CN115006583A

CN115006583A - Medical dressing, preparation method and application

Info

Publication number: CN115006583A
Application number: CN202210787095.0A
Authority: CN
Inventors: 蔡晓军; 杨超; 李林; 陈冬帆; 陈佳乐
Original assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Current assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2022-09-06
Anticipated expiration: 2042-07-04

Abstract

The invention relates to the technical field of medical dressings, in particular to a medical dressing, a preparation method and application, wherein the medical dressing comprises a base material, wherein the surface of the base material contains a quaternary ammonium salt antibacterial agent and a gelatin gel layer; the quaternary ammonium salt antibacterial agent is stably adsorbed on the surface of the base material through a mussel bionic chemical strategy, the gelatin gel is deposited on the base material attached with the quaternary ammonium salt antibacterial agent through physical adsorption, and the quaternary ammonium salt antibacterial agent is respectively connected with the gelatin gel layer and the base material through hydrogen bonds, covalent bonds and pi-pi interaction to form a gel microporous structure on the surface of the base material. The medical dressing has the functions of quick hemostasis and high-efficiency antibiosis, and a gel micropore structure is formed on the surface of the base material through the quaternary ammonium salt antibacterial agent and the gelatin gel, so that the dressing can absorb water quickly to promote hemostasis and exert high-efficiency antibiosis capability, and meanwhile, cells can be effectively gathered, moisture is maintained, and wound healing is promoted.

Description

Medical dressing, preparation method and application

Technical Field

The invention relates to the technical field of medical dressings, in particular to a medical dressing, a preparation method and application thereof.

Background

The primary principle of clinical management of acute wounds is rapid hemostasis and debridement disinfection, although the commonly used cotton non-woven dressing can wrap and protect the wound in the form of a bandage and exert the hemostatic effect by rapidly absorbing blood and activating platelets through aggregation; it also shields the wound and promotes scabbing by gas exchange and absorption of exudate. However, the traditional cotton non-woven fabric dressing has slow hemostasis and no antibacterial effect, and easily causes excessive blood loss and bacterial growth, so that infection is induced, the wound healing process is delayed, and especially after infection of multidrug resistant bacteria, huge risks such as septicemia, organ failure and death are caused. Therefore, trying to improve the hemostatic and antibacterial properties of cotton nonwoven dressings is crucial for the repair of acute bleeding wounds, reducing bacterial infections and reducing medical costs.

Although such as oxidized regenerated cellulose gauze has been developed

Carboxymethyl cotton fiber

Kaolin/non-woven fabric

And the like; various antibacterial cotton yarn dressings are developed by utilizing various antibacterial agents; however, there is little research and development on cotton yarn dressings that can simultaneously rapidly stop bleeding and effectively resist bacteria, and they are used for the treatment of acute wounds. Therefore, if the hemostatic and antibacterial agents can be stably integrated into the cotton yarn dressing by using new technologies or processes, a structure may be formedThe multifunctional cotton yarn dressing can simultaneously realize rapid hemostasis and high-efficiency antibiosis.

To achieve the above goals, mussel biomimetic chemistry may be a simple, practical and fruitful functional modification strategy. The strategy can be realized by chemically grafting a small molecular compound containing catechol group onto a hemostatic agent or an antibacterial agent respectively, and then integrating the hemostatic agent and the antibacterial agent onto the cotton dressing simultaneously by using catechol-mediated interaction (such as hydrogen bond, metal coordination, Michael addition, Schiff base reaction and the like). In fact, mussel biomimetic chemistry has been investigated for surface functionalization modification of cotton yarn dressings as a simple, efficient surface functionalization modification strategy. For example, researchers use catechol/amino chemistry inspired by mussel to easily and firmly fix amphoteric particles poly (carboxyl betaine-co-dopamine methacrylamide) @ silver nanoparticles (PCBDA @ Ag NPs) on amino-modified medical cotton gauze through covalent and non-covalent actions, so that the antibacterial cotton gauze with excellent antibacterial capability is constructed, and the antibacterial cotton gauze not only has excellent bacterial anti-adhesion and bactericidal activity, but also has good blood compatibility and cell compatibility, and is beneficial to wound and healing.

Disclosure of Invention

Aiming at the problems of poor hemostasis and wound infection existing in acute wounds treated by cotton non-woven fabrics, the invention aims to provide the cotton non-woven fabrics with the rapid hemostasis and antibiosis functions, which are simple to prepare, rich in functions and capable of promoting healing, so that the cotton non-woven fabrics can be used for acute wound care, achieve the effects of rapid hemostasis, high-efficiency antibiosis, seepage absorption, wound moistening and healing environment maintenance and wound healing promotion.

According to a first aspect of the object of the present invention, there is provided a medical dressing comprising a substrate, further comprising:

a quaternary ammonium salt antimicrobial agent that is stably adsorbed on the substrate surface by a mussel biomimetic chemical strategy;

a gelatin gel layer that deposits a gelatin gel on the base material attached with the antibacterial agent by physical adsorption;

the antibacterial agent is respectively connected with the gelatin gel layer and the base material through hydrogen bonds, covalent bonds and pi-pi interaction, and a gel micropore structure is formed on the surface of the base material.

Preferably, the mass ratio of the gelatin to the quaternary ammonium salt antibacterial agent is 5:3, and the quaternary ammonium salt antibacterial agent is catechol quaternary ammonium salt chitosan;

preferably, the base material is a cotton non-woven fabric, and the cotton non-woven fabric is pretreated by absolute ethyl alcohol.

According to a second aspect of the object of the present invention, there is provided a method for preparing the aforementioned medical dressing, comprising the steps of:

s1, ultrasonically cleaning a cotton non-woven fabric (NF) by using absolute ethyl alcohol, removing surface impurities, and then drying for later use;

s2, immersing the NF obtained by processing the S1 into a quaternary ammonium salt antibacterial agent solution dissolved in a Tris-HCl buffer solution, depositing for 24 hours at 37 ℃, then taking out the non-woven fabric, cleaning for 3 times by using ethanol and deionized water, and drying for 2 hours at 60 ℃ to obtain a CQCS modified NF sample CQCS @ NF (CNF);

s3, immersing the CNF prepared by the S2 in gelatin (Gel) solution dissolved by PBS, shaking for a certain time under the condition of water bath at a certain temperature, uniformly depositing, then taking out a sample, extruding to the same wet weight, placing in a refrigerator at 4 ℃ for 5 hours to form a CQCS/Gel hydrogel layer, finally immersing in deionized water for 10S, and removing hydrogel which is not attached on non-woven fabrics. And freeze-drying to obtain the Gel coated CQCS modified cotton non-woven fabric composite dressing Gel-CQCS @ NF (GCNF).

Preferably, in step S1, the absolute ethanol washing time is 20min, and the drying temperature is 60 ℃.

Preferably, in the step S2, the Tris-HCI buffer solution has a pH of 8.5 and a solution concentration of 10 mMol.

Preferably, in step S2, the quaternary ammonium salt antibacterial agent solution is a catechol quaternary ammonium salt chitosan solution, the concentration of the quaternary ammonium salt antibacterial agent solution is 10 to 40g/L, and the bath ratio of NF to the catechol quaternary ammonium salt chitosan solution is 1: 30-50.

Preferably, in the step S3, the certain temperature is 40 to 60 ℃ and the certain time is 1.5 to 24 hours.

Preferably, the preparation of the catechol-based quaternary ammonium salt chitosan comprises the following steps:

x1, dissolving chitosan in glacial acetic acid to prepare a suspension, continuously stirring at a first temperature interval, slowly adding a glycidyltrimethylammonium chloride (GTMAC) solution into the suspension, continuously reacting for 18-24h, taking out the mixture, centrifuging at room temperature to remove insoluble polymers, filtering a supernatant through a Buchner funnel under reduced pressure, precipitating a filtrate in precooled absolute ethyl alcohol, filtering, washing for three times, and then placing the filtrate in a vacuum oven for drying for 3 days to obtain a light yellow quaternary ammonium salt chitosan (QCS) solid;

x2, dissolving QCS in deionized water to prepare a solution, adding a 3, 4-dihydroxyphenyl propionic acid (DHPA) solution in deionized water, and adjusting the pH of the solution to 5.5. Weighing carbodiimide (EDC), dissolving in a mixed solution of deionized water and absolute ethyl alcohol (1:1, v/v), adding into the reaction solution, adjusting pH to 5.5, stirring vigorously at room temperature for 4h, adjusting pH once every 1h, and keeping the pH at about 5.5. After the reaction, the mixture was dialyzed for 48 hours in deionized water having a pH of 5.5 using a dialysis bag having a molecular weight of 3500 standard, and freeze-dried to obtain catechol quaternary ammonium salt chitosan (CQCS).

