CN112741927A

CN112741927A - Antibacterial healing-promoting nanofiber scaffold and nanofiber scaffold patch prepared from same

Info

Publication number: CN112741927A
Application number: CN202110260034.4A
Authority: CN
Inventors: 颜廷安; 秦承雪; 刘玉军; 辛泉军
Original assignee: SHANDONG HANFANG PHARMACEUTICAL CO Ltd
Current assignee: SHANDONG HANFANG PHARMACEUTICAL CO Ltd
Priority date: 2020-07-07
Filing date: 2021-03-10
Publication date: 2021-05-04
Also published as: CN111701070A

Abstract

The invention relates to the technical field of medicine and pharmacology, in particular to an antibacterial healing-promoting nanofiber scaffold and a nanofiber scaffold patch prepared by adopting the scaffold. The antibacterial healing-promoting nanofiber scaffold comprises polyvinyl alcohol, silk fibroin, carboxymethyl cellulose and an antibacterial traditional Chinese medicine composition; the raw materials are prepared into a nanofiber membrane with the fiber diameter of 100nm-500nm by adopting an electrostatic spinning method and are subjected to physical crosslinking treatment. The nanofiber scaffold patch prepared by the nanofiber scaffold can remarkably improve the performance of absorbing wound seepage, and the slowly formed gel layer is applied on the wound, so that the minimum change of a microenvironment in the wound healing process can be ensured, the drug slow release is facilitated, the healing period is shortened, and particularly, the secondary injury to the wound can not be caused during replacement.

Description

Antibacterial healing-promoting nanofiber scaffold and nanofiber scaffold patch prepared from same

Technical Field

The invention relates to the technical field of medicine and pharmacology, in particular to an antibacterial healing-promoting nanofiber scaffold and a nanofiber scaffold patch prepared by adopting the scaffold.

Background

The chronic wound is commonly seen in various clinical departments, has complex etiology and long course of disease, is a great problem in the new century, has obvious negative effects on the life quality of patients, causes medical burden to society due to high dressing change cost, and is difficult to heal due to infection, and the wound which is difficult to heal can be infected at the same time.

The medical dressing is one of the most common products in hospitals, is a material for covering wounds, mainly comprises traditional dressings such as gauze and absorbent cotton and a new generation of polymer material dressing, can protect the wounds from external mechanical injury, prevent wound infection and promote the wounds to heal as soon as possible. However, wound healing is a complex process, and is affected by many factors, such as the temperature, humidity, bacterial infection, etc. of the wound microenvironment. When the parameters of these local factors are in a proper range, the wound surface is more favorable for healing.

The existing medical dressing for the wound surface is difficult to balance the change of the microenvironment of the wound surface, can play a role in protecting the wound and diminishing inflammation to a certain extent in use, but has poor absorption on wound surface seepage liquid, and the wound surface seepage liquid is easy to scab.

In addition, in order to increase the air permeability between the dressing patch and the wound surface, an antibacterial fiber film product prepared by an electrostatic spinning process is adopted, the fiber diameter is generally smaller than 100nm and smaller size is pursued, and the production process tends to solve the problem that how to spin the fiber film product to be thinner, so that the defect that the fiber film product is thin and fragile is made up by the subsequent chemical crosslinking preparation step to ensure the strength of the fiber film product.

Disclosure of Invention

The invention provides an antibacterial healing-promoting nanofiber support and a nanofiber support patch prepared by adopting the nanofiber support, on the basis of air permeability, the permeability of a wound surface can be obviously improved, a gel layer can be slowly formed to contact the wound surface in use, the minimum change of a microenvironment in the healing process of the wound surface is ensured, the healing period is shortened, and particularly, the secondary damage to the wound surface can not be caused during replacement; the antibacterial and anti-inflammatory healing effect is obvious; the preparation is simple, the fiber membrane is breathable and stable in performance, chemical crosslinking treatment is not needed, the operation is simple, and the problems in the prior art are solved.

The technical scheme adopted by the invention is as follows:

an antibacterial healing-promoting nanofiber scaffold comprises the following raw materials in parts by weight: 7.5-15 parts of polyvinyl alcohol, 0.5-1.5 parts of silk fibroin, 0.5-1.5 parts of carboxymethyl cellulose and 1.0-1.5 parts of an antibacterial traditional Chinese medicine composition;

the raw materials are prepared into a nanofiber membrane with the fiber diameter of 100nm-500nm by adopting an electrostatic spinning method and are subjected to physical crosslinking treatment to obtain the nanofiber scaffold.

Further, the antibacterial healing-promoting nanofiber scaffold comprises the following raw materials in parts by weight: 15 parts of polyvinyl alcohol, 1 part of silk fibroin, 1 part of carboxymethyl cellulose and 1.2 parts of an antibacterial traditional Chinese medicine composition.

