CN117138104A

CN117138104A - Antioxidant protein peptide-based nano hydrogel for burn wound surface

Info

Publication number: CN117138104A
Application number: CN202311198710.5A
Authority: CN
Inventors: 任珊珊; 杨玉昌; 刘媛媛
Original assignee: Qingdao Situo Xinyuan Cell Medicine Co ltd; Affiliated Hospital of Weifang Medical University
Current assignee: Qingdao Situo Xinyuan Cell Medicine Co ltd; Affiliated Hospital of Weifang Medical University
Priority date: 2023-09-18
Filing date: 2023-09-18
Publication date: 2023-12-01

Abstract

The invention discloses an antioxidant protein peptide-based nano hydrogel for burn wound, which relates to the technical field of burn hydrogel, and adopts the technical scheme that selenium nano particles (Sepps) are subjected to covalent modification in acetic acid buffer solution, and are further activated by using a silane coupling agent, the modified Sepps and protein peptide are coupled through EDC and Sulfo-NHS, the compound is mixed with Hyaluronic Acid (HA), and is subjected to first network crosslinking through sulfur dioxide, PVA and boric acid are added to form a second network, and Tween 20 is added as an auxiliary agent to improve gel performance, and the invention HAs the beneficial effects that: the invention improves the dispersibility and mechanical strength of the hydrogel through covalent modification and double-network structure of the selenium nano particles (Senps). The protein peptide-nanoparticle composite increases biocompatibility and antioxidation effect, and is helpful for burn healing.

Description

Antioxidant protein peptide-based nano hydrogel for burn wound surface

Technical Field

The invention relates to the technical field of burn hydrogels, in particular to an antioxidant protein peptide-based nano-hydrogel for burn wounds.

Background

Burns are a common severe wound, and their treatment not only requires wound healing, but also requires antioxidant treatment of the wound to reduce inflammation and other complications. Hydrogels are well known for their good biocompatibility, wettability and breathability to find widespread use in wound therapy, and in particular, they provide a sustained moist environment for the wound surface, thereby promoting wound healing. However, how to introduce oxidation resistance into these hydrogels to further improve the therapeutic effect on burn wounds has become a hot spot of current research.

In order to provide sustained antioxidant protection to burn wounds, methods have been known in the art to incorporate antioxidant nanoparticles such as selenium nanoparticles (SeNPs) into hydrogels. However, these nanoparticles tend to deposit or aggregate easily in the gel, resulting in uneven release thereof, reducing the therapeutic effect. In addition, the prior art often suffers from the problems of poor compatibility among materials, poor crosslinking effect and the like when preparing the antioxidant protein peptide-based nano hydrogel, so that the stability, the antioxidant property and the biocompatibility of the gel are difficult to be fully ensured.

In view of the above, the present invention aims to provide a novel antioxidant protein peptide-based nano-hydrogel for burn wound and a preparation method thereof, which are expected to solve the problem of dispersibility of nanoparticles in the hydrogel and to improve the crosslinking effect and compatibility of the gel. In addition, it is desirable to optimize the preparation conditions so that the resulting hydrogels have more durable, uniform antioxidant release characteristics, thereby more effectively promoting healing of burn wounds.

Disclosure of Invention

In order to achieve the above object, the present invention provides an antioxidant protein peptide-based nano hydrogel for burn wound, which comprises the following steps:

a) Covalent modification of selenium nanoparticles (SeNPs):

putting Senps into acetic acid buffer solution, stirring until the SeNPs are completely dispersed, adding silane coupling agent, continuously stirring, separating modified nano particles by using a centrifuge after the reaction is completed, washing the nano particles by using deionized water, and finally suspending the nano particles in the deionized water;

b) Linking the nanoparticle to the protein peptide:

uniformly mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (Sulfo-NHS) with modified Senps, gradually adding protein peptide, continuously stirring the mixture, and centrifuging after the reaction is finished to obtain a protein peptide-nanoparticle compound;

c) Forming a first network:

uniformly mixing the hyaluronic acid solution and the protein peptide-nanoparticle compound, gradually adding sulfur dioxide, and stirring for reaction;

d) Forming a second network:

adding boric acid solution into the mixture of c), and continuously stirring for reaction;

e) And (3) introducing an auxiliary agent:

tween 20 was added to the reaction product of d) to obtain a hydrogel.