Preferably, in the step S1, the chitosan solution concentration is 30g/L, and the glacial acetic acid solution concentration is 0.2%.

Preferably, in step S1, the first temperature range of the reaction is 60-75 ℃, and the mass ratio of the glycidyl trimethyl ammonium chloride to the chitosan is 1.2: 1.

preferably, in step S1, the centrifugation speed in the purification process is 5000rpm, and the centrifugation time is 10 min.

Preferably, in the step S2, the concentration of the quaternized chitosan solution is 20g/L, the concentration of the 3, 4-dihydroxyphenyl propionic acid solution is 100g/L, the concentration of the carbodiimide solution is 50g/L, and the molar ratios of the three materials are 5: 2.5: 1.

according to a third aspect of the object of the present invention, there is provided a use of the aforementioned medical dressing in the fields of rapid hemostasis and high-efficiency antibiosis.

According to a fourth aspect of the object of the present invention, there is provided a use of the aforementioned medical dressing in the field of wound repair.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the medical dressing, the catechol quaternary ammonium salt chitosan and the gelatin are sequentially deposited on the surface of the cotton non-woven fabric by adopting a simple layer-by-layer deposition process, the catechol quaternary ammonium salt chitosan can be connected with a cellulose unit and a gelatin long chain of the cotton non-woven fabric through hydrogen bonds and electrostatic interaction, the interiors of molecules of the catechol quaternary ammonium salt chitosan and the gelatin long chain can also be connected through hydrogen bonds, covalent bonds and pi-pi interaction, and a stable cross-linked gel structure is formed on the surface of the cotton non-woven fabric, so that water molecules are effectively absorbed and diffused, the aggregation and activation of erythrocytes and platelets are promoted, the formation of blood clots is accelerated, and hemostasis is realized; meanwhile, the microporous structure suitable for the gelatin gel layer can ensure the good antibacterial effect of the quaternary ammonium salt antibacterial agent, and can retain water through water absorption and expansion, so that the moist environment required by wound healing is maintained, the wound infection chance is effectively reduced, and the healing is promoted.

2. According to the medical dressing, the gelatin and the quaternary ammonium salt antibacterial agent form a cross-linked gel structure on the surface of the cotton non-woven fabric, the quaternary ammonium salt antibacterial agent is wrapped in the three-dimensional network, exposure of partial quaternary ammonium cations is shielded, the surface layer gelatin gradually falls off and degrades along with the prolonging of the contact time, the shielded quaternary ammonium cations are gradually exposed to participate in an antibacterial process, and the catechol has a stable structure and strong adhesion, so that the deposited quaternary ammonium cations can be adhered for a long time, and the dressing has stable and lasting antibacterial activity.

3. The invention combines the excellent antibacterial property of the cationic polymer with the natural biocompatibility of the gelatin, ensures that the functionalized cotton non-woven fabric has excellent cationic membrane-penetrating sterilization performance, has good biological safety and does not influence cell proliferation.

4. The novel cotton non-woven fabric with rapid hemostasis and high-efficiency antibacterial performance can be prepared by a simple layer-by-layer deposition and dipping drying process without an external cross-linking agent, and the novel cotton non-woven fabric is simple in preparation process, low in raw material cost, excellent in performance and good in safety.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

Fig. 1 is a flow chart of the preparation of a medical dressing of the present invention.

FIG. 2 is a schematic diagram of the synthesis of catechol quaternary ammonium salt chitosan according to the present invention

FIG. 3a is an FT-IR spectrum of CS, QCS, and CQCS in example 2.

FIG. 3b is a UV-vis spectrum of CS, QCS, and CQCS in example 2.

FIG. 3c is of CS, QCS, and CQCS in example 2 ¹ HNMR spectrogram.

FIG. 4a is the FT-IR spectra of NF, CNF, GCNF-1, GCNF-5 and GCNF-10 in example 3.

FIG. 4b is an XPS spectrum of NF, CNF, GCNF-1, GCNF-5 and GCNF-10 from example 3.

FIG. 4c is the fluorescent labeling and SEM images of NF, CNF, GCNF-1, GCNF-5 and GCNF-10 in example 3.

FIG. 5 is a graph showing liquid absorption measurements of NF, CNF, GCNF-1, GCNF-5, GCNF-10 and MHS in example 4

FIG. 6a is the BCI assay results for NF, CNF, GCNF-1, GCNF-5, GCNF-10, and MHS in example 5.

FIG. 6b is the hemodynamic behavior of NF, CNF, GCNF-1, GCNF-5, GCNF-10 and MHS in example 5.

FIG. 6c is the in vitro hemostatic effect of NF, CNF, GCNF-1, GCNF-5, GCNF-10, and MHS of example 5.

FIG. 6d is the surface red blood cell/platelet adhesion behavior of NF, CNF, GCNF-1, GCNF-5, GCNF-10, and MHS in example 5.

FIG. 7a shows the inhibition zones of NF, CNF and GCNF-5 against Staphylococcus aureus and Escherichia coli in example 6.

FIG. 7b is the results of absorbance of NF, CNF and GCNF-5 against Staphylococcus aureus in example 6.

FIG. 7c is the results of absorbance of NF, CNF and GCNF-5 on E.coli in example 6.

FIG. 7d is the dot plate count of NF, CNF and GCNF-5 against S.aureus and E.coli in example 6.

FIG. 7e is the colony count results of CNF and GCNF-5 against S.aureus and E.coli in example 6 within 30 min.

FIG. 7f is the colony counts of NF, CNF and GCNF-5 on S.aureus and E.coli in example 6 over 24 h.

FIG. 8a is the elution rate change for example 7 after soaking CNF in PBS at different pH values for 7 days.

FIG. 8b is the elution rate change for example 7 after soaking GCNF-5 in PBS at different pH values for 7 days.

FIG. 8c shows the change in fluorescence labeling of GCNF-5 soaked with PBS at different pH for 7 days in example 7.

FIG. 9a is a graph showing the change in the antibacterial rate against Staphylococcus aureus when GCNF-5 was soaked with PBS at different pH values for 7 days in example 7.

FIG. 9b is the change of the antibacterial rate against E.coli in example 7 after soaking GCNF-5 in PBS at different pH values for 7 days.

FIG. 9c shows the BCI results for example 7 with varying pH of PBS soaking GCNF-5 for 7 days.

FIG. 10a is the actual hemolysis results for NF, CNF and GCNF-5 in example 8.

FIG. 10b is the results of the hemolysis rates of NF, CNF and GCNF-5 in example 8.

FIG. 10c is the live and dead staining results of L929 cells and EA.hy926 cells treated with NF, CNF and GCNF-5 in example 8.

FIG. 10d is a graph comparing the cell viability of L929 cells treated with NF, CNF and GCNF-5 in example 8.

FIG. 10e is a graph comparing cell viability of EA.hy926 cells treated with NF, CNF and GCNF-5 in example 8.

FIG. 11a is a schematic diagram of the mouse liver injury model in example 9 and the hemostatic effects of NF, CNF, GCNF-5 and MHS.

FIG. 11b is a graph comparing the blood loss in the NF, CNF, GCNF-5, and MHS-treated liver injury models in example 9.

FIG. 11c is a graph comparing the hemostatic time of the NF, CNF, GCNF-5 and MHS-treated liver injury models in example 9.

FIG. 11d is a schematic representation of the mouse tailgating hemorrhage model in example 9 and the hemostatic effects of NF, CNF, GCNF-5 and MHS are shown.

FIG. 11e is a graph comparing blood loss for NF, CNF, GCNF-5, and MHS treated tailed bleeding models in example 9.

FIG. 11f is a graph comparing the hemostatic time for NF, CNF, GCNF-5, and MHS treatment of the tailed bleeding model in example 9.

FIG. 12a is a graph of the macroscopic change in healing of wounds over time following the action of NF, CNF, GCNF-5 and MHS in example 10.

FIG. 12b is a graph comparing the closure rate of wounds following the action of NF, CNF, GCNF-5 and MHS in example 10.

FIG. 12c is a graph of the tissue colonization effect of skin wounds following the action of NF, CNF, GCNF-5 and MHS in example 10.

FIG. 12d is a graph comparing the tissue colony counts of skin wounds following the action of NF, CNF, GCNF-5 and MHS in example 10.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In this disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. Embodiments of the present disclosure are not necessarily intended to include all aspects of the invention. It should be appreciated that the various concepts and embodiments described above, as well as those described in greater detail below, may be implemented in any of numerous ways.

