CN115957178A - External anti-inflammatory and bacteriostatic gel spray and preparation method thereof - Google Patents
External anti-inflammatory and bacteriostatic gel spray and preparation method thereof Download PDFInfo
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- CN115957178A CN115957178A CN202211331456.7A CN202211331456A CN115957178A CN 115957178 A CN115957178 A CN 115957178A CN 202211331456 A CN202211331456 A CN 202211331456A CN 115957178 A CN115957178 A CN 115957178A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses an external anti-inflammatory and bacteriostatic gel spray and a preparation method thereof. The gel spray takes acid polysaccharide Hyaluronic Acid (HA) as a main material, and utilizes carboxyl on a polysaccharide structure of the HA to graft with Polylysine (PLL) rich in an amino structure through amidation reaction to obtain HA-PLL; the main component of catechin, epigallocatechin gallate (EGCG), is synthesized into EGCG dimer through Bayer acid catalytic condensation, and is coupled with hyaluronic acid to obtain HA-EGCG, wherein the EGCG can generate active quinone under physiological conditions due to the structure containing pyrogallol by tyrosinase (SA _ Ty) mediated oxidation, and the generated active quinone can be covalently combined with amino on HA-PLL to form stable hydrogel.
Description
Technical Field
The invention relates to an external composition with an anti-inflammatory effect, in particular to an external anti-inflammatory and bacteriostatic gel spray and a preparation method thereof.
Background
Skin abrasions are a very common type of trauma in everyday life, which is extremely common in sports activities. Skin abrasion refers to damage caused by the friction of a rough foreign object against the epidermis layer of the skin. In addition, skin abrasion may be accompanied by blunt blow, high fall, traffic accidents, and the like.
Improper treatment of abrasions can lead to ulceration and suppuration of the skin. If the foreign matters (such as dust, gravel and the like) in the wound are not completely removed, traumatic tattoos, scars and the like are left after healing, and if the bruises are positioned on the face or joints and the like, the patients can be seriously stressed by influencing the appearance or limiting the movement. Thus, timely treatment of skin abrasions is extremely important for wound healing.
After the bruise happens, the wound needs to be cleaned in time in the first step, and then the medicine is applied and is properly wrapped according to the degree of the wound. The existing external use medicine has various types, and has classical disinfectants such as mercurochrome, gentian violet, iodophor and the like, and also has a spray preparation added with various pain-relieving and inflammation-diminishing medicines. The classic disinfectant is gradually not accepted by the public because the wound is colored and not easy to clean after being coated on the wound, the beauty of the human body is affected and the like. In contrast, spray formulations are more popular with the public due to their ease of use. However, the spray preparation is liable to lose efficacy with water evaporation at the wound, the duration of efficacy is short, multiple continuous administrations are required, and in the case of severe bruising, dressing treatment may be required for preventing bacterial infection after spray treatment, and repeated administration may be required due to the short duration of efficacy, which is a relatively large challenge to patient compliance.
In summary, the conventional scratch treatment has the following disadvantages:
1. the traditional lotion of red and purple colors can seriously color the skin, is not easy to clean and affects the beauty;
2. the spraying preparation has short drug effect time and needs to be sprayed for many times;
3. some traditional bandaging methods, such as woundplasts and gauzes, are easily adhered to the wound due to direct contact with the wound, and easily tear the wound to cause secondary injury when being torn off.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an external gel spray with the functions of diminishing inflammation and healing wounds and a preparation method thereof.
The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme. According to the external anti-inflammatory and bacteriostatic gel spray provided by the invention, two materials of HA-PLL and HA-EGCG are used as main bodies to form stable gel;
the HA-PLL is obtained by grafting acid polysaccharide hyaluronic acid and polylysine;
the HA-EGCG is obtained by synthesizing epigallocatechin gallate dimer through Bayer acid catalytic condensation and coupling with hyaluronic acid.