Further, the antibacterial traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-25 parts of phellodendron, 35-45 parts of fructus forsythiae, 15-25 parts of honeysuckle, 15-25 parts of dandelion and 1-1.5 parts of centipede.

Further, the antibacterial traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20 parts of phellodendron, 40 parts of forsythia, 20 parts of honeysuckle, 20 parts of dandelion and 1.2 parts of centipede.

Further, the physical crosslinking treatment is thermal crosslinking treatment.

Further, the preparation process of the nanofiber scaffold comprises the following operation steps:

1) extracting with water to obtain decoction of the antibacterial Chinese medicinal composition, filtering, concentrating to obtain paste with relative density of 1.10-1.15, precipitating with 70% alcohol solution, collecting filtrate, concentrating, and evaporating to obtain extract A;

2) mixing polyvinyl alcohol, carboxymethyl cellulose and silk fibroin in parts by weight, dissolving in water bath, and cooling for later use;

3) taking the solution cooled in the step 2), adding a proper amount of the extract A in the step 1), and stirring until the extract A is completely dissolved to obtain a spinning solution;

4) preparing the spinning solution in the step 3) into a nanofiber membrane with the fiber diameter of 100nm-500nm by adopting an electrostatic spinning method;

5) and (3) placing the nanofiber membrane obtained in the step 4) in an oven at 150 ℃ for 10-30min for thermal crosslinking treatment.

Further, the preparation process of the decoction in the step 1) is as follows: decocting the raw materials of the antibacterial traditional Chinese medicine composition in water for 3 times, 60 minutes for the first time, 45 minutes for the second time and 30 minutes for the third time, mixing the decoctions, and filtering to obtain the antibacterial traditional Chinese medicine composition;

polyvinyl alcohol in step 2): carboxymethyl cellulose: the weight ratio of the silk fibroin is preferably 15:1: 1; the water for the water bath is deionized water; the water bath temperature is 85 ℃, the dissolving time is 60min, and the stirring speed is 60R/min;

adding the extract A in the step 3) according to the part ratio of the extract A to the polyvinyl alcohol of 1.2: 15;

in the step 4), all parameters of electrostatic spinning are set as follows: the spinning distance is 155mm, the voltage is 32kV, the liquid supply speed is 0.8mL/min, the temperature is 20 ℃, and the humidity is 42%.

An antibacterial healing-promoting nanofiber scaffold patch is prepared by compounding the antibacterial healing-promoting nanofiber scaffold with a back lining and an isolation layer.

The antibacterial traditional Chinese medicine liquid used as the raw material has the following functions in the nano-fiber scaffold:

phellodendron bark: clearing heat and eliminating dampness, purging fire and removing steam, and removing toxic substance and treating sore; the main effective components are as follows: berberine, magnoflorine, phellodendrine, tetrandrine and other alkaloids; the pharmacological action is as follows: has obvious bacteriostatic effect on staphylococcus aureus, staphylococcus albus, pneumococcus, dysentery bacillus, bacillus subtilis, pseudomonas aeruginosa and other bacteria; the action mechanism is as follows: can inhibit the production and secretion of cytokines such as IFN-gamma, IL-1, TNF-alpha, IL-2, etc. to inhibit immunoreaction, relieve inflammation injury, eliminate oxygen free radical to play antioxidation role, enhance phagocytosis of monocyte, and improve nonspecific immunity of organism.

Fructus forsythiae: clearing away heat and toxic materials, relieving swelling, dispersing pathogen accumulation, and dispelling pathogenic wind and heat; the main effective components are as follows: forsythoside, phillyrin, oleanolic acid, ursolic acid, etc.; the pharmacological action is as follows: has antioxidant and broad-spectrum antibacterial activity, and can inhibit pathogenic bacteria such as Staphylococcus aureus and Bacillus dysenteriae; the action mechanism is as follows: antagonize the activity of endotoxin, obviously inhibit the over-expression of inflammatory factors induced by the endotoxin, and directly or indirectly prevent the damage of the endotoxin to organisms. Honeysuckle flower: clearing away heat and toxic materials, and dispelling pathogenic wind-heat; the main effective components are as follows: organic acids, flavonoids and triterpenoid saponins; the pharmacological action is as follows: has inhibiting effect on various pathogenic bacteria; the action mechanism is as follows: destroy the fine structure of endotoxin, has obvious anti-inflammatory effect for external application, can gradually enhance the phagocytic function of leucocyte, obviously enhances the antibody generating capacity of damaged lymphocyte, and simultaneously has the functions of removing free radicals and resisting oxidation reaction.

Dandelion: clearing away heat and toxic materials, relieving swelling, dispersing pathogen accumulation, inducing diuresis, and treating stranguria; the main effective components are as follows: sterols, taraxacin; the pharmacological action is as follows: resisting pathogenic microorganism and immunity;

the action mechanism is as follows: enhancing phagocytic system of macrophage, improving immunity of cell, and inhibiting oxidative stress and inflammatory reaction.