With the combination of figure 1, the invention provides a medical dressing, which adopts a simple layer-by-layer deposition process to sequentially deposit catechol quaternary ammonium salt chitosan and gelatin on the surface of a cotton non-woven fabric, wherein the catechol quaternary ammonium salt chitosan can be connected with a cellulose unit and a gelatin long chain of the cotton non-woven fabric through hydrogen bonds and electrostatic interaction, the interiors of molecules of the catechol quaternary ammonium salt chitosan and the gelatin long chain can also be connected through hydrogen bonds, covalent bonds and pi-pi interaction, a stable cross-linked gel structure is formed on the surface of the cotton non-woven fabric, the good antibacterial effect of a quaternary ammonium salt antibacterial agent is ensured, water can be retained through water absorption and expansion, the moist environment required for wound healing is further maintained, the wound infection chance is effectively reduced, and the healing is promoted.

In a particular embodiment, a medical dressing is provided, comprising a substrate having a surface comprising a quaternary ammonium salt antimicrobial agent and a gelatin gel layer; the quaternary ammonium salt antibacterial agent is stably adsorbed on the surface of the base material through a mussel bionic chemical strategy, the gelatin gel is deposited on the base material attached with the quaternary ammonium salt antibacterial agent through physical adsorption, and the quaternary ammonium salt antibacterial agent is respectively connected with the gelatin gel layer and the base material through hydrogen bonds, covalent bonds and pi-pi interaction to form a gel microporous structure on the surface of the base material.

In a preferred embodiment, the mass ratio of the gelatin to the quaternary ammonium salt antibacterial agent is 5:3, and the quaternary ammonium salt antibacterial agent is catechol quaternary ammonium salt chitosan.

In a preferred embodiment, the substrate is a cotton non-woven fabric, and the cotton non-woven fabric is pretreated by absolute ethyl alcohol.

In another preferred embodiment, there is provided a method for preparing the medical dressing, comprising the steps of:

and S1, ultrasonically cleaning the cotton non-woven fabric (NF) by using absolute ethyl alcohol, removing surface impurities, and then drying for later use.

S2, immersing the NF obtained by the treatment of S1 in a quaternary ammonium salt antibacterial agent solution dissolved in a Tris-HCl buffer solution, depositing for 24 hours at 37 ℃, taking out the non-woven fabric, washing for 3 times by using ethanol and deionized water, and drying for 2 hours at 60 ℃ to obtain a CQCS modified NF sample CQCS @ NF (CNF).

S3, immersing the CNF prepared in the S2 in gelatin (Gel) solution dissolved in PBS, shaking for a certain time under the condition of water bath at a certain temperature to deposit uniformly, then taking out a sample to be extruded to the same wet weight, placing the sample in a refrigerator at 4 ℃ for 5 hours to form a CQCS/Gel hydrogel layer, finally immersing in deionized water for 10S, and removing hydrogel which is not attached to non-woven fabrics. And freeze-drying to obtain the Gel coated CQCS modified cotton non-woven fabric composite dressing Gel-CQCS @ NF (GCNF).

In a preferred embodiment, in the step S1, the absolute ethanol washing time is 20min, and the drying temperature is 60 ℃.

In another preferred embodiment, in the step S2, the Tris-HCI buffer has a pH of 8.5 and a solution concentration of 10 mMol.

In a preferred embodiment, in step S2, the quaternary ammonium salt antibacterial agent solution is a catechol quaternary ammonium salt chitosan solution, the concentration of the catechol quaternary ammonium salt chitosan solution is 10-40g/L, and the bath ratio of NF to the catechol quaternary ammonium salt chitosan solution is 1 (30-50).

In another preferred embodiment, in the step S3, the dissolution temperature of the gelatin solution is 60 ℃, the dissolution concentration is 10-100g/L, and the bath ratio of the CNF to the gelatin solution is 1 (30-50).

In a preferred embodiment, in the step S3, the certain temperature is 40-60 ℃ and the certain time is 1.5-24 h.

With reference to fig. 2, the invention provides an antibacterial agent for medical dressings, wherein the antibacterial agent is catechol-treated quaternary ammonium salt chitosan, the chitosan is firstly subjected to quaternization to obtain quaternary ammonium salt chitosan, and then catechol groups are introduced through amidation reaction, so that the chitosan has good cationic antibacterial property, and simultaneously can show strong adhesion by virtue of the catechol groups, thereby being effectively used for surface modification of cotton dressings.

In a specific embodiment, a medical dressing is provided, comprising an antimicrobial agent, wherein the antimicrobial agent is catechol quaternary ammonium salt chitosan; the antibacterial agent is prepared by quaternizing chitosan to obtain quaternary ammonium salt chitosan, and then introducing catechol groups through amidation reaction, wherein the antibacterial agent contains cationic quaternary ammonium groups and catechol groups, has good cationic antibacterial property and strong adhesion, and is easy to modify cotton dressings.

In a preferred embodiment, a method for preparing a quaternary ammonium salt antibacterial agent is provided, comprising the following steps

And X1, dissolving chitosan in glacial acetic acid to prepare a suspension, continuously stirring at a first temperature interval, slowly adding a glycidyltrimethylammonium chloride (GTMAC) solution into the suspension, continuing to react for 18-24h, taking out the mixture, centrifuging at room temperature to remove insoluble polymers, filtering a supernatant through a Buchner funnel under reduced pressure, precipitating a filtrate in precooled absolute ethanol, filtering, washing for three times, and then placing the filtrate in a vacuum oven for drying for 3 days to obtain a light yellow quaternary ammonium salt chitosan (QCS) solid.

X2, dissolving QCS in deionized water to prepare a solution, adding a 3, 4-dihydroxyphenyl propionic acid (DHPA) solution in deionized water, and adjusting the pH of the solution to 5.5. Weighing carbodiimide (EDC), dissolving in a mixed solution of deionized water and absolute ethyl alcohol (1:1, v/v), adding into the reaction solution, adjusting the pH to 5.5, stirring vigorously at room temperature for 4h, adjusting the pH once every 1h, and keeping the pH at about 5.5; after the reaction, the mixture was dialyzed for 48 hours in deionized water having a pH of 5.5 using a dialysis bag having a molecular weight of 3500 standard, and freeze-dried to obtain catechol quaternary ammonium salt chitosan (CQCS).

In a preferred embodiment, in the step S1, the chitosan solution concentration is 30g/L, and the glacial acetic acid solution concentration is 0.2%.

In step S1, the first temperature range of the reaction is 60-75 ℃, and the mass ratio of the glycidyl trimethyl ammonium chloride to the chitosan is 1.2: 1.

In step S1, the centrifugation speed in the purification process is 5000rpm, and the centrifugation time is 10 min.

In another preferred embodiment, in the step S2, the concentration of the quaternized chitosan solution is 20g/L, the concentration of the 3, 4-dihydroxyphenyl propionic acid solution is 100g/L, the concentration of the carbodiimide solution is 50g/L, and the molar ratio of the three materials is 5: 2.5: 1.

in another preferred embodiment, the application of the medical dressing in the fields of rapid hemostasis and high-efficiency antibiosis is provided, so that the dressing has good water absorption and diffusion capacity, can rapidly absorb blood moisture, promotes the aggregation and activation of red blood cells and blood platelets, accelerates the formation of blood clots, and realizes hemostasis; and the effective exposure of the quaternary ammonium salt antibacterial agent can be ensured based on the appropriate micropore structure of the gelatin gel layer, so that the high-efficiency antibacterial effect is shown.

In another preferred embodiment, the application of the medical dressing in the field of wound repair is further provided, so that the dressing can retain moisture through water absorption and expansion, and further maintain a moist environment required by wound healing, effectively reduce the infection chance of the wound and promote healing.

The preparation of the aforementioned medical dressings and the effects thereof will be exemplified and compared with specific examples and tests as follows. Of course, the embodiments of the invention are not limited thereto.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents, and the like used in the following embodiments are commercially available unless otherwise specified.

[ example 1 ]

Preparation of GCNF

Step 1, ultrasonically cleaning a cotton non-woven fabric (NF) for 20min by using absolute ethyl alcohol, removing surface impurities, and then drying in an oven at 60 ℃ for later use.

And 2, immersing the pretreated NF into a quaternary ammonium salt antibacterial agent solution (30g/L) dissolved in a Tris-HCl buffer solution (10.0mM, pH8.5) according to a bath ratio of 1:30, oscillating and depositing at 37 ℃ for 24 hours, taking out the non-woven fabric, washing with ethanol and deionized water for 3 times, and drying at 60 ℃ for 2 hours to obtain a CQCS modified NF sample CQCS @ NF (CNF).