The invention provides a preparation method of the external anti-inflammatory and bacteriostatic gel spray, which comprises the following steps:
1) Preparation of HA-PLL:
weighing sodium hyaluronate, dissolving in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adjusting pH, standing, adding N-hydroxysuccinimide, continuously stirring, weighing polylysine, adding into the solution, adjusting pH, reacting, dialyzing the solution, and freeze-drying to obtain HA-PLL;
2) Preparation of HA-EGCG:
dissolving aminoacetaldehyde diethyl acetal in a mixture of cold methane sulfonic acid and tetrahydrofuran; dissolving EGCG in tetrahydrofuran and cold methane sulfonic acid, slowly adding the mixed solution under stirring, and stirring for 12h at room temperature in a dark place; concentrating the obtained mixture by a rotary evaporator for 15-30 min, and vacuum-drying for 24h at room temperature; extracting and purifying the product, and performing rotary evaporation to obtain EGCG dimer;
coupling dimerized EGCG to hyaluronic acid by EDC-NHS chemistry to form HA-EGCG;
3) Preparation of hydrogel:
preparing an HA-PLL aqueous solution; dissolving tyrosinase in the HA-PLL aqueous solution, and marking as a solution A; preparing HA-EGCG aqueous solution, and marking as solution B; and uniformly mixing the solution A and the solution B to form stable gel.
Further, in the preparation method of the external gel spray for anti-inflammation and bacteriostasis, in the step 1), the sodium hyaluronate is dissolved in water, and each 1g of sodium hyaluronate is dissolved in 50mL of water; the mass ratio of the sodium hyaluronate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:0.8 to 1.2; the mass ratio of the sodium hyaluronate to the N-hydroxysuccinimide is as follows: 2:0.8 to 1.2; the mass ratio of the sodium hyaluronate to the polylysine is as follows: 2:0.8 to 1.5.
In a further aspect, in the step 1), the mass ratio of the sodium hyaluronate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:1; the mass ratio of the sodium hyaluronate to the N-hydroxysuccinimide is as follows: 2:1; the mass ratio of the sodium hyaluronate to the polylysine is as follows: 2:1.
further, in the preparation method of the external anti-inflammatory and bacteriostatic gel spray, in the step 1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is adjusted to be 5.0-6.0, and the standing time is 15-30 min; adding N-hydroxysuccinimide, and stirring for 15-30 min; the polylysine is weighed and added, the pH is adjusted to 7.4-10.0, and the reaction lasts for 12-24 h.
Furthermore, in the preparation method of the gel spray for external use with anti-inflammatory and antibacterial effects, in the step 1), after the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is adjusted to 5.5; after the polylysine was added, the pH was adjusted to 8.0.
Further, in the preparation method of the gel spray for external use for anti-inflammation and anti-bacteria, in the step 1), the dialysis is performed for 1 day in a sodium hydroxide solution with pH =10, and then is performed for 3 days in pure water.
Further, in the preparation method of the gel spray for external use with anti-inflammatory and bacteriostatic effects, in the step 2), the volume ratio of the aminoacetaldehyde diethyl acetal to the mixture of the cold methane sulfonic acid and tetrahydrofuran is 29;
in the mixture of the cold methane sulfonic acid and the tetrahydrofuran, the volume ratio of the cold methane sulfonic acid to the tetrahydrofuran is 1:5;
the EGCG dissolution ratio is 0.8-1.2 g/mL, and the volume ratio of tetrahydrofuran to cold methane sulfonic acid is 1500-3000: 1;
the extraction is to dissolve the product in 10mL of pure water and extract and purify the product for 5 to 10 times by using 10mL of ethyl acetate;
the coupling of dimerised EGCG to hyaluronic acid comprises the steps of: weighing hyaluronic acid, dissolving the hyaluronic acid in pure water, adding excessive 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, standing for 15-30 min, then adding N-hydroxysuccinimide with the same molar amount as the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, continuously stirring for 15-30 min, adding EGCG dimer, stirring for reacting for 12-18 h, dialyzing the product, and freeze-drying to obtain HA-EGCG;
still further, in the above method for preparing a gel spray for external use for anti-inflammation and anti-bacteria, the dimerized EGCG is coupled to hyaluronic acid, and the sodium hyaluronate is dissolved in water at a ratio of 50mL per 1g of sodium hyaluronate; the mass ratio of the sodium hyaluronate to the EGCG dimer is 2; the dialysis is performed by dialyzing the product against a dialysis membrane in pure water for 3 days.