Centipede: calming endogenous wind, relieving spasm, dredging collaterals, relieving pain, removing toxic substances, and resolving hard mass; the main effective components are as follows: various amino acids, small molecular peptides, sterols and other active substances; the pharmacological action is as follows: antithrombotic property, protecting vascular endothelial cells, inhibiting capillary permeability in early inflammation, improving hemodynamics, and obviously having antibacterial activity.

The invention has the beneficial effects that:

1. the cellulose membrane of the nanofiber scaffold prepared by the invention has good air permeability and stable performance, can be contacted with wound seepage and can absorb the seepage, and then is converted into a gel layer to be applied on the wound. The gel layer keeps the three-dimensional structure of the nanofiber membrane extracellular matrix, has the effect of bacterium resistance and is beneficial to the creeping growth of cells on the wound surface. Compared with the conventional breathable thin cellulose membrane which needs chemical crosslinking treatment to enhance performance after electrostatic spinning preparation, the nanofiber scaffold provided by the invention adopts physical crosslinking of thermal crosslinking in preparation, is simple to prepare, has a slow gel layer forming speed and gradually forms a gel layer compared with the preparation without thermal crosslinking treatment, and is more beneficial to slow release of a medicament, prolongs medicament aging and shortens a healing period. The obtained nanofiber scaffold can remarkably improve the performance of absorbing the seepage of a wound surface after being combined with the wound surface by adopting the specific raw material combination for preparation, a gel layer with a certain area is gradually formed to contact the wound surface according to the amount of the seepage, and the defect that the existing medical dressing is easy to adhere to the wound in use and cause secondary mechanical damage in replacement is overcome under the protection of the gel layer in the replacement process.

2. The raw materials used by the nanofiber scaffold have good biocompatibility and are not irritant; the solvent used in the spinning process is deionized water, so that the use amount of the organic solvent is reduced, and the method is economical and environment-friendly.

3. The nano fiber scaffold is beneficial to wound healing, inflammation diminishing and bacteria resisting; the carboxymethyl cellulose blended in the nano-fiber and the antibacterial traditional Chinese medicine liquid are slowly released through a gel layer formed by permeation, and jointly act on a wound surface to generate antibacterial and anti-inflammatory effects and better promote healing. The antibacterial traditional Chinese medicine composition adopts the golden cypress, the fructus forsythiae, the honeysuckle, the dandelion and the centipede in a specific ratio, has obvious antibacterial effect and anti-inflammatory effect on various bacteria, and is beneficial to wound healing. The nanofiber scaffold has the diameter of the nanofiber with the diameter of 100nm-500nm, has larger specific surface area, and the pore size is matched with the size of a cell to form a three-dimensional network structure similar to a natural extracellular matrix, so that more attachment points can be provided for the adhesion of the cell, the cell is more stretched, and the growth of the cell is facilitated.

Drawings

FIG. 1 is a graph of fiber sem and diameter statistics for example 1 of the present invention;

FIG. 2 is a graph of fiber sem and diameter statistics for example 2 of the present invention;

FIG. 3 is a graph of fiber sem and diameter statistics for example 3 of the present invention;

FIG. 4 is a graph of fiber sem and diameter statistics for example 4 of the present invention;

FIG. 5 is a graph of fiber sem and diameter statistics for example 5 of the present invention;

FIG. 6 is a graph of fiber sem and diameter statistics for example 6 of the present invention;

FIG. 7 is a graph of fiber sem and diameter statistics for example 7 of the present invention;

FIG. 8 shows the result of macrophage phagocytosis assay after 12hr of treatment with the sample of example 1;

FIG. 9 shows the result of macrophage phagocytosis assay after 12hr treatment with the sample of example 2;

FIG. 10 shows the phagocytosis assay of macrophages after 12hr of treatment with the sample of example 5;

FIG. 11 is a statistical plot of water contact angle tests for various embodiments of the present invention;

FIG. 12 is a statistical chart of mechanical property testing for various embodiments of the present invention;

FIG. 13 is a drug dissolution profile of example 2 of the present invention;

FIG. 14 is a drug dissolution profile of example 3 of the present invention;

FIG. 15 is a drug dissolution profile of example 4 of the present invention;

FIG. 16 is a drug dissolution profile of example 5 of the present invention;

FIG. 17 is a graph of the drug dissolution profile of example 6 of the present invention;

FIG. 18 is a drug dissolution profile of example 7 of the present invention.

Detailed Description

In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings.

Example 1

An antibacterial healing-promoting nanofiber scaffold patch is prepared from the following raw materials: 15g of polyvinyl alcohol, 1g of silk fibroin and 1g of carboxymethyl cellulose.