And 3, immersing the prepared CNF into a gelatin (Gel) solution (1%, 5%, 10% (w/v)) dissolved in PBS (60 ℃, pH6.8) according to a bath ratio of 1:30, shaking for 12h under the water bath condition of 50 ℃, uniformly depositing, then taking out a sample, extruding to the same wet weight, placing in a refrigerator at 4 ℃ for 5h to form a CQCS/Gel hydrogel layer, finally immersing in deionized water for 10s, and removing the hydrogel not attached to the non-woven fabric. After freeze drying, the Gel-coated CQCS modified cotton non-woven fabric composite dressing with different concentrations is obtained, wherein the Gel is coated with 1% Ge-CQCS @ NF, 5% Ge-CQCS @ NF and 10% Ge-CQCS @ NF (GCNF-1, GCNF-5 and GCNF-10).

The test samples used below were CNF, GCNF-1, GCNF-5 and GCNF-10 obtained in example 1.

[ example 2 ]

Cqcs synthesis and characterization

Step 1, placing 1g of chitosan into 36mL of 0.2% glacial acetic acid to prepare a suspension, continuously stirring for 30min at 60 ℃, slowly adding 2mL of glycidyltrimethyl ammonium chloride (GTMAC) solution (0.6g/mL), continuing to react for 18h at 75 ℃, taking out the mixture, centrifuging the mixture for 10min at 5000rpm at room temperature to remove insoluble polymers, filtering the supernatant through a Buchner funnel under reduced pressure, precipitating the filtrate in precooled absolute ethanol, filtering, washing for three times, placing the filtrate in a vacuum oven for drying for 3 days to obtain a light yellow quaternary ammonium salt chitosan (QCS) solid, grinding and bagging for later use.

Step 2, QCS (3.25mmol,500mg) was first dissolved in 25mL deionized water to make a solution, followed by the addition of 3, 4-dihydroxylpropyl acid (DHPA, 1.62mmol, 294mg) in 3mL deionized water and adjusting the solution pH to 5.5. Carbodiimide (EDC, 0.65mmol, 124.6mg) was weighed again and dissolved in 4mL of a mixed solution of deionized water and absolute ethanol (1:1, v/v), added to the reaction solution, adjusted to pH 5.5, stirred vigorously at room temperature for 4h, during which the pH was adjusted at 1h intervals, and kept at around 5.5. After the reaction is finished, dialyzing in deionized water solution with pH of 5.5 for 48h by using a dialysis bag with molecular weight of 3500 specification, freezing and drying to obtain catechol quaternary ammonium salt chitosan (CQCS), and storing in a vacuum drier at 4 ℃ in a dark place.

To verify the successful synthesis of CQCS, the chemical composition and structural characteristics of CS, QCS and CQCS were recorded using a fourier infrared spectrometer, an ultraviolet spectrophotometer and a nuclear magnetic resonance spectrometer.

The results of the characterization are shown in FIG. 3, and first of all, by analyzing the chemical composition of each sample by FT-IR and UV-vis (FIGS. 3a, b), it can be seen that CS is only 1595cm ^-1 Occurrence ascribed toThe C ═ O stretching vibration peak of the non-deacetylated part, QCS 1483cm ^-1 And 1651cm ^-1 And 2920cm ^-1 Shows 3 new absorption peaks which are respectively-CH on quaternary ammonium groups ₃ The deformation vibration and stretching vibration peaks of QCS, which demonstrates successful synthesis of QCS; for CQCS, it is at 1558cm ^-1 A stretching vibration peak ascribed to C ═ C on the aromatic group appeared, indicating that the catechol group had been successfully grafted onto the QCS. In addition, as shown in fig. 3b, CQCS also exhibited a characteristic absorption peak at 280nm, which was attributed to the catechol group, further indicating that the catechol group was successfully grafted to the QCS and that the absorption peak did not undergo a blue shift, indicating that the catechol group was not yet oxidized. In addition, of FIG. 3c ¹ H NMR shows that the CQCS has a proton peak belonging to a C-2 position on a main chain sugar ring at a position delta 2.65 and also has a proton peak belonging to a benzene ring at about delta 7.4, and further shows that the successful introduction of the catechol group, namely the successful synthesis of the CQCS.

[ example 3 ]

FT-IR, XPS and SEM morphology characterization

In order to verify that the functionalized cotton non-woven fabric is successfully prepared, the surface information of each modified non-woven fabric is recorded by using a Fourier infrared spectrometer, the surface element composition of each gauze sample is analyzed by using x-ray photoelectron spectroscopy (XPS), a recorded object diagram is shot, the deposition condition of a sample is visually displayed by using a fluorescence labeling method (FITC labels CQCS and Cy5.5-NHS labels Gel), and finally the surface appearance of each sample is observed by using a scanning electron microscope.

The characterization result is shown in fig. 4, and firstly, the successful synthesis of the CPCG is preliminarily determined through the change of the FT-IR spectrogram. As shown in FIG. 4a, both CNF and GCNF were 1528cm ^-1 A stretching vibration peak ascribed to C ═ C of the benzene ring skeleton appears nearby, and CNF is 1652cm ^-1 The expansion vibration peak attributed to C ═ O appears nearby, is obviously reduced compared with NF, and is reduced to 1644cm after Gel is further deposited ^-1 The reduction of the stretching vibration peak is attributed to the existence of hydrogen bonds, while the transmission strength of each peak in GCNF is increased and ranges from 2500 cm to 3500cm ^-1 A broad and strong absorption band occurs, which is attributed toThe significantly enhanced O-H hydrogen bonding interactions in GCNF suggest that CQCS and Gel were successfully physically deposited onto the NF surface.

The results of XPS analysis are shown in FIG. 4b, where the peaks at 284eV, 397eV and 531eV of binding energy correspond to C1s, N1s and O1s photoelectrons, respectively. Both GCNF and CNF have a distinct N1s photoelectron peak at 397eV compared to NF, and as the Ge concentration increases, the C1s, N1s and O1s photoelectron peaks all increase gradually due to the large number of amino groups on CQCS and Gel, indicating that both CQCS and various concentrations of Ge were successfully deposited onto the NF surface.

The physical image of each sample is shown in fig. 4c, and the color of NF slightly yellows after CQCS deposition, which further demonstrates that CQCS has oxidized on the NF surface. In addition, after further deposition of Gel on the CNF surface, a layer of increasingly dense gelatin sponge was clearly observed with increasing concentration of gelatin deposition. After labeling CQCS and Gel with FITC and cy5.5, respectively, we further recorded the deposition of CQCS and Gel on the NF surface using laser confocal. As shown in fig. 4d, the CNF surface showed strong green fluorescence due to the deposition of FITC-labeled CQCS, the GCNF surface showed strong yellow fluorescence due to the deposition of cy 5.5-labeled Gel, and the color of GCNF gradually changed from yellow to orange as the deposition concentration of Gel increased, which also indicates that the GCNF surface deposited more and more cy 5.5-labeled Gel. Next, we performed surface morphology and surface elemental composition analysis on GCNF using SEM-EDX. As shown in fig. 4e and f, the NF surface is uniform and smooth, and the CNF surface forms a thin chitosan film between the gaps and the partial fiber surface due to the deposition of CQCS. The GCNF surface forms a unique Gel microporous structure due to the deposition of Gel, and the densification degree of the Gel sponge is more densified along with the increase of the concentration of the Gel, for example, the GCNF-1 surface has a large number of pores and a loose structure, while the GCNF-10 surface has almost no pores and a dense structure, and the result is consistent with the physical condition of each dressing. Taken together, these results further clearly demonstrate the successful conjugation and adsorption of PHMG and CS on the CG surface, as well as the successful preparation of the medical dressing CPCG.