Further, in the preparation method of the external anti-inflammatory and bacteriostatic gel spray, in the step 3), the preparation concentration of the HA-PLL aqueous solution is 0.1g/mL; the preparation concentration of tyrosinase in the solution A is 1mg/mL; the preparation concentration of the HA-EGCG aqueous solution is 0.04-0.1 g/mL; the solution A and the solution B are mixed according to a volume ratio of 1.
Furthermore, in the preparation method of the external gel spray for anti-inflammation and bacteriostasis, in the step 3), the preparation concentration of the HA-EGCG aqueous solution is 0.08g/mL.
By means of the technical scheme, the invention at least has the following advantages:
1) HA-PLL is rich in amino, can react with active quinone to generate crosslinking, and meanwhile, PLL is a natural biological metabolic product and HAs good bactericidal capacity;
2) EGCG in the HA-EGCG HAs good antioxidant activity, generates active quinone under the catalysis of tyrosinase, can be adhered to tissues while being crosslinked with HA-PLL, prevents the invasion of bacteria, and avoids the direct contact between a binding material and a wound;
3) The hydrogel disclosed by the invention has better biocompatibility, oxidation resistance and antibacterial activity, can provide good physiological conditions for wound healing, has good adhesion performance, can be adhered to the skin in a longer time period, and can play a role in isolating foreign matters such as bacteria while avoiding repeated administration.
Drawings
FIG. 1 is a schematic representation of example 5 before and after forming a gel;
FIG. 2 shows the results of the biocompatibility test of the hydrogels of the examples.
Detailed Description
The gel spray takes acid polysaccharide Hyaluronic Acid (HA) as a main material, and utilizes carboxyl on a polysaccharide structure of the HA-HA to graft (graft through covalent bonds) with Polylysine (PLL) rich in an amino structure through amidation reaction to obtain HA-PLL; the main component of catechin, epigallocatechin gallate (EGCG), is synthesized into EGCG dimer through Bayer acid catalytic condensation, and is coupled with hyaluronic acid to obtain HA-EGCG, the EGCG can be oxidized into active quinone due to the structure containing pyrogallol, which is similar to melanin chemistry and mussel induced adhesion chemistry, and the EGCG can generate active quinone under physiological conditions through tyrosinase (SA _ Ty) mediated oxidation, and HAs no cytotoxicity.
The preparation method of the hydrogel material with the function of promoting bone repair comprises the following steps:
1) Preparation of polylysine grafted hyaluronic acid (HA-PLL)
Weighing 2g of sodium hyaluronate (molecular weight of 100000) and dissolving in 100mL of water, adding 1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) after dissolving, adjusting the pH to 5.5, activating carboxyl groups on the hyaluronic acid, adding 1g of N-hydroxysuccinimide (NHS) after 15min, continuing stirring for 15min, weighing 1g of PLL and adding into the solution, adjusting the pH to 8.0, reacting overnight, filling the solution into a dialysis bag (molecular weight cut-off: 12-14 kDa), dialyzing in sodium hydroxide solution with pH =10 for 1 day, dialyzing in pure water for 3 days, and freeze-drying to obtain polylysine grafted hyaluronic acid (HA-PLL).
2) Preparation of EGCG grafted hyaluronic acid (HA-EGCG)
mu.L of aminoacetaldehyde diethyl acetal was dissolved in 1.2mL of a mixture of cold methanesulfonic acid (MSA) and Tetrahydrofuran (THF) (1, 5,v/v). EGCG (2.29 g) was dissolved in 3.8mL THF and 1.7. Mu.L MSA, and the above mixed solution was slowly added with stirring and stirred overnight at room temperature in the dark. The resulting mixture was concentrated by rotary evaporator for 20 minutes and dried under vacuum at room temperature overnight. The product is dissolved in 10mL of pure water, extracted and purified by 10mL of ethyl acetate for about 5-10 times, and rotary evaporated to obtain EGCG dimer.
