CN116607266A

CN116607266A - Asymmetric nanofiber membrane containing giant salamander skin secretion and preparation method and application thereof

Info

Publication number: CN116607266A
Application number: CN202310588938.9A
Authority: CN
Inventors: 顾振; 黄哲; 安恒; 温永强
Original assignee: University of Science and Technology Beijing USTB
Current assignee: University of Science and Technology Beijing USTB
Priority date: 2023-05-24
Filing date: 2023-05-24
Publication date: 2023-08-18

Abstract

The invention relates to the technical field of medical dressing, and provides an asymmetric nanofiber membrane containing giant salamander skin secretion, and a preparation method and application thereof. According to the invention, polylactic acid electrostatic spinning solution and gelatin giant salamander mixed spinning solution are respectively subjected to electrostatic spinning to prepare polylactic acid nanofiber membranes and gelatin giant salamander mixed nanofiber membranes, a part of gelatin giant salamander mixed nanofiber membranes are crosslinked, and the gelatin giant salamander mixed nanofiber membranes, the crosslinked gelatin giant salamander mixed nanofiber membranes and the polylactic acid nanofiber membranes are sequentially subjected to hot pressing in a superimposed manner, so that the asymmetric nanofiber membrane containing skin secretion of the giant salamander is obtained. The asymmetric nanofiber membrane dressing provided by the invention can be automatically adhered to the surface of a wound to repair the wound, the wound can be removed from the surface of the skin after extremely short-time cooling is performed on the surface of the dressing, and painless dressing replacement can be realized while pain of the wound of a patient is relieved in a short cooling process.

Description

Asymmetric nanofiber membrane containing giant salamander skin secretion and preparation method and application thereof

Technical Field

The invention relates to the technical field of medical dressings, in particular to an asymmetric nanofiber membrane containing giant salamander skin secretion, and a preparation method and application thereof.

Background

Skin defects are common injuries in daily life work, and not only need to stop bleeding in time, but also need to recover the functions of regenerated tissues. This places greater demands and challenges on current wound dressings. The ideal wound dressing should possess excellent overall properties. Firstly, the dressing should be soft and adaptable, create a moist environment at the skin window and isolate the wound from the external adverse environment, so as to promote wound repair and prevent secondary injury or bacterial infection of the wound. In addition, the dressing should have good cell compatibility to promote cell proliferation and cell migration processes.

In recent years, some new wound dressings have been designed and proposed, such as hydrogels, cellulose sponges, nanofibers, and the like. Among these new wound dressings, nanofibers produced by electrospinning have attracted considerable attention. Compared with the traditional fiber, the nanofiber has smaller, softer and larger specific surface area, and better air permeability is also a great advantage of the nanofiber dressing.

To solve the problems associated with the use of surgical sutures, or the problem of simple wound repair procedures in which the dressing can be adhered directly to the wound surface without external fixation, materials have been developed to date that have very strong adhesive properties, mainly adhesives in the form of hydrogels, that provide better mechanical properties and good biological properties than commercial adhesives used in clinical applications while having a certain adhesion. However, to avoid pain during dressing changes and to maximize the protection of the wound from secondary injury, a clinical problem still exists today with painless changes to the wound dressing.

Disclosure of Invention

The invention aims to provide an asymmetric nanofiber membrane containing giant salamander skin secretion, and a preparation method and application thereof, so that a dressing film can be easily attached to a wound, the in-vivo tissue regeneration process can be accelerated, and meanwhile, no unnecessary reaction can be caused in the wound repair process. Painless replacement is also achieved.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of an asymmetric nanofiber membrane containing giant salamander skin secretion, which comprises the following steps:

(1) Preparing a polylactic acid electrostatic spinning solution and a gelatin giant salamander mixed spinning solution;

(2) Respectively carrying out electrostatic spinning on the polylactic acid electrostatic spinning solution and the gelatin giant salamander mixed spinning solution to prepare a polylactic acid nanofiber membrane and a gelatin giant salamander mixed nanofiber membrane;

(3) Crosslinking a part of gelatin giant salamander mixed nanofiber membrane to obtain a crosslinked gelatin giant salamander mixed nanofiber membrane;

(4) And sequentially superposing and hot-pressing the gelatin giant salamander mixed nanofiber membrane, the crosslinked gelatin giant salamander mixed nanofiber membrane and the polylactic acid nanofiber membrane to obtain the asymmetric nanofiber membrane containing skin secretion of the giant salamander.