[ example 4 ]

Liquid absorption Performance test

The liquid absorption rate is an important index for evaluating the hemostatic material, and the high liquid absorption rate is favorable for concentrating red blood cells, platelets and blood coagulation factors so as to improve the hemostatic reaction. Liquid absorption tests were performed on NF, CNF, GCNF-1, GCNF-5 and GCNF-10 using deionized water, physiological saline (NS), PBS (pH6.8) and Whole Blood (white Blood, WB) as test solutions and commercial hemostatic sponge (MHS) as a control. The specific method comprises the following steps: first, a clean sample (1cm x 1cm) is dried completely in an oven at 60 ℃, and then taken out for pre-weighing (W) ₁ ) Then, an equal volume of the test solution was added, and the mixture was soaked at 37 ℃ for 30min, after which the sample was taken out, and the surface residual liquid was carefully absorbed on the filter paper by the vertical dropping method to determine the wet weight (W) ₂ ). The above procedure was repeated 3 times for each type of sample. The liquid absorption (Lar) is calculated as follows:

as can be seen from fig. 5, 1) the absorption rates of CNF and GCNF for all liquids are significantly higher than NF, because the wettability of NF is reduced after deposition of CQCS and Gel, but the hydrophilicity of NF is enhanced, which is beneficial to absorption and retention of water molecules; 2) the liquid absorption rate of GCNF is obviously superior to that of CNF, which is mainly attributed to the excellent water swelling property of Gel, and can absorb and diffuse water molecules to a great extent; 3) in all cotton yarn dressings, GCNF-5 has the best liquid absorption rate, the absorption rate for four test liquids is maintained at 900-; the GCNF-10 surface has a compact gelatin layer and low porosity, so that the permeation and absorption of liquid are prevented to a certain extent. It is worth noting that MHS, thanks to its porous structure, has the highest absorption rate for all the tested liquids in all the tested dressings, about 200% higher than GCNF-5, but also the high liquid absorption rate does not mean the best hemostatic effect, since excessive loss of blood tends to increase the chance of disability and mortality significantly.

[ example 5 ]

Evaluation of in vitro hemostatic Properties

The in vitro clotting effects of GCNF complex dressings were evaluated by measuring the clotting index (BCI). Fresh blood was first collected from the mouse eyeball, then GCNF was cut into squares (1cm x 1cm) and placed in a petri dish to preheat for 5min at 37 ℃. Then, 100. mu.L of activated citric acid whole blood (containing 10. mu.L of 0.2M CaCl) was added dropwise to each sample ₂ ) Standing for different periods (1,3,5,10,20min), adding 5mL deionized water, standing for 10min to wash unbound blood cells, transferring 200 μ L of the wash water into a 96-well plate, and recording the absorbance value (Is) of each sample at 540nm with a microplate reader. Using a no sample plate (TCP), 100. mu.L of blood was added as Control group (Ic), and BCI values were calculated using the following formula:

in addition, to explore the interfacial interaction between the red blood cells/platelets and gauze, fresh blood collected from mouse eyeballs was processed by centrifugation (2500rpm,10min) and layered in PBS to give a red blood cell/platelet suspension. Then, 200. mu.L of each suspension was dropped onto the surface of gauze (1 cm. times.1 cm), after which the treated gauze was immersed in PBS containing 2.5 wt% glutaraldehyde at room temperature for 2 hours to fix red blood cells/platelets, and finally, the red blood cells/platelets were dehydrated for 15 minutes using a series of graded ethanol-PBS solutions (30,40,50,60,70,80,90, 100%, v/v), and vacuum oven-dried at room temperature for 24 hours, and then the adhesion of red blood cells/platelets was observed using SEM.

Characterization results as shown in fig. 6, it can be seen from the BCI assay results (fig. 6a) that the BCI of NF is as high as 83%, comparable to the TCP group, indicating that the pure cotton yarn dressing hardly stopped bleeding. After CQCS is deposited, the BCI value of NF is reduced to 55 percent, which shows that the hemostatic performance is improved, mainly due to the hydrophilic water absorption of CQCS and the electrostatic interaction between surface quaternary ammonium cation and red blood cell, the CQCS can absorb blood, promote the aggregation of red blood cell and platelet, accelerate the formation of blood clot and achieve the purpose of hemostasis; interestingly, the hemostatic effect of NF was further significantly improved after deposition of a suitable concentration of gelatin, especially the BCI value of GCNF-5 had been reduced to 34%, slightly above 32.6% of MHS, indicating that GCNF-5 has excellent hemostatic potential. In addition, the BCI value of GCNF-10 with higher gelatin concentration is 55%, which is probably because the surface of GCNF-10 has a denser gelatin layer which prevents the infiltration and absorption of blood to a certain extent, so the hemostatic effect is obviously inferior to that of GCNF-5 with proper gelatin concentration and porous structure on the surface. FIG. 6b shows the dynamic behavior of blood coagulation for each sample. Overall, all the modified cotton yarn dressings and MHS showed a continuous decrease in BCI values with longer standing time of the hemostatic material and whole blood, and GCNF-5 showed the fastest decrease in BCI values, which decreased to 21% at 10 minutes, which is equivalent to 22% of MHS. In addition, the BCI values of GCNF-5 and GCNF-1 at 1 minute are reduced to 51 percent and 57 percent respectively and are far lower than 72 percent of MHS, and the result shows that GCNF-5 and GCNF-1 can start coagulation faster than MHS, which is mainly attributed to the good wettability and dynamic water absorbability of GCNF-5 and GCNF-1, can slowly absorb blood moisture after blood drops are contacted, promote the aggregation and adhesion of red blood cells/platelets and accelerate the coagulation process, but because the GCNF-1 has larger porosity, blood cells are easy to diffuse and permeate along with the moisture, the aggregation and adhesion quantity is reduced, and the BCI value is slightly smaller than GCNF-5.

Figure 6c shows the hemostatic effect of GCNF in vitro. TCP set was diluted rapidly to a red solution and diffused throughout the dish after slow drop-wise addition of deionized water as the blood did not coagulate. The cleaning solution of the NF group was also relatively red, indicating that only a small amount of blood had coagulated. The CNF group had a slightly lighter cleaning solution than the NF group, indicating that CQCS imparted some clotting properties to CNF, but was not sufficient to completely stop bleeding. The final wash of the GCNF group was only slightly red compared to the NF and CNF groups, especially the GCNF-5 group was almost colorless, comparable to the MHS group, which indicates that GCNF-5 was indeed able to exert hemostatic effects by rapidly coagulating blood. After confirming that GCNF-5 can exert a hemostatic effect by rapidly coagulating blood, we further examined the adhesion behavior of erythrocytes and platelets on the surface of GCNF-5 by scanning electron microscopy. As shown in fig. 6d, each dressing surface was able to aggregate adherent red blood cells and had a normal morphology, with NF and CNF having a smaller number of adhesions due to their smoother surface and larger pores; the GCNF-1 has less Gel deposition, so the change of the adhesion quantity is not obvious; GCNF-5 has good surface wettability, proper porosity and complete blood moisture diffusion, and a large number of red blood cells are adhered to the tiny fibers and holes, so that the activation of a blood coagulation system is facilitated; although a large number of red blood cells are also accumulated on the surface of GCNF-10, most of the red blood cells are stacked and accumulated mutually (have weak interaction with GCNF-10) and are easy to wash and shed; MHS, however, benefits from a unique sponge structure, where a large collection of red blood cells adhere to the internal cavity, also contributing to the activation of the coagulation system. The adhesion condition of the platelets on the surfaces of the dressings is similar to that of red blood cells, the NF and CNF surfaces can only adhere few platelets, the porosity of GCNF-1 is reduced due to deposited gelatin, so that the adhesion quantity of the platelets is improved, the adhesion quantity of GCNF-10 is reduced due to the fact that the surfaces of the GCNF-10 are too dense, however, GCNF-5 still has the most platelet adhesion due to proper wettability and porosity, the quantity of the platelet adhesion is even better than that of MHS, and probably, the internal fiber structure of the GCNF-5 is more beneficial to the intertwined adhesion of the platelets compared with the internal sponge cavity structure of the MHS, so that the platelets are effectively activated, and hemostasis is promoted. In general, both GCNF-5 and MHS have good liquid absorbability, a low BCI value and rapid hemostatic ability, and can effectively aggregate red blood cells/platelets and promote hemostasis.

[ example 6 ]

In vitro antimicrobial Performance testing

As the wound dressing needs to have high-efficiency antibacterial activity, the wound dressing can effectively avoid wound infection; therefore, the in-vitro antibacterial performance of the functionalized cotton non-woven fabric is evaluated by adopting an antibacterial ring method, an absorbance method and a dot-panel counting method.

The bacteriostatic ring method comprises the following steps: the surface of a dish into which 15ml of solid LB medium was poured was inoculated with 100. mu.L of PBS-washed Escherichia coli and Staphylococcus aureus (10) ⁸ CFU/ml), and use disposableThe bacterial coating rod is evenly smeared, after the bacterial liquid is dried, a sterilized sheet sample (0.5cm x 0.5cm) is placed on the surface of a culture dish by using sterile forceps, is carefully compacted, and then 10 mu L of sterile PBS is dripped on the sample to promote the diffusion of the antibacterial component. Then, the culture dish is placed in a constant-temperature incubator at 37 ℃ upside down for incubation for 24 hours, and the size of the inhibition zone around the sample is observed. The bacteriostasis performance of the sample can be determined by the area of the bacteriostasis ring around the sample, the larger the area is, the better the bacteriostasis performance is, and otherwise, the poorer the bacteriostasis performance is.

