CN115177778A

CN115177778A - Composite wound dressing, preparation method and application

Info

Publication number: CN115177778A
Application number: CN202210875485.3A
Authority: CN
Inventors: 王春华; 侯桂革; 李诚博; 高中飞
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2022-07-25
Filing date: 2022-07-25
Publication date: 2022-10-14

Abstract

The invention discloses a composite wound dressing, a preparation method and application. The composite wound dressing comprises an electrostatic spinning structure on the outer layer and a sponge structure on the inner layer; the electrostatic spinning structure is obtained by electrostatic spinning of poly-epsilon-caprolactone and quaternized silicone, and the sponge structure is obtained by freeze-drying and self-assembling of fish skin collagen and quaternized chitosan; the mass ratio of the poly-epsilon-caprolactone to the quaternized silicone is 7; the mass ratio of the fish skin collagen to the quaternized chitosan is 6. The composite wound dressing can rapidly remold epidermis and promote the growth of fibroblasts, so that the composite wound dressing has multiple functions of wound hemostasis, wound repair, antibiosis and anti-inflammation, scar repair and the like, and solves the problems of single function, poor healing and scar repair quality and the like of a wound repair product in the prior art.

Description

Composite wound dressing, preparation method and application

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a multifunctional wound repair film, a preparation method and application thereof.

Background

In recent years, the incidence rate of various traumatic events is remarkably increased, and the requirements of patients on wound repair are higher and higher. However, the clinical medical dressing has the problems of insufficient antibacterial activity and single function, so that the wound infection is frequent and the scar repairing effect is poor. Quaternization has been widely used for various polysaccharides such as chitosan, pectin, konjac glucomannan, starch, and cellulose. Researches show that the antibacterial capacity of the natural polysaccharide can be improved by quaternization. The polysaccharide containing quaternary ammonium salt groups has hydrophilicity, biodegradability, bacteriostasis, and can support certain components of skin and hair. These polymers have many applications in many fields, including paper and textile, food, cosmetic, chemical and pharmaceutical industries.

Electrospinning is a practical technique for manufacturing fibrous scaffolds that mimic natural tissues. Fibrous bandages having a nano/micro structure offer a more promising wound care potential than traditional bandages, as they are able to partially reproduce the inherent cytoplasmic matrix structure, facilitating the adhesion, growth and migration of fibroblasts, thus promoting the regeneration of skin tissue at the wound site. The highly interconnected porous structure of the wound dressing prepared by the electrospinning technology enables the wound dressing to permeate gas, exchange substances with the outside, prevent dehydration of a wound area and promote wound treatment.

The research results of the applicant team on the multifunctional bacteriostatic repair material include CN202010846293, CN202010846307, CN201710859662, CN201510080616 and the like. Aiming at the existing research results, the following problems need to be solved, firstly, the repair performance of the repair material such as antibiosis, wound healing, scar removal and the like needs to be further improved; secondly, the performance of active components in the repair material, such as toxicity, antibacterial and anti-inflammatory effects, needs to be further improved; thirdly, the structure and the components of the repair material are to be further optimized.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems in the prior art, the invention takes two common wound repair materials of chitosan and silicone as an entry point, overcomes the compatible quaternization modification technology of high antibacterial activity and low cytotoxicity, further develops a composite wound dressing by taking the quaternization modification technology as a main raw material, and provides a preparation method and application of the wound dressing. The invention is based on fiber-based medical composite materials such as electrostatic spinning and self-assembly, and endows the material with multiple functions of hemostasis repair, antibiosis and anti-inflammation, scar repair and the like.

The technical scheme is as follows: the composite wound dressing is of a layered structure and comprises an electrostatic spinning structure at an outer layer and a sponge structure at an inner layer; the electrostatic spinning structure is obtained by electrostatic spinning of poly-epsilon-caprolactone and quaternized silicone, and the sponge structure is obtained by freeze-drying and self-assembling of fish skin collagen and quaternized chitosan; the mass ratio of the poly-epsilon-caprolactone to the quaternized silicone is 7; the mass ratio of the fish skin collagen to the quaternized chitosan is 6; the thickness of the electrostatic spinning structure is 0.02-0.05mm, the thickness of the sponge structure is 0.5-0.8mm, and the mass ratio of the electrostatic spinning structure to the sponge structure is 1; the average diameter of the fibers in the electrostatic spinning structure is 150-200 nm; the pore diameter of the sponge structure is 30-300 mu m; the surface porosity of the composite wound dressing is 18-23%.

In a preferred embodiment of the present invention, the structure of the quaternized chitosan is as follows:

the substitution degree of the quaternized chitosan is 16.4-17.2%.

In a preferred embodiment of the present invention, the quaternized silicone has the following structure:

wherein n is the degree of polymerization of polymerized units of silicone, and the degree of substitution of the quaternized silicone is from 25.6 to 27.6%.

As a preferred embodiment of the present invention, the composite wound dressing is prepared by a two-step freeze-drying method, comprising the steps of:

(1) Pouring a blending solution prepared from fish skin collagen and quaternized chitosan into a mould, ensuring that the blending solution is tightly attached to an electrostatic spinning structure, enabling the liquid level height of the blending solution to be 4-5mm, performing gradient freezing for 3-4 h at-20 ℃ and-80 ℃, then transferring into a freeze dryer at-80 ℃, and continuously performing freeze-drying for 45-50 h;

(2) And (2) taking out the freeze-dried dressing obtained in the step (1), washing with deionized water, and carrying out secondary freeze-drying for 45-50 h after pre-freezing. And (5) flatly pressing the composite support to be plastic, and regularly trimming to obtain the composite wound dressing.

As a preferred embodiment of the present invention, the spinning parameters of the electrospinning structure are: the voltage is 19-20kV, the injection rate is 0.8-0.9mL/h, and the receiving distance is 13-14cm; the environmental temperature is 25-35 ℃, and the relative humidity is 35-45%.

The preparation method of the composite wound dressing comprises the following steps:

(1) Weighing poly-epsilon-caprolactone and quaternary ammonium silicone, dissolving in hexafluoroisopropanol, stirring for dissolving, performing ultrasonic defoaming to prepare a homogeneous spinning solution, and spinning, wherein the spinning parameters are as follows: the voltage is 19-20kV, the bolus injection rate is 0.8-0.9mL/h, and the receiving distance is 13-14cm; the environmental temperature is 25-35 ℃, and the relative humidity is 35-45%;

(2) Pouring a blending solution prepared from fish skin collagen and quaternized chitosan into a mould, ensuring that the blending solution is tightly attached to the electrostatic spinning structure obtained in the step (1), enabling the liquid level height of the blending solution to be 4-5mm, performing gradient freezing for 3-4 h at-20 ℃ and-80 ℃, and then transferring into a freeze dryer at-80 ℃ for continuous freeze-drying for 45-50 h;

(3) And (3) taking out the freeze-dried dressing obtained in the step (2), washing with deionized water, and carrying out secondary freeze-drying for 45-50 h after pre-freezing. And (4) flatly pressing the composite support for plasticity, and regularly trimming to obtain the composite wound dressing.

The composite wound dressing prepared above was sterilized by irradiation (20 kGy) before use.

As a preferred embodiment of the invention, the quaternized silicone is prepared by the following method:

(S1) dissolving polymethylhydrosiloxane in toluene with the total volume of 1/3 under the nitrogen atmosphere, quickly transferring to a reaction kettle, starting mechanical stirring, and controlling the temperature in the kettle to be 25 ℃; diluting 6-bromine-1-hexene with toluene with the total volume of 1/3, uniformly dividing the diluted toluene into three times, adding the mixture into a reaction kettle, uniformly mixing a catalyst Karstedts catalyst with the toluene with the total volume of 1/3, uniformly adding the mixture into the reaction system for three times, adjusting and controlling the reaction temperature to 65 ℃ after the addition is finished, keeping the reaction temperature for 48 hours, ensuring that nitrogen is always introduced, cooling the reaction liquid to 40 ℃, adding cold methanol with the temperature of-20 ℃, collecting bottom-layer oily precipitate, adding active carbon powder for decolorization, filtering black substances by taking diatomite as a filter aid medium, collecting filtrate, and carrying out vacuum distillation on bromopolymethylsiloxane (MHSB) at the temperature of 70 ℃;

and (S2) dissolving the bromopolymethylsiloxane obtained in the step (S1) in 1/2 of the total volume of DMF, transferring the bromopolymethylsiloxane into a reaction kettle, controlling the temperature to be 70 ℃, dissolving N-dodecyl diethanol amine in 1/2 of the total volume of DMF, then adding the mixture into the reaction kettle in several times, controlling the temperature to be 70 ℃ after feeding is finished, continuously reacting for 24 hours, after the reaction is finished, carrying out vacuum distillation on reaction liquid at 70-80 ℃, removing most of the solvent to obtain a yellow gelatinous quaternized silicone crude product, dissolving the obtained quaternized silicone crude product in absolute ethyl alcohol, carrying out ultrafiltration, and carrying out reduced pressure distillation to obtain the quaternized silicone.

In the synthesis of an intermediate MHSB, karstedts catalyst is used as a catalyst, and the added materials are diluted by a solvent and then are added in 3 batches, so that the reaction intensity is reduced, and the safety is ensured; and methanol with the temperature of minus 20 ℃ is used as a precipitator to purify reaction products, so that the energy consumption is reduced. In the synthesis step of QP12, DMF is used as a solvent, and the reaction product is subjected to ultrafiltration purification after distillation, so that the use of organic solvents in the purification step is reduced, and a product with higher purity is obtained.

As a preferred embodiment of the present invention, the quaternized chitosan is prepared by the following method:

(S1) under the condition that the relative humidity is less than or equal to 55%, controlling the reaction temperature to be 65-75 ℃, dissolving N-methyldiethanolamine in 1/2 of the total volume of anhydrous DMF, transferring the N-methyldiethanolamine into a reaction kettle, dissolving bromododecane in 1/2 of the total volume of anhydrous DMF, putting the obtained solution into the reaction kettle in batches, continuously reacting for 6 hours, and pouring acetone to precipitate a reaction product after the reaction is finished to obtain dodecyl quaternary ammonium salt;

(S2) controlling the temperature of the reaction kettle to be-5 to-3 ℃, dissolving dodecyl quaternary ammonium salt by using 1/3 of the total volume of anhydrous DMF, transferring the dodecyl quaternary ammonium salt into the reaction kettle, uniformly mixing triethylamine and 1/6 of the total volume of anhydrous DMF, transferring the mixture into the reaction kettle, dissolving a catalyst 4-dimethylaminopyridine in 1/6 of the total volume of anhydrous DMF, transferring the mixture into the reaction kettle, slowly dripping DMF solution of paratoluensulfonyl chloride when the temperature of a mixture in the reaction kettle is about-10 ℃, raising the temperature to 22-26 ℃ after dripping is finished, and carrying out heat preservation reaction for 4 hours to obtain dodecyl quaternary ammonium salt sulfonate;

(S3) controlling the reaction temperature to be 68-72 ℃, dissolving chitosan in an acetic acid solution and transferring the acetic acid solution to a reaction kettle, dissolving benzaldehyde in absolute ethyl alcohol with the same volume as the chitosan solution, transferring all the benzaldehyde to the reaction kettle, continuously reacting for 4 hours, after the reaction is finished, adjusting the pH value to 6-7 by using a NaOH solution, precipitating, filtering, and washing by using ethanol to obtain N-phenylmethylene chitosan;

(S4) putting the N-benzylidene chitosan into 1/2 of isopropanol in the total reaction volume, transferring the mixture to a reaction kettle, stirring until the N-benzylidene chitosan is completely swelled, keeping the temperature in the kettle at 78-82 ℃, adding KOH into the reaction kettle, continuously stirring and mixing for 2h, dissolving dodecyl quaternary ammonium salt sulfonate into the isopropanol in the total reaction volume of 1/2, adding the mixture in 3-5 times, keeping the temperature for 24h after dropwise addition, filtering, washing with acetone, collecting filter cakes, drying in vacuum, sealing and storing to obtain the quaternized chitosan.