The preparation method of the antibacterial healing-promoting nanofiber scaffold patch comprises the following steps:

(1) adding the polyvinyl alcohol, the carboxymethyl cellulose and the silk fibroin into a double-layer glass reaction kettle, and dissolving in a water bath at 85 ℃ for 60 minutes by adopting a high-temperature circulating oil bath kettle; in the process, the magnetic stirring speed is set to be 60R/min to form spinning solution;

(2) cooling the prepared spinning solution, and transferring the cooled spinning solution to a liquid storage tank of electrostatic spinning equipment for electrostatic spinning; the electrostatic spinning conditions were: spinning distance of 155mm, voltage of 32kV and liquid supply speed of 0.8mL/min to obtain the antibacterial healing-promoting nanofiber membrane;

(3) placing the nanofiber membrane on the electrostatic spinning receiving plate in an oven at 150 ℃ for 10min for thermal crosslinking treatment; obtaining a nanofiber membrane after heat exchange;

(4) cutting the nanofiber membrane into different specifications of 7.5cm, 5cm, 1.5cm and the like according to different requirements, then compounding the nanofiber membrane with a back lining on an automatic dressing packaging machine KC-FF- | II, compounding the back lining with an isolating layer, and packaging into a product.

Example 2

An antibacterial healing-promoting nanofiber scaffold patch is prepared from the following raw materials: 15g of polyvinyl alcohol, 1g of silk fibroin, 1g of carboxymethyl cellulose, and 1.2g of an antibacterial traditional Chinese medicine composition prepared from phellodendron, fructus forsythiae, honeysuckle, dandelion and centipedes;

(1) taking 20g of phellodendron, 40g of forsythia, 20g of honeysuckle, 20g of dandelion and 1.2g of centipede, mixing, adding water, decocting for 3 times, wherein the first time is 1 hour, the second time is 45 minutes, and the third time is 30 minutes, combining decoctions, and filtering; then concentrating the filtrate to obtain fluid extract with relative density of 1.10-1.15(50 deg.C); adding ethanol into the fluid extract, wherein the mass of the ethanol is 2-3 times of that of the fluid extract, standing for 24h, filtering, and concentrating the filtrate to dryness to obtain dark reddish brown solid; detecting to obtain berberine hydrochloride content of 0.16 wt% and phillyrin content of 0.72 wt% to obtain extract A;

(2) adding the polyvinyl alcohol, the carboxymethyl cellulose and the silk fibroin into a double-layer glass reaction kettle, and dissolving in a water bath at 85 ℃ for 60 minutes by adopting a high-temperature circulating oil bath kettle; in the process, the magnetic stirring speed is set to be 60R/min to form spinning solution;

(3) cooling the prepared spinning solution, adding 1.2g of the extract A, continuously stirring until the extract A is completely dissolved, and transferring the spinning solution to a liquid storage tank of electrostatic spinning equipment for electrostatic spinning; the electrostatic spinning conditions were: spinning distance of 155mm, voltage of 32kV and liquid supply speed of 0.8mL/min to obtain the antibacterial healing-promoting nanofiber membrane;

(4) placing the nanofiber membrane on the electrostatic spinning receiving plate in a drying oven at 150 ℃ for 20min for thermal crosslinking treatment; after the heat exchange is finished, a drug-loaded nanofiber membrane is obtained;

(5) cutting the nanofiber membrane into different specifications of 7.5cm, 5cm, 1.5cm and the like according to different requirements, then compounding the nanofiber membrane with a back lining on an automatic dressing packaging machine KC-FF- | II, compounding the back lining with an isolating layer, and packaging into a product.

Example 3

An antibacterial healing-promoting nanofiber scaffold patch is the same as the antibacterial healing-promoting nanofiber scaffold patch of embodiment 2, except that carboxymethyl cellulose is removed, and the antibacterial healing-promoting nanofiber scaffold patch is prepared from the following raw materials: 15g of polyvinyl alcohol, 1g of silk fibroin and 1.2g of an antibacterial traditional Chinese medicine composition prepared from phellodendron, fructus forsythiae, honeysuckle, dandelion and centipede;

(1) mixing 20g of phellodendron, 40g of forsythia, 20g of honeysuckle, 20g of dandelion and 1.2g of centipede, adding water, decocting for 3 times, wherein the first time is 1 hour, the second time is 45 minutes, and the third time is 30 minutes, combining decoctions, and filtering; then concentrating the filtrate to obtain fluid extract with relative density of 1.10-1.15(50 deg.C); adding ethanol into the fluid extract, wherein the mass of the ethanol is 2-3 times of that of the fluid extract, standing for 24 hours, filtering, and concentrating the filtrate to dryness to obtain dark reddish brown solid; detecting to obtain berberine hydrochloride content of 0.16 wt% and phillyrin content of 0.72 wt% to obtain extract A;

