermosensitive hydrogel.

10. A chronic wound dressing, characterized in that, It includes the thermosensitive hydrogel of claim 8 and linagliptin and curcumin loaded in the thermosensitive hydrogel.

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