The invention also provides application of the composite wound dressing in wound healing and scar repair materials.

The invention also provides application of the composite wound dressing in tissue engineering dressings, antibacterial materials and medical instruments.

In the present invention, "%" is a mass percentage unless otherwise specified.

Has the advantages that: (1) The preparation process of the quaternized silicone (QP 12) and the quaternized chitosan is optimized, and the obtained quaternized silicone has the characteristics of higher substitution degree, better antibacterial performance, low toxicity, broad-spectrum antibacterial property and the like, and meets the biological safety standard of medical antibacterial materials; the quaternized chitosan has antibacterial property and simultaneously promotes HacaT cell proliferation; (2) The double-layer composite wound dressing QCP is prepared by an electrostatic spinning technology and a freeze-drying self-assembly technology, wherein the outer layer is an electrostatic spinning structure (MQP) taking poly epsilon-caprolactone (PCL) as an excipient and QP12 as an active ingredient, and the inner layer is a sponge structure (QCC) taking QCS12 to compound fishskin Collagen (COL), so that the wound dressing disclosed by the invention is high in biological safety and mutually compatible with the physical and chemical properties of human skin, can effectively prevent wound infection caused by microbial invasion in the wound healing process, and is superior to the similar preparations sold in the aspects of promoting skin wound healing and scar repair; (3) The QCP of the wound dressing prepared by the invention has good mechanical property and thermal stability; in the antimicrobial challenge test, after 48h of action, little microbes invaded the interior of the QCP; the QCP of the wound dressing has no in-vivo cytotoxicity, the hemolysis rate is lower than 5 percent, and the QCP has no skin irritation and conforms to the biological safety standard of the wound dressing; in-vivo effectiveness experiments show that the wound healing condition of 14 days is obviously superior to that of the wound dressing sold on the market, the wound healing rate is over 90 percent, and no visible raised strip-shaped or ellipsoidal scar exists; the histomorphology research further finds that the number of skin blood vessels and hair follicles after QCP repair is more abundant, and the collagen deposition is more ordered.

Drawings

FIG. 1 is a schematic diagram of the preparation of QCPs by electrospinning and self-assembly techniques;

FIG. 2 is a NMR spectrum of three batches of quaternized silicone of example 1 of the present invention;

FIG. 3 is the HaCaT cell viability at different concentrations of QP 12;

FIG. 4 is a NMR hydrogen spectra characterization of three batches of quaternized chitosan of example 2 herein;

FIG. 5 shows the survival of HaCaT cells at different concentrations of QCS12 and CS;

FIG. 6 is a scanning electron microscope image of electrospun membranes of different concentrations and proportions;

fig. 7 is a topographical characterization of a composite dressing, wherein (a) is a plot of micro-and macro-topography of a QCP of a composite dressing; (b) The figure is a scanning electron microscope image of an electrostatic spinning membrane before and after freeze-drying and other treatments;

FIG. 8 is a graph of infrared spectra of different materials;

fig. 9 is a graph of the determination of the performance of a wound dressing prepared according to the present invention, wherein (a) is the determination of porosity, (b) is the determination of water absorption, (c) is the determination of water retention, (d) is the determination of water vapor transmission, GS is gelfoam, p <0.05, p <0.01, n =3;

FIG. 10 shows the results of mechanical property testing of a wound dressing, wherein (a) and (b) are plots of stress (stress) -strain (strain) for different materials; (c) Tensile elastic modulus for films PCL, QCC, MQP and QCP (n = 3);

FIG. 11 is a graph of the thermal stability evaluation of the QCP of the mouth dressing, wherein (a) is a DSC plot for PCL, MQP, QCC and QCP, and (b) is a TGA plot for PCL, MQP, QCC and QCP;

FIG. 12 shows the relative proliferation rates of HaCaT cells cultured in different concentrations of leaching solution for 24h, 48h, 72 h;

FIG. 13 shows the result of a blood compatibility test of a wound dressing, wherein (a) the chart shows hemolysis; (b) The plot shows the hemolysis rate of different materials, { fraction (p) } p <0.001, } p <0.01, } p <0.05, n =5;

FIG. 14 is an observation of intradermally stimulated erythema and edema status;

FIG. 15 shows the weight change (NS: physiological saline; QCP/NS: leachate) at various times (4 h, 24h, 48h, 72 h);

FIG. 16 is SEM images of the PCL and QCP membranes after 8h, 24h and 48h incubation with Escherichia coli, staphylococcus aureus and Candida albicans, respectively;

fig. 17 is the coagulation index (n = 5) of each sample. * Significant differences from other groups (. < 0.05); * P <0.001;

fig. 18 is a result of a hemostatic performance test of a wound dressing, in which (a) is a schematic diagram of a mouse tail-biting model and a liver bleeding model; (b) the amount of bleeding in a mouse tail-off model within 5 min; (c) the corresponding hemostasis time; (d) The plots are the amount of bleeding in the mouse model of liver bleeding, { fraction (p) } p <0.001, { fraction (p) } 0.01, { fraction (p) } 0.05.N =4;

FIG. 19 shows the measurement results of the performance of promoting wound healing and scar repair, wherein (a) is a macroscopic image of the wound surface of each group of 0d (when a model is built), 3d,7d, 14d and 21 d; FIG. 14d is a schematic illustration of a wound healing site within FIG. 14 b;

fig. 20 is a graph of the wound healing rate assay after surgery, wherein (a) is a graph of the wound healing rates at postoperative 0d,3d,7d and 14d, (b) is a graph of the heat map analysis results, (n = 4);

FIG. 21 is the observation of HE staining in post-operative histomorphology, wherein (a) is the pathological image of rat skin wounds (HE staining), and (b) is the thickness of granulation tissue at the wound site at 14d,. P.0.001; # ##, significant differences from other groups (p < 0.001);

FIG. 22 shows the results of MASSON trichrome stain observation of post-operative tissue morphology, wherein (a) shows the pathological images of rat skin wounds (MASSON trichrome stain), and (b) shows the amount of collagen deposition at the wound site,. About.p <0.001,. About.p < 0.05;

fig. 23 is a pathological image of rat skin wound (CD 31 immunohistochemical staining).

Detailed Description

Example 1: preparation of quaternized silicones

(1) Synthesis of Bromopolymethylsiloxane (MHSB)

(S1) pumping the 10L reaction kettle to the vacuum degree of about-0.1 MPa by using a vacuum air pump (the air pumping speed is 2L/S), and blowing dry nitrogen until the vacuum degree is reduced to about 0 MPa. The process was repeated 3 times to keep the autoclave completely under nitrogen atmosphere. The whole reaction process is carried out under the condition of nitrogen blowing.

(S2) Polymethylhydrosiloxane (PMHS) (230.2 g, 3.51mol) is dissolved in 1.7L toluene and quickly transferred to a reaction kettle, mechanical stirring is started (200 rpm), and the temperature in the kettle is controlled to be 25 ℃.

(S3) 6-bromo-1-hexene (801.3 g, 4.91mol) was diluted with 1.7L of toluene and added to the reactor in three portions (10 min intervals).

(S4) uniformly mixing 500 +/-10 ppm of catalyst Karstedts catalyst and 1.7L of toluene, and uniformly adding the mixture into the reaction system in three times (the interval time is about 15min depending on the intensity of the reaction). After the addition was complete, the reaction temperature was adjusted and controlled at 65 ℃ for 48h and it was ensured that nitrogen was always introduced.

(S5) after the reaction is finished, transferring the reaction liquid into a material barrel, cooling to 40 ℃, adding cold methanol at the temperature of minus 20 ℃, observing obvious layering, collecting oily sediment at the bottom layer, adding 2% (w/v) of activated carbon powder, decoloring for 20min at the temperature of 50 ℃, and repeating the decoloring process for 2 times. And filtering off black substances by using kieselguhr as a filter aid, collecting filtrate, and distilling at 70 ℃ in vacuum to obtain a crude MHSB.

(2) Preparation of crude Quaternary Silicone (QP 12)

(S1) all the MHSB obtained was dissolved in 1.75L of DMF solvent and transferred to a 10L reactor, mechanical stirring (200 rpm) was turned on, and the temperature was adjusted and controlled at 70 ℃.

(S2) dissolving N-dodecyl diethanol amine (LA 12) (990.2g, 3.63mol) in 1.75L of DMF, adding into the reaction kettle for 2 times (at an interval of 20 min), and after the feeding is finished, controlling the temperature in the kettle at 70 ℃ and continuously reacting for 24h.

(S3) after the reaction is finished, the reaction solution is distilled in vacuum at 70-80 ℃ to remove most of the solvent. A yellow gel with poor fluidity was obtained as crude quaternized silicone (QP 12).

(3) Ultrafiltration purification of crude quaternized silicone (QP 12)

Dissolving the obtained crude QP12 in anhydrous ethanol to obtain 10% (w/v) solution with molecular weight cutoff of less than or equal to 3000Da and cut-off area of 0.46m ² The ultrafiltration membrane of (2) is used as a filtration medium, and an industrial peristaltic pump provides a driving force (the rotating speed is 120rpm, and the flow is converted into 1800mL/min under the default condition). The environmental temperature is 23-25 ℃, and the working pressure of the system is less than or equal to 0.1MPa. And (4) performing ultrafiltration purification for 8-10h, observing that the yellow color of the concentrated solution becomes light, and removing the solvent by reduced pressure distillation to obtain a refined product QP12.

(4) Performance characterization and evaluation of QP12 refined product

(1) Yield of three batches of QP12

Three batches of experiments were carried out according to the method of example 1, and the yields of the three batches of the final refined product QP12 are listed in table 1. The yield of the crude product exceeds 100 percent because the crude product contains a part of solvent and excessive micromolecules such as reactant LA 12. After small molecules are removed through ultrafiltration membrane filtration, the purification yield (the mass ratio of purified products to purified products) is 49.2%, 50.2% and 47.4%, and the difference between batches is less than 5%; the final yields (ratio of mass after purification to theoretical crude yield) were 51.5%, 50.5%, 48.2%, respectively, with less than 5% batch-to-batch variation. The above results show that the pilot synthesis of different batches of product has a certain stability.

TABLE 1 yield of three batches of Pilot QP12

(2) Nuclear magnetic resonanceResonance hydrogen spectrum ( ¹ H-NMR) characterization

Three batches of samples were subjected to DMSO-d ⁶ After dissolution, the spectra were collected using a nuclear magnetic resonance apparatus (Bruker Avance 600, germany). The substitution degree (DS,%) was calculated by substituting the signal area of the peak into formula (1).