(2) adding the polyvinyl alcohol and the silk fibroin with the weight into a double-layer glass reaction kettle, and dissolving in water bath at 85 ℃ for 60 minutes by adopting a high-temperature circulating oil bath kettle; in the process, the magnetic stirring speed is set to be 60R/min to form spinning solution;

(4) placing the nanofiber membrane on the electrostatic spinning receiving plate in a 150 ℃ oven for 25min for thermal crosslinking treatment; after the heat exchange is finished, a drug-loaded nanofiber membrane is obtained;

Example 4

An antibacterial healing-promoting nanofiber scaffold patch is the same as the antibacterial healing-promoting nanofiber scaffold patch of embodiment 2, except that silk fibroin is removed, and the patch is prepared from the following raw materials: 15g of polyvinyl alcohol, 1g of carboxymethyl cellulose and 1.2g of an antibacterial traditional Chinese medicine composition prepared from phellodendron, forsythia, honeysuckle, dandelion and centipede;

(2) adding the polyvinyl alcohol and the carboxymethyl cellulose into a double-layer glass reaction kettle, and dissolving in a water bath at 85 ℃ for 60 minutes by adopting a high-temperature circulating oil bath kettle; in the process, the magnetic stirring speed is set to be 60R/min to form spinning solution;

(3) cooling the prepared spinning solution, adding 1.2g of the extract A, continuously stirring until the extract A is completely dissolved, and transferring the spinning solution to a liquid storage tank of electrostatic spinning equipment for electrostatic spinning; the electrostatic spinning conditions were: spinning distance is 155mm, voltage is 32kV, liquid supply speed is 0.8mL/min, and a drug-loaded nanofiber membrane is obtained;

Example 5

An antibacterial healing-promoting nanofiber scaffold patch is prepared from the following raw materials: 7.5g of polyvinyl alcohol, 0.5g of silk fibroin, 0.5g of carboxymethyl cellulose and 1.2g of an antibacterial traditional Chinese medicine composition prepared from phellodendron, fructus forsythiae, honeysuckle, dandelion and centipede.

(1) mixing 20g of phellodendron, 40g of forsythia, 20g of honeysuckle, 20g of dandelion and 1.2g of centipede, adding water, decocting for 3 times, wherein the first time is 1 hour, the second time is 45 minutes, and the third time is 30 minutes, combining decoctions, and filtering; then concentrating the filtrate to obtain clear paste with the relative density of 1.10(50 ℃); adding ethanol into the fluid extract, wherein the mass of the ethanol is 2-3 times of that of the fluid extract, standing for 24 hours, filtering, and concentrating the filtrate to dryness to obtain dark reddish brown solid; detecting to obtain berberine hydrochloride content of 0.16 wt% and phillyrin content of 0.72 wt% to obtain extract A;

(3) cooling the prepared spinning solution, adding 1.2 parts of extract A, continuously stirring until the extract A is completely dissolved, and transferring the spinning solution to a liquid storage tank of electrostatic spinning equipment for electrostatic spinning; the electrostatic spinning conditions were: spinning distance of 155mm, voltage of 32kV and liquid supply speed of 0.8mL/min to obtain the antibacterial healing-promoting nanofiber membrane;

Example 6

An antibacterial healing-promoting nanofiber scaffold patch, which is the same as the antibacterial healing-promoting nanofiber scaffold patch of example 2;

the preparation method of the antibacterial healing-promoting nanofiber scaffold patch is different from that of the antibacterial healing-promoting nanofiber scaffold patch in example 2, and comprises the following steps:

(3) cooling the prepared spinning solution, adding 1.2g of the extract A, continuously stirring until the extract A is completely dissolved, and transferring the spinning solution to a liquid storage tank of electrostatic spinning equipment for electrostatic spinning; the electrostatic spinning conditions were: the spinning distance is 155mm, the voltage is 32kV, and the liquid supply speed is 0.8 mL/min; obtaining a drug-loaded nanofiber membrane;

(4) cutting the drug-loaded nanofiber membrane into different specifications of 7.5cm, 5cm, 1.5cm and the like according to different requirements, compounding the drug-loaded nanofiber membrane with a back lining on an automatic dressing patch packaging machine KC-FF- |, and packaging into a product after an isolation layer is compounded.

Example 7

The antibacterial healing-promoting nanofiber scaffold patch and the preparation method thereof are the same as those of the antibacterial healing-promoting nanofiber scaffold patch and the preparation method of the antibacterial healing-promoting nanofiber scaffold patch in example 2, except that the thermal crosslinking in the step (4) is replaced by chemical crosslinking treatment of glutaraldehyde.

And (4) observing the effect:

comparison of Water absorption

Water absorption rate determination method:

instrument for measuring the position of a moving object

1 culture dish with diameter of 90 + -5 mm.

2 laboratory drying cabinet with forced air circulation, temperature can be kept at (37 + -1) degree centigrade.

