CN113786512B - Low-pressure in-situ anti-bacteria repair-promoting electrospinning dressing and preparation method thereof - Google Patents

Abstract

The invention discloses a low-pressure in-situ bacteriostasis repair-promoting electrospinning dressing and a preparation method thereof. The method comprises the steps of completely dissolving polymers PVP and PVB by using absolute ethyl alcohol, then adding the absolute ethyl alcohol or ultrapure water to dissolve a functional medicine solution, adding the prepared spinning precursor solution into a low-pressure portable electrostatic spinning device, and adjusting spinning parameters to enable the bacteriostatic repair-promoting dressing to be deposited at a wound in situ. The electrostatic spinning fiber membrane prepared by the invention has good air permeability and degradability, and is high in safety by adopting low-pressure equipment. The copper peptide and the organic polyquaternium-73 have a synergistic antibacterial effect, are not easy to generate drug resistance, can promote wound repair, and the wound can better keep a moist environment due to the addition of the humectant without causing secondary damage to the skin.

Description

Low-pressure in-situ anti-bacteria repair-promoting electrospinning dressing and preparation method thereof

Technical Field

The invention belongs to the field of biomedical dressings, and particularly relates to a low-pressure in-situ antibacterial repair-promoting electrospun dressing and a preparation method thereof.

Background

Acute wounds are wounds that are formed suddenly and heal within 4-8 weeks, with pathological progression following the classical wound repair process. Once the wound healing process is hindered by certain factors, such as ischemia, infection, dryness, etc., the wound healing process is delayed or even arrested, and a chronic refractory wound is finally formed. The generation of acute wounds is often accompanied by severe trauma and bleeding, life safety is easily threatened, and timely treatment and nursing are not easy.

Maintaining a good local wound environment is critical to wound healing. Studies have shown that a moist wound environment prevents cell dehydration, stimulates cell migration and thus promotes epidermal repair. Common dressing products that create a moist environment for wound healing include films, hydrogels, gels, alginates, and foam dressings. However, the dressing has the problems of adhesion to skin, accumulation of seepage, generation of unpleasant odor, possible stimulation of overgrowth of granulation tissue and the like, and the dressing needs to be changed frequently. It has been thought that prophylactic topical drug use can reduce the infection rate of superficial acute wounds and promote healing, and many ointments can create a moist wound environment, thus increasing the frequency of use. However, frequent use of the same drug can increase the resistance of the bacteria, with serious consequences.

Compared with the traditional fiber preparation technology, the micro-nano fiber prepared by electrostatic spinning has the characteristics of better continuity, higher porosity, smaller fiber diameter, larger specific surface area and the like, has strong surface activity, is easy for surface modification, meets the requirements of small pore diameter, high porosity, physical topological structure similar to extracellular matrix and lightness and softness of a dressing film, is beneficial to the adsorption growth of cells, can effectively filter bacteria to prevent wound infection and quickens the wound healing speed. Chinese patent No. cn201310574818.x discloses the preparation of a chitosan-based electrospun composite wound dressing. Chinese patent CN201610584679.2 discloses a preparation method of a polymer PLCL drug-loaded nanofiber membrane. Chinese patent CN201710076435.8 discloses a preparation method of an antibacterial nanofiber wound dressing. However, the prepared electrostatic spinning dressing has the defects that the safety problem is easily caused by using larger voltage (more than 10 KV); the electrospinning solvent is usually selected from toxic and pungent odor solvents such as dichloromethane, chloroform, acetone, hexafluoroisopropanol and the like, and the dressing is usually required to be prepared in specified equipment; the prepared electro-spinning dressing needs to be used for wound care in two steps, a dressing sheet with a specified shape is prepared by a special electro-spinning machine and then is used for fixing the wound of a patient by using an adhesive plaster, and secondary damage is easily caused to the wound. Therefore, a preparation method of the portable real-time electrostatic spinning dressing with high safety is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a low-voltage in-situ bacteriostasis repair-promoting electrospinning dressing and a preparation method thereof, wherein the low voltage improves the use safety, and can be timely adjusted according to the shape and size of a wound, and the dressing can be better attached and absorbed by the wound by direct in-situ deposition.

