CN114392388A

CN114392388A - Hydrogel composition and application thereof

Info

Publication number: CN114392388A
Application number: CN202111595876.1A
Authority: CN
Inventors: 郭瑞; 刘玉
Original assignee: Guangzhou Bioscience Co ltd
Current assignee: Guangzhou Bioscience Co ltd
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-04-26

Abstract

The present invention relates to a hydrogel composition and use thereof, wherein the composition comprises: (1) a first component, wherein the first component comprises a Bmkn2 polypeptide and an aldehydized pluronic; (2) a second component, wherein the second component comprises sanguinarine and aminated hyaluronic acid. The hydrogel composition has good biocompatibility and antibacterial function, and is aldehyde-based pluronic (F127-CHO)/Aminated Hyaluronic Acid (AHA)/BmKn 2/sanguinarine hydrogel.

Description

Hydrogel composition and application thereof

Technical Field

The invention belongs to the technical field of medical biomaterials, and particularly relates to a hydrogel composition and application thereof.

Background

Bacterial infections are one of the most common and challenging diseases facing the human health and medical community. With the progress of the technology, development of novel biomaterials having the effects of preventing external contamination and tissue infection and promoting tissue regeneration has been a hot spot of research. Hydrogels are polymers with a three-dimensional network structure that have been widely used in regenerative medicine and biomedical applications over the last 20 years. Since hydrogels do not readily adhere to proteins and other substances, they exhibit good biocompatibility and non-toxicity to humans when exposed to blood, body fluids, and human tissues. In addition, hydrogels closely resemble the elasticity and texture of natural tissue and can be used as implants in humans to reduce adverse reactions. Thus, hydrogels are not only a promising material for biomedical applications, such as drug carriers, tissue scaffolds, and biomedical devices (soft contact lenses), but also prevent further tissue damage and bacterial invasion. The antibacterial hydrogel has the advantages of promoting tissue regeneration and resisting bacteria, and can meet the requirements of biological materials. Traditional antibiotic-loaded hydrogel materials that are directly loaded with antibiotics may develop antibiotic resistance, which in turn generates multi-resistant microorganisms or "superbacteria". Therefore, the search for new antibacterial strategies is imperative. Although conventional antibacterial dressings such as nano silver dressings and the like have good antibacterial performance, due to the existence of nano silver, certain damage to wound surfaces is inevitably caused due to high cytotoxicity. Meanwhile, gauze dressings are easy to adhere to wounds, and when the dressings are uncovered after the wounds heal, secondary damage to the wounds is easy to cause.

Disclosure of Invention

The present invention provides in one aspect a hydrogel composition comprising: (1) a first component, wherein the first component comprises a Bmkn2 polypeptide and an aldehydized pluronic; (2) a second component, wherein the second component comprises sanguinarine and aminated hyaluronic acid. The invention prepares stable hydrogel on natural composite matrix through chemical crosslinking reaction (Schiff base reaction), has good swelling performance and biocompatibility, slowly releases antibacterial substance BmKn2 polypeptide and sanguinarine on wound surface, and has long-acting antibacterial effect.

In some embodiments, the Bmkn2 polypeptide is a Bmkn2 polypeptide-loaded polydopamine nanoparticle (PDA-Bmkn 2).

In some embodiments, the mass ratio of the BmKn2 polypeptide to the polydopamine nanoparticle is BmKn2 polypeptide: the poly-dopamine nanoparticle is 1: 1-3. In some embodiments, the mass ratio of the BmKn2 polypeptide to the polydopamine nanoparticle is BmKn2 polypeptide: polydopamine nanoparticles 1: 2.

In some embodiments, the pluronic is F127.

In some embodiments, the mass ratio of the BmKn2 polypeptide to the polydopamine nanoparticle is BmKn2 polypeptide: polydopamine nanoparticles 1: 2. In some embodiments, the mass ratio of the hydrogel aldehydized pluronic to the aminated hyaluronic acid is aldehydized pluronic: aminated hyaluronic acid-5: 2, 5:1 or 15: 2.