3 test solution A consists of a solution of sodium chloride and calcium chloride, which contains 142mmol of sodium ions and 2.5mmol of calcium ions. The ion content of the solution is equivalent to human serum or wound exudate. 8.298g of sodium chloride and 0.3689 g of calcium chloride dihydrate were dissolved in deionized water in a volumetric flask and diluted to 1L.

4 balance, weighing 100g with accuracy of 0.0001 g.

The method comprises the following steps:

1 placing a sample of known mass 0.5g in a petri dish;

2 adding test solution preheated to 37 +/-1 ℃ and the mass of the test solution is 40 times of that of the test material +/-0.5 g;

3 moving the mixture into a drying box, and keeping the mixture at the temperature of 37 +/-1 ℃ for 30 min;

4, clamping one corner or one end of the sample by using a pair of tweezers, suspending for 30s, and weighing;

5 repeat steps 1 to 4 for other samples.

Calculation of results

The absorption is expressed as the average mass of absorbing solution per gram of sample. The absorption capacity of each example is shown in table 1 below.

TABLE 1

As is clear from table 1, the water absorption effect of each example is the most excellent as in example 2, and then example 5. The water absorption effects of the example 1 without the addition of the antibacterial traditional Chinese medicine composition, the examples 3 and 4 without individual components and the example 7 with the chemical crosslinking treatment are all inferior to the antibacterial healing-promoting nanofiber scaffold patch of the invention.

Second, comparison of bacteriostatic Effect

And (3) detecting the antibacterial performance: the antibacterial performance of each example was measured by an oscillation method with reference to FZ/T73023-2006 antibacterial knitwear, the used strain was Staphylococcus aureus, and the antibacterial performance of the sample was expressed by bacteriostatic rate. The bacteriostatic rate of each example is shown in the following table 2.

TABLE 2

As can be seen from table 2, in the aspect of the bacteriostatic effect, the bacteriostatic rate is greatly improved in each embodiment in which the bacteriostatic traditional Chinese medicine composition is added, compared with embodiment 1 in which the bacteriostatic traditional Chinese medicine composition is not added. The bacteriostatic rate of the embodiment 2 is the highest, and compared with other embodiments, the bacteriostatic rate of the embodiment 3 without methyl cellulose is reduced.

Thirdly, the comparison of the promotion of wound healing is shown in the following table 3:

TABLE 3

As can be seen from table 3, the wound healing rate was the most effective as in example 2, followed by example 5. The wound healing rate of the example 7 which changes the thermal crosslinking mode is obviously reduced compared with the examples 2-6, and even the effect is worse than that of the example 6 which does not adopt the thermal crosslinking treatment.

Fourth, protection of wound surface for secondary injury comparison

The common wound dressing and the wound contact layer are mostly pu isolating films, when the dressing is replaced, the dry wound is easily subjected to secondary damage, the granulation tissue is damaged, and the wound recovery time is delayed while the pain is brought to a patient.

The existing nanofiber membrane medical dressing has air permeability and moisture absorption, and can absorb wound seepage in time, but secondary damage to a certain degree is still easily caused when the dressing is replaced after a contact surface is dried, and wound healing is not facilitated.

According to the invention, after the antibacterial healing-promoting nanofiber scaffold patch is contacted with the wound surface, the wound surface seepage liquid is absorbed, the electrostatic spinning film of the contact layer is rapidly changed into a gel layer, and the amount of formed gel is different from the area of the gel layer according to the difference of the amount of absorbed seepage liquid. The gel layer is applied on the wound surface, the release of effective components is further slowed down, and in the replacement process, the lining layer of the nanofiber bracket paste is separated from the functional layer which becomes the gel layer, so that the wound surface is indirectly protected, and the wound surface is prevented from being mechanically damaged.

The antibacterial healing-promoting nanofiber scaffold material and the conventional medical dressing of the scaffold patches in the embodiments are used for wrapping the wound surface on the back of a hand of a person, and the influence on the wound surface and the healing condition of the wound surface are compared as shown in the following table 4 when the dressing is replaced.

TABLE 4

In table 4 above, the wound healing cycle length is a statistic performed relative to the healing cycles of examples 2 and 5. The wound healing period of example 6 is long, and considering that no physical crosslinking treatment is adopted in the preparation, so that the gel layer is rapidly formed when contacting the exudate and the drug is suddenly released, the drug of the nanofiber scaffold of example 6 fails within several hours, and the effect cannot be persistently exerted and influenced in the wound healing period.

Fifth, fiber morphology analysis

The fiber sem chart and diameter statistical chart of fig. 1-7 show the morphology and diameter distribution of the nanofibers of examples 1-7. Among them, examples 1, 3 and 4 all have droplets or beads, and examples 3 and 4 are particularly obvious. Other examples 2, 5, 6 and 7 with silk fibroin, carboxymethyl cellulose and antibacterial Chinese medicinal extracts can obviously improve the appearance of the nanofiber, and no liquid drop or bead exists in the sem picture of the fiber. From the diameter statistical chart, the diameter distribution of the example 2 is more concentrated and is a better dressing material than that of the examples 5, 6 and 7.