DS(％)＝S ₁ /S ₂ X 100% of formula (1);

in the formula: s ₁ -CH at the dodecyl end of QP12 ₃ Peak area;

S ₂ -backbone polymerizing part of QP12 Si-CH ₃ Peak area.

The nmr hydrogen spectra of the three batches of crude product before and after purification are shown in figure 2. After purification, the signal peak (A) of the solvent DMF and the unreacted-CH of N-dodecylethanolamine ₃ The signal peak (B) disappears or weakens, further proving the reliability of the ultrafiltration purification method. Each batch of samples contained a silicone backbone and a peak characteristic of LA12, e.g., about 0ppm Si-CH ₂ 、Si-CH ₃ And characteristic peaks of alkyl hydrogens at 0.8, 1.2ppm, the above results indicate successful grafting of LA12 to give quaternized silicone (QP 12), the degree of substitution of the three batches of samples was 26.8%, 27.6% and 25.6%, respectively, indicating the feasibility and stability of the optimization method of the present invention.

(3) Analysis of antibacterial Activity

1) Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC):

preparing a liquid medicine to be detected: a10 mg/mL DMSO solution of quaternized silicone was prepared, and benzalkonium Bromide (BZK) was used as a positive control and MHSB was used as a negative control, and the mixture was filtered through a 0.22 μm filter and sterilized to obtain a mother solution. Based on the mother liquor, the dilution step by step is 8000 ug/mL, 4000 ug/mL, 2000 ug/mL, 1000 ug/mL, 500 ug/mL, 250 ug/mL, 125 ug/mL, 62.5 ug/mL, 31.25 ug/mL, 0 ug/mL.

Preparation of cell suspension: selecting single colony after rejuvenation and purification, shake culturing in culture solution for 4-8h, washing with sterile PBS, centrifuging, resuspending, and correcting absorbance at 625nm to 0.08-0.1, i.e. concentration of 1.5 × 10 ⁸ CFU/mL (phase)At 0.5 McLeod standard turbidity), and diluting by 100 times to obtain 10 ⁶ CFU/mL of test bacterial liquid.

Microdilution method: adding 90 mu L of test bacteria liquid into a 96-hole polystyrene flat plate, sequentially adding 10 mu L of drug solutions with different concentrations into the bacteria liquid of a pore plate, parallelly measuring 5 multiple pores in each concentration, shaking uniformly, and culturing at the constant temperature of 37 ℃ for 18h. In the experiment, the blank group (0 concentration) is turbid, which indicates that the group of culture solution is bred with microorganisms; part of the culture broth of the drug group was clear and transparent, indicating that there was almost no proliferation of microorganisms therein. And (3) interpretation of MIC: and taking the minimum concentration as the MIC value on the premise that the culture solution is in a clear and transparent state. Sucking all clear and transparent pore plate culture solution to a culture medium, uniformly coating, and placing in an incubator at 37 ℃ for inverted culture for 24h. Less than 5 colonies in the plate and the lowest concentration is recorded as the lowest bactericidal concentration (MBC) of the sample to be tested.

The antimicrobial activity of QP12 is shown in table 2, with the MIC and MBC of QP12 being higher for all strains than benzalkonium Bromide (BZK), but less than the product MHSB before the quaternary ammonium substitution reaction, indicating the antimicrobial advantage of quaternized silicone (QP 12). Meanwhile, QP12 showed broad-spectrum antibacterial performance, but varied greatly from strain to strain. QP12 values of MIC and MBC for Staphylococcus aureus were 50. Mu.g/mL and 80. Mu.g/mL respectively, for Escherichia coli were 200. Mu.g/mL and 400. Mu.g/mL respectively, and for Candida albicans were 100. Mu.g/mL and 100. Mu.g/mL respectively, suggesting that QP12 may have better antibacterial properties against gram-positive bacteria and fungi.

TABLE 2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of quaternized silicones in μ g/mL

2) Determination of the bacteriostatic Rate

The bacteriostatic rate of the quaternized silicone is tested according to the description of GB/T20944.3 on the evaluation of antibacterial performance by an oscillation method, a negative control group is polymethyl siloxane (PDMS), a blank control group is additionally arranged, and each group is subjected to parallel measurement for 3 times. The evaluation requirements of the antibacterial result are as follows: the bacteriostatic rate of the sample on staphylococcus aureus and escherichia coli is more than or equal to 70 percent, or the bacteriostatic rate on candida albicans is more than or equal to 60 percent, and the sample has antibacterial performance. The results of the QP12 bacteriostasis test are shown in table 3. The negative control PDMS has a bacteriostasis rate of less than 15% to each strain and has no antibacterial effect. The inhibition rates of QP12 after co-culture with staphylococcus aureus, escherichia coli and candida albicans are 85.43 +/-4.14%, 74.75 +/-4.76% and 81.32 +/-6.75%, respectively, and the results prove that QP12 has antibacterial property, and the differences of the antibacterial effects on different floras are similar to those described in Table 2.

TABLE 3 inhibition ratio (in%)

(4) Cytotoxicity assays

The test strains are: human immortalized keratinocytes (HacaT) purchased from institute of biotechnology, hama, north nah.

Preparing a liquid medicine to be detected; stock solutions were prepared by dissolving 10mg of quaternized silicone (QP 12) in 1mL of DMSO. The mother liquor was diluted to the following gradient concentrations in order: 1000. Mu.g/mL, 800. Mu.g/mL, 400. Mu.g/mL, 200. Mu.g/mL, 100. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, 12.5. Mu.g/mL, and 6.25. Mu.g/mL. Benzalkonium Bromide (BZK) was selected as a positive control, and the blank control was 10% broth.

The test method comprises the following steps: the cytotoxicity of the quaternized silicone was measured by the CCK-8 method (cell viability assay kit (CCK-8), beijing lanjieko technologies ltd), and the results are shown in fig. 3 and table 4.

TABLE 4 IC50 values for QP12 and BZK

^* Has significant difference with positive group (BZK), p is less than 0.05

The invention optimizes the production process of the quaternized silicone (QP 12) by optimizing key factors such as the feed ratio, the processing method, the purification and the like to obtain three batches of QP12 products with the purity of more than 90 percent. The substitution degree of the quaternary ammonium is more than 25 percent through a nuclear magnetic resonance hydrogen spectrum method test. The results of tests on the bacteriostatic concentration and the bacteriostatic rate of the three strains show that the three strains have certain antibacterial performance, and especially have better antibacterial effect on staphylococcus aureus. By taking a small-molecule drug BZK as a control, haCaT cytotoxicity examination finds that QP12 half inhibition concentration (IC 50) is 433.22 +/-27.34 mu g/mL, and good cell compatibility is shown.

Example 2: synthesis of quaternized Chitosan (QCS 12)

(1) Preparation of dodecyl Quaternary Ammonium Salt (QAS)

Ensuring that the reaction is carried out under the condition that the Relative Humidity (RH) is less than or equal to 55 percent, and adjusting and controlling the temperature of the reaction kettle to be 70 +/-2 ℃; methyldiethanolamine (MDEA) (5081.0 g, 42.64mol) was dissolved in 3L of anhydrous DMF and transferred in one portion to a 30L reactor, with mechanical stirring (300 rpm) turned on for rapid heat transfer; bromododecane (C) ₁₂ H ₂₅ Br) (11812.0 g, 47.36mol) was dissolved in 3L of anhydrous DMF and the solution was added to the reactor in three portions (10 min intervals); when the temperature stabilized at 70 + -2 deg.C, the reaction was continued for 6h and the progress of the reaction was monitored by silica gel Thin Layer Chromatography (TLC). After the reaction is finished, cooling the reaction liquid to 40 ℃, pouring the reaction liquid into acetone with at least 3 times of volume, mechanically stirring (200 rpm) for 15min, hermetically standing at 4 ℃ for 12h, crushing, caking and precipitating by using a grinding dispersion machine (200 rpm, running for 15 min), performing suction filtration, washing with acetone for 3 times, performing vacuum drying on the precipitated product at 24 ℃ for 24h, and placing the obtained dodecyl quaternary ammonium salt at 4 ℃ for hermetic storage.

(2) Preparation of dodecyl quaternary ammonium salt sulfonate (QAS-Ts)

The reactor temperature was adjusted and controlled at-5. + -. 2 ℃ QAS (8019.5g, 20.23mol) was dissolved in 1.6L anhydrous DMF and transferred to a 30L reactor with mechanical stirring (200 rpm). The homogeneously mixed triethylamine (2447.0 g, 24.36mol) and 1.6L of anhydrous DMF were transferred to a reaction vessel; catalyst 4-Dimethylaminopyridine (DMAP) (634.4 g,5.10 mol) is dissolved in 1.6L of anhydrous DMF and transferred to a reaction kettle, DMF solution of paratoluensulfonyl chloride (TsCl) (3777.0 g, 33.90mol) is slowly dripped through a dripping funnel when the temperature of the mixture in the reaction kettle is about-10 ℃, the temperature rise rate of the reaction liquid is accelerated due to violent reaction in the reaction process, the stirring rate (400 rpm) is adjusted at the moment, the temperature rises to 24 +/-2 ℃ after the dripping is completed, the temperature is kept for 4h, and the TLC tracks the reaction progress.

(3) Synthesis of N-benzylidene Chitosan (CSBA) intermediate

Adjusting and controlling the temperature of the reaction kettle at 70 +/-2 ℃, dissolving 503.8g of Chitosan (CS) in 2.0L of 10% (w/w) acetic acid solution, transferring the solution to a 10L reaction kettle, and starting mechanical stirring (200 rpm); benzaldehyde (1014.41g, 9.56mol) is dissolved in 2.0L of absolute ethyl alcohol, all the benzaldehyde is transferred to a reaction kettle, the temperature is kept at 70 +/-2 ℃, the reaction is continued for 4 hours, after the reaction is finished, the reaction liquid is transferred to a material barrel after being cooled to 40 ℃, the pH value is adjusted to 6-7 by using 40% (w/v) NaOH solution, precipitation and filtration are carried out, ethanol is used for dispersing and washing for 3 times, so as to remove redundant benzaldehyde, and the N-benzylidene chitosan is obtained.

(4) Synthesis of quaternized Chitosan (QCS 12)

498.72g of CSBA is put into 7.5L of isopropanol and transferred to a 30L reaction kettle, the temperature is 24 ℃, the mechanical stirring is carried out at 200rpm for at least 4 hours until the CSBA is completely swelled, after the CSBA is swelled, the temperature in the kettle is controlled and kept at 80 +/-2 ℃, KOH powder (112.31g, 2.00mol) is weighed and directly added into the reaction kettle, and the mixture is continuously stirred and mixed for 2 hours. Controlling and maintaining the temperature in the kettle at 80 +/-2 ℃. And (3) dissolving dodecyl quaternary ammonium sulfonate (2624.50g, 4.76mol) in 7.5L isopropanol solution, adding the solution for 3-5 times, keeping the temperature for reacting for 24 hours after the dropwise addition is finished, filtering, washing with acetone, collecting a filter cake, drying in vacuum, sealing and storing to obtain the quaternized chitosan.