In another aspect, the present invention provides a hydrogel formed from the reaction of the first and second components of any of the above hydrogel compositions. The invention prepares stable hydrogel on natural composite matrix through chemical crosslinking reaction (Schiff base reaction), has good swelling performance and biocompatibility, slowly releases antibacterial substance BmKn2 polypeptide and sanguinarine on wound surface, and has long-acting antibacterial effect.

In some embodiments, the first component and the second component are formed after reaction in a buffer solution. In some embodiments, the concentration of Bmkn2 in the first mixture is 200 μ g/mL. In some embodiments, the concentration of the sanguinarine polypeptide in the second cocktail is 1 μ g/mL.

In some embodiments, the buffer solution is PBS.

In some embodiments, the concentration of sanguinarine after the reaction is 0.5 μ g/mL. In some embodiments, the concentration of the Bmkn2 polypeptide after the reaction is 100 μ g/mL.

In a further aspect, the present invention provides a dressing in which the hydrogel is formed by reaction of the first and second components of any of the hydrogel compositions described above. The invention prepares stable hydrogel on natural composite matrix through chemical crosslinking reaction (Schiff base reaction), has good swelling performance and biocompatibility, slowly releases antibacterial substance BmKn2 polypeptide and sanguinarine on wound surface, and has long-acting antibacterial effect.

In some embodiments, the dressing is used for an open infected wound.

In a further aspect, the present invention provides the use of any one of the above hydrogel compositions or any one of the above hydrogels for the preparation of a hemostatic aid having antimicrobial effect. The invention prepares stable hydrogel on natural composite matrix through chemical crosslinking reaction (Schiff base reaction), has good swelling performance and biocompatibility, slowly releases antibacterial substance BmKn2 polypeptide and sanguinarine on wound surface, and has long-acting antibacterial effect.

In some embodiments, the microorganism is at least one of staphylococcus aureus and escherichia coli.

In a further aspect, the present invention provides a method for preparing any of the above hydrogels, the method comprising the steps of: (1) dissolving the first component in a first buffer solution to obtain a first mixed solution; (2) dissolving the second component in a second buffer solution to obtain a second mixed solution; and mixing the first mixed solution and the second mixed solution to obtain the hydrogel.

In some embodiments, the volume of the first mixed liquor and the second mixed liquor is 1: 1. In some embodiments, the concentration of sanguinarine in the first mixed solution is 1 μ g/mL. In some embodiments, the concentration of the Bmkn2 polypeptide in the second cocktail is 200 μ g/mL.

The invention prepares stable hydrogel on natural composite matrix through chemical crosslinking reaction (Schiff base reaction), has good swelling performance and biocompatibility, slowly releases antibacterial substance BmKn2 polypeptide and sanguinarine on wound surface, and has long-acting antibacterial effect.

Drawings

FIG. 1 is a graph showing the appearance of the 10% F127-CHO/2% AHA hydrogel of example 2 before and after gelation.

FIG. 2 is an electron transmission electron micrograph of PDA-Bmkn 2.

FIG. 3 shows the swelling performance of F127-CHO/AHA hydrogels with different ratios.

FIG. 4 in vitro degradation performance of F127-CHO/AHA hydrogel in the presence or absence of lysozyme.

FIG. 5 in vitro release profile of sanguinarine, PDA-Bmkn 2.

Detailed Description

Bmkn2 polypeptide

The Bmkn2 polypeptide (sequence FIGAIARLLSKIF) is an antibacterial peptide existing in scorpion venom, and is reported to have effective antibacterial performance on gram-negative and gram-positive bacteria, so that a potential new way for treating bacterial infection is opened. However, most antimicrobial peptides are water soluble and rapidly hydrolyze and fail in the absence of other carriers.

Sanguinarine

Sanguinarine is a phenanthroline alkaloid, has molecular weight of 367.8, and has good biological effects of resisting bacteria, inflammation and tumor. In addition, sanguinarine also has broad-spectrum bioactivity, and has good antibacterial, anti-inflammatory and anti-tumor effects on escherichia coli, pseudomonas and the like.

Example 1.