Preferably, in step a), the silane coupling agent is used in an amount of 5-10% by weight of the nanoparticles. Preferably, the particle size of the SeNPs in step a) is from 10 to 50 nm;

preferably, in step a), the acetate buffer concentration is 0.1-0.5mg/ml;

preferably, in step a) the pH of the acetate buffer used is in the range of 4.5-6.0;

preferably, in step a), stirring is continued and the reaction is carried out for 2-4 hours;

preferably, in step b), EDC: sulfo-NHS: the molar ratio of the SeNPs is (0.5-2): (8-12).

Preferably, the amount of protein peptide added in step b) is 2-5% by weight of SeNPs.

Preferably, in step b), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (Sulfo-NHS) and the modified SenPS are mixed homogeneously and stirred continuously for 10-20 minutes to activate EDC and Sulfo-NHS;

preferably, in step b), the protein peptide is gradually added, the pH is adjusted to 7-8, and the mixture is continuously stirred for 4-6 hours;

preferably, the concentration of hyaluronic acid in step c) is in the range of 0.5-3% (w/v).

Preferably, the sulfur dioxide used in step c) is added in an amount of 1-5% by weight of hyaluronic acid.

Preferably, the stirred reaction of step C) is carried out at 25-40 ℃.

Preferably, in step c) sulfur dioxide is gradually added, stirring is continued and the mixture is allowed to react for 2-4 hours at a pH of about 7;

preferably, in step d), a PVA solution having a molecular weight of 30,000-90,000,5-10% (w/v) is selected and stirred at 25℃for 30 minutes;

preferably, in step d), the concentration of the boric acid solution is in the range of 0.3-3% (w/v);

preferably, in step d), the pH of the mixture is adjusted to a range of 8-9 and the reaction is continued with stirring at a temperature of 37℃for 4-6 hours;

preferably, tween 20 is used in an amount of 0.1-1% of the total weight of the hydrogel in step e);

preferably, tween 20 is selected in an amount of 0.01-0.1% (v/v);

preferably, in step e), the resulting hydrogel is cooled at 4 ℃ for 12 hours and then left at room temperature for at least 24 hours to ensure complete crosslinking.

a) Covalent modification of selenium nanoparticles (SeNPs):

the chemical modification of the SeNPs is realized by utilizing the surface reaction of the silane coupling agent and the SeNPs, and the step ensures that the surface of the SeNPs is modified, thereby creating conditions for the subsequent combination with protein peptides.

The specific particle size of the SeNPs, the concentration of the acetic acid buffer solution and the pH range ensure the optimal reaction of the silane coupling agent and the SeNPs, realize more uniform and stable modification of the SeNPs and provide a more stable nano-base for the subsequent steps.

Naked SeNPs are easily oxidized or reacted with other substances in the environment, so that the structural and functional properties of the naked SeNPs are changed, and a protective layer can be provided for the naked SeNPs through modification, so that the stability of the naked SeNPs is improved; in order to ensure compatibility of SeNPs with biological systems, surface modification is required; the surface charge of SeNPs can affect its dispersibility in liquids, and the modification can optimize its dispersibility, thereby improving its uniform distribution in hydrogels.

b) Linking the nanoparticle to the protein peptide:

EDC and Sulfo-NHS can activate the surface carboxyl group of the SENPs, form an amide bond with amino groups in the protein peptide, form stable combination of the protein peptide and the SENPs, and increase the biological activity of the nano particles.

Activation of carboxyl groups on the surface of SeNPs can be maximized by selecting the specific molar ratios of EDC, sulfo-NHS and SeNPs. In addition, by controlling the addition amount of the protein peptide and the reaction pH, the maximum binding efficiency of the protein peptide and the Senps can be ensured, a more stable and high-density protein peptide-nanoparticle complex is formed, and higher bioactivity and oxidation resistance are ensured.

c) Forming a first network:

sulfur dioxide reacts with hyaluronic acid to cause cross-linking of the hyaluronic acid to form a network structure; ensures the structural stability of the hydrogel and increases the adhesiveness to burn wound surfaces.