(5) Characterization and evaluation of QCS12 Performance

(1) Nuclear magnetic resonance hydrogen spectrum ( ¹ H-NMR)

The nmr spectra of the three batches of samples with verified purity are shown in fig. 4. Samples from different batches all contained a chitosan pyran ring (4.0-5.0 ppm) and a quaternary ammonium salt long chain alkyl-CH ₂ CH ₃ Characteristic peaks (0.9, 1.1 ppm) indicating successful grafting of quaternary ammonium salt to C-6 OH of chitosan, with degrees of substitution of 16.9%, 17.2% and 16.4%, respectively.

(2) Analysis of antibacterial Activity

1) The chitosan and quaternized chitosan were dissolved in 1% acetic acid solution, respectively, and MIC and MBC were measured in sequence according to the method described in example 1, and the results are shown in table 5.

TABLE 5 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of quaternized chitosan in units of μ g/mL

2) Determination of the bacteriostatic Rate

Add 25mL sterile PBS to the flask, pipette the chitosan or quaternized chitosan solution to a final concentration of 200. Mu.g/mL (i.e., 200 ppm) and add 5X 10 ⁵ CFU/mL of bacterial suspension 1mL. The culture was performed at 37 ℃ for 18 hours with shaking at 150 rpm. Methods for coating plates, colony counting rules and calculating bacteriostatic ratio refer to the methods cited in example 1.

TABLE 6 bacteriostatic ratio (unit%) of QCS12

The quaternary ammonium-substituted chitosan antimicrobial results are shown in table 5. Chitosan (CS) acts similarly on Escherichia coli and Staphylococcus aureus, but has greater lethality against Candida albicans. The MIC and MBC of the QCS12 to staphylococcus aureus are 100 mug/mL and 140 mug/mL respectively, the MIC and MBC to escherichia coli are 100 mug/mL and 160 mug/mL respectively, no obvious antibacterial difference exists, the antibacterial effect is probably determined by the structure of chitosan, the antibacterial effect to candida albicans is 50 mug/mL and 50 mug/mL, namely the QCS12 shows excellent antibacterial effect to candida albicans, and the MIC and MBC of the QCS12 to three strains are smaller than CS, which shows that the antibacterial performance of quaternary ammonium substituted chitosan is obviously improved, but the different strains have larger difference. The results of the bacteriostatic ratio test (table 6) also confirm the above conclusion.

(3) Cytotoxicity assays

The results of the survival rates of QCS12 and CS solution after 24h of co-culture with HaCaT cells are shown in the figure. Overall, the IC50 of CS was > 2500. Mu.g/mL, whereas QCS12 was 1681.31. + -. 210.69. Mu.g/mL, with slightly enhanced toxicity but still some cell compatibility. When the concentration is less than or equal to 312.5 mu g/mL, the survival rate of the CS group cells is obviously higher than that of the QCS12 group cells; when the concentration is 156.3 mug/mL, the survival rate of the QCS12 and CS to the cells exceeds 90 percent, and the difference between the two is not significant.

Example 3: preparation of composite wound dressing (sponge dressing)

(1) Preparation of polycaprolactone/quaternized silicone electrospun fiber Membranes (MQPs)

Weighing a certain mass of polycaprolactone (Mw is approximately equal to 80000) and quaternized silicone (QP 12) and dissolving in Hexafluoroisopropanol (HFIP), dissolving by magnetic stirring at 24 ℃, and performing ultrasonic defoaming to prepare a homogeneous PCL/QP12 spinning solution (prepared at present). According to the above method, a spinning solution having a mass volume percentage concentration (total mass of PCL and QP 12) of 6% and a PCL/COL mass ratio of 100, 90, 80, 20, 70, 30, 60. Similarly, a spinning solution having a PCL/QP12 mass ratio of 70, and concentrations of 4%, 6%, and 8% was prepared.

Adding the treated spinning solution into a 10mL disposable plastic injector (connected with a metal flat-head needle as a nozzle, the inner diameter of the needle head is 0.60 mm), and fixing the injector on a micro-injection pump; the anode of the high-voltage power supply clamps the metal spray head, and the cathode of the high-voltage power supply is connected with a cylindrical roller (rotating speed of 120rpm and externally connected with a ground wire) which takes the tin foil paper as a receiving medium; and finally applying a high-voltage electrostatic field in the horizontal direction. The voltage intensity is adjusted to be 19-20kV through preliminary experiments, the injection rate is 0.8-0.9mL/h, the receiving distance is 13-14cm, the spinning temperature is 28 +/-3 ℃, and the ambient relative humidity is 40 +/-5%. The prepared spun film was vacuum dried at 24 ℃ for 72h to dissipate the residual solvent. Sealing the dry electrostatic spinning film, and storing at room temperature in a dark place.

The micro-morphology of the electrospun membranes with different concentrations and proportions is shown in fig. 6, the concentration of the spinning solution is fixed at 6%, when the mass proportion of QP12 is increased from 20% to 30%, the fiber surface is smooth, the diameter tends to be average, and the generation of beads is improved; when the QP12 proportion reaches 40%, the fiber diameter is increased, but the fibers with uneven thickness are also increased, which is probably because the QP12 with higher viscosity enables more polymers to be condensed among the whole fibers, and meanwhile, part of the QP12 is separated out to enable firm adhesion to be generated among the fibers, so the component mass ratio is preferably PCL: QP12= 70.

In addition, when the concentration of the spinning solution is less than 4%, the spinning solution forms jet flow, and a solvent with high content is not completely volatilized in the jet flow whipping process, so that the collected fibers are in a bead string structure with different sizes, the breakage of the fibers is aggravated by the presence of the bead strings, and the overall physical strength is reduced; when the concentration of the spinning solution exceeds 10%, the viscosity is too high, so that the fibers are adhered and wound, the diameter difference is increased, and the appearance state is deteriorated. Through exploration and exploration, the optimal concentration of the spinning solution is finally determined to be 6%.

(2) Preparation of polycaprolactone/quaternized silicone electrospun film/composite collagen/quaternized chitosan sponge dressing (QCP)

The electric spinning membrane composite collagen sponge dressing QCP is prepared by adopting a two-step freeze drying method. Firstly, using 0.5% acetic acid as a solvent, respectively preparing a fish skin collagen (Mw ≈ 300000)/quaternized chitosan mass ratio of 80. Spreading an electrostatic spinning membrane MQP at the bottom of a freezing platform mould, tightly attaching the spinning membrane to a square hollow mould with a certain height of 25mm multiplied by 25mm, and slowly pouring a certain amount of blending liquid into the mould to ensure that the liquid level is 4-5mm. Then the mould is frozen in a gradient way at the temperature of minus 20 ℃ and minus 80 ℃ for 4 hours respectively, and then the mould is transferred into a freeze drier at the temperature of minus 80 ℃ for continuous freeze drying for 48 hours.

The freeze-dried scaffolds were removed and washed 3 times with deionized water to remove residual acetate, 10min each. After pre-freezing, secondary freeze-drying is carried out for 48 hours. The composite stent is flat-pressed and plastic, trimmed, sealed and packaged, and then irradiated and sterilized with 20kGy dosage.

In the preparation of quaternized chitosan/collagen sponges, the effect of the ratio of quaternized chitosan (QCS 12) and fish skin Collagen (COL) on the sponge morphology was mainly examined. In this experiment, the fixed concentration was 1%, and the freeze-drying self-assembly was performed using mixed solutions of different mass ratios. QCS12 is an amorphous yellow powder, which easily falls off when its mass exceeds 50% of the total mass of solute, and may be lost due to the lack of stable chemical forces between collagen and QCS12 molecules. By the search screening, the optimum condition was finally determined as a 1% concentration blend solution at a mass ratio COL: QCS12= 60.

As shown in fig. 7 (a), after the sponge dressing is subjected to the treatment processes of washing, secondary freeze-drying, flat pressing and the like, the thickness of the electrospun layer is 0.02-0.05mm, the thickness of the sponge layer is 0.5-0.8mm, and the mass ratio of the electrospun layer to the sponge layer is about 1. The sponge layer is light yellow in color, the surface layer is soft and loose, the structure is porous (the aperture is 30-300 mu m), and the pores are tightly arranged; the fiber diameter change of the electrospun layer before and after the series of processing procedures is shown in fig. 7 (b), the average diameter of 130.91nm is increased to 167.53nm, and the porosity of the surface is reduced from 20.23 +/-2.22% to 12.12 +/-1.45%, because in the process of secondary freeze-drying and flat compression, the sponge undergoes secondary swelling and mechanical extrusion to cause partial fiber adhesion and local flattening, thereby causing the fiber diameter to be increased and the porosity to be reduced.

(3) Performance test

(1) Trimming a sample polycaprolactone fiber film (PCL), a quaternized silicone/polycaprolactone fiber film (MQP), a quaternized chitosan/collagen sponge (QCC) and a wound dressing QCP (differentiate double faces) into a light-transmitting sheet dressing film, additionally taking a small amount of fish skin collagen fine powder, and acquiring a corresponding scanning curve by using a Fourier transform infrared spectrum tester. The scanning wave number range is 4000-400cm ^-1 Scanning frequency of 4cm ^-1 . The results are shown in FIG. 8. From the results of fig. 8, it can be seen that the MQP and the electrospun layered spectrum in the composite film are consistent, and both contain all the stretching vibration peaks in QP12, such as 2900cm ^-1 、2800cm ^-1 About the dodecyl vibration peak at 1050cm ^-1 Nearby Si-O-Si stretching vibration peak; 1728cm in PCL film ^-1 、1296cm ^-1 、1239cm ^-1 The C = O, C-C and C-O key stretching vibration peaks at three positions also have signal correspondence in MQP. The spectra of the quaternized chitosan/collagen sponge collagen (QCC) and sponge layer in the composite membrane were consistent except 2928cm ^-1 The sum of the vibration peaks of dodecyl group and 1050cm ^-1 Left and right chitosan skeleton vibration peak, 1636cm in fish skin Collagen (COL) ^-1 (ii) amide I band (carbonyl stretching peak) at 1518cm ^-1 Amide II band (N-H bending vibration peak) and 1236cm ^-1 The absorption peak of the amide III band (C-H stretching vibration and N-H bending vibration) also has corresponding signals on the spectrums of the two. The above results are combined to show that the components coexist in different materials in the form of physical mixtures.

(2) Wound dressing QCP porosity, water absorption, water retention and water vapor permeability analysis

1) Porosity of the material

The test method comprises the following steps: about 20mg of a regular sample (W) was weighed ₀ ) The length, width and thickness of the sample are measured by a vernier caliper and the volume (V) is calculated _s ). Immersing in absolute ethyl alcohol at room temperature, and repeatedly vacuumizing and pressurizing to completely fill the pores of the material with the ethyl alcohol. After removing the sample, the hanging and non-falling droplets were wiped off and weighed immediately (W) _e ). Each sample was assayed in 3 replicates. The Porosity (Porosity,%) of the material was calculated according to equation (2), where ρ is the absolute ethanol relative density (0.789, 20 ℃ C.)

An ideal sponge-type dressing would have a high porosity (> 90%) and would absorb more wound exudate. The good pore structure and high porosity can increase the internal surface area, provide more space for cell attachment, growth and proliferation, and accelerate hemostasis and tissue repair. As shown in fig. 9 (a), the QCP porosity measured by the present invention was 93.81 ± 3.19%, which was not statistically significantly different from the commercially available collagen sponge (GS).