Sixthly, macrophage phagocytosis test

1. Experimental procedure

1.1 isolation of macrophages from bone marrow:

1. the ultraviolet irradiation of the biological safety cabinet is carried out 30min in advance.

2. Mice were sacrificed (decapped) and sterilized by spraying with 75% ethanol solution.

3. Using sterilized scissors and forceps to cut the skin along the thigh ring at one side, and peeling the skin towards the claw direction.

4. The leg muscles were separated with forceps to expose the femur and tibia (care was taken not to damage the bones).

5. The upper and lower sides of the femur were cut with sterile scissors, and the bone marrow was flushed out by aspirating pre-cooled MEM-alpha medium with a 1ml syringe until the marrow cavity became white.

6. The suspension was centrifuged at 1500rpm for 5 min.

7. Discarding the supernatant, adding a proper amount of erythrocyte lysate into a centrifuge tube, uniformly blowing by using a gun head, uniformly mixing, standing at room temperature for 2min, quickly adding 10 times of culture medium for neutralization at 1500rpm for 5min, and discarding the supernatant.

8. Resuspending cells in MEM complete culture medium (only adding 10% FBS and 1X double antibody), uniformly inoculating the cells into a cell culture dish, adding M-CSF to enable the final mass concentration to be 15-20ng/ml, and culturing in an incubator for 3d to obtain the fully differentiated mouse bone marrow-derived macrophages.

1.2 detection of phagocytic potency of cells:

1. the culture medium in the dish was discarded and adherent cells were washed with PBS.

2. Preheating 0.25% Trypsin-EDTA pancreatin digestive juice at 37 deg.C for 15 min, adding pancreatin digestive solution (2ml), standing at room temperature for 3-5min, and observing digestive state under the microscope.

3. After digestion is completed, 5-8ml of complete culture medium is added, digestion is stopped, and the cells are collected in a 15ml centrifuge tube and centrifuged at 1500rpm for 5 min.

4. The supernatant was discarded and the cells were resuspended in complete medium and counted.

5. Adjusting the density of 4-5 × 105/hole on 6-pore plate, sticking wall for 3-4h, adding different kinds of nanofiber membranes, respectively shearing, ultraviolet irradiating for 30min, respectively adding 6-pore plate, and culturing for 12 h.

6. Adding red fluorescent latex beads with the diameter of 2um and the concentration of 6-8 ul/well, culturing at 37 ℃ for 3 hours, collecting cells (the action is light when collecting the cells, and the cells are prevented from being damaged),

7. washing with precooled PBS for 3 times, digesting cells with pancreatin for 3-5min, neutralizing and washing with Facs buffer, and collecting cells at 1300rpm for 5 min. The cells were collected by flow tube filtration.

8. And (6) performing detection on the machine.

2. The results of the experiments are shown in FIGS. 8-10.

Mouse bone marrow-derived mononuclear macrophages were induced in vitro, and the samples of examples 1, 2 and 5 were added and treated for 12hr, followed by macrophage phagocytosis assay. The results show that the phagocytic capacity of macrophages can be remarkably improved when the extract A is contained. The effect is more obvious in example 2, and in example 5, when the content of the extract A is too high, the phagocytosis of macrophages is inhibited.

Water contact angle test

The material with small water contact angle has better hydrophilicity, and is beneficial to the creeping and growth of cells. As can be seen from fig. 11, the water contact angles of examples 2 to 7 become smaller compared to example 1, with examples 2, 3, 5, 6 being more hydrophilic. Therefore, the water contact angle experiment can be influenced by the bacteriostatic traditional Chinese medicine composition added into the bacteriostatic traditional Chinese medicine composition, in the four embodiments with better hydrophilicity, the content of the bacteriostatic traditional Chinese medicine composition in the embodiment 5 is the highest, the water contact angle is the smallest, compared with the embodiment 2, the embodiment 6 is not crosslinked, the hydrophilicity is not reduced, and the embodiment 7 adopts chemical crosslinking, so that the hydrophilicity is greatly changed, and the cell crawling and growth are not facilitated.

Eighthly, mechanical property test

The samples of examples 1 to 7 were cut into 1cm × 3cm strips, the thickness thereof was measured, and the strips were placed in a tensile tester to test the mechanical properties thereof, with the results shown in fig. 12. As can be seen from fig. 12, the overall mechanical properties are the best among all the materials in example 2. Compared with example 2, example 5 has small stress and large strain, and the material is slightly soft. While the stress is large and the strain is small in the embodiment 1, the material is too hard and brittle; examples 3 and 4 lack the addition of silk fibroin or cellulose, the material strength is poor, and the material cannot bear large force; example 6 no crosslinking was performed, the material stress and strain indicators were poor; example 7, which employs chemical crosslinking, has improved ductility but worsens stress.