Preferably, the preparation method of the polylactic acid electrostatic spinning solution in the step (1) comprises the following steps: dissolving polylactic acid in V _{Chloroform (chloroform)} ：V _{Dimethylformamide} =6 to 8: and 2-4 to obtain the polylactic acid electrostatic spinning solution with the mass concentration of 5-15%.

Preferably, the gelatin giant salamander mixed spinning solution in the step (1) is: dissolving giant salamander skin secretion in a gelatin glacial acetic acid solution with the mass volume concentration of 15-25% to obtain a gelatin giant salamander mixed spinning solution with the volume concentration of 1-5%.

Preferably, in the step (2), the electrostatic spinning adopts a 12-18 mL injector to push spinning solution, the voltage is 50-70 kv, the solution flow rate is 0.8-2 mL/h, the needle receiver is non-woven fabric, the distance between the needle tip of the injector and the receiver is 15-19 cm, the indoor temperature is 22-28 ℃, the relative humidity is 40-60%, and the spinning time is 20-30 min.

Preferably, the diameter of the polylactic acid nanofiber membrane and the gelatin giant salamander mixed nanofiber membrane is 345-405 nm.

Preferably, the crosslinking in step (3) is performed using glutaraldehyde solution: the volume concentration of the glutaraldehyde solution is 45-55%; .

Preferably, the temperature of the hot pressing is 90-110 ℃; the pressure of the hot pressing is 0.4-0.6 MPa.

The invention provides an asymmetric nanofiber membrane containing giant salamander skin secretion.

The invention provides an application of an asymmetric nanofiber membrane containing giant salamander skin secretion as a wound dressing.

According to the invention, the polylactic acid electrostatic spinning solution is prepared, and the gelatin giant salamander mixed spinning solution is prepared; respectively carrying out electrostatic spinning on the polylactic acid electrostatic spinning solution and the gelatin giant salamander mixed spinning solution to prepare a polylactic acid nanofiber membrane and a gelatin giant salamander mixed nanofiber membrane, crosslinking part of the gelatin giant salamander mixed nanofiber membrane, sequentially superposing and hot-pressing the gelatin giant salamander mixed nanofiber membrane, the crosslinked gelatin giant salamander mixed nanofiber membrane and the polylactic acid nanofiber membrane, and obtaining the asymmetric nanofiber membrane containing skin secretion of the giant salamander.

The asymmetric nanofiber membrane dressing provided by the invention can be automatically adhered to the surface of a wound to repair the wound, so that the regeneration process of in-vivo tissues is accelerated, and any unnecessary reaction is not caused. After the wound repair is completed, the gelatin giant salamander mixed nanofiber membrane layer is gradually decomposed on the applied surface, so that no trace is left on the tissue micro-morphology, the tissue micro-morphology is not changed, the gelatin giant salamander mixed nanofiber membrane layer can be taken down from the skin surface after extremely short-time cooling is performed on the dressing surface, and the painless dressing replacement can be realized while the pain of the wound of a patient is relieved in a short cooling process.

The invention utilizes the electrostatic spinning method, and the prepared polylactic acid nanofiber outer layer can isolate external water from penetrating to prevent wound pollution and bacterial breeding. The inner layer has strong adhesiveness, can be attached to the surface of a wound, has the functions of stopping bleeding, resisting bacteria and the like, and promotes the wound to heal quickly. Gelatin, giant salamander powder and polylactic acid used for the nanofiber dressing are all derived from natural animals and plants, have good biocompatibility and degradability, and are environment-friendly dressing. The dressing simultaneously satisfies the functions of coagulation, antibiosis and antiphlogosis and promotes tissue repair, the nanofiber dressing is easy to operate, reasonable in structural design, mature and easy to operate in manufacturing process, can still maintain good mechanical properties under the condition of different moisture contents, can be widely applied to dynamic wound management of joints and the like to complete related tasks of skin wound treatment, does not need to be fixed by supporting materials, and has good application prospects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a scanning electron microscope image of a gelatin giant salamander mixed fiber membrane prepared by using a gelatin giant salamander mixed spinning solution containing skin secretions of giant salamander with different concentrations under different spinning voltages in the embodiment 1 of the invention;

FIG. 2 is a schematic illustration of a thermal compression process for the asymmetric nanofiber membrane dressing of example 2;

FIG. 3 is a graph showing the results of mechanical property testing of different nanofiber membranes according to example 3 of the present invention;