2) Water absorption and water retention

The test method comprises the following steps: accurately weighing the sample mass (m) ₀ ) Immersed in PBS (37 ℃). Soaking for 24 hr, swelling, holding the sample corner with fine tweezers, standing in air for 1min, removing water on the surface, and weighing (m) ₁ ) (ii) a Subsequently, the sample was centrifuged at 500rpm for 3min and weighed again (m) ₂ ). Each sample was tested in parallel 3 times. The Water absorption ratio (g/g) and Water retention ratio (g/g) of the sample were calculated by the formula (3) and the formula (4), respectively.

The absorption performance of the sponge on wound exudate is mainly simulated and investigated. Under certain conditions, the stronger the absorption capacity of the sponge on exudates, the less the adverse effect on healthy tissues around the focus is, thus being more beneficial to wound healing and tissue repair. As shown in fig. 9 (b), QCP can absorb 15 times its own weight of water, which is 2 times the water absorption capacity of commercially available sponges.

Water retention is an important criterion for evaluating whether an application type wound dressing can maintain the humidity of the wound surface and maintain a microenvironment for good cell growth. As shown in fig. 9 (c), QCP has higher water retention than commercial gelatin sponge, and there is a significant difference (p < 0.05). Therefore, QCP can better cope with wound exudate in the early stage of trauma, maintaining moist microenvironment of the wound.

3) Water vapor transmission rate

The test method comprises the following steps: 9mL of 0.01M PBS was added to a 10mL centrifuge tube, and the mixture was weighedTotal weight of centrifuge tube and PBS (Experimental group W) _i The blank control group is denoted as W _r ). Cutting the sample into the size of a pipe orifice, completely covering the bottle opening, covering with an open cover, and sealing the joint, wherein the blank control is not covered with any covering. The treated device was transferred to a 37 ℃ oven and allowed to stand. After 24h, the sample was removed and weighed (test set W) _i "W, blank control group _r "). Each sample was assayed in 3 replicates. The water vapor transmission rate (WVTR,%) was calculated according to formula (5).

Generally, when the water vapor transmission capacity of a wound dressing is too low, wound healing may be hindered due to poor exudate drainage. When the air permeability is too high, the wound surface may become dry due to fluid loss. As can be seen from the graph (d) in FIG. 9, QCP has a water vapor transmission rate of 54.05. + -. 3.05%, which is less than that of a commercially available collagen sponge (61.00. + -. 2.04%), but satisfies the balance between wound exudate penetration and moisture retention.

(3) Mechanical property test of wound dressing QCP

The test method comprises the following steps: using the electrospun membranes PCL, MQP, the freeze-dried sample QCC and the composite membrane QCP as test samples, punching dumbbell-shaped diaphragms with the size of 20mm multiplied by 2mm by using a manual punching machine and a die, measuring the thickness of 5 positions by using a thickness gauge, and taking the average value as the actual thickness. Environmental conditions: the temperature is 20-25 ℃, and the relative humidity is 40-50%. The two ends of the diaphragm are fixed by a clamp of an electronic universal test tensile machine, the stretching speed is set to be 10mm/min, the gauge length is set to be 20mm, the diaphragm is stretched in a single direction until the sample is broken, and a stress-strain curve is recorded. Each sample was assayed in 3 replicates. Tensile strength at break (in MPa) and Elongation at break (in%) were calculated from the values, respectively.

Tensile strength(MPa)＝F _max Formula (6);

in the formula: f _max -maximum tensile force at break in N;

s-initial cross-sectional area, in mm ² 。

In the formula: l is a radical of an alcohol ₁ -the length of the membrane before breaking, in mm;

L ₀ the initial length of the membrane in mm.

The mechanical strength of the wound dressing can ensure the stability of practical application and provide a favorable biomechanical environment before new tissue is formed. As can be seen from FIG. 10 and Table 7, the addition of QP12 has no effect on the elongation at break of MQP compared to the fibrous film PCL, while the tensile strength is increased to 5.31. + -. 0.54MPa, the tensile modulus is increased to 114.11. + -. 18.02kPa, and the mechanical strength and mechanical toughness are improved as a whole. The breaking elongation and tensile strength of the sponge dressing QCC are respectively 12.61 +/-1.23% and 0.77 +/-0.14 MPa, the tensile modulus and PCL are not significantly different, but the overall tensile property of the sponge dressing QCC is inferior to that of the fiber membrane PCL, probably because the nano-scale fiber membrane is continuous in a larger range, and the micron-scale material cannot be continuously formed into fibers in a large range due to the preparation method. Compared with a simple sponge QCC, the QCP compounded with the MQP film has almost unchanged breaking elongation, slightly improved tensile strength (1.02 +/-0.08 MPa) and similar tensile modulus (104.09 +/-9.60 kPa) to that of MQP.

TABLE 7 tensile Strength and elongation at Break (n = 3) for each sample

(3) Evaluation of thermal stability of QCP of wound dressing

The test method comprises the following steps: an aluminum crucible containing 5 +/-2 mg of dry uniform membrane samples (PCL, MQP, QCC and QCP) is placed in a thermogravimetry and synchronous thermal analyzer (TGA/DSC 3 +) to measure the thermal processes of the material, such as heat flow change, thermal effect and the like so as to evaluate the thermal change stability of the material. In the testing process, the sample chamber is in a high-purity nitrogen protection atmosphere (40 mL/min), the testing temperature interval is set to be 25-500 ℃, the heating rate is controlled to be 10K/min, the sample can be measured after the base line is corrected, and a data curve is acquired.

FIG. 11 (a) is a DSC curve of four materials. The pure PCL fiber film has an endothermic peak related to the melting temperature at 63.1 ℃, the peak position is shifted to 59.5 ℃ in MQP, and the intensity is slightly increased; the broad peak around 400 ℃ is caused by heat desorption and heat absorption of PCL, and the corresponding endothermic peak in MQP is enhanced, which is probably because the melting point of the MQP fiber membrane is reduced due to the doping of QP12 with wider melting range, and the thermal decomposition effects of PCL and QP12 are superposed. In the sponge type materials QCC and QCP, the peak position of QP12 and PCL generating heat effect disappears because the proportion of the spinning film in QCP is small (< 25%), so that the QCC and QCP heat effect is basically consistent, and the wide and blunt endothermic peak is caused by collagen triple helix conformation change and further denaturation at 71 ℃.

The thermogravimetric change is shown in the TGA curve of FIG. 11 (b). PCL 25.0-125.1 degrees C is the first phase loss of weight (0.12 wt%) caused by water loss; the second stage (336.1-441.6 ℃) was the major weight loss stage (94.22 wt%). Compared to PCL, MQP is divided into three weight loss phases: first losing about 0.13wt% of water at 25.0-209.8 ℃; the temperature is 210.0-352.6 ℃, QP12 is decomposed slowly by opening chain, and the weight loss is about 16.61wt%; the third stage (330.6-488.2 ℃) is a process of rapidly decomposing PCL and QP12, the weight loss is about 73.34wt%, the total weight loss of the three stages is 90.08wt%, and is 4.26wt% less than that of PCL, and the part is mainly silicon dioxide ash obtained by oxidizing and decomposing QP12. QCC and QCP weightlessness phases are similar: the first stage is 25.0-112.7 ℃, and the weight loss is 10.65wt% due to water dispersion and the space conformation of collagen is destroyed and degradation occurs; the most predominant weight loss (58.57 wt%) stage is at 185.3-500.0 ℃, which is associated with substantial thermal degradation of PCL and COL.

From the above results, it can be seen that the components in the QCP prepared by the invention exist in a blended form, and have good tensile modulus (104.09 ± 9.60 kPa) although being relatively fragile, and simultaneously have certain thermal stability; the QCP has the porosity of more than 90%, water absorption capacity which is 15 times of that of the QCP per se and certain water retention and water vapor permeability, can fully absorb wound exudate, can keep the wettability and air permeability of a wound surface, and provides a favorable microenvironment for adhesion, migration and proliferation of cells, so that the tissue repair progress is accelerated.

Example 4: safety and effectiveness evaluation of QCP (quaternary ammonium phosphate) of composite wound dressing

(1) Cytotoxicity testing of wound dressing QCP

The test method comprises the following steps: (1) preparation of dressing extraction stock solution: trimming the dressing paste into a film with a proper size, fumigating for 24 hours by 75% ethanol, sterilizing for 30min by ultraviolet irradiation, and cleaning the surface by sterile PBS. With reference to GB/T16886.12-2017, the continuous extraction was carried out with a ratio of 0.1g (membrane PCL or QCP) to 1mL (DMEM serum-free medium) of extraction and with the additional addition of the membrane solvent uptake. The leaching conditions are 37 +/-1 ℃ and 72 +/-2 h. After leaching, the extract was centrifuged and sterilized by filtration through a 0.22 μm filter. (2) Collecting HaCaT cells in logarithmic growth phase, digesting with pancreatin for 5min, counting, and making into 5 × 10 concentration ⁴ Cell suspension per mL. Inoculating the cell suspension in a 96-well plate at a volume of 200. Mu.L per well, 37 ℃, 5% ₂ Cells were incubated for 24h. (3) Examining the cells proliferated at the bottom of the 96-well plate under a microscope, after the cells are completely attached to the wall, removing the old culture solution by a pipette, adding 100 mu L of extraction stock solution and diluent thereof, setting 5 re-wells per concentration, and taking a DMEM culture medium containing 0.6% of phenol as a positive control group. 37 ℃ and 5% of CO ₂ Cells were incubated for 24h. (4) The solution in the well plate was discarded, 200. Mu.L of the test solution (10-fold dilution of CCK-8) was added, and incubation was continued for 1 hour. The ELISA detects the absorbance value of the liquid in each hole at 450 nm. The cell growth rate (RGR) was calculated by substituting the result of equation (8).

In the formula: OD _control -absorbance measurements of the blank set;

OD _sample -absorbance measurements of the experimental group.

TABLE 8 grading of relative cell proliferation

TABLE 9 cytotoxicity evaluation of different materials

The CCK-8 cytotoxicity test method is used for measuring the proliferation and toxicity of HaCaT cells in different material leaching solutions, and the results after 24h, 48h and 72h of culture are shown in figure 12. Toxicity evaluations were carried out on the PCL and QCP extracts (concentration: 100%) and 0.6% phenol according to the cytotoxicity classification specified in United states Pharmacopeia of Table 8, and the results are shown in Table 9. The cells in each group are influenced by metabolic waste, living space and the like along with the increase of the culture time, and the number of the cells is relatively reduced. 0.6% phenol denatures or coagulates proteins to damage cells, is extremely cytotoxic, and is graded for toxicity as 3 or 4. After the cells infiltrated in the QCP and PCL membrane leaching liquor are cultured for a period of time, the cells are still in a polygonal epithelial cell shape and grow in an attached manner uniformly. The survival rate in QCP group was higher than 24h for 48h, which is probably due to the fact that cells predominantly adhere to the adsorbent during the early culture phase. Similar to the negative control, the cell relative proliferation rate of the QCP 100% leaching solution is more than 75%, the cell survival rate of the 50% leaching solution is higher than that of the 100% leaching solution, the toxicity is graded to be 0 or 1 or 2, and the QCP is qualified after being evaluated, so that the QCP has no potential cytotoxicity and has good cell compatibility.