Preparation of aldehyde-substituted Pluronic (F127-CHO): 12g of Pluronic F127 (. about.0.001 mol) was weighed into a 250mL flask, 30mL of toluene was added for azeotropic drying, and most of the toluene was removed by rotary evaporation after drying. 50mL of anhydrous dichloromethane and 2.78mL of triethylamine (. about.0.02 mol) were added to the flask, and after completion of dissolution, the flask was cooled to 0 ℃ and 10mL of a dichloromethane solution of p-toluenesulfonyl chloride (3.82g, 0.02mol) was slowly added dropwise over 30min with stirring, followed by reaction at room temperature for 24 hours. After the reaction, 100mL of water was added and extracted with 25mL of 5 dichloromethane. The organic layer was washed with 1M hydrochloric acid solution and saturated brine (25mL × 5 times), respectively, and the organic layer was collected, dried over anhydrous magnesium sulfate, filtered, concentrated, precipitated with cold diethyl ether, and dried in a vacuum oven at 40 ℃ to obtain pluronic p-toluenesulfonate. Pluronic p-toluenesulfonate (12g, 0.001mol) obtained in the above reaction was dissolved in 60mL of N, N-Dimethylformamide (DMF), and 1.22g of p-hydroxybenzaldehyde (0.01mol) and 1.38g of anhydrous potassium carbonate (0.01mol) were weighed into the above solution. And (2) stirring the mixture at 80 ℃ for reaction for 3 days, then cooling to room temperature, adding 50mL of water, extracting with 25mL of multiplied by 5 dichloromethane, collecting an organic layer, drying with anhydrous magnesium sulfate, filtering, concentrating, precipitating with cold ether, and drying the final aldehyde-group product in a vacuum drying oven at 40 ℃ to obtain F127-CHO.

Preparation of Aminated Hyaluronic Acid (AHA): 0.5g of sodium Hyaluronate (HA) and 5g of dihydrazide Acetate (ADH) are weighed, dissolved in 100mL of deionized water, and stirred uniformly. 0.8g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.7g of 1-hydroxybenzotriazole hydrate (HOBt) were weighed, dissolved in a mixture of dimethylsulfoxide and water (v/v ═ 1:1, 5mL each), added dropwise to the above solution, adjusted to pH 5.0 with dilute hydrochloric acid, and reacted at room temperature for 1 day. After the reaction, the reaction mixture was dialyzed against deionized water using a dialysis bag (Kw 12000), and the product was lyophilized and stored at 4 ℃.

Preparation of BmKn 2-loaded polydopamine nanoparticles (PDA-BmKn 2): preparation of PDA (polydopamine) nanoparticles: measuring 90mL of deionized water, 40mL of ethanol and 2mL of ammonia water at room temperature, stirring and mixing uniformly for 30min, quickly weighing 0.5g of dopamine hydrochloride, dissolving the dopamine hydrochloride in 10mL of water, slowly and dropwise adding the dopamine hydrochloride into the mixed solution, changing the solution from colorless to light yellow, then changing the solution to black, reacting for 4h, and dropwise adding dilute hydrochloric acid to adjust the pH of the solution to about 6.5. After the reaction is finished, centrifuging the solution at 14000r/min at 4 ℃ to collect precipitates, washing the precipitates with water for three times, dispersing the precipitates with deionized water, centrifuging at 4000r/min to remove macromolecular PDA, and freeze-drying the upper solution to obtain the PDA nano-particles. Preparation of PDA-BmKn2 antibacterial nanoparticles: weighing 10mg of BmKn2 antibacterial peptide (purchased from Shanghai Chu peptide Biotechnology Co., Ltd.) and 10mg of EDC, dissolving in 5mL of PBS buffer solution with pH 5.5, stirring at room temperature for 15min, weighing NHS15mg, adding into the solution, adding 10mL of PBS buffer solution with pH 7.4, stirring for 15min, dissolving 20mg of weighed PDA in 10mL of mixed solution of water/DMSO (v/v is 3:1), slowly dropping into the previous solution, reacting for 4h under ice bath condition, dialyzing the reaction solution for 4h with dialysis bag with Kw of 2000Da, and freeze-drying to obtain PDA-BmKn2, wherein the storage is carried out at 4 ℃.