By selecting specific concentrations of hyaluronic acid and sulfur dioxide, and controlling the reaction temperature and pH, optimal cross-linking of hyaluronic acid can be ensured, forming a first network structure with high elasticity and stability, providing a solid basis for the subsequent steps.

d) Forming a second network:

the boric acid reacts with PVA to form a stable boric acid-polyol complex, so that the net structure is further enhanced, the mechanical strength of the hydrogel is further enhanced, and the sufficient supporting effect on the burn wound surface is ensured.

By selecting PVA and boric acid of specific molecular weight and concentration, and controlling the reaction temperature and pH, the crosslinking between boric acid and PVA can be maximized, a stable second network structure is formed, and the mechanical strength and stability of the hydrogel are further enhanced.

First a first network is formed in which the protein peptide is immobilized and protected, ensuring its stability and functionality. Thus, the protein peptide is not affected by possible adverse reaction conditions when the formation of the second network is performed, the first network being responsible for providing the biological activity, while the second network is more concerned with the physical properties; thus, each network has a definite purpose, rather than a mix of the two, possibly sacrificing certain characteristics; by sequentially forming the two networks, the respective preparation conditions can be optimized respectively, the process is simplified, and the complexity possibly brought by adding all the components simultaneously is avoided.

e) And (3) introducing an auxiliary agent:

tween 20 is used as a nonionic surfactant and uniformly dispersed in the hydrogel, so that the fluidity of the hydrogel is improved, the lubricity of the hydrogel is increased, the hydrogel is easier to smear on burn wounds, meanwhile, the wound can be kept moist, the wound healing is accelerated, the Tween 20 with specific concentration is selected, the Tween 20 is uniformly distributed in the hydrogel, the lubricity and the adhesiveness of the hydrogel are increased, and the hydrogel is easier to smear and uniformly cover the burn wounds.

The technical scheme provided by the embodiment of the invention has the beneficial effects that:

improving the dispersibility of the nano particles: through covalent modification of selenium nanoparticles (SeNPs), the dispersibility of the nanoparticles in the hydrogel is remarkably improved, and the deposition and aggregation of the nanoparticles are effectively prevented. This ensures a uniform distribution of nanoparticles in the hydrogel, providing a sustained and uniform antioxidant protection for the burn wound.

Enhancing the crosslinking effect: by utilizing the crosslinking reaction of hyaluronic acid and sulfur dioxide and combining the interaction of boric acid and PVA, a stable and uniform dual-network structure is formed. This not only enhances the mechanical strength of the hydrogel, but also greatly improves its stability.

Optimizing biocompatibility: the formation of the protein peptide-nanoparticle complex ensures the stable existence of the protein peptide and improves the biocompatibility of the whole hydrogel. This means that the hydrogel is not easy to cause adverse reaction when in use, and is more beneficial to healing of burn wound surfaces.

Durable and uniform antioxidant release: in combination with the modification of the nanoparticles and the optimized crosslinking technique, the invention ensures the sustained release of selenium nanoparticles in hydrogels. The burn wound surface is provided with continuous and uniform antioxidation protection, is helpful for reducing oxidative stress injury and accelerating wound healing.

And (3) optimizing preparation conditions: the invention considers the optimal conditions of each raw material and each step, thereby ensuring that the prepared hydrogel reaches an optimal state in physical, chemical and biological properties, and simultaneously ensuring the economical efficiency and the practicability of the hydrogel in production and practical application.

Comprehensively solve multiple problems: the invention comprehensively considers the dispersibility, the crosslinking effect, the compatibility and the antioxidation release characteristics of the nano particles, and provides a comprehensive and efficient solution for burn wound treatment.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. Of course, the specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.

Example 1

Covalent modification of selenium nanoparticles (SeNPs):

senps with a particle size of 30 nm were placed in 0.3mg/ml acetic acid buffer at pH 5.0.

After stirring to complete dispersion, a silane coupling agent was added, corresponding to 7.5% by weight of the nanoparticles.