Next, dimerized EGCG was coupled to hyaluronic acid by EDC-NHS chemistry to form EGCG grafted hyaluronic acid (HA-EGCG): weighing 1g of hyaluronic acid, dissolving the hyaluronic acid in 50mL of pure water, adding excessive EDC, activating carboxyl for 15min, then adding NHS with the same molar amount as EDC, continuing stirring for 15min, adding 0.5g of EGCG dimer, stirring for reacting for 6h, dialyzing the mixture in pure water for 3 days by using a dialysis membrane (12-14 kDa), and freeze-drying to obtain HA-EGCG.
The scheme takes two materials of HA-PLL and HA-EGCG as main bodies, the HA-EGCG generates active quinone under the catalysis of tyrosinase (SA _ Ty), the active quinone can react with amino on the HA-PLL to generate an imine structure to generate crosslinking, the active quinone can also combine with groups on wound tissues to play a certain adhesion performance, and a formed gel layer can effectively isolate the invasion of bacteria so as to accelerate the healing of wounds.
The hydrogel disclosed by the invention has better biocompatibility, oxidation resistance and antibacterial activity, can provide good physiological conditions for wound healing, has good adhesion performance, can be adhered to the skin in a longer time period, and can play a role in isolating foreign matters such as bacteria while avoiding repeated administration.
Example 1
As an example of the gel spray with wound healing promotion function of the present invention, the present example includes the following components in the following concentrations: 0.05g/mLHA-PLL, 0.02g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel described in this example includes the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.04g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Example 2
As an example of the gel spray with wound healing promotion function of the present invention, the present example includes the following components in the following concentrations: 0.05g/mL HA-PLL, 0.025g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel described in this example includes the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.05g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Example 3
As an example of the gel spray with wound healing promotion function of the present invention, the present example includes the following components in the following concentrations: 0.05g/mL HA-PLL, 0.03g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel of the embodiment comprises the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.06g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Example 4
As an example of the gel spray with wound healing promotion function according to the present invention, the present example includes the following components in the following concentrations: 0.05g/mL HA-PLL, 0.035g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel of the embodiment comprises the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing solution A with the concentration of 1mg/mL;
3. preparing 0.07g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Example 5
As an example of the gel spray with wound healing promotion function according to the present invention, the present example includes the following components in the following concentrations: 0.05g/mL HA-PLL, 0.04g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel described in this example includes the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.08g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Schematic diagrams before and after the glue is formed in the embodiment are shown in fig. 1.
Example 6
As an example of the gel spray with wound healing promotion function of the present invention, the present example includes the following components in the following concentrations: 0.05g/mL HA-PLL, 0.045g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel described in this example includes the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.09g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Example 7
As an example of the gel spray with wound healing promotion function of the present invention, the present example includes the following components in the following concentrations: 0.05g/mL HA-PLL, 0.05g/mL HA-EGCG, 0.5mg/mL tyrosinase.
The hydrogel of the embodiment comprises the following steps:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.1g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Eg. comparative example
Preparation of hydrogel:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL; marking as a solution A;
2. preparing 0.08g/mL HA-EGCG aqueous solution, and marking as solution B;
3. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Eg. comparative example
Preparation of hydrogel:
preparing 0.08g/mL HA-EGCG aqueous solution, adding tyrosinase to prepare 1mg/mL HA-EGCG aqueous solution; a stable gel was formed.
Eg. comparative example 1
Preparation of polylysine-grafted hyaluronic acid (HA-PLL):
weighing 2g of sodium hyaluronate (molecular weight of 100000) and dissolving in 100mL of water, adding 1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) after dissolving, adjusting the pH to 5.5, activating carboxyl groups on the hyaluronic acid, adding 1g of N-hydroxysuccinimide (NHS) after 15min, continuing stirring for 15min, weighing 1g of PLL and adding into the solution, adjusting the pH to 8.0, reacting overnight, filling the solution into a dialysis bag (molecular weight cut-off: 12-14 kDa), dialyzing in sodium hydroxide solution with the pH =10 for 1 day, dialyzing in pure water for 3 days, and freeze-drying to obtain polylysine grafted hyaluronic acid (HA-PLL).