(2) Blood compatibility

The determination method comprises the following steps: cutting the application into square film of 10mm × 10mm, and sterilizing. The experimental group comprises an electrostatic spinning film PCL, MQP, a freeze-dried sponge QCC and a composite film QCP; the positive control group is sterile ultrapure water; the negative control group was sterile saline. All liquids were used after ultrasonic degassing.

(1) Preparing anticoagulant rabbit blood: 10mL of blood is obtained from the far-end of the vein at the outer edge of the rabbit ear, 1mL of 2% potassium oxalate normal saline solution is immediately added to be used as an anticoagulant, and the normal saline is diluted in half to obtain the newly-prepared anticoagulant rabbit blood.

(2) And (3) incubation: 10mL of physiological saline is measured and added into a centrifuge tube with a sterilized membrane, the positive control group and the negative control group are only respectively added with the same volume of the sterile water and the physiological saline, and after the treatment is finished, the mixture is transferred into a 37 ℃ water bath for rewarming for 30min. 200 μ L of freshly prepared anticoagulated rabbit blood was added to each group in parallel, and after shaking slowly, the group was covered and incubated in a water bath at 37 ℃ for 60min.

(3) Centrifuging and measuring: each group of samples was centrifuged at 800g for 5min and 100. Mu.L of supernatant fluid was pipetted into a 96-well plate, with 5 replicate wells per group. The absorbance (OD value) at 540nm was measured, and the average value was calculated and substituted into the formula (9) to calculate the Hemolysis rate (%).

In the formula: OD _i -absorbance measurements corresponding to the experimental groups;

OD _n -absorbance measurements of the negative control group;

as shown in FIG. 13 (a), the positive group was hemolyzed by the swelling of erythrocytes due to water absorption, and formed a transparent and uniform dark red liquid. The other groups have different degrees of erythrocyte sedimentation after centrifugation, which indicates that different materials have certain biocompatibility to blood cells.

The result of the hemolysis rate test is shown in (b) of fig. 13, the hemolysis rate of the PCL electrospun membrane is 4.09 ± 0.56%, and the incorporation of collagen with abundant amino acids allows sufficient and effective contact of red blood cells without changing the osmotic pressure of the infiltration environment, thereby reducing the hemolysis ability of the collagen sponge QCC (the hemolysis rate is 1.24 ± 0.65%). Most pure silicones are not hemolytic, while the hemolysis rate of MQP fiber membranes is 8.54 + -2.68% which is 2 times higher than that of PCL membranes, probably because too much cation aggregation in QP12 causes erythrocyte disruption. But because the QP12 content in the QCP is relatively less, the hemolysis rate of the QCP and the QCC has no statistically significant difference and meets the use requirement that the hemolysis rate is less than 5%.

(3) Skin irritation

The test method comprises the following steps: according to the description in GB/T16886.10-2017, new zealand albino rabbits weighing 2.0-2.5kg in early adulthood, were used as subjects, and 2 solvents (polar and non-polar) were selected as extraction medium for intradermal injection, and scored according to a scoring table to evaluate the potential skin irritation of the composite QCP.

TABLE 10 intradermal response Scoring System

TABLE 11 evaluation of intradermal stimulation of QCC and QCP membranes (Total score of statistics)

FIG. 14 shows Erythema (ER) and Edema (ED) of rabbit back skin after intradermal injection of each sample for 24h, 48h and 72 h. The results were judged according to the relevant descriptions in table 10 to obtain the stimulation scores for the QCC and QCP experimental and control groups (table 11). Each statistic was the total score of the test for two rabbits (total 10 injection sites). After 72h observation and scoring, the erythema and edema scores of each group are added, and then the ratio operation is carried out on the total of 12[2 (animal number, 2 animals in each group) × 3 (observation period, 24h, 48h and 72 h) × 2 (score type, erythema and edema) ] to calculate the comprehensive average score of each sample leaching liquor and the corresponding leaching medium. If the difference between the two comprehensive average scores is within 1.0, the test requirement is met, and the material is proved to have no skin irritation. If the reaction of the sample experimental group is suspected to be larger than that of the solvent control group in the observation period, the experiment is required to be carried out again.

For quaternized chitosan/collagen sponges (QCC), the experimental group scored 35 total, and the composite average scored 2.91; the control group was assigned a corresponding score of 27 and 2.25, respectively. For the composite QCP, the experimental components total 34, the ensemble average score was 2.83; the control group is correspondingly divided into 3 and 2.5. The difference between the comprehensive average scores of the test group and the control group of QCC and QCP is 0.66 and 0.33 respectively, and the difference is less than 1.0 specified in GB/T16886.10, which shows the safety standard of the subcutaneous irritation toxicity of the QCC and QCP.

(4) Acute systemic toxicity

The test method comprises the following steps: in vivo toxicity testing of membrane QCP was performed according to the Industrial Standard YBB00042003-2015 acute systemic toxicity test method.

After mice were injected with samples 4h, 24h, 48h and 72h in tail vein, they were identified and recorded according to the observation indexes in table 12, such as digestion, respiration, motor system and weight change. The animal death or the stress reaction above the moderate degree is not observed in the experimental group and the volume control group at each time, the number of the animals with the reaction degree of 'no' and 'light' is shown in the table 13, and the result is shown in the figure 15, and the body weight of all the animals is in the trend of stable increase; only 1-2 animals have mild reaction within 24h, and the number of the animals with mild reaction in the experimental group does not exceed that of the animals in the control group within the observation period, which indicates that QCP meets the safety standard in YBB 00042003.

Table 12 animal response observations after injection.

TABLE 13 animal response levels and quantitative records at various times after tail vein injection

(5) Wound dressing QCP bioavailability analysis

(1) Ability to resist microbial attack

In the experiment, 3 representative non-drug-resistant strains, namely escherichia coli (gram-negative bacteria), staphylococcus aureus (gram-positive bacteria) and candida albicans (dermatophyte) are selected as test strains. The composite dressing was evaluated for antimicrobial invasion resistance by culturing the strain on one side of the dressing and observing the number and state of microorganisms in a microscopic state.

(1) Pretreatment of dressing films: cutting into square membrane of 10mm × 10mm, sterilizing, and attaching the sponge layer to the surface of agar culture medium.

(2) Co-culturing microorganisms and membranes: the preparation concentration is 1.5 multiplied by 10 ⁶ CFU/mL ofAnd (4) bacterial suspension. Sucking 50 mu L of bacterial liquid, uniformly coating the bacterial liquid on the surface of the membrane, inverting the plate at 37 ℃, and culturing for 8h, 24h and 48h at constant temperature.

(3) At the corresponding time point, carefully remove the membrane material with forceps and separate the inner and outer layers, wash the residual bacteria, metabolites and culture medium with PBS, and immerse in 2.5% glutaraldehyde and fix the microorganism morphology for at least 4h (or overnight at 4 ℃). The remaining fixative was replaced with PBS and the membrane was dehydrated in a series of graded alcohols (50%, 70%, 75%, 85%, 95%, 100%) (10 min each). CO 2 ₂ Drying at critical point, removing ethanol, spraying gold on surface, and capturing image with scanning electron microscope system.

The antibacterial performance of the wound dressing is that the wound dressing QCP is used as an experimental group, polycaprolactone fiber membrane (PCL) is used as a control group to observe the form and the quantity of microorganisms and the capability of resisting biofilm formation, and an SEM image is shown in figure 16. The dressing QCP compounded by the electrostatic spinning sponge and the freeze-dried sponge has a breathable porous three-dimensional structure to deal with gas and liquid exchange in the processes of wound healing and scar repairing: on the other hand, the inner and outer layers contain antibacterial components such as quaternary ammonium type cations and the like, and have certain performance of resisting invasion of pathogenic microorganisms so as to reduce or avoid negative reactions such as inflammation, infection and the like caused by the antibacterial components. As can be seen from FIG. 16, after the material was co-cultured with the microorganisms for 8 hours, the growth state of the three microorganisms on the PCL membrane was good, no biofilm was formed, and the number of microorganisms invading the PCL membrane was large. The escherichia coli is short and straight single rods or exists in pairs, and the surface is fine and smooth; the diameter of the staphylococcus aureus is 0.8-1 mu m, and the staphylococcus aureus is distributed in an irregular bead-string manner; candida albicans is spherical or ellipsoidal, and hyphae formed by budding are adhered to part of the material and penetrate into the material. The number of viable bacteria in the outer layer and the inner layer of the QCP membrane is obviously less than that of the PCL membrane, the shape of the bacteria is poor, and some bacteria shrink and are adhered to each other. When the bacteria are further co-cultured for 24h, the number of the inner and outer layer bacteria is obviously increased, and the tendency of biofilm formation on the PCL membrane is gradually increased. The secretion of the outer escherichia coli wraps the escherichia coli and adheres to the contact surface to form a local bacterial aggregation membrane sample; candida albicans is gathered, and thalli deep into the dressing membrane are increased rapidly; on the PCL membrane of Staphylococcus aureus, the bacteria still accumulated but in smaller numbers. On the QCP membrane, the number of bacteria on the surface is smaller than that on the surface, and only a part of the bacteria invades the inside. After 48h of co-culture, the PCL film has formed a biofilm of three microorganisms. The escherichia coli invading the interior also shows a certain growth trend; the clustered candida albicans completely invade to the deep layer, and the growth trend of the microorganisms on the inner layer is similar to that of escherichia coli; the growth state of staphylococcus aureus lags behind the first two. In contrast, the inner and outer QCP layers, although containing a large number of attached microorganisms, are significantly less abundant than PCL and grow dispersively without forming a dense biofilm. It can thus be seen that QCP has a good resistance against microorganisms, which may be associated with the addition of quaternising ingredients.

(6) Blood coagulation and hemostasis properties

(1) In vitro coagulation performance testing of wound dressing QCP

The test method comprises the following steps: the sterilized 10mm × 10mm square pieces were placed on the bottom of a 25mL conical flask and preheated at 37 ℃ for 5min. Aspirate 200. Mu.L of fresh anticoagulated rabbit whole blood (containing 0.25M CaCl) ₂ Concentration in whole blood of 10 mM) was dropped uniformly on the upper surface of the membrane and incubated in a water bath at 37 ℃. After 10min, 10mL of deionized water was added and the unbound erythrocytes were lysed by shaking at 37 ℃ for 20min at 40 rpm. After centrifugation at 800g for 5min, 100. Mu.L of the supernatant was aspirated to determine the absorbance at 542nm (A) _i ). The blank control group is a sufficient dilution of 200. Mu.L rabbit anticoagulated whole blood and 10mL deionized water, and the measured value is the absorbance at the time of complete hemolysis (A) ₀ ) The clotting index was recorded as 100%. The in vitro blood coagulation index (BCI,%) of each sample was calculated according to equation (10).