Absorbance method: the sample (1 cm. times.1 cm) was immersed in an EP tube containing 1ml of the diluted bacterial solution and cultured for 24 hours at 37 ℃ in a constant temperature shaker at 180 rpm. At intervals (0,3,6,9,12,24h), 100. mu.l of the cell suspension was transferred to a 96-well plate, and OD at 600nm of the cell suspension was measured by a microplate reader.

Dot plate counting method: the sample is placed in a container containing S.aureus or E.coli (10) after being sterilized by ultraviolet radiation ⁸ CFU/mL) in PBS (pH7.4), culturing at 37 ℃, transferring 100 μ l of cultured bacterial suspension into a 96-well plate at different time intervals (1,10,20,30min,1,3,6,9,12,24h), sequentially diluting to 10CFU/mL in a gradient manner, further adding a spot plate on an LB culture plate according to concentration, placing the spot plate in a 37 ℃ incubator for culturing for 24h, and counting the bacterial number on each sample culture plate, wherein the PBS liquid without adding a sample is a control group.

In vitro antibacterial evaluation results are shown in fig. 7, and the inhibition zone results (fig. 7a) show that the inhibition zones (21 and 19mm, respectively) formed after the co-incubation of GCNF-5 with staphylococcus aureus and escherichia coli for 24h are almost completely consistent with the inhibition zones (21 and 19mm, respectively) formed after the co-incubation of CNF with staphylococcus aureus and escherichia coli for 24h, which indicates that the antibacterial activity of the CNF is not affected after a certain concentration of gelatin is deposited on the surface of the CNF, and the results also mean that GCNF-5 can realize efficient antibacterial while completing rapid hemostasis. It is worth noting that the inhibition zones formed by GCNF-5 and CNF are obviously larger than the contact area of the cotton yarn dressing and bacteria, which shows that GCNF-5 and CNF not only can exert antibacterial action by directly contacting with bacteria, but also can form wider inhibition zones by diffusion action. The contact antibacterial effect of the CQCS and the chitosan is mainly from the antibacterial effect of quaternary ammonium salt groups on the CQCS and the chitosan, and the diffusion inhibition effect is caused by the four-week diffusion of the desquamated CQCS.

To explore the antibacterial potential of GCNF-5 in depth, we further investigated by OD ₆₀₀ The assay and plate counting systems evaluated the bacteriostatic and antibacterial activity of GCNF-5. As shown in FIGS. 7b, c, NF did not inhibit bacterial growth because of OD ₆₀₀ The value of (c) increases with increasing incubation time. However, both GCNF-5 and CNF were able to inhibit the growth of S.aureus and E.coli with high efficiency, and the OD of both ₆₀₀ The value begins to decrease slowly after only 1 minute of contact with the bacteria, indicating that the growth of the bacteria has been inhibited with high efficiency. FIG. 7d shows the plate count results for GCNF-5 and CNF after different times of exposure to bacteria. Consistent with the results of 7b, c, both GCNF-5 and CNF were able to exert excellent antibacterial attention after only 1 minute of contact with the bacteria, e.g. GCNF-5 killed 99.998% and 99.993% of the bacteria after 1 minute of contact with s.aureus and e.coli, respectively, even cleared 99.999% of the bacteria after 20 minutes (fig. 6e, f), and cleared all the bacteria after 30 minutes (the colony count had been reduced to 0CFU/mL, fig. 7 e). In summary, the above results indicate that GCNF-5 has excellent bacteriostatic and antibacterial activity, and also has excellent persistence of its bacteriostatic and antibacterial activity, i.e., its antibacterial activity is maintained at 100% throughout the subsequent 24h (FIG. 7 f).

In conclusion, CQCS can significantly improve the antibacterial activity of NF, and the antibacterial mechanism thereof is mainly attributed to the following points: 1) the electrostatic interaction of the quaternary ammonium salt group with positive charge and the amino group on the CQCS and the bacterial cell membrane causes the severe rupture of the bacterial cell membrane; 2) with severe rupture of the bacterial cell membrane, electrostatic interaction between CQCS and bacterial cell membrane further causes massive leakage of intracellular proteins and at the same time significantly inhibits intracellular ATP synthesis. In addition, it is worth noting that the antibacterial activity of CQCS is not significantly reduced after gelatin with appropriate concentration is further deposited on the surface of CNF, and this characteristic enables GCNF-5 to achieve rapid hemostasis and simultaneously exert high-efficiency antibacterial effect.

[ example 7 ]

Deposition adsorptionFastness and Performance testing

Although the above results have confirmed that GCNF-5 has important potential for rapid hemostasis and high-efficiency antibiosis, since GCNF-5 is mainly prepared by physical deposition, the complex microenvironment around the wound requires that the wound dressing has excellent performance and can keep stable properties without influencing wound healing. Therefore, PBS buffers with different pH values are selected to simulate the in-vivo wound microenvironment, and the deposition stability of CQCS and Ge on the NF surface and the corresponding antibacterial and hemostatic property changes are measured by a weighing method. Briefly, the mass of CNF before and after CQCS deposition was first recorded as m ₀ ,m ₁ Then, the CNF was soaked in PBS solution at pH6.8 and pH7.4 for different times at 37 ℃ (3,6,12,24,48,72,96,120,144,168h), and then the CNF was taken out, washed, dried and weighed (m is 120,144,168h) ₂ ) Finally, the stability of the (Δ E) reaction coating was quantified by calculating the elution of CQCS under these conditions, as follows:

and (3) taking the CNF after the soaking treatment, and measuring the change of the antibacterial effect after the CNF reacts with the bacteria for 6 hours according to the point-plate counting method. For the Ge-deposited CNF, Cy5.5-NHS-labeled GCNF-5 was selected as the test sample, also pre-weighed and dip-dried, the gelatin elution (Δ w) was recorded, and the fluorescence change was recorded using confocal laser microscopy.

As shown in fig. 8a, after CNF is soaked in PBS solutions with different pH values for 7 days, the elution rate of CQCS is only about 3.5%, and the elution rate is higher in an acidic environment (pH6.8), because the acidic environment weakens the hydrogen bonding between CQCS and NF, so that CQCS partially fall off, but as time goes on, CQCS fall off under the acidic condition tends to be balanced, because catechol groups oxidized under the acidic condition are not easy to change, and strong adhesion can be continuously maintained by virtue of hydrogen bonding, covalent bonding and pi-pi interaction between deposited CQCS and a large amount of hydrogen bonding between CQCS and NF. And GCNF-5P at pH6.8 and 7.4After the BS solution is soaked for 7 days (figure 8b), the elution rate is respectively 54.4 and 60.2 percent, which shows that the Gel layer can fall off due to the gradual degradation of Gel under the long-time treatment of the physiological environment at 37 ℃, but the degradation speed is relatively slow under the alkaline environment, because the interaction of carboxyl and amino among molecules of the Gel is stronger under the alkaline environment, the Gel structure is relatively stable, and the hydrogen bond interaction between the Gel and CNF can be caused by-NH under the acidic condition ₂ Protonation and weakening. The change in the overall fluorescence of the fluorescence-labeled GCNF-5 after the soaking treatment was recorded by confocal laser microscopy (fig. 8 c). It can be seen that both PBS-treated GCNF-5 had significant green fluorescence throughout due to FITC-labeled CQCS, whereas red fluorescence due to Cy5.5-NHS-labeled Gel gradually decreased with the increase of soaking time and was more significantly decreased in the buffer solution at pH6.8, since Gel itself is readily soluble and degradable at 37 ℃ and hydrogen bonding is readily attenuated by protonation under acidic conditions. The result shows that when GCNF-5 acts on a wound-like microenvironment for a long time, CQCS based on super-strong adhesion of catechol can be stably attached to the surface of GCNF-5, so that high-efficiency and durable antibacterial activity is provided, but a large amount of Ge gel is obviously dropped due to temperature, time and other problems, and the subsequent moisturizing and healing of the wound can be influenced.

The antibacterial and hemostatic performance changes of the PBS soaked GCNF-5 were further evaluated by dot plate counting and BCI values. The antimicrobial rate curves are shown in fig. 9a, b, and it can be seen that the antimicrobial rates of both PBS-treated CNFs on s.aureus and e.coli decreased from the first 99.999% and 99.998% to the last 99.992% and 99.92% after 7 days of soaking due to the slight shedding of CQCS. It was demonstrated that CNF had a high antibacterial effect even though a trace amount of CQCS fell off after the soaking treatment. The change situation of the BCI value (fig. 9c) shows that the BCI value of GCNF-5 also increases basically with the increase of the soaking time, and after the speed increase of the first 24 hours is greater than 24 hours, the reason is that the peeling of the surface layer Gel at the early stage is due to the fracture caused by hydrogen bond protonation in the solution, and the peeling rate under the acidic condition is obviously greater than that under the alkaline condition, so the change of the BCI value under the acidic condition is more obvious, while the change of the surface layer Gel at the later stage is mainly due to the degradation of the Gel, the degradation speed is gradually slowed down, the change trend of the corresponding BCI value is slowed down, but both 36% and 40%, and still has certain blood coagulation potential.