The reaction was stirred at room temperature for 3 hours.

And separating the modified nano particles by using a centrifugal machine, and washing the nano particles with deionized water until the nano particles are clean.

Finally, the nano particles are suspended in deionized water for standby.

Linking the nanoparticle to the protein peptide:

mixing EDC, sulfo-NHS with modified Senps, wherein EDC: sulfo-NHS: the molar ratio of Senps was 1:1:10.

Stirring was uniform and continued for 15 minutes to activate EDC and Sulfo-NHS.

The protein peptide was gradually added in an amount of 3.5% by weight relative to the weight of SeNPs.

The pH was adjusted to 7.5 and the mixture was stirred for 5 hours.

And obtaining the protein peptide-nanoparticle complex by centrifugation.

Forming a first network:

a hyaluronic acid solution with a concentration of 2% (w/v) was mixed with the protein peptide-nanoparticle complex uniformly.

At 30 ℃, sulfur dioxide was gradually added, corresponding to 3% by weight of hyaluronic acid.

At a pH of about 7, stirring was continued and the mixture was allowed to react for 3 hours.

Forming a second network:

a7.5% (w/v) PVA solution having a molecular weight of 60,000 was selected at 25℃and was mixed with the above mixture and stirred for 30 minutes.

A1.5% (w/v) boric acid solution was gradually added.

The pH of the mixture was adjusted to 8.5 and the reaction was continued with stirring at 37℃for 5 hours.

And (3) introducing an auxiliary agent:

to the above reaction was added 0.05% (v/v) Tween 20.

Uniformly mixing to obtain the hydrogel.

The resulting hydrogel was cooled at 4 ℃ for 12 hours and then left at room temperature for 24 hours to ensure complete crosslinking.

Example 2

All other steps according to example 1 are distinguished in that the particle size of the SeNPs in step a) is 10 nm.

Example 3

All other steps according to example 1 are distinguished in that the particle size of the SeNPs in step a) is 50 nm.

Example 4

All other steps according to example 1 are distinguished in that the silane coupling agent is used in step a) in an amount of 5% by weight of the nanoparticles.

Example 5

All other steps according to example 1 are distinguished in that the silane coupling agent is used in step a) in an amount of 10% by weight of the nanoparticles.

Example 6

All other steps according to example 1 are distinguished in that in step b) EDC: sulfo-NHS: the molar ratio of SeNPs was 0.5:0.5:8.

Example 7

All other steps according to example 1 are distinguished in that in step b) EDC: sulfo-NHS: the molar ratio of Senps was 0.5:0.5:12.

Example 8

Example 9

All other steps according to example 1 are distinguished in that in step b) EDC: sulfo-NHS: the molar ratio of Senps was 1:1:8.

Example 10

All other steps according to example 1 are distinguished in that in step b) EDC: sulfo-NHS: the molar ratio of Senps was 1:0.5:8.

Example 11

All other steps according to example 1 are distinguished in that in step b) EDC: sulfo-NHS: the molar ratio of SeNPs was 2:2:12.

Example 12

All other steps according to example 1 are distinguished in that the amount of protein peptide added in step b) is 2% by weight of SeNPs.

Example 13

All other steps according to example 1 are distinguished in that the amount of protein peptide added in step b) is 5% by weight of Senps.

Example 14

All other steps according to example 1 are distinguished in that the concentration of hyaluronic acid in step c) is 0.5% (w/v).

Example 15

All other steps according to example 1 are distinguished in that the concentration of hyaluronic acid in step c) is 3% (w/v).

Example 16

All other steps according to example 1 are distinguished in that the amount of sulphur dioxide added in step c) is 1% by weight of hyaluronic acid.

Example 17

All other steps according to example 1 are distinguished in that sulphur dioxide is added in step c) in an amount of 5% by weight of hyaluronic acid.

Example 18

All other steps according to example 1, except that the stirring reaction of step C) was carried out at 25 ℃.

Example 19

All other steps according to example 1, except that the stirring reaction of step C) was carried out at 40 ℃.

Example 20

All other steps according to example 1 are distinguished in that in step d) the concentration of the boric acid solution is 0.3% (w/v).