In order to achieve the above object, the present invention adopts the following technical solutions.

A preparation method of a low-pressure in-situ anti-bacteria repair-promoting electrospun dressing comprises the following steps:

adding the electrostatic spinning solution into a portable electrostatic spinning machine for electrostatic spinning to obtain the anti-bacteria repair-promoting electro-spinning dressing; the electrostatic spinning solution is a mixed solution of a polymer solution, a functional medicine solution and a humectant.

Preferably, the electrostatic spinning solution comprises 8-15 wt% of polymer, 0.3-3.5 wt% of functional drug and 1-2wt% of humectant.

Preferably, the functional drug comprises one or more of copper-coated peptide, organic polyquaternium-73 and borneol.

Preferably, the electrospinning solution comprises 0.3wt% of the blue copper peptide and 0.3wt% of the organic polyquaternium-73.

Preferably, the electrostatic spinning parameters are spinning voltage of 3-6KV, temperature of 15-35 ℃, relative humidity of 30% -90% and receiving distance of 5-20 cm.

Preferably, the parameters of the electrostatic spinning are spinning voltage of 5KV, temperature of 25 ℃, relative humidity of 42% and receiving distance of 12 cm.

Preferably, the electrostatic spinning injector is a 5-10ml medical injector, and the needle head is a flat head metal needle head with the specification of 18-22G; the portable electrostatic spinning machine is a low-pressure handheld in-situ electrospinning device or a low-pressure fixed in-situ electrospinning device.

Preferably, the polymer is polyvinylpyrrolidone PVP and polyvinyl butyral PVB; the humectant is at least one of glycerol, xylitol and glycerol glucoside.

Preferably, the preparation of the electrostatic spinning solution by using the polyvinyl butyral is preceded by a pretreatment comprising the following steps:

adding polyvinyl butyral into hot water of 70-75 ℃ for water washing, centrifugally dewatering after water washing to remove supernatant, repeatedly washing for 3-5 times, drying collected wet powder for 6-10h under the vacuum drying condition of 75-80 ℃, grinding and sieving to obtain polyvinyl butyral powder.

Preferably, the concentration of the polymer solution is 6 to 15wt%, preferably 8 wt%.

The antibacterial repair-promoting electrospun dressing prepared by the preparation method of any one of the above.

The anti-bacteria repair-promoting electro-spinning dressing is directly electro-spun and deposited on a wound without directly contacting the wound with hands or other apparatuses.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the low-voltage electrostatic spinning formula provided by the invention has no strict condition limitation, and can be normally carried out under the conditions of 4-6KV voltage, 15-35 ℃ ambient temperature and 30-90% relative humidity. The direct spinning to the body surface can be realized by adjusting the distance between the needle head and the wound surface, the fiber membrane covering area and the diameter and thickness of the spun fiber membrane can be controlled, the spinning speed is high, and the wound dressing is nontoxic and pollution-free. Can be applied to hospitals, families, battlefields and other places.

(2) The invention selects PVP and PVB as high molecular polymer base materials, the PVP is easy to dissolve in water, and the biocompatibility is good; PVB is insoluble in water and stable in wet environments. After the wound tissue fluid is absorbed by the dressing, PVP absorbs water rapidly and degrades, and PVB provides a skeleton structure for the dressing, so that the dressing is not prone to rapid collapse and has strong water absorption capacity.

(3) The invention selects the absolute ethyl alcohol as the spinning solvent, various medicines can be dissolved in the ethyl alcohol, and the PVP and the PVB have good dissolubility in the ethyl alcohol. The ethanol is volatile, safe and nontoxic in the spinning process, does not cause secondary damage to wounds due to stimulation, and does not have toxic solvent residual body surface.

(4) The selected copper peptide and the organic polyquaternium-73 can play a role in synergistic antibacterial action, are less prone to drug-resistant bacteria caused by multiple times of administration compared with traditional antibacterial drugs, and can achieve a better antibacterial effect at a lower concentration. Meanwhile, copper ions in the copper-coated peptides can play a role in resisting oxidation, promoting collagen proliferation, assisting wound healing and simultaneously realizing synergistic antibacterial function.