[ example 8 ]

Blood and cell compatibility assays

Although GCNF-5 has excellent hemostatic ability and in vitro antibacterial activity, it is clinically applied on the premise of excellent biocompatibility. Thus, hemolysis and cytotoxicity of GCNF-5 was evaluated using mouse whole blood, mouse fibroblasts (L929) and human umbilical vein cell fusion cells (EA.hy926).

First, 1mL of fresh blood was collected from mouse eyeballs, Red Blood Cells (RBCs) were collected by centrifugation (2500rpm,4 ℃,10min), and RBC suspension (4%, v/v) was prepared in PBS (pH 6.8). A piece of sample (0.5 cm. times.0.5 cm) was then placed in a centrifuge tube, 900. mu.L of PBS and 100. mu.L of RBC suspension were added, and incubated at 37 ℃ for 1 h. The supernatant was then centrifuged at 3000rpm for 5min, and the apparent density of the supernatant was measured at 560nm using a microplate reader, and hemolysis was recorded by optical photography. The hemolysis rate was calculated using the following formula, and RBC suspensions treated with ultrapure water and physiological saline (NS) were used as positive and negative controls, respectively. NF, CNF and GCNF-5 as material control group

Then 10 pieces of sample (0.5cm x 0.5 cm/piece) were soaked in 10ml PBS for 24 hours at 37 deg.C, and then the sample was taken out to obtain a leaching solution for use. The L929 cells were then plated in 96-well plates containing DMEM medium and placed in humidified incubator (37 ℃, 5% CO) ₂ ) Incubated for 24h, then the leachate was added and incubated together for 24h, then stained with AM and PI and observed under an inverted fluorescence microscope. After the cells were washed three times with PBS, 10. mu.L of CCK-8 reagent was added, and incubated at 37 ℃ in the dark for 1h, and finally the OD of each well at 450nm was recorded using a microplate reader. Hy926 cells were treated in the same way. In which untreated cells were used as negative controls, wells containing no cells but mediumUsed as a blank control. Cell viability was calculated from the following formula:

as shown in fig. 10, the hemolysis results (fig. 10a, b) show that CNF alone caused slight hemolysis after 1h incubation of each dressing with erythrocyte suspension, with hemolysis rate of about 4.3%, and GCNF-5 and NF did not cause hemolysis (hemolysis rates of 2.4% and 2.2%, respectively). CNF causes hemolysis mainly because of its fiber surface carrying a large number of quaternary ammonium salt groups and amino groups, which may destroy the integrity of the erythrocyte membrane to some extent, thereby causing hemolysis. But after deposition of gelatin, its blood compatibility is further improved, probably due to the weakening of the quaternary ammonium salt groups and the interaction of the amino groups with the erythrocyte membranes. Live \ dead cell staining and CCK-8 detection further confirm that GCNF-5 has good cell compatibility, as shown in FIG. 10c, both L929 and EA.hy926 cells can maintain normal cell morphology after being incubated with GCNF-5 for 24h, and no PI-labeled dead cell (red fluorescence signal) is detected, which indicates that GCNF-5 has good cell compatibility. The results of CCK-8 detection were consistent with the results of live and dead cell staining, as shown in FIG. 10d, e, the survival rates of GCNF-5 treated L929 and EA.hy926 cells were as high as 99% and 97%, respectively, which was almost identical to the cell survival rate of PBS group. In conclusion, the results of hemolysis and cytotoxicity experiments show that GCNF-5 has good biocompatibility and important application potential as a hemostatic material.

[ example 9 ]

In vivo hemostatic effect detection

Although GCNF-5 has excellent and rapid in-vitro hemostatic ability, the in-vivo hemostatic effect is not clear, so the in-vivo hemostatic ability of GCNF-5 is detected by establishing a mouse liver injury model and a tail-broken hemorrhage model.

Mouse liver injury model: first, the abdomen was shaved, skin tissue was incised, and the abdominal cavity was dissected to expose the liver. Serum surrounding the liver was then carefully removed and pre-weighed filter paper placed under the liver to accurately estimate blood loss. A20G sterile needle is selected to induce liver bleeding, each sample (1cm x 1cm) is placed on a bleeding part after natural bleeding for 10s, and the bleeding stopping time and the blood loss are recorded.

Mouse tail-breaking bleeding model: two thirds of the tail length was cut with surgical scissors and the cut wound was exposed for 15 seconds to ensure normal blood loss. Each sample was then used to stop bleeding, and the time to hemostasis and the amount of blood lost were recorded. Each model was tested 3 times, with untreated lesion model as a control.

As shown in fig. 11, the real-time hemostatic effect of GCNF-5 on the mouse liver injury model (fig. 11a) shows that NF has almost no hemostatic effect, and after contacting with the bleeding liver, it can not stop the liver bleeding rapidly, and the blood loss is severe and spread all over. Compared with NF, CNF has certain hemostatic effect, and the blood loss is relatively less, which is similar to the in vitro hemostatic result. It is worth noting that GCNF-5 indeed has an excellent in vivo hemostatic effect, and can rapidly exert a hemostatic effect after contacting a bleeding liver, thereby effectively reducing blood loss and diffusion. The results in fig. 11b, c further demonstrate the excellent hemostatic effect of GCNF-5, which is only 52.50 ± 3.67s to stop bleeding quickly, much faster than 178 ± 6.5s in control group, 167.5 ± 2s in NF group, and 117.5 ± 3.7s in CNF group, even slightly faster than 69 ± 4.9s in MHS group; in addition to the fast hemostasis time, the blood loss of the GCNF-5 group was much less than that of the control, NF and CNF groups, the former was only 58.4 + -1.8 mg, while the latter was as high as 125.5 + -3.7 mg, 112 + -7.5 mg and 103.9 + -8.2 mg, and the GCNF-5 was also slightly less than MHS, the latter was 71.5 + -4.4 mg.

The results of the mouse tail-severed model (fig. 11d) show that GCNF-5 rapidly stopped bleeding after exposure to the bleeding mouse tail, and the amount of blood loss was significantly less than in the Control, NF, and CNF groups. As shown in FIG. 11e, f, the calculation results show that the hemostasis time of GCNF-5 is only 67.7 + -2.2 s, slightly faster than 70.7 + -5.3 s of MHS group, but significantly faster than 199 + -13.5 s of Control group, 158.3 + -10.8 s of NF group, and 124.3 + -10.4 s of CNF group; in addition, the blood loss of GCNF-5 was 70.7. + -. 3.6mg, also slightly less than 71. + -. 2.6mg for MHS, but significantly less than 246.3. + -. 10mg for Control, 195.00. + -. 5.7mg for NF, and 106.00. + -. 12.4mg for CNF.

In conclusion, the experimental results of the two bleeding model experiments prove that GCNF-5 has excellent hemostatic effect, the hemostatic effect is obviously superior to NF, and even the hemostatic effect is slightly superior to MHS in terms of hemostatic time and blood loss. The excellent hemostatic properties of GCNF-5 are related to CQCS and Gel, which are sequentially deposited on the NF surface. After contacting with a bleeding wound, GCNF-5 can firstly utilize the strong water absorption of a unique gel micropore structure and the hydrophilicity of surface amino, hydroxyl and other groups to quickly absorb blood and vertically diffuse water so as to promote the aggregation and activation of red blood cells and platelets; subsequently, GCNF-5 can further effectively adhere red blood cells and platelets through the electrostatic interaction of positive charge groups on the surface and blood cells, activate the blood coagulation system, generate blood coagulation factors and thrombin, induce the conversion of fibrillar proteins into fibrin, promote fibrin monomers to aggregate to form stable fibrin polymers, promote the formation of blood clots, and reduce blood loss. Furthermore, it is worth particular emphasizing that GCNF-5, which is constructed on the basis of a cotton nonwoven, not only has better softness and comfort than MHS, but also can absorb blood, aggregate red blood cells and platelets more rapidly upon contact with blood and exert a hemostatic effect. Therefore, we believe that GCNF-5 is a novel hemostatic dressing with great potential, with hemostatic potential comparable to, or even slightly superior to, MHS.