Preparation of EGCG grafted hyaluronic acid (HA-EGCG):
coupling EGCG to hyaluronic acid by EDC-NHS chemistry to form EGCG grafted hyaluronic acid (HA-EGCG): weighing 1g of hyaluronic acid, dissolving the hyaluronic acid in 50mL of pure water, adding excessive EDC, activating carboxyl for 15min, then adding NHS with the molar quantity equal to that of EDC, continuing stirring for 15min, adding 0.5g of EGCG, stirring for reacting for 6h, dialyzing the mixture in pure water for 3 days by using a dialysis membrane (12-14 kDa), and freeze-drying to obtain HA-EGCG.
Preparation of hydrogel:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.08g/mL HA-EGCG aqueous solution, and marking as solution B;
4. and uniformly mixing the solution A and the solution B according to a volume ratio of 1.
Eg. comparative example 2
Preparation of polylysine-grafted hyaluronic acid (HA-PLL):
weighing 2g of sodium hyaluronate (molecular weight 100000) and dissolving in 100mL of water, adding 1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) after dissolving, adjusting the pH to 5.5, activating carboxyl on hyaluronic acid, adding 1g of N-hydroxysuccinimide (NHS) after 15min, continuously stirring for 15min, weighing 1g of PLL and adding into the solution, adjusting the pH to 8.0, reacting overnight, filling the solution into a dialysis bag (molecular weight cut-off: 12-14 kDa), dialyzing in sodium hydroxide solution with the pH =10 for 1 day, dialyzing in pure water for 3 days, and freeze-drying to obtain polylysine grafted hyaluronic acid (HA-PLL).
Preparation of hydrogel:
1. preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL;
2. dissolving tyrosinase in the HA-PLL solution, and preparing a solution A with the concentration of 1mg/mL;
3. preparing 0.08g/mL EGCG aqueous solution, and marking as solution B;
4. the solution A and the solution B are uniformly mixed according to the volume ratio of 1, and the method cannot form gel.
Pure EGCG HAs a relatively small molecular weight, can react with HA-PLL after being oxidized by tyrosine, but cannot form a cross-linked network, and finally can exist in a flocculent precipitate form, so that gel cannot be formed.
Eg. comparative example 3
Preparation of polylysine-grafted hyaluronic acid (HA-PLL):
weighing 2g of sodium hyaluronate (molecular weight of 100000) and dissolving in 100mL of water, adding 1g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) after dissolving, adjusting the pH to 5.5, activating carboxyl groups on the hyaluronic acid, adding 1g of N-hydroxysuccinimide (NHS) after 15min, continuing stirring for 15min, weighing 1g of PLL and adding into the solution, adjusting the pH to 8.0, reacting overnight, filling the solution into a dialysis bag (molecular weight cut-off: 12-14 kDa), dialyzing in sodium hydroxide solution with the pH =10 for 1 day, dialyzing in pure water for 3 days, and freeze-drying to obtain polylysine grafted hyaluronic acid (HA-PLL).
Preparation of hydrogel: preparing an HA-PLL aqueous solution with the concentration of 0.1g/mL; tyrosinase was dissolved in the above HA-PLL solution, and this method failed to gel.
Example of Effect verification
1. Hydrogel gel formation time test
Testing the gel forming time of the hydrogel by adopting an inversion method, uniformly mixing the solution A and the solution B, pouring the mixture into a glass bottle, repeatedly inverting the glass bottle until the mixed solution does not flow any more, and recording the gel forming time of the hydrogel.
Table 1 shows the results of the gel-forming time tests of the examples, from which it can be seen that when the concentration of HA-PLL is fixed, the gel-forming time decreases with increasing concentration of HA-EGCG, since the proportion of EGCG is also higher in high concentrations of HA-EGCG, which increases in proportion and rate of oxidation to active quinone under the catalysis of tyrosinase, thereby accelerating the crosslinking and formation of gel. The gelling time of example 5 is 38.73 +/-8.36 s, and the quick gelling characteristic makes the gel spray prepared.