Example 2.

F127-CHO/AHA hydrogel preparation: weighing 0.2g of AHA prepared according to example 1, dissolving the AHA in 5mL of PBS to prepare an initial solution with the concentration of 0.04g/mL, similarly, weighing a certain amount of F127-CHO prepared according to example 1, dissolving the F127-CHO in PBS to prepare a solution with the concentration of 0.1g/mL, uniformly mixing the prepared AHA solution and the F127-CHO solution according to the volume ratio of 1:1, pouring the mixture into a 48-well plate, and waiting for 3min to obtain 5% F127-CHO/2% AHA gel.

Weighing 0.2g of AHA prepared according to the embodiment 1, dissolving the AHA in 5mL of PBS to prepare an initial solution with the concentration of 0.04g/mL, similarly, weighing a certain amount of F127-CHO prepared according to the embodiment 1, dissolving the F127-CHO in the PBS to prepare a solution with the concentration of 0.2g/mL, uniformly mixing the prepared AHA solution and the F127-CHO solution according to the volume ratio of 1:1, pouring the mixture into a 48-hole plate, and waiting for 3min to obtain the 10% F127-CHO/2% AHA group hydrogel.

Weighing 0.2g of AHA prepared according to the embodiment 1, dissolving the AHA in 5mL of PBS to prepare an initial solution with the concentration of 0.04g/mL, similarly, weighing a certain amount of F127-CHO prepared according to the embodiment 1, dissolving the F127-CHO in the PBS to prepare a solution with the concentration of 0.3g/mL, uniformly mixing the prepared AHA solution and the F127-CHO solution according to the volume ratio of 1:1, pouring the mixture into a 48-hole plate, and waiting for 3min to obtain the 15% F127-CHO/2% AHA group hydrogel.

Example 3.

F127-CHO/AHA/PDA-Bmkn2 hydrogel preparation: a certain amount of PDA-BmKn2 is weighed and dispersed in the F127-CHO solution prepared in the above example 2, the F127-CHO solution and the AHA solution are uniformly mixed according to the volume ratio of 1:1, and then the mixture is poured into a 48-well plate, and after the mixture is solidified, 10% F127-CHO/2% AHA/sanguinarine/PDA-BmKn 2 hydrogel is obtained, wherein the concentration of the PDA-BmKn2 in the hydrogel is 100 mug/mL.

Example 4.

F127-CHO/AHA/sanguinarine aqueous gel preparation: weighing a certain amount of sanguinarine, dissolving the sanguinarine in a prepared 4% AHA solution, uniformly mixing the F127-CHO solution and the AHA solution according to the volume ratio of 1:1, pouring the mixture into a 48-pore plate, and obtaining 10% F127-CHO/2% AHA/sanguinarine hydrogel after the mixture is coagulated, wherein the final concentration of the sanguinarine in the hydrogel is 0.5 mu g/mL.

Example 5.

F127-CHO/AHA/sanguinarine/PDA-Bmkn 2 hydrogel preparation: weighing a certain amount of PDA-BmKn2 to be dispersed in the F127-CHO solution prepared in the above example 2, weighing a certain amount of sanguinarine to be dissolved in the prepared 4% AHA solution, uniformly mixing the F127-CHO solution and the AHA solution according to the volume ratio of 1:1, pouring the mixture into a 48-pore plate, waiting for 3min to form gel, and obtaining 10% F127-CHO/2% AHA/sanguinarine/PDA-BmKn 2 hydrogel, wherein the concentration of the PDA-BmKn2 in the hydrogel is 100 mu g/mL, and the final concentration of the sanguinarine in the hydrogel is 0.5 mu g/mL.

Example 6 gel Performance testing

1. Transmission Electron Microscopy (TEM) test

1mg of PDA-Bmkn2 (prepared according to example 1) was weighed, dispersed in 1mL of water, filtered through a 0.45um filter, dropped onto a copper mesh carbon support membrane, allowed to dry naturally in air, and placed in a high resolution transmission electron microscope to observe the overall morphology of the nanoparticles. FIG. 2 is an electron transmission electron micrograph of the PDA-Bmkn2 prepared in example 1, which shows that the prepared PDA-Bmkn2 has better dispersibility, uniform particle size and particle size of about 200 nm.