(5) The moisture-retaining component is added in the preparation, so that the dressing can be better attached to the body surface, the moisture environment of the wound is kept, and the transdermal absorption of the medicine and the recovery of the wound are more facilitated.

Drawings

Fig. 1 is a scanning electron microscope SEM picture of the drug-loaded electrospun fiber prepared in example 1 of the present invention.

Fig. 2 is a scanning electron microscope SEM picture of the drug-loaded electrospun fiber prepared in example 2 of the present invention.

Fig. 3 is a scanning electron microscope SEM picture of the drug-loaded electrospun fiber prepared in example 3 of the present invention.

Fig. 4 is a picture of the bacteriostatic and repair promoting effects of the drug-loaded electrospun fibers prepared in examples 1-3 of the invention.

Fig. 5 is a picture of a mouse skin defect repair of the drug-loaded electrospun fiber prepared in example 1 of the invention.

Fig. 6 is a picture of the bacteriostatic and repair promoting effect of the drug-loaded electrospun fiber prepared in example 4 of the invention.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

The reagents used in the examples are commercially available without specific reference.

PVP is purchased from Shanghai Aladdin Biotechnology GmbH PVP, K88-96 M.W.1300000; PVB is purchased from Shanghai Aladdin Biotechnology Co., Ltd, M.W.90000-120000; the organic polyquaternium-73 is purchased from Hangzhou forest biotechnology limited; the lyophilized powder of the blueberries was purchased from Xianminlang Biotechnology Ltd. Borneol is purchased from Anhui tung flower Tang Chinese herbal pieces science and technology Limited. The examples employ devices (chinese patent application No.: 201210229010.3 or 201510214221.3).

Example 1:

step 1: weighing 5g of PVB, adding the PVB into 200ml of hot water at 70 ℃ for washing, centrifugally dewatering after washing to remove supernatant, repeatedly washing for 5 times, carrying out vacuum drying at 75 ℃ for 8 hours, and grinding and sieving to obtain pretreated PVB powder;

step 2: weighing 0.24g of PVP powder, weighing 0.56g of PVB powder obtained in the step 1, completely dissolving the PVB powder in 5g of absolute ethyl alcohol, and stirring for 2 hours to obtain a uniformly mixed polymer spinning solution;

and step 3: weighing 0.2g of bluecopper peptide, dissolving in 0.1g of ultrapure water, stirring until the bluecopper peptide is completely dissolved, weighing 0.1g of borneol and 50mg of organic polyquaternium-73, dissolving in 3.65g of absolute ethyl alcohol, and stirring until the organic polyquaternium-73 is completely dissolved;

and 4, step 4: and (3) adding all the solution in the step (3) into the spinning solution prepared in the step (2), and continuously stirring until the solution is completely mixed to obtain a spinning precursor solution with the mass of 8 wt% of high molecular solute, 2wt% of blue copper peptide, 1 wt% of borneol and 0.5 wt% of organic polyquaternium-73.

And 5: and adding 0.1g of glycerol glucoside into the spinning solution, and stirring for 30min to obtain a spinning solution.

Step 6: adding the spinning solution prepared in the step 5 into a 5ml injector, selecting a needle head with the specification of 20G, installing the needle head into a low-voltage electrostatic spinning device, turning on a switch, adjusting the voltage to 5KV, the room temperature to 25 ℃, the relative humidity to 45%, pressing a push rod of the injector to enable the distance between a wound and an injection port to be 12cm, and directly covering the affected part with the bacteriostatic repair-promoting electrospun fibrous membrane. The morphology is shown in FIG. 1. The fiber diameter can reach 413 +/-39 nm.