FIG. 4 is a graph showing the results of the adhesion performance test of PGSGS in example 4 of the present invention;

FIG. 5 is a graph showing the contact angle performance test results of PLA, GS, and GS (c) in example 5 of the present invention;

FIG. 6 is a statistical chart of cytotoxicity test results of gelatin, GS (c) and PLA in example 6 of the present invention;

FIG. 7 is a live/dead status chart of cells co-cultured with dressing taken from the confocal of GS and PGSGS in example 6 of the present invention.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

And preparing gelatin giant salamander mixed spinning solution with the skin secretion concentration of 0%, 1%, 3%, 5% and 7%. Specific: and dissolving skin secretion of the giant salamander in the proportion into gelatin glacial acetic acid solution with the mass volume concentration of 20%, and heating and magnetically stirring for 5 hours at 50 ℃ to obtain gelatin giant salamander mixed spinning solution with the concentration.

And (3) adopting a 15ml syringe to push the spinning solution for electrostatic spinning to prepare the gelatin giant salamander mixed nanofiber membrane (GS). When spinning is carried out on the gelatin giant salamander mixed spinning solution, three voltages (50, 60 and 70 kv) are respectively adopted for spinning the five spinning solutions. Other spinning conditions were unchanged. The flow rate of the solution is 1mL/h, the receiver is non-woven fabric, the distance between the needle tip of the injector and the receiver is 17cm, the indoor temperature is 25 ℃, the relative humidity is 50%, and the spinning time is 30min.

And observing the physical appearance of the gelatin giant salamander mixed nanofiber membrane/nanofiber wound dressing prepared by different giant salamander secretion concentrations according to different spinning voltages by adopting a scanning electron microscope (SEM, HITACHI-8010, japan), wherein the acceleration voltage is set to be 5kV. Before the scanning electron microscope scanning test is carried out, a thin layer of gold is sputtered on the surface of the optical fiber to prevent abnormal charging. It can be obviously observed that the nanofiber morphology is the same when the content of giant salamander powder in the system is different. The results are shown in FIG. 1.

As can be seen from fig. 1, dressing concentrations of 0%, 1%, 3%, 5% can form nanofibers to varying degrees. Whereas a dressing with a concentration of 7% does not form nanofibers. The appearance is the best is that the skin secretion concentration of giant salamander is 5%, and the spinning condition is selected to be 60kv, and the obtained nanofiber is the best. The fiber diameter was 375.+ -.30 nm.

Example 2

Preparing polylactic acid electrostatic spinning solution: dissolving polylactic acid in V _{Chloroform (chloroform)} ：V _{Dimethylformamide} =7: 3, heating and magnetically stirring the mixed solution at 80 ℃ for 5 hours to obtain polylactic acid electrostatic spinning solution with the mass concentration of 10%; the electrostatic spinning was performed under the spinning conditions in example 1 (spinning voltage: 60 kv) to prepare a polylactic acid nanofiber membrane (PLA) having a fiber diameter of 375±30nm.

Meanwhile, a part of GS prepared at a spinning voltage of 60kv of the mixed spinning solution of the gelatin giant salamander with the concentration of 5% in example 1 was crosslinked. Specific: and (3) performing airtight crosslinking treatment by using glutaraldehyde steam with the volume concentration of 50% to enhance the mechanical property and reduce the solubility, and placing the crosslinked polylactic acid nanofiber membrane and gelatin giant salamander mixed nanofiber membrane in a vacuum drying oven at 40 ℃ for standing and drying for 3d to remove the influence of glutaraldehyde and acetic acid solvent. The crosslinked gelatin giant salamander mixed nanofiber membrane (GS (c)) is obtained.

According to the hot pressing method shown in fig. 1, with PLA as the bottom layer, sequentially superposing GS (c) and GS, and hot pressing PLA, GS (c) and GS at 100deg.C under 0.5MPa to obtain asymmetric nanofiber membrane (PGSGS) containing skin secretion of giant salamander (shown in fig. 2).

Example 3

Elastic tensile tests were performed on GS prepared in example 1 and PLA, GS (c), PGSGS prepared in example 2, and GS (c) GS composite films without PLA bottom layer.

In order to ensure the uniformity of the test results, the above samples were all subjected to a hot press treatment (100 ℃ C., 0.5 MPa) before the mechanical property test was performed. The test results are shown in FIG. 3.