TABLE 1 gel formation time for the examples
2. Adhesion test
Cutting fresh pigskin into small pieces with the length multiplied by 1cm, scraping off a fat layer, cleaning, wiping off excessive water by using gauze, uniformly coating a thin layer of hydrogel liquid on the 2 multiplied by 1cm part of the center of one pigskin (marked as P1), slightly covering one end of the other pigskin (marked as P2) on an area containing the hydrogel liquid, placing the pigskin in a refrigerator at 4 ℃, taking out the pigskin after 30min, fixing the pigskin P1 on a universal mechanical tester platform, clamping the other end of the pigskin P2 by using a clamp, performing tensile stripping at an angle of 90 degrees with the angle of P2, recording the change of force along with the stripping length in the stripping process, integrating the curve of the force and the stripping length, and calculating the average stripping strength.
The results of the tack test for each example are shown in table 2 below.
TABLE 2 tack test results for each example
As can be seen from table 2, the viscosity of the hydrogel increases with the increase of the concentration of HA-EGCG, because the proportion of EGCG increases, and the pyrogallol can be combined with some surface groups on skin tissues through chemical bonding and intermolecular force while participating in gelling after being oxidized into active quinone, so as to present a certain adhesiveness.
3. Test for Oxidation resistance
By the use of H 2 O 2 Detection kit for detecting hydrogen peroxide (H) 2 O 2 ) The concentration was decreased to evaluate the antioxidant capacity of the hydrogel. First, H is established within a suitable concentration range 2 O 2 Standard Curve, adding 100. Mu.L of hydrogel to 1mL of H 2 O 2 In solution (25 mM). After 30min of reaction, the remaining H was determined 2 O 2 And (4) concentration.
The damaged part is often accompanied by the rise of Reactive Oxygen Species (ROS), the ischemia injury caused by the ROS rise causes the obvious increase of proinflammatory cytokines such as TNF-alpha and IL-6, thereby causing the inflammation infiltration of ischemic tissues, and the gel clearance activity is tested forOxygen capacity, tested by the present invention against H 2 O 2 The results of the antioxidant performance test of each example are as follows
Shown in table 3.
TABLE 3 antioxidant Performance test results of the examples
As can be seen from Table 3, the active oxygen scavenging capacity of the gel is enhanced along with the increase of the HA-EGCG ratio, and the appropriate HA-EGCG concentration can effectively relieve the inflammatory symptoms at the wound and accelerate the healing of the wound.
4. Antibacterial property
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. The number of culturable colonies was counted after 24h incubation at 37 ℃ using the following formula:
AR(%)=(Nc-Ns)/Nc×100;
the Antibacterial Ratio (AR) was calculated, where Nc is the average number of colonies of the control sample and Ns is the average number of bacterial colonies of the hydrogel sample.
The results of the in vitro antibacterial testing for each example are shown in table 4 below.
TABLE 4 in vitro antibacterial test results for each example
As seen from table 4, the hydrogel exhibited better antibacterial properties because the antibacterial properties of the gel were reduced when the concentration of HA-EGCG was increased to a certain degree due to the presence of PLL in the gel, which may be that a part of amino groups in HA-PLL were bonded, thereby decreasing the antibacterial properties of the gel.
5. Biocompatibility
Cultured human dermal fibroblasts were digested with 0.25% trypsin and suspended at a density of 2X 10 per well 4 one/mL cell suspension was seeded in 48-well plates. Culturing for 12 hr, taking out stock culture solution, transferring hydrogel sample to 48-well plate, inoculating 10-contained hydrogel 5 Cell suspension 100. Mu.L/mL. Each group is provided with at least 5 holes. The liquid is changed every 24h, and three time points of 1d, 4d and 7d are set in the experiment. The specific operation method comprises the following steps:
cell survival rate: cell viability was quantified using CCK 8. The corresponding well plate was removed at the indicated time intervals, 100. Mu.L of CCK8 working solution was added to each well, and carbon dioxide incubator (containing 5% CO) was maintained at a constant temperature of 37 ℃ 2 ) After incubation for 1-2 h, measuring the absorbance (OD) at the wavelength of 450nm by using an enzyme-labeling instrument, and calculating the cell survival rate according to the formula:
cell survival (%) = OD experimental group/OD control group × 100%.