Ninth, drug Release test

Test protocol

1) Selecting the sample of example 2, accurately weighing 0.5g of the sample, and placing the sample in a container containing 50mL of PBS buffer (pH 7.2, 0.01M);

2) placing the container in a thermostatic water bath with the temperature of 37 ℃ and shaking at the speed of 90 rpm;

3) accurately measuring 3mL of solution at 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h and 72h (adding 3mL of PBS buffer solution into the rest solution after measuring), and detecting the content of phillyrin and berberine hydrochloride by high performance liquid chromatography.

Determination of content

2.1 Forsythiaside

The measurement is carried out according to high performance liquid chromatography (0512 in 2015 edition of the general rules of Chinese pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (25: 75) as mobile phase; the detection wavelength was 278 nm. The number of theoretical plates is not less than 3000 calculated according to phillyrin peak.

Preparation of reference solution A proper amount of phillyrin reference is precisely weighed, and methanol is added to make into solution containing 0.1mg per 1 ml.

Preparation of test solution samples at each dissolution sampling time point were taken as test solutions.

The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

2.2 Berberine hydrochloride

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.033 mol/L potassium dihydrogen phosphate solution (35: 65) is used as a mobile phase; the detection wavelength was 347 nm. The number of theoretical plates is not less than 3000 calculated according to berberine hydrochloride peak.

Preparation of reference substance solution taking appropriate amount of berberine hydrochloride reference substance, precisely weighing, and adding methanol to obtain solution containing 8 μ g per 1 ml.

3. The test results are shown in fig. 13 to 18.

Experiments show that the forsythin dissolution rate of 4h and the berberine hydrochloride dissolution rate of 35% in the nano-fibers in the embodiment 2 reach 80%. Phillyrin and berberine hydrochloride both reach the dissolution plateau.

Further comparison of the test dissolution curves of other examples shows that the curves of example 2 and example 5 of the present invention are relatively flat, which indicates that the drug is slowly released from the nanofiber membrane, and the final drug dissolution rate of example 2 is higher than that of example 5. Example 6 the steepest curve, the fastest drug release, the sudden drug release due to no crosslinking; example 7 employs chemical crosslinking, and the drug release rate is faster than that of examples 2 and 5 employing thermal crosslinking, whereas examples 3 and 4 have poor drug release effects due to the lack of cellulose and silk fibroin in the nanofibers, respectively. In summary, the sustained release effect of example 2 was the best in the drug release test, and that of example 5 times, that of example 6, which was not crosslinked, was the worst.

Claims

1. The antibacterial healing-promoting nanofiber scaffold is characterized by comprising the following raw materials in parts by weight: 7.5-15 parts of polyvinyl alcohol, 0.5-1.5 parts of silk fibroin, 0.5-1.5 parts of carboxymethyl cellulose and 1.0-1.5 parts of an antibacterial traditional Chinese medicine composition;

2. The antibacterial healing-promoting nanofiber scaffold according to claim 1, comprising the following raw materials in parts by weight: 15 parts of polyvinyl alcohol, 1 part of silk fibroin, 1 part of carboxymethyl cellulose and 1.2 parts of an antibacterial traditional Chinese medicine composition.

3. The antibacterial healing-promoting nanofiber scaffold according to claim 1 or 2, wherein the antibacterial traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-25 parts of phellodendron, 35-45 parts of fructus forsythiae, 15-25 parts of honeysuckle, 15-25 parts of dandelion and 1-1.5 parts of centipede.

4. The antibacterial healing-promoting nanofiber scaffold according to claim 3, wherein the antibacterial traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20 parts of phellodendron, 40 parts of forsythia, 20 parts of honeysuckle, 20 parts of dandelion and 1.2 parts of centipede.

5. The antimicrobial healing-promoting nanofiber scaffold according to claim 1, wherein the physical cross-linking treatment is a thermal cross-linking treatment.

6. The antibacterial healing-promoting nanofiber scaffold according to claim 1, wherein the preparation process of the nanofiber scaffold comprises the following operation steps:

7. The antibacterial healing-promoting nanofiber scaffold according to claim 6, wherein the preparation process of the decoction in the step 1) is as follows: decocting the raw materials of the antibacterial traditional Chinese medicine composition in water for 3 times, 60 minutes for the first time, 45 minutes for the second time and 30 minutes for the third time, mixing the decoctions, and filtering to obtain the antibacterial traditional Chinese medicine composition;

8. An antibacterial healing-promoting nanofiber scaffold patch, which is characterized by being prepared by compounding the antibacterial healing-promoting nanofiber scaffold according to any one of claims 1 to 7 with a backing and an isolation layer.