Cutting the nanofiber membrane prepared in the step 6 into a 0.5cm round piece, placing the round piece on the surface of a solid culture medium coated with bacterial liquid escherichia coli (8099) and staphylococcus aureus (ATCC6538), culturing the round piece in a constant-temperature incubator at 37 ℃ for 24 hours, observing the size of a bacteriostatic zone, and arranging another group of control groups as blank paper pieces to eliminate the influence of other factors. The prepared fiber membrane wafer is placed in a culture dish, a cell migration experiment test is carried out by utilizing a scratch experiment of L929 cells, and the cell migration rates of 0h, 12h and 24h are observed. The bacteriostasis and repair promoting effect is shown in figure 4, the bacteriostasis zone of escherichia coli is 10.0 +/-0.3 cm, the bacteriostasis zone of staphylococcus aureus is 17.3 +/-0.5 cm, the cell migration rate is 12h 57 percent and 24h 79 percent.

According to the invention, a 6-8 week male mouse of a Babl/C mouse is used, and hairs on the back are fixedly removed after anesthesia, so that the skin is fully exposed. The mouse back was sterilized with 75% alcohol, and then a 1cm × 1cm full skin wound surface was created on the mouse back with surgical scissors, divided into 3 groups: the control group was bandaged with ordinary gauze; the substrate group is PVP/PVB fiber in-situ deposition without carrying medicine; the drug-loaded group was the in situ deposition of the fibers under the formulation of example 1. The mouse wound healing conditions are observed on the 3 rd, 7 th and 10 th days respectively, and the wound healing rate is finally calculated, as shown in fig. 5, the drug-loaded medicine has the best healing effect, wherein the healing rate in the 3 th day is 27%, the healing rate in the 7 th day is 83%, and the healing rate in the 10 th day is 96%.

Example 2:

step 1: weighing 5g of PVB, adding the PVB into 200ml of 70 ℃ hot water for washing, centrifugally dewatering after washing to remove supernatant, repeatedly washing for 3 times, carrying out vacuum drying for 6 hours at 75 ℃, and grinding and sieving to obtain pretreated PVB powder;

and 2, step: weighing 0.6g of PVP powder, weighing 0.9g of PVB powder obtained in the step 1, completely dissolving the PVB powder in 5g of absolute ethyl alcohol, and stirring for 2 hours to obtain a uniformly mixed polymer spinning solution;

and step 3: weighing 0.1g of bluecopper peptide, dissolving in 0.1g of ultrapure water, stirring until the bluecopper peptide is completely dissolved, weighing 50mg of borneol and 50mg of organic polyquaternium-73, dissolving in 3.1g of absolute ethyl alcohol, and stirring until the materials are completely dissolved;

and 4, step 4: and (3) adding all the solution in the step (3) into the spinning solution prepared in the step (2), and continuously stirring until the solution is completely mixed to obtain a spinning precursor solution with the mass of a high molecular solute of 15wt%, the mass of the blue copper peptide of 1 wt%, the mass of the borneol of 0.5 wt% and the mass of the organic polyquaternium-73 of 0.5 wt%.

And 5: adding 0.1g of glycerol into the spinning solution, and stirring for 30min to obtain a spinning solution.

Step 6: adding the spinning solution prepared in the step 5 into a 5ml syringe, selecting a needle with the specification of 20G, installing the syringe into a low-voltage electrostatic spinning device, turning on a switch, adjusting the voltage to 6KV, the room temperature to 30 ℃, the relative humidity to 45%, pressing a push rod of the syringe to enable the distance between a wound and a jet orifice to be 8cm, directly covering an anti-bacteria repair-promoting electrospun fiber membrane on an affected part, wherein the appearance is shown in figure 2, and the fiber diameter can reach 589 +/-27 nm.

And (3) cutting the nanofiber membrane prepared in the step (6) into a 0.5cm round piece, placing the round piece on the surface of a solid culture medium coated with bacterial liquid, culturing the round piece in a constant-temperature incubator at 37 ℃ for 24 hours, and observing the size of a bacteriostatic circle. And another group of control group is blank paper sheets to eliminate the influence of other factors. The prepared fiber membrane wafer is placed in a culture dish, a cell migration experiment test is carried out by utilizing a scratch experiment of L929 cells, and the cell migration rates of 0h, 12h and 24h are observed. The bacteriostasis and repair promoting effect is shown in figure 4, the bacteriostasis zone of escherichia coli is 7.9 +/-0.4 cm, the bacteriostasis zone of staphylococcus aureus is 12.8 +/-0.2 cm, the cell migration rate is 12h 49 percent and 24h 66 percent.