Example 21

All other steps according to example 1 are distinguished in that in step d) the concentration of the boric acid solution is 3% (w/v).

Example 22

All other steps according to example 1, except that Tween 20 was used in an amount of 0.1% by weight based on the total weight of the hydrogel in step e).

Example 23

All other steps according to example 1, except that Tween 20 was used in an amount of 1% by total weight of the hydrogel in step e).

Example 24

Covalent modification of selenium nanoparticles (SeNPs):

senps with a particle size of 10 nm were placed in 0.1mg/ml acetic acid buffer at pH 4.5.

After stirring to complete dispersion, a silane coupling agent was added in an amount equivalent to 5% by weight of the nanoparticles.

The reaction was stirred at room temperature for 2 hours.

Finally, the nano particles are suspended in deionized water for standby.

Linking the nanoparticle to the protein peptide:

mixing EDC, sulfo-NHS with modified Senps, wherein EDC: sulfo-NHS: the molar ratio of SeNPs was 0.5:0.5:8.

Stirring was uniform and continued for 10 minutes to activate EDC and Sulfo-NHS.

The protein peptide was gradually added in an amount of 2% by weight relative to the Senps.

The pH was adjusted to 7 and the mixture was stirred for 4 hours.

And obtaining the protein peptide-nanoparticle complex by centrifugation.

Forming a first network:

a hyaluronic acid solution with a concentration of 0.5% (w/v) was mixed uniformly with the protein peptide-nanoparticle complex.

At 25 ℃, sulfur dioxide was gradually added in an amount corresponding to 1% by weight of hyaluronic acid.

Forming a second network:

a5% (w/v) PVA solution having a molecular weight of 30,000 was selected at 25℃and mixed with the above mixture and stirred for 30 minutes.

A solution of boric acid was gradually added at 0.3% (w/v).

The pH of the mixture was adjusted to 8 and the reaction was continued with stirring at 37℃for 4 hours.

And (3) introducing an auxiliary agent:

to the above reaction was added 0.01% (v/v) Tween 20.

Uniformly mixing to obtain the hydrogel.

Example 25

Covalent modification of selenium nanoparticles (SeNPs):

senps with a particle size of 50 nm were placed in 0.5mg/ml acetic acid buffer, pH 6.

After stirring to complete dispersion, a silane coupling agent was added in an amount equivalent to 10% by weight of the nanoparticles.

The reaction was stirred at room temperature for 4 hours.

Finally, the nano particles are suspended in deionized water for standby.

Linking the nanoparticle to the protein peptide:

mixing EDC, sulfo-NHS with modified Senps, wherein EDC: sulfo-NHS: the molar ratio of SeNPs was 2:2:12.

Stirring was uniform and continued for 20 minutes to activate EDC and Sulfo-NHS.

The protein peptide was gradually added in an amount of 5% by weight relative to the Senps.

The pH was adjusted to 8 and the mixture was stirred for 6 hours.

And obtaining the protein peptide-nanoparticle complex by centrifugation.

Forming a first network:

a hyaluronic acid solution with a concentration of 3% (w/v) was mixed uniformly with the protein peptide-nanoparticle complex.

At 40 ℃, sulfur dioxide was gradually added in an amount of 5% by weight relative to the weight of hyaluronic acid.

At a pH of about 7, stirring was continued and the mixture was allowed to react for 4 hours.

Forming a second network:

a10% (w/v) PVA solution having a molecular weight of 90,000 was selected at 25℃and was mixed with the above mixture and stirred for 30 minutes.

A3% (w/v) boric acid solution was gradually added.

The pH of the mixture was adjusted to 9 and the reaction was continued with stirring at 37℃for 6 hours.

And (3) introducing an auxiliary agent:

to the above reaction was added 0.1% (v/v) Tween 20.

Uniformly mixing to obtain the hydrogel.

Comparative example 1

All other steps according to example 1 were followed, except that unmodified SeNPs were used directly without addition of a silane coupling agent.

Comparative example 2

All other steps according to example 1 are distinguished in that the protein peptide is directly added in the step of not linking the nanoparticle to the protein peptide.