The whole blood coagulation experiment researches the coagulation capacity of different materials (inner and outer layers of QCC sponge, spinning membrane PCL, MQP and composite membrane QCP) on blood, and the lower the in vitro coagulation index (BCI) value of the hemoglobin solution is, thus prompting that the hemoglobin solution has higher potential hemostatic performance. In fig. 17, the coagulation index of the PCL film is 57.56 ± 0.67%, and the PCL raw material itself has no coagulation performance but increases a dense pore structure after electrospinning film formation, which may cause a certain coagulation phenomenon. MQP has a coagulation index of 48.09 +/-2.64%, is significantly different from a blank group (p is less than 0.05), and has a poor coagulation effect similar to that of PCL. The coagulation index of the collagen-based sponge QCC is 29.46 ± 0.43%, which is significantly different from the spinning film MQP (p < 0.01) because collagen can directly activate platelets in the anticoagulation system to promote blood coagulation. The coagulation indexes of the outer layer (56.96 +/-1.47%) and the inner layer (29.91 +/-1.63%) in the QCP are not significantly different from those of MQP and QCC respectively, which indicates that the original coagulation performance of the QCP is kept after a series of preparation and post-treatment processes, and also indicates that the coagulation function of the QCP is mainly derived from quaternized chitosan/collagen sponge.

(2) Hemostasis Performance test

Hemostatic ability of the composite dressing QCP in a tailgating bleeding model and a liver bleeding model was evaluated using a Kunming mouse with a weight of 20-25g at six weeks of age as a subject and commercially available sterile Gauze (Gauze) and Gelatin Sponge (GS) as controls.

(1) Experimental grouping and disposal: preliminary experiments (n = 4) show that the established sequence, bleeding degree and hemostatic effect of the broken tail and liver bleeding models of the same individual are greatly different, so that only a single bleeding model is established for one individual. Animals were randomly divided into 3 groups (Gauze, GS and QCP), 8 animals per group, and 4 tailgating bleeding models and 4 liver bleeding models were established, respectively. Raising for 3 days before experiment to adapt to environment; after the observation period is finished, the treatment is carried out according to the GB/T39741 laboratory animal euthanasia guideline.

(2) Model of bleeding after broken tail: the mice are fixed on an operation board in a prone position after being anesthetized, and the tail is vertically cut off along the position 50% of the length of the tail by using an operation scissors to form a flat bleeding surface. The section is suspended in the air for 15s, and if the bleeding is normal, the model is successfully established. Immediately applying the dressing to be pre-weighed, moving the dressing away every 10-20s to observe whether bleeding occurs, ending the experiment if the bleeding does not occur within 1min, immediately weighing the dressing with the blood until the bleeding ends, and calculating the blood volume. In order to prevent physiological dysfunction and even death of animals caused by excessive blood loss, if bleeding still occurs after 5min, compression hemostasis is carried out on the bleeding part.

Liver bleeding model: the mice under general anesthesia are fixed on a laboratory bench in supine position, and after being sterilized by alcohol, a 3-5cm longitudinal incision is made along the abdominal midline to penetrate into the abdominal cavity, and the liver lobes are separated and exposed. Thoroughly cleaning secretion of the liver and the periphery with gauze, inducing a bleeding point with a 20G needle, immediately applying a pre-weighed dressing, moving the dressing away every 10-20s to observe whether bleeding continues, if the bleeding does not occur within 1min, ending the experiment, immediately weighing the dressing with the blood until the bleeding ends, and calculating the blood volume (formula 11). If bleeding still occurs after 5min, compression hemostasis is carried out on the bleeding part to prevent the animal from being adversely affected by excessive blood loss.

Bleeding amount (mg) = net weight of material before hemostasis-total weight of material after hemostasis equation (11);

the coagulation property of the material in vitro suggests that it may have the same hemostatic effect in vivo. As shown in fig. 18, bleeding models were established in the tail and liver of mice, respectively, and hemostatic properties of composite dressing QCP, sterile Gauze (Gauze), and commercially available Gelatin Sponge (GS) were evaluated.

In a tail hemostasis experiment, bleeding at bleeding points cannot be effectively inhibited within 5min after the covering dressing is pasted, the gauze absorbs 402.80 +/-72.44 mg of blood within 5min, and compared with the sponge material GS with the blood absorption amount being 1/3 (140.32 +/-30.25 mg) of that of the gauze, the organism damage caused by excessive bleeding is reduced, and the result is similar to the in vitro coagulation result of collagen-based sponge; QCP has a blood uptake of only 72.20 ± 9.04mg, probably due to the more compact porous structure and more porous lamellar structure resulting from the addition of quaternized chitosan.

Similarly, in the liver hemostasis study, different materials can make the covered surface not bleed within 5min, but the bleeding inhibiting time and the bleeding amount are greatly different. The gauze absorbs 204.86 +/-8.07 mg of liver blood at 120.0 +/-16.9 s, the GS controls the blood loss at 27.79 +/-3.29 mg at 73.2 +/-27.8 s, and the gauze has significant difference (p is less than 0.001 and p is less than 0.05) in hemostasis time and bleeding amount, thereby showing the advantage of the collagen sponge in resisting bleeding. QCP hemostasis time is 74.4 +/-3.9 s, and is basically consistent with GS; however, the outer surface of the QCP is covered with the electrospun fiber membrane with high specific surface area and high porosity, so that the bleeding amount is as low as 15.17 +/-2.56 mg and is about 1/2 of GS, the dressing with the composite structure has excellent hemostasis performance, in the hemostasis process, the inner layer sponge is partially dissolved after contacting blood, and forms a hemostatic scab with rapidly aggregated blood platelets, and the outer layer of the electrospun fiber membrane is matched to prevent the blood from being rapidly lost, so that the hemostasis is rapidly and effectively realized.

(7) Promoting wound healing and scar repairing performance

The self-made quaternized chitosan collagen sponge (QCC) and the compound dressing (QCP) are taken as an experimental group, and the commercially available sterile Gauze (Gauze) and the commercially available breathable dressing (Tegaderm) are taken as an experimental group ^TM ) And a commercial Scar plaster (Scar gel) as a control group. Dressings QCC and QCP were used after double-sided radiation sterilization and commercially available dressings were applied directly. Performing surgical anesthesia: before anesthesia, the patient is fasted for 12h, and is administered with 10% chloral hydrate by intraperitoneal injection according to the dosage of 0.3-0.4mL/100g so as to cause general anesthesia.

After general anesthesia, dorsal pili (4 cm. Times.6 cm) inside the scapula were shaved off in rats, and after disinfection with iodine tincture, alcohol was deiodinated to reduce skin mucosal irritation. 2 circular incisions were made up to the fascia using biopsy punches on both sides of the spinal back, and skin edges were trimmed with surgical scissors, a scalpel, with 2cm spacing between wound edges. The secretion of the wound is cleaned by normal saline, and the residual liquid is wiped off by sterile gauze.

After the model was established, the wound surface was completely covered with various types of wound dressings and moderately fixed with two types of dressings. All material coverage was observed and recorded daily to ensure that the dressing did not come off or become damaged, and the dressing was changed every 3 days.

At 3, 7, 14, 21d post-surgery, the dressing was removed to expose the Wound surface, a standard ruler was attached and an electronic photograph of the Wound was taken, and the area of the Wound surface was measured by Image J software and Wound healing rate (percent) was calculated according to equation (11).

The wound surface was covered with 3d,7d, 14d and 21d of each type of scaffold material, and the repair of the wound and surrounding skin tissue was observed, as shown in fig. 19 (a), and fig. 19 (b) is a schematic view of the wound during observation. The result shows that the healing promoting effect of the compound wound dressing QCP is superior to that of other groups, animals normally move and eat within the first 3d, the mental state is good, and wounds have no pathological changes such as infection, suppuration or tissue necrosis; at 7d, the granulation tissue generated by the edge is full in shape and minimal in swelling degree and quickly fills the wound; at 14d, the skin of the wound part is rapidly epithelialized, and the number of new blood vessels is increased; the scab completely falls off at 21 days, the wound position is smooth and tidy, and no obvious linear scar is left.

Generally, 14d after surgery is the major period of skin healing, and hyperproliferating tissue forms scars on the skin surface after 14 d. The rats in each group have the phenomena of dressing adhesion, local scabbing and the like after 3 days of operation, and granulation tissues gradually grow from the edge. After 7d, the wound is reduced in different degrees, the healing degree of the QCP group is more obvious, and uneven scar bulges are arranged in the center of the wound of other groups. At 14d, the wounds of the QCC and QCP groups are basically closed, the color and luster are consistent with those of normal skin, and the fur grows, so that the corium layer is prompted to basically recover the normal function; the wounds of the gauze control group and the breathable application control group are in an irregular edge incomplete healing state and are accompanied by suspected erythema symptoms, and the wounds are possibly caused by immune reactions such as local inflammation and the like; the scar gel group showed a slight edema with erythema due to its poor air permeability. In the graph (b) of fig. 19, it is visually observed that QCC and QCP have better wound healing promoting performance according to the wound profile change in each group of the first 14 d. When the number of the cells is 21d, the surface of the skin of the QCP group is smooth and has no abnormal scar bulges, and the surface of the healing position of the QCC group is proliferated with strip scars, which indicates that the scar repairing effect comes from the outer layer of the spinning fibrous membrane; the scar conditions of the gauze group, the breathable application group and the scar gel adhesion group are the same as those of the QCC group, but the presumed reasons can be that the healing period is long in the early stage, the collagen deposition time is prolonged, or the scar repair property is not possessed in the later stage, so that the scars cannot be repaired. The results show that the QCP is obviously superior to other similar products sold in the market in the treatment effects of wound healing and scar repair, and the structures of the inner layer sponge and the outer layer spinning film can inhibit or remove the existing scar while the wound is healed, so that the normal physiological function of the skin is recovered.

Analysis of wound healing rate for post-operative 14d is shown in fig. 20. 3d, the breathable dressing can meet the requirement of exchanging materials between the wound and the outside to achieve the purpose of shrinking the edge, for example, the wound healing rates of gauze, the breathable application Tegaderm and the QCC are respectively 31.28 +/-7.76%, 34.76 +/-4.17% and 30.70 +/-5.66% which are obviously higher than those of scar gel, particularly, the healing rate of QCP is 44.08 +/-3.80%, and the breathable dressing has significant difference (p is less than 0.05) with the gauze, the breathable application Tegaderm and the QCC. After 7d, the healing rate of each group is increased, the difference between the groups is similar to that of the group 3d, in the control group, due to the characteristic that the breathable material Tegaderm is thin and soft, the removal is easy, the healing is not influenced by the fact that gauze is easily adhered to tissues, and the healing rate is higher than that of gauze and scar gel (47.97 +/-2.58%); whereas there was no significant difference in healing between the QCC and QCP groups, it is likely that granulation tissue had substantially covered the wound and collagen deposition was the predominant process, both of which had better effects on wound healing than the three control materials. The QCP healing rate after 14d was 96.28 + -2.94%, slightly higher than QCC (92.51 + -2.14%), and the wound closure was less than 90% for the control group. This result demonstrates that QCP can significantly accelerate the progress of wound healing.

(8) Tissue morphology analysis

The test method comprises the following steps: on the 7 th, 14 th and 21 th days after operation, the material is taken from the whole layer of skin with the diameter of 1cm at the center of the pathological tissue after anesthesia, and the whole layer of skin is immersed in 4 percent paraformaldehyde solution for shape fixation after being cleaned by normal saline. After dehydration with gradient ethanol and xylene clearing, the tissue was embedded into paraffin tissue blocks, and 4 μm tissue sections were prepared for HE staining, masson trichrome staining and CD31 immunohistochemical staining, respectively. Indexes such as hair follicle growth, vascular proliferation and collagen fiber distribution are observed under a microscope, and photos are collected and subjected to data statistics.