2. Swelling Rate test

The weight of the hydrogel was weighed using balance and recorded as W₀The hydrogel was placed in PBS (pH 7.4), and at a specific time point, the surface of the hydrogel was quickly wiped off with a wet filter paper (wet non-traumatic gel), and W was immediately weighed_t. Each sample was subjected to three experiments in parallel, and the average value thereof was determined, and the swelling ratio of the sample was calculated according to the formula (4-1):

XR＝(W_t－W₀)/W₀x 100% formula (4-1)

Wherein, W₀The weight of the hydrogel; w_tIs the weight of the hydrogel after water absorption; XR is the swelling ratio of the hydrogel.

FIG. 3 shows the results of in vitro swelling performance tests of three groups of hydrogels of 5% F127-CHO/2% AHA, 10% F127-CHO/2% AHA, and 10% F127-CHO/2% AHA prepared in example 2. As shown in the figure, all three groups of hydrogels have good swelling ratios, and the swelling ratio of the hydrogel decreases with the increase of the concentration of F127-CHO, because excess amino groups remain in the AHA at low concentration of F127-CHO, and the excess amino groups of the AHA are reacted to the end with the increase of the concentration of F127-CHO, so that the degree of crosslinking of the hydrogel increases, and the swelling ratio of the hydrogel decreases.

3. In vitro degradation experiments

A portion of the hydrogel prepared in example 2 was freeze-dried and weighed to obtain a mass W₀Then, the initial hydrogel was soaked in a PBS solution (pH 7.4) and a PBS solution (pH 7.4) containing 1000U/mL of lysozyme, respectively, and placed on a constant temperature shaker (37 ℃, 70 rpm). At the time point of measurement, the hydrogel was taken out, washed with ultrapure water and lyophilized, which was freeze-dried, and after drying, the mass was accurately weighed and recorded (W)_t). The weight loss of the hydrogel was calculated using equation (4-2) and is reported as X:

X＝{1－[(W_t－W₀)/W₀]formula (4-2) } × 100%

FIG. 3 shows the results of in vitro degradation tests of 10% F127-CHO/2% AHA hydrogels prepared in example 2 with and without lysozyme, and it can be seen from the figure that the hydrogels were completely degraded after 9 days without lysozyme, the degradation speed of the hydrogels was increased after lysozyme addition, and the hydrogels were completely degraded after 8 days.

4. In vitro release profile of sanguinarine, PDA-Bmkn2

(1) Standard curve

Precisely weighing antibacterial peptide BmKn 21 mg, dissolving in 1mL PBS solution to prepare a mother solution with the concentration of 1mg/mL, diluting the solution 1 in series to prepare solutions of 50 [ mu ] g/mL, 40 [ mu ] g/mL, 30 [ mu ] g/mL, 20 [ mu ] g/mL, 10 [ mu ] g/mL, 5 [ mu ] g/mL, 2.5 [ mu ] g/mL and 1.25 [ mu ] g/mL, placing 0.5mL of each solution in a quartz cuvette, measuring the absorbance value at 204.5nm by using an ultraviolet spectrophotometer, and drawing a standard curve; precisely weighing sanguinarine 1mg, dissolving in 1mL PBS solution to obtain 1mg/mL mother solution, diluting the mother solution to obtain 25 μ g/mL, 20 μ g/mL, 15 μ g/mL, 10 μ g/mL, 5 μ g/mL, and 2.5 μ g/mL solutions, placing 0.5mL of each solution in quartz cuvette, measuring absorbance at 276nm with ultraviolet spectrophotometer, and drawing standard curve

(2) Hydrogel in vitro drug release test

Adding hydrogel loaded with 2200 μ g of Bmkn and 200 μ g of sanguinarine into 10mL of PBS solution, placing the solution in a constant-temperature shaking table at 37 ℃, taking out 1mL of solution at a specific time point, adding 1mL of new PBS to keep the volume of the 10mL of PBS solution unchanged, measuring the concentration of the drug in the taken-out solution by using an ultraviolet spectrophotometer, measuring the release behavior of the drug-loaded hydrogel for 7 days continuously, measuring the release amount of Bmkn2 and the sanguinarine according to a standard curve, and drawing an accumulated release curve.