Example 3:

step 1: weighing 5g of PVB, adding the PVB into 200ml of 75 ℃ hot water for washing, centrifugally dewatering after washing to remove supernatant, repeatedly washing for 5 times, carrying out vacuum drying for 6 hours at 75 ℃, and grinding and sieving to obtain pretreated PVB powder;

step 2: weighing 0.3g of PVP powder, weighing 0.7g of PVB powder obtained in the step 1, completely dissolving the PVB powder in 5g of absolute ethyl alcohol, and stirring for 2 hours to obtain a uniformly mixed polymer spinning solution;

and step 3: weighing 60mg of bluecopper peptide, dissolving the bluecopper peptide in 0.1g of ultrapure water, stirring until the bluecopper peptide is completely dissolved, weighing 0.1g of borneol and 50mg of organic polyquaternium-73, dissolving in 3.49g of absolute ethyl alcohol, and stirring until the organic polyquaternium-73 is completely dissolved;

and 4, step 4: and (3) adding all the solution in the step (3) into the spinning solution prepared in the step (2), and continuously stirring until the solution is completely mixed to obtain a spinning precursor solution with the mass of 10 wt% of high molecular solute, 0.6 wt% of blue copper peptide, 1 wt% of borneol and 0.5 wt% of organic polyquaternium-73.

And 5: and adding 0.2g of glycerol glucoside into the spinning solution, and stirring for 30min to obtain a spinning solution.

Step 6: adding the spinning solution prepared in the step 5 into a 5ml injector, selecting a needle head with the specification of 18G, installing the needle head into a low-voltage electrostatic spinning device, turning on a switch, adjusting the voltage to 4KV, the room temperature to 30 ℃, the relative humidity to 56%, pressing a push rod of the injector to enable the distance between a wound and an injection port to be 10cm, directly covering an anti-bacteria repair-promoting electrospun fiber membrane on an affected part, wherein the appearance is shown in figure 3, and the fiber diameter can reach 507 +/-37 nm.

And (3) cutting the nanofiber membrane prepared in the step (6) into a 0.5cm round piece, placing the round piece on the surface of a solid culture medium coated with bacterial liquid, culturing the round piece in a constant-temperature incubator at 37 ℃ for 24 hours, and observing the size of a bacteriostatic circle. And another group of control group is blank paper sheets to eliminate the influence of other factors. The prepared fiber membrane wafer is placed in a culture dish, a cell migration experiment test is carried out by utilizing a scratch experiment of L929 cells, and the cell migration rates of 0h, 12h and 24h are observed. The bacteriostasis and repair promoting effect is shown in figure 4, the bacteriostasis zone of escherichia coli is 6.3 +/-0.7 cm, the bacteriostasis zone of staphylococcus aureus is 11.2 +/-0.4 cm, the cell migration rate is 12h 52 percent and 24h 71 percent.

Example 4:

step 1: weighing 5g of PVB, adding the PVB into 200ml of 75 ℃ hot water for washing, centrifugally dewatering after washing to remove supernatant, repeatedly washing for 5 times, carrying out vacuum drying for 6 hours at 75 ℃, and grinding and sieving to obtain pretreated PVB powder;

and 2, step: weighing 0.3g of PVP powder, weighing 0.7g of PVB powder obtained in the step 1, completely dissolving the PVB powder in 5g of absolute ethyl alcohol, and stirring for 2 hours to obtain a uniformly mixed polymer spinning solution;

and step 3: weighing 30mg of bluecopper peptide, dissolving the bluecopper peptide in 0.1g of ultrapure water, stirring until the bluecopper peptide is completely dissolved, dissolving 30mg of organic polyquaternium-73 in 3.64g of absolute ethyl alcohol, and stirring until the organic polyquaternium-73 is completely dissolved to obtain a solution A; weighing 60mg of bluecopper peptide, dissolving in 3.74g of ultrapure water, and stirring until the bluecopper peptide is completely dissolved to obtain a solution B; 60mg of organic polyquaternium-73 is dissolved in 3.74g of absolute ethyl alcohol and stirred until the solution is completely dissolved to obtain solution C;

and 4, step 4: adding the three solutions prepared in the step (3) into the spinning solution prepared in the step (2), and continuously stirring until the solutions are completely mixed to obtain a spinning precursor solution A, B, C;

and 5: and adding 0.2g of glycerol glucoside into the spinning solution, and stirring for 30min to obtain a spinning solution.