Comparative example 3

All other steps according to example 1 were followed, except that PVA and boric acid were not used, and only hyaluronic acid and sulfur dioxide were relied upon to form a network.

Comparative example 4

All other steps according to example 1 are followed, except that Tween 20 is not added in the step of not introducing an auxiliary agent.

Comparative example 5

Commercially available wet burn and scald ointment

1. Viscosity test

Placing the prepared hydrogel sample into a sample cup of a viscometer to ensure complete coverage of the rotor; after the viscometer was turned on and the instrument was left to stabilize, the viscosity values at the set rotational speed were recorded, each sample was measured at least three times, and then the average was taken.

Temperature: 25 ℃ C (room temperature) ensures a constant temperature during the test.

Rotational speed: rotational speed ranges such as 50rpm, 100rpm and 200rpm were selected to evaluate the viscosity change under different shear conditions.

TABLE 1 results of viscosity test

Project	Viscosity (mPas) 50rpm	Viscosity (mPas) 100rpm	Viscosity (mPas) 200rpm
				Example 1	212	196	183
Example 4	205	190	177
				Example 5	215	199	185
Example 22	210	194	181
				Example 23	208	193	180
Comparative example 1	220	204	190
				Comparative example 4	214	198	184

Examples 4 and 5 show the relationship between the concentration of the silane coupling agent and the viscosity, indicating that the concentration of the silane coupling agent contributes to better dispersion.

Examples 22 and 23 demonstrate the effect of increasing Tween 20 concentration on viscosity. The viscosity was slightly changed depending on the amount of Tween 20, but similar to example 1.

Comparative example 1 has the highest viscosity, which may mean that unmodified SeNPs aggregate more easily, resulting in poor dispersibility.

Comparative example 4, without the addition of Tween 20, had a slightly higher viscosity than example 1, which may mean slightly less dispersibility.

2. Determination of crosslink Density:

weighing an antioxidant protein peptide-based nano hydrogel sample, and recording the initial weight;

preparing a large volume of deionized water to ensure that the water is fresh and clean;

completely immersing the prepared hydrogel sample into deionized water;

samples were taken at predetermined time intervals and the surface was gently wiped off with a paper towel.

Re-weigh the sample and record the weight at each time point

The water absorption was calculated using the following formula:

water absorption (%) = (Wt-Wo)/wo×100%

W t: the weight of the sample after water absorption; wo: initial weight of sample.

Table 2 experimental data

Examples/comparative examples	Water absorption (%)
		Example 1	55.0
Example 14	66.7
		Example 15	48.3
Example 16	72.1
		Example 17	57.8
Example 18	59.0
		Example 19	53.4
Example 20	64.2.0
		Example 21	49.5
Comparative example 3	81.9

Examples 1, 14, 15 demonstrate that cross-linking of hyaluronic acid and sulfur dioxide forms a first network, the primary function of which is to provide structural stability and adhesion; high concentration of hyaluronic acid results in high degree of crosslinking, low water absorption, and low concentration of hyaluronic acid results in low degree of crosslinking, high water absorption.

Examples 1, 20, 21, comparative example 3 demonstrate that crosslinking of boric acid and PVA forms a second network, providing mainly mechanical strength. Small changes in boric acid concentration may not significantly affect water absorption because the second network affects mainly mechanical strength rather than water absorption properties; comparative example 3 shows a water absorption of 81.9%, which is the highest among all samples. This is because PVA and boric acid are not used, resulting in the lowest degree of crosslinking. While this provides it with excellent water absorption properties, doing so may compromise the structural stability and mechanical strength of the hydrogel, such high water absorption also means that the hydrogel is not sufficiently stable, especially in situations requiring long-term or high-strength applications. Meanwhile, the lack of PVA and boric acid means that this comparative example lacks a second network structure, which is generally used to increase mechanical strength and stability. The dual network system of the embodiments provides adhesion and structural stability through the first network and mechanical strength through the second network, comprehensively optimizing the performance of the hydrogel. This staged optimization strategy simplifies the manufacturing process and potentially reduces the mutual interference between ingredients, allowing finer control of the properties.