Fig. 2 shows the result of the biocompatibility test of the hydrogel of each example, and it can be seen from fig. 2 that when the concentration of HA-EGCG is lower, the gel shows weak toxicity, which may be due to more residual amino groups remaining in the gel, and the biocompatibility of the gel shows a certain gain effect as the proportion of HA-EGCG increases, which indicates that HA-PLL and HA-EGCG in proper proportion are almost non-toxic to human cells, which provides an advantage for using the hydrogel as a gel spray.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, so that any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (10)
1. An external gel spray for anti-inflammation and bacteriostasis, which is characterized in that: two materials of HA-PLL and HA-EGCG are used as main bodies to form stable gel;
the HA-PLL is obtained by grafting acid polysaccharide hyaluronic acid and polylysine;
the HA-EGCG is obtained by synthesizing epigallocatechin gallate dimer through Bayer acid catalytic condensation and coupling with hyaluronic acid.
2. The preparation method of the gel spray for external use with anti-inflammation and bacteriostasis is characterized by comprising the following steps:
1) Preparation of HA-PLL:
weighing sodium hyaluronate, dissolving in water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adjusting pH, standing, adding N-hydroxysuccinimide, continuously stirring, weighing polylysine, adding into the solution, adjusting pH, reacting, dialyzing the solution, and freeze-drying to obtain HA-PLL;
2) Preparation of HA-EGCG:
dissolving aminoacetaldehyde diethyl acetal in a mixture of cold methane sulfonic acid and tetrahydrofuran; dissolving EGCG in tetrahydrofuran and cold methanesulfonic acid, slowly adding the mixture of the cold methanesulfonic acid and the tetrahydrofuran in which aminoacetaldehyde diethyl acetal is dissolved under stirring, and stirring for 12 hours at room temperature in a dark place; concentrating the obtained mixture by a rotary evaporator for 15-30 min, and vacuum-drying for 24h at room temperature; extracting and purifying the product, and performing rotary evaporation to obtain EGCG dimer;
coupling dimerized EGCG to hyaluronic acid by EDC-NHS chemistry to form HA-EGCG;
3) Preparation of hydrogel:
preparing an HA-PLL aqueous solution; dissolving tyrosinase in the HA-PLL aqueous solution to obtain a solution A; preparing HA-EGCG aqueous solution, and marking as solution B; and uniformly mixing the solution A and the solution B to form stable gel.
3. The method of claim 2, wherein: in the step 1), the sodium hyaluronate is dissolved in 50mL of water per 1g of sodium hyaluronate;
the mass ratio of the sodium hyaluronate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:0.8 to 1.2;
the mass ratio of the sodium hyaluronate to the N-hydroxysuccinimide is as follows: 2:0.8 to 1.2;
the mass ratio of the sodium hyaluronate to the polylysine is as follows: 2:0.8 to 1.5.
4. The production method according to claim 3, characterized in that: in the step 1), the mass ratio of the sodium hyaluronate to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 2:1;
the mass ratio of the sodium hyaluronate to the N-hydroxysuccinimide is as follows: 2:1;
the mass ratio of the sodium hyaluronate to the polylysine is as follows: 2:1.
5. the method of claim 2, wherein: in the step 1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is adjusted to be 5.0-6.0, and the standing time is 15-30 min;
adding N-hydroxysuccinimide, and stirring for 15-30 min;
the polylysine is weighed and added, the pH value is adjusted to 7.4-10.0, and the reaction time is 12-24 h.
6. The method of claim 5, wherein: in the step 1), after the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the pH is adjusted to 5.5;
after the polylysine was added, the pH was adjusted to 8.0.
7. The method of claim 2, wherein: in the step 1), the dialysis is performed for 1 day in a sodium hydroxide solution with pH =10, and then for 3 days in pure water.