Step 6: adding the spinning solution prepared in the step 5 into a 5ml injector, selecting a needle head with the specification of 20G, installing the needle head into a low-voltage electrostatic spinning device, turning on a switch, adjusting the voltage to 5KV, the room temperature to 25 ℃, the relative humidity to 45%, pressing a push rod of the injector to enable the distance between a wound and a jet orifice to be 12cm, and receiving the electrospun fiber membrane on an aluminum foil.

And (3) cutting the nanofiber membrane prepared in the step (6) into a 0.5cm round piece, placing the round piece on the surface of a solid culture medium coated with bacterial liquid, culturing the round piece in a constant-temperature incubator at 37 ℃ for 24 hours, and observing the size of a bacteriostatic circle. As can be seen from FIG. 6, the inhibition zone of the spinning film A added with both the blue-copper peptide and the organic polyquaternium-73 is obviously larger than that of the pure blue-copper peptide film B and the organic polyquaternium-73 film C under the same mass ratio, the Escherichia coli group A is 8.2cm, the B is 3.9cm and the C is 5.4 cm; the staphylococcus aureus group A is 11.1cm, the staphylococcus aureus group B is 6.0cm and the staphylococcus aureus group C is 9.4cm, and the two medicaments have synergistic antibacterial effects.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. The preparation method of the low-pressure in-situ anti-bacteria repair-promoting electrospun dressing is characterized by comprising the following steps of:

adding the electrostatic spinning solution into a portable electrostatic spinning machine for electrostatic spinning to obtain the antibacterial repair-promoting electrospun dressing; the electrostatic spinning solution is a mixed solution of a polymer solution, a functional medicine solution and a humectant; the functional drug comprises copper-coated peptides and organic polyquaternium-73.

2. The method of claim 1, wherein the electrospinning solution comprises 8 to 15wt% of the polymer, 0.3 to 3.5wt% of the functional drug, and 1 to 2wt% of the humectant.

3. The method of claim 2, wherein the functional drug further comprises borneol.

4. The method according to any one of claims 1 to 3, wherein the electrospinning solution comprises 0.3wt% of the blue-copper peptide and 0.3wt% of the organic polyquaternium-73.

5. The method of claim 4, wherein the electrospinning parameters are spinning voltage 4-6kV, temperature 15-35 ℃, relative humidity 30% -90%, and take-up distance 5-20 cm.

6. The method of claim 5, wherein the electrospinning parameters are a spinning voltage of 5kV, a temperature of 25 ℃, a relative humidity of 42%, and a take-up distance of 12 cm.

7. The preparation method of claim 4, wherein the electrostatic spinning syringe is a 5-10mL medical syringe, and the needle is a flat head metal needle with the specification of 18-22G; the portable electrostatic spinning machine is a low-pressure handheld in-situ electrospinning device or a low-pressure fixed in-situ electrospinning device.

8. The method of claim 4, wherein the polymers are polyvinylpyrrolidone and polyvinylbutyral; the humectant is at least one of glycerol, xylitol and glycerol glucoside.

9. The method of claim 8, wherein the step of pre-treating the polyvinyl butyral prior to preparing the electrospinning solution comprises the steps of:

adding polyvinyl butyral into hot water of 70-75 ℃ for water washing, centrifugally dewatering after water washing to remove supernatant, repeatedly washing for 3-5 times, drying collected wet powder for 6-10h under the vacuum drying condition of 75-80 ℃, grinding and sieving to obtain polyvinyl butyral powder.

10. An antibacterial repair-promoting electrospun dressing prepared by the preparation method of any one of claims 1-9.

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