3. Antioxidant Activity test

DPPH (2, 2-diphenyl-1-pyridinehydrazine) radical scavenging test and ABTS (2, 2' -diaminobis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging test were used. The following are the detailed steps of the experiment:

DPPH radical scavenging test

Preparing a DPPH solution of 0.1. 0.1 mM using ethanol as a solvent;

the hydrogel sample and DPPH solution were mixed, left to stand in the dark for 20-30 minutes, and absorbance was measured using a spectrophotometer at 517 nm.

The DPPH radical scavenging rate was calculated.

ABTS free radical scavenging assay

Abts+ solution was prepared, the hydrogel sample and abts+ solution were mixed using water as a solvent, left to stand for 6 minutes, and absorbance was measured using a spectrophotometer at 734 nm.

The clearance of ABTS free radicals was calculated.

TABLE 3 experimental data

Test item	DPPH clearance (%)	ABTS clearance (%)
			Example 1	95	93
Example 2	78	82
			Example 3	81	76
Example 6	68	71
			Example 7	72	65
Example 8	77	70
			Example 9	79	80
Example 10	67	74
			Example 11	83	85
Example 12	74	72
			Example 13	89	88
Comparative example 2	50	54
			Comparative example 5	45	48

Example 1 used 30 nm selenium nanoparticles (SeNPs), while examples 2 and 3 used 10 nm and 50 nm particles, respectively. Particle size can affect surface area and thus antioxidant activity. In example 1, an optimal balance between surface area and reactivity was achieved.

The molar ratios of EDC, sulfo-NHS and SenPs also varied during the step of linking the nanoparticles to the protein peptides. The 1:1:10 molar ratio used in example 1 is most advantageous for the formation of stable and active complexes. EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and Sulfo-NHS (N-hydroxysulphonic acid sulfoxide) are used to activate carboxyl groups to form amide bonds, the molar ratio of these coupling agents being critical, the molar ratio having a direct effect on the efficiency of the reaction and on side reactions.

Comparative example 2: in this example, the protein peptide was added directly without conjugation to the nanoparticle. This explains its relatively low antioxidant activity.

Comparative example 5: as a commercial product, the raw materials and production process used are different from those of laboratory products, which are the reasons for the low antioxidant activity.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. An antioxidant protein peptide-based nano hydrogel for burn wound surface, the preparation method comprises the following steps:

a) Covalent modification of selenium nanoparticles (SeNPs):

b) Linking the nanoparticle to the protein peptide:

c) Forming a first network:

uniformly mixing a Hyaluronic Acid (HA) solution and a protein peptide-nanoparticle compound, gradually adding sulfur dioxide, and stirring for reaction;

d) Forming a second network:

gradually adding PVA solution into the crosslinked HA network, stirring at high speed, adding boric acid solution into the mixture, and continuously stirring for reaction;

e) And (3) introducing an auxiliary agent:

tween 20 was added to the reaction product of d) to obtain a hydrogel.

2. The hydrogel of claim 1, wherein in step b), EDC: sulfo-NHS: the molar ratio of the SeNPs is (0.5-2): (8-12).

3. The gel of claim 1, wherein in step a), the silane coupling agent is used in an amount of 5 to 10% by weight of the nanoparticles.

4. The hydrogel of claim 1, wherein the SeNPs in step a) have a particle size of 10 to 50 nm.

5. The hydrogel of claim 1, wherein the amount of protein peptide added in step b) is 2-5% by weight of SeNPs.

6. The hydrogel according to claim 1, wherein the concentration of hyaluronic acid in step c) is in the range of 0.5-3% (w/v).

7. The hydrogel of claim 1, wherein in step d) the boric acid solution has a concentration in the range of 0.3-3% (w/v).

8. The hydrogel according to claim 1, wherein the sulfur dioxide used in step c) is added in an amount of 1-5% by weight of hyaluronic acid.

9. The hydrogel of claim 1, wherein the stirring reaction of step C) is carried out at 25 ℃ to 40 ℃.

10. The hydrogel according to claims 1 to 9, wherein Tween 20 is used in an amount of 0.1 to 1% of the total weight of the hydrogel in step e).