In the section, researches on pathologies such as HE staining, MASSON trichrome staining, CD31 immunohistochemical staining and the like are mainly carried out on the tissue of a rat back wound, pathological structures and forms are observed under a microscope, and the causes of tissue form generation are further analyzed. In the study, QCC and QCP are used as experimental groups, and three commercial materials are used as a control observation group, namely sterile Gauze (Gauze), sterile transparent dressing (Tegaderm) and Scar Gel plaster (Scar Gel).

The HE staining of the pathological tissue section of the wound surface is shown in (a) of FIG. 21, and the epithelialization degree and granulation tissue formation of the wound can be observed. 7d after the operation, a small amount of capillary vessels appear in the sterile gauze group, a large amount of inflammatory cells around tissues are infiltrated, and epithelization is started; the inflammatory reaction of Tegaderm and Scar Gel groups is similar to gauze, and Scar tissues are formed respectively; collagen-type sponges QCC, QCP group have fewer inflammatory cells and a relatively high degree of epithelialization, but the tissue margins have different degrees of raised scarring, probably because early scarring contributes to the formation of granulation tissue and thus promotes wound healing. At 14d, all wound tissues showed increased thickness of squamous epithelium and epidermal layers; the gauze group still infiltrates a small amount of inflammatory cells; the surfaces of the Tegaderm and Scar Gel groups are rugged and uneven and have Scar bulges; the QCP group had thick and flat granulation tissue, no scar hyperplasia, and increased number of hair follicles, which is substantially consistent with the image information in fig. 19 (a), indicating that the QCP group had the best wound healing effect. The average thickness of 14d granulation tissue is shown in fig. 21 (b), where QCP thickness is the largest, 43.56 ± 6.34 μm, statistically significant different from other groups (p < 0.001). At 21d, the sterile gauze and Tegaderm groups still had a small amount of inflammatory cells, but fewer new hair follicles appeared in the new tissues than at 14d, and the three control groups all had scar hyperplasia of different degrees; QCP group hair follicle quantity is more abundant, and the epidermal layer roughness is higher. The above results indicate that QCP can rapidly achieve epithelialization within two weeks and effectively inhibit scar hyperplasia while promoting healing.

MASSON trichrome staining of wound pathological tissue sections is shown in FIG. 22 (a), and collagen deposition on granulation tissue can be observed, with quantitative statistics shown in FIG. 22 (b). 7d after the dressing is applied, the gauze, tegaderm and Scar Gel groups have a large amount of inflammatory cells and erythrocytes, and the collagen deposition amount is small; the QCC and QCP groups had less inflammatory cell distribution, more than 50% collagen deposition, and significant differences from the control group (p < 0.001). At 14d post-surgery, the collagen deposition increased in the control group and the collagen decreased relatively in the QCC and QCP groups, probably due to the remodeling of the regenerated granulation tissue at the wound of the epithelial tissue after most of the wounds healed, but the content level was not significantly different from that of gauze and the collagen alignment was more oriented, because the sterile environment created by the quaternized components in QCP promoted the secretion of normal fibroblast collagen. At 21d, collagen distribution of the control group is disordered, scar hyperplasia exists on the surface layer of the skin, the amount of collagen of the gauze group is reduced, and the collagen of the other two control groups is slightly increased; the deposition amount of the collagen in the QCC and QCP groups is not changed greatly, but the QCP shows the characteristic of more orderly and orderly arrangement of the collagen and has more consistency with the form and orientation of substances in normal skin, and the scar repairing effect of an outer layer spinning film is shown.

Platelet endothelial cell adhesion molecule (CD 31), a membrane glycoprotein. Belongs to the immunoglobulin superfamily and is expressed on the cell membrane of all continuous endothelia but not expressed on discontinuous blood sinus endothelium. Megakaryocytes, platelets and some T and B cells are also expressed. The expression quantity reflects the generation degree of new vessels. At the early stage of healing, the earlier the appearance time of the new blood vessels is, the better the healing efficiency is; in the later healing period, the number of blood vessels for supplying nutrition is reduced, which is beneficial to epidermal remodeling and skin surface flattening, and scar formation is reduced. The expression result is shown in FIG. 23. The number of vessels in each group tended to increase and then decrease, the gauze and Scar Gel group had a lower number of vessels in 7d than the other groups and a slower neogenesis rate to 14 d: after 21d, the number of blood vessels in the gauze is minimum, and the wound is not completely closed; tegaderm, which is comparable in density to the vascular group of Scar Gel, may focus on raised Scar tissue; the QCP group vascular expression was less than that of the QCC group, suggesting that it could have an effective anti-scarring effect.

The results of wound healing effect, HE dyeing, masson dyeing and CD31 immunohistochemical expression after the dressing is comprehensively applied show that the QCP of the composite dressing can synergistically play double roles of healing wounds and repairing scars while inhibiting bacteria and resisting inflammation. The possibility that QCP could accelerate the cause of wound healing in 14d is presumed to be as follows: collagen in the material is dissolved and deposited on the wound surface after contacting the wound, and granulation tissues are accelerated to fill the gap; the quaternized chitosan of the membrane component is beneficial to the generation of new blood vessels and the secretion of collagen of fibroblasts, and the collagen is combed to be distributed in tissues in order. Whereas QCP has scar repair properties from spun films, including its good hydrophobic properties to maintain a sufficiently moist environment to inhibit scarring, QP12 is modified while retaining the well-recognized anti-scarring effect of the silicone component.

Claims

1. The composite wound dressing is characterized by comprising an electrostatic spinning structure at an outer layer and a sponge structure at an inner layer; the electrostatic spinning structure is obtained by electrostatic spinning of poly-epsilon-caprolactone and quaternized silicone, and the sponge structure is obtained by freeze-drying and self-assembling of fish skin collagen and quaternized chitosan; the mass ratio of the poly-epsilon-caprolactone to the quaternized silicone is 7; the mass ratio of the fish skin collagen to the quaternized chitosan is 6; the thickness of the electrostatic spinning structure is 0.02-0.05mm, the thickness of the sponge structure is 0.5-0.8mm, and the mass ratio of the electrostatic spinning structure to the sponge structure is 1; the average diameter of the fibers in the electrostatic spinning structure is 150-200 nm; the pore diameter of the sponge structure is 30-300 mu m; the surface porosity of the composite wound dressing is 18-23%.

2. A composite wound dressing according to claim 1, wherein the quaternized chitosan has the structure shown below:

the substitution degree of the quaternized chitosan is 16.4-17.2%.

3. A composite wound dressing according to claim 1, wherein the quaternised silicone has the structure:

the degree of substitution of the quaternized silicone is 25.6 to 27.6%.

4. Composite wound dressing according to claim 1, wherein the composite wound dressing is prepared by a two-step freeze-drying process comprising the steps of:

(2) And (2) taking out the freeze-dried dressing obtained in the step (1), washing the freeze-dried dressing by using deionized water, and carrying out secondary freeze-drying for 45-50 hours after pre-freezing. And (4) flatly pressing the composite support for plasticity, and regularly trimming to obtain the composite wound dressing.

5. Composite wound dressing according to claim 4, wherein the electrospun structure has spinning parameters of: the voltage is 19-20kV, the bolus injection rate is 0.8-0.9mL/h, and the receiving distance is 13-14cm; the environmental temperature is 25-35 ℃, and the relative humidity is 35-45%.

6. Method for producing a composite wound dressing according to claim 1, comprising the following steps:

(2) Pouring a blending solution prepared from fish skin collagen and quaternized chitosan into a mould, ensuring that the blending solution is tightly attached to the electrostatic spinning structure obtained in the step (1), enabling the liquid level height of the blending solution to be 4-5mm, performing gradient freezing for 3-4 h respectively at-20 ℃ and-80 ℃, then transferring into a freeze dryer at-80 ℃, and continuously performing freeze-drying for 45-50 h;

(3) And (3) taking out the freeze-dried dressing obtained in the step (2), washing the freeze-dried dressing by using deionized water, and carrying out secondary freeze-drying for 45-50 hours after pre-freezing. And (4) flatly pressing the composite support for plasticity, and regularly trimming to obtain the composite wound dressing.

7. A method of preparing a composite wound dressing according to claim 6, wherein the quaternised silicone is prepared by:

(S1) dissolving polymethylhydrosiloxane in toluene with the total volume of 1/3 under the nitrogen atmosphere, quickly transferring to a reaction kettle, starting mechanical stirring, and controlling the temperature in the kettle to be 25 ℃; diluting 6-bromine-1-hexene with toluene with the total volume of 1/3, then equally dividing the diluted toluene into three times, adding the mixture into a reaction kettle, uniformly mixing a catalyst Karstedts catalyst with the toluene with the total volume of 1/3, equally dividing the mixture into three times, adding the mixture into the reaction system, after the addition is finished, adjusting and controlling the reaction temperature to 65 ℃, keeping the reaction temperature for 48 hours, ensuring that nitrogen is always introduced, after the reaction is finished, cooling the reaction liquid to 40 ℃, adding cold methanol at-20 ℃, collecting bottom-layer oily precipitate, decoloring the mixture with active carbon, filtering out black substances by taking diatomite as a filter aid medium, collecting filtrate, and carrying out vacuum distillation at 70 ℃ to obtain a crude bromopolymethylsiloxane product;

8. A method of making a composite wound dressing according to claim 6, wherein the quaternized chitosan is prepared by:

(S1) under the condition that the relative humidity is less than or equal to 55%, controlling the reaction temperature to be 65-75 ℃, dissolving N-methyldiethanolamine in 1/2 of the total volume of anhydrous DMF, transferring the N-methyldiethanolamine into a reaction kettle, dissolving bromododecane in 1/2 of the total volume of anhydrous DMF, putting the mixture into the reaction kettle in batches, continuously reacting for 6 hours, and pouring acetone to precipitate a reaction product after the reaction is finished to obtain dodecyl quaternary ammonium salt;

(S3) controlling the reaction temperature to be 68-72 ℃, dissolving chitosan in an acetic acid solution and transferring the solution to a reaction kettle, dissolving benzaldehyde in absolute ethyl alcohol and then directly transferring the solution to the reaction kettle, continuously reacting for 4 hours, adjusting the pH value to 6-7 by using a NaOH solution after the reaction is finished, precipitating and filtering, and dispersing and washing by using ethyl alcohol to obtain N-benzylidene chitosan;

(S4) adding N-benzylidene chitosan into 1/2 of the total reaction volume of isopropanol, transferring the isopropanol into a reaction kettle, stirring until the N-benzylidene chitosan is completely swelled, keeping the temperature in the kettle at 78-82 ℃, adding KOH into the reaction kettle, continuously stirring and mixing for 2h, dissolving dodecyl quaternary ammonium salt sulfonate into the 1/2 of the total reaction volume of isopropanol, adding the mixture in 3-5 times, keeping the temperature for 24h after the dropwise addition is finished, filtering, washing with acetone, collecting a filter cake, and drying in vacuum and storing in a sealed manner to obtain the quaternized chitosan.

9. Use of a composite wound dressing according to claim 1 in a wound healing and scar repair material.

10. Use of a composite wound dressing according to claim 1 in tissue engineering dressings, antimicrobial materials and medical devices.