[ example 10 ]

Evaluation of in vivo antibacterial and wound healing Performance in animals

The invention adopts a simple layer-by-layer deposition process to prepare the functional cotton non-woven fabric dressing capable of rapidly stopping bleeding and efficiently resisting bacteria, and evaluates the in-vivo antibacterial and wound healing conditions of the novel cotton non-woven fabric dressing by constructing a staphylococcus aureus infected mouse whole skin wound.

The specific implementation method comprises the following steps: mice were anesthetized and shaved, and then a circular full-thickness skin wound (8mm x 8mm) was created on the back of each mouse, followed by inoculation of 10 μ L of staphylococcus aureus solution (10) onto the wound ⁸ CFU/mL). After 10min, all mice were randomly assigned to 5 groups (n ═ 5), and the wounds were covered with NF, MHS, CNF, GCNF-5 as a control group and uncovered with any dressing as a blank group, respectively. The various dressings were changed every 2 days throughout the treatment to keep the wound clean. And simultaneously photographing and recording the area of the wound surface, wherein the wound surface closure rate (WCR) is calculated according to the following formula:

wherein S ₀ The wound area of the wound surface on the day is shown, and S is the wound area of the shooting record day.

To evaluate the antibacterial activity of GCNF-5 for wound treatment, mice were sacrificed on

days

2 and 14, infected tissues were collected, weighed, placed in sterile PBS, disrupted with an ultrasonic cell disrupter (UP-250, zhejiang minder instruments, china), and homogenized. After centrifugation at 2500rpm for 5 minutes at 4 ℃,100 μ L of the supernatant was inoculated on LB agar plates for CFU counting.

The results of the animal experiments are shown in fig. 12, where macroscopic healing of wound tissue treated with different gauzes over time (fig. 12a) and corresponding wound closure rates (fig. 12b) show that the healing rate of GCNF-5 and CNF-treated wounds is significantly faster than MHS, NF and controls, and 68% of the wound area has been repaired at day 6. The MHS and NF groups had a slower rate of wound healing until 35% and 28% of the wound area remained unrepaired and there was also wound scarring, respectively, after 10 days of treatment. In addition, although the CNF group has a wound healing rate similar to that of GCNF-5, the early healing effect is poor because the CNF has no gelatin gel layer on the surface and only relies on CQCS to stop bleeding rapidly and absorb wound exudate, so as to create a suitable wound healing environment. Therefore, the condition of the CNF-treated wound is only slightly better than that of NF and MHS groups at 2 days, partial effusion is still accumulated on the surface of the wound, the bacteria are easily nourished, and the wound abscess infection is induced. The GCNF-5 dressing can quickly sterilize, absorb seepage, promote cell proliferation and repair 28% of wound areas within 2 days of treatment, can repair 68% of wound areas within 6 days, and enables the wound healing area to reach 99.99% on the 14 th day, thereby showing excellent performance of promoting wound healing.

The colony counts of wound tissue (FIG. 12c, d) showed that, as with the in vitro antibacterial results, MHS and NF were not as effective as the in vivo antibacterial effect, and the bacterial count at the wound site was still as high as 10 after 48h treatment ⁶ CFU/mg, with little antibacterial effect. But after GCNF-5 treatment for 6h, the antibacterial rate reaches 99%, and after further treatment for 24h, the bacterial number of the wound part is sharply reduced to 10 ^2.8 CFU/mg, the antibacterial rate is as high as 99.9%. In addition to being highly effective, GCNF-5 was also effective in inhibiting bacteria, as evidenced by the gradual reduction in the bacterial count at the wound site to 2CFU/mg over the next 14 days. In conclusion, the above results clearly demonstrate that GCNF-5 also has excellent anti-infective properties in vivo, and the antibacterial effect thereof is not significantly different from CNF, indicating that depositing gelatin at an appropriate concentration on the surface of CNF does not significantly affect the antibacterial activity of CQCS.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A medical dressing comprising a substrate, and further comprising:

wherein the antibacterial agent is respectively connected with the gelatin gel layer and the base material through hydrogen bonds, covalent bonds and pi-pi interaction, and a gel microporous structure is formed on the surface of the base material.

2. A medical dressing according to claim 1, wherein: the mass ratio of the gelatin to the quaternary ammonium salt antibacterial agent is 5: 3.

3. A medical dressing according to claim 1, wherein: the base material is cotton non-woven fabric.

4. A method of making a medical dressing according to any one of claims 1 to 3, comprising the steps of:

s2, immersing the cotton non-woven fabric obtained by the S1 treatment in a quaternary ammonium salt antibacterial agent solution dissolved in a Tris-HCl buffer solution, depositing for 24 hours at 37 ℃, taking out the non-woven fabric, washing for 3 times by using ethanol and deionized water, and drying for 2 hours at 60 ℃ to obtain a CQCS modified NF sample CQCS @ NF (CNF);

s3, immersing the CNF prepared in the S2 in gelatin (Gel) solution dissolved in PBS, shaking for 1.5-24h under the water bath condition of 40-60 ℃, depositing uniformly, taking out a sample, extruding to the same wet weight, placing in a refrigerator of 4 ℃ for 5h to form a CQCS/Gel hydrogel layer, finally immersing in deionized water for 10S, and removing hydrogel not attached to the non-woven fabric. And freeze-drying to obtain the Gel coated CQCS modified cotton non-woven fabric composite dressing Gel-CQCS @ NF (GCNF).

5. The method of claim 4, wherein: in the step S1, the absolute ethyl alcohol washing time is 20min, and the drying temperature is 60 ℃.

6. The method of claim 4, wherein: in step S2, the Tris-HCI buffer solution has a pH of 8.5 and a solution concentration of 10 mMol.

7. The method of claim 4, wherein: in the step S2, the quaternary ammonium salt antibacterial agent solution is a catechol quaternary ammonium salt chitosan solution, the concentration of the quaternary ammonium salt antibacterial agent solution is 10 to 40g/L, and the bath ratio of NF to the catechol quaternary ammonium salt chitosan solution is 1: 30-50.

8. The method of claim 7, wherein: in the step S3, the dissolution temperature of the gelatin solution is 60 ℃, the dissolution concentration is 10-100g/L, and the bath ratio of the CNF to the gelatin solution is 1: 30-50.

9. The method for preparing chitosan of catechol-based quaternary ammonium salt according to claim 7, wherein the chitosan of catechol-based quaternary ammonium salt is prepared by quaternizing and catechol-based modifying chitosan, and the method comprises the following steps:

x1, dissolving chitosan in glacial acetic acid to prepare a suspension, continuously stirring at 60-75 ℃, slowly adding a glycidyltrimethylammonium chloride (GTMAC) solution into the suspension, continuously reacting for 18-24h, taking out the mixture, centrifuging at room temperature to remove insoluble polymers, filtering the supernatant through a funnel under reduced pressure, precipitating the filtrate in precooled absolute ethanol, filtering, washing for three times, and then placing in a vacuum oven for drying for 3 days to obtain a light yellow quaternary ammonium salt chitosan (QCS) solid;

x2, firstly, dissolving QCS in deionized water to prepare a solution, then adding a 3, 4-dihydroxyphenyl propionic acid (DHPA) solution dissolved in the deionized water, and adjusting the pH of the solution to 5.5; weighing carbodiimide (EDC), dissolving in a mixed solution of deionized water and absolute ethyl alcohol (1:1, v/v), adding into the reaction solution, adjusting the pH to 5.5, stirring vigorously at room temperature for 4h, adjusting the pH once every 1h, and keeping the pH at 5.5; after the reaction is finished, dialyzing in deionized water solution with pH of 5.5 for 48h by using a dialysis bag with molecular weight of 3500 specification, and freeze-drying to obtain pyrocatechol quaternary ammonium salt chitosan (CQCS).

10. The method of claim 9, wherein: in the step X1, the concentration of the chitosan solution is 30g/L, and the concentration of the glacial acetic acid solution is 0.2%.

11. The method of claim 9, wherein: in the step X1, the mass ratio of the glycidyl trimethyl ammonium chloride to the chitosan is 1.2: 1.

12. the method of claim 9, wherein: in the step X1, the centrifugation speed in the purification process is 5000rpm, and the centrifugation time is 10 min.

13. The method of claim 9, wherein: in the step X2, the concentration of the quaternized chitosan solution is 20g/L, the concentration of the 3, 4-dihydroxyphenyl propionic acid solution is 100g/L, the concentration of the carbodiimide solution is 50g/L, and the molar ratios of the three materials are 5: 2.5: 1.

14. use of the medical dressing according to any one of claims 1-3 in the fields of rapid hemostasis and highly effective antisepsis.

15. Use of a medical dressing according to any of claims 1 to 3 in the field of wound repair.