Figure 5 is a cumulative release profile of BmKn2 loaded and sanguinarine loaded hydrogels. On day 1 of drug release, the release rate of sanguinarine is about 59%, the release rate of BmKn2 antibacterial peptide is about 47%, and compared with sanguinarine, the release process is slower because the BmKn2 antibacterial peptide is connected with PDA molecules. Both drugs were essentially not released by day 4, with a total sanguinarine release rate of about 68% and a total BmKn2 antibacterial peptide release rate of about 65%, both of which were not completely released, and it was expected that some of the drugs were inactivated over time.

5. In vitro antibacterial test

The antibacterial performance of the hydrogel was evaluated with gram-positive staphylococcus aureus and gram-negative escherichia coli. The OD value of the bacteria in the antibacterial test was adjusted to 0.1. Co-culturing the hydrogel sample and the bacterial suspension in a biochemical incubator at 37 ℃, and measuring the OD value of the blended bacterial liquid after 12 hours. 100 μ L of the bacterial suspension was diluted and plated on LB agar plates. After culturing at 37 ℃ for 24h, counting the number of culturable colonies according to the formula (4-3)

AR(％)＝(N_c-N_s) Nc 100 formula (4-3)

Calculating the Antibacterial Rate (AR), wherein N_cAverage colony number for control samples, N_sThe average bacterial colony number of the hydrogel sample.

Table 1: results of in vitro antibacterial testing of different hydrogels

Table 1 shows the in vitro antibacterial test results of the hydrogels prepared in examples 2 (i.e., 10% F127-CHO/2% AHA), 3, 4 and 5, and it can be seen from the table that the antibacterial rate of the hydrogel without adding PDA-BmKn2 and sanguinarine is negative, which may be the reason that pure hydrogel material provides nutrient for bacteria, and the hydrogels with PDA-BmKn2 and sanguinarine added alone have certain antibacterial performance, but the antibacterial rate is not high, and when both are added at the same time, the antibacterial rate reaches more than 90%, which indicates that the combination of PDA-BmKn2 and sanguinarine can better exert antibacterial effect.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A hydrogel composition, wherein the composition comprises:

(1) a first component, wherein the first component comprises a Bmkn2 polypeptide and an aldehydized pluronic;

(2) a second component, wherein the second component comprises sanguinarine and aminated hyaluronic acid.

2. The hydrogel composition of claim 1, wherein the Bmkn2 polypeptide is a Bmkn2 polypeptide-loaded polydopamine nanoparticle.

3. The hydrogel composition of claim 2, wherein the mass ratio of the BmKn2 polypeptide to the polydopamine nanoparticles is BmKn2 polypeptide: the poly-dopamine nanoparticle is 1: 1-3.

4. The hydrogel composition of claim 1, wherein the pluronic is F127.

5. The hydrogel composition according to any one of claims 1 to 4, wherein the mass ratio of the hydrogel aldehyde-modified pluronic to the aminated hyaluronic acid is: the aminated hyaluronic acid is 5-15: 1-2.

6. A hydrogel formed by the reaction of the first and second components of the hydrogel composition of any one of claims 1 to 5.

7. The hydrogel of claim 6, wherein the concentration of sanguinarine after the reaction is 0.5 μ g/mL.

8. The hydrogel of claim 6, wherein the concentration of the Bmkn2 polypeptide after the reaction is 100 μ g/mL.

9. A dressing, wherein the hydrogel is formed by reacting the first and second components of the hydrogel composition of any one of claims 1 to 5.

10. Use of a hydrogel composition according to any one of claims 1 to 5 or a hydrogel according to any one of claims 6 to 8 for the preparation of a haemostatic aid having an antimicrobial effect.