8. The production method according to claim 2, characterized in that: in the step 2), the volume ratio of the aminoacetaldehyde diethyl acetal to the cold methane sulfonic acid and tetrahydrofuran mixture is 29;
in the mixture of the cold methane sulfonic acid and the tetrahydrofuran, the volume ratio of the cold methane sulfonic acid to the tetrahydrofuran is 1:5;
the EGCG dissolution ratio is 0.8-1.2 g/mL, and the volume ratio of tetrahydrofuran to cold methane sulfonic acid is 1500-3000: 1;
the extraction is to dissolve the product in 10mL of pure water and extract and purify the product for 5 to 10 times by using 10mL of ethyl acetate;
the coupling of dimerized EGCG to hyaluronic acid comprises the steps of: weighing hyaluronic acid, dissolving the hyaluronic acid in pure water, adding excessive 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, standing for 15-30 min, adding N-hydroxysuccinimide with the same molar amount as the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, continuously stirring for 15-30 min, adding EGCG dimer, stirring for 12-18 h, dialyzing the product, and freeze-drying to obtain HA-EGCG;
coupling the dimerized EGCG to hyaluronic acid, said sodium hyaluronate dissolved in water being dissolved in 50mL of water per 1g of sodium hyaluronate; the mass ratio of the sodium hyaluronate to the EGCG dimer is 2; the dialysis is performed by dialyzing the product against a dialysis membrane in pure water for 3 days.
9. The method of claim 2, wherein the HA-PLL aqueous solution is prepared at a concentration of 0.1g/mL in step 3);
the preparation concentration of tyrosinase in the solution A is 1mg/mL;
the preparation concentration of the HA-EGCG aqueous solution is 0.04-0.1 g/mL;
the solution A and the solution B are mixed according to a volume ratio of 1.
10. The method for preparing the compound of claim 9, wherein the HA-EGCG aqueous solution is prepared at a concentration of 0.08g/mL in the step 3).
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090142309A1 (en) * | 2003-01-10 | 2009-06-04 | The Cleveland Clinic Foundation | Hydroxyphenyl cross-linked macromolecular network and applications thereof |
CN106902392A (en) * | 2017-03-01 | 2017-06-30 | 西南交通大学 | Carry the preparation method of heparin/poly-D-lysine nano-particle hyaluronic acid gel |
CN111732741A (en) * | 2020-06-24 | 2020-10-02 | 华熙生物科技股份有限公司 | Method for crosslinking hyaluronic acid and polylysine, composite crosslinked product obtained by method and application of composite crosslinked product |
CN112190763A (en) * | 2020-06-15 | 2021-01-08 | 南京工业大学 | Hyaluronic acid/epsilon-polylysine antibacterial hydrogel and preparation method and application thereof |
CN112778772A (en) * | 2020-12-31 | 2021-05-11 | 中山大学附属第一医院 | Antibacterial composite hydrogel and preparation method and application thereof |
CN113559314A (en) * | 2021-08-06 | 2021-10-29 | 西北大学 | Extracellular matrix-simulated hydrogel dressing for diabetic foot ulcer and preparation method thereof |
-
2022
- 2022-10-28 CN CN202211331456.7A patent/CN115957178A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090142309A1 (en) * | 2003-01-10 | 2009-06-04 | The Cleveland Clinic Foundation | Hydroxyphenyl cross-linked macromolecular network and applications thereof |
CN106902392A (en) * | 2017-03-01 | 2017-06-30 | 西南交通大学 | Carry the preparation method of heparin/poly-D-lysine nano-particle hyaluronic acid gel |
CN112190763A (en) * | 2020-06-15 | 2021-01-08 | 南京工业大学 | Hyaluronic acid/epsilon-polylysine antibacterial hydrogel and preparation method and application thereof |
CN111732741A (en) * | 2020-06-24 | 2020-10-02 | 华熙生物科技股份有限公司 | Method for crosslinking hyaluronic acid and polylysine, composite crosslinked product obtained by method and application of composite crosslinked product |
CN112778772A (en) * | 2020-12-31 | 2021-05-11 | 中山大学附属第一医院 | Antibacterial composite hydrogel and preparation method and application thereof |
CN113559314A (en) * | 2021-08-06 | 2021-10-29 | 西北大学 | Extracellular matrix-simulated hydrogel dressing for diabetic foot ulcer and preparation method thereof |
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