As can be seen from fig. 3, the composition of PLA significantly improves the mechanical properties of GS or GS (c) and its composite film, which are originally poor in mechanical properties. This is critical for application in the actual wound repair process. And it can also be observed from fig. 3 that the mechanical properties of the composite film PGSGS are also significantly higher than those of PLA. The composite film better retains the mechanical properties of PLA.

Example 4

Adhesion performance test on PGSGS prepared in example 2

Pigskin is selected for adhesion testing, and PGSGS is adhered to the surface of pigskin for adhesion testing. The nanofiber membrane is adhered to the surface of pigskin tissue, the adhesion area is 10mm multiplied by 10mm, and the measurement is carried out by two modes of cutting and stretching respectively. As shown in FIG. 4, the adhesive strength at room temperature (26 ℃ C.) was 20KPa, and after standing at 4 ℃ C. For 20s, the adhesive strength was reduced to 4KPa, and the pig skin surface was easily removed. The characteristic enables the PGSGS auxiliary material to be easily removed from the wound by adopting a low-temperature treatment mode at 4 ℃ without causing secondary injury to the wound.

Example 5

The PLA and GS (c) were each subjected to contact angle testing, and the PLA and GS (c) were each adhered to a slide glass using a droplet shape analyzer device and placed on a stage. A drop of PBS solution was dropped on the sample with a syringe, and the solubility was observed. As a result, it was found that the PLA layer was hydrophobic, while the GS layer was hydrophilic, and the GS droplets dissolved in a very short time. After GS cross-linking (GS 5 means GS (c)) swelling behavior, PBS was absorbed but not dissolved.

In the use process, after the GS layer is dissolved, the swelling behavior of the GS (c) layer can enable the adhesion of the system to be maintained, because after the GS (c) layer is crosslinked, the GS (c) layer cannot be dissolved because the internal molecular chains are crosslinked, and at the moment, the adhesion strength is higher because the molecular chains are spread, so that the adhesion of the system is maintained. After low-temperature treatment, the gelatin chain can shrink, so that the molecular chains of the gelatin giant salamander are gathered, the adhesive strength of the gelatin giant salamander contacted with a tissue interface is reduced, and the gelatin giant salamander is easy to take down. As shown in fig. 5.

Example 6

In vitro biocompatibility experiments

Mouse 3T3 fibroblasts were cultured in DMEM (Hyclone, USA) supplemented with 5% fetal bovine serum. Gel (gelatin) (gelatin nanofiber, nanofiber obtained by electrostatic spinning of 20% gelatin spinning solution, solvent acetic acid)), GS (c) and PLA were weighed respectively with equal mass (5 mg). Sterilizing with ultraviolet rays for 30 minutes, soaking in DMEM for 3 days, and then co-culturing with cells in a 96-well plate for 24 hours, wherein since PLA and GS (c) are insoluble, they are spread on the cells, GS and Gel are dissolved, and their lysates are co-cultured with the cells. The CCK-8 method determines cell viability. The effect of various fibrous membrane materials on cells was observed (cell culture alone served as a blank). The statistical results are shown in fig. 6. As a result, the raw materials used in the invention have good biocompatibility, and cytotoxicity meets the conditions, and the invention belongs to the category of green wound dressings.

Mouse 3T3 fibroblasts were seeded on nanofiber membranes (PGSGS and GS) and blank plates (as controls) for 24 hours and then adherent cells were fixed with 4% paraformaldehyde. Cell morphology was observed by staining for cellular actin. The living/dead state of the cells was photographed using a laser scanning confocal microscope (OLYMPUS FV1000, japan), wherein black represents cell death, and after the dead staining was changed, if cell death occurred, red color appeared and cell survival was green. Grey figures are another method of imaging when cells survive. As can be seen from FIG. 7, GS and PGSGS have proliferation effects on cells.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The preparation method of the asymmetric nanofiber membrane containing giant salamander skin secretion is characterized by comprising the following steps of:

2. The method of claim 1, wherein the method for preparing the polylactic acid electrospinning solution of step (1) comprises the steps of: dissolving polylactic acid in V _{Chloroform (chloroform)} ：V _{Dimethylformamide} =6 to 8: and 2-4 to obtain the polylactic acid electrostatic spinning solution with the mass concentration of 5-15%.

3. The method of claim 2, wherein the gelatin giant salamander mixed dope of step (1) is: dissolving giant salamander skin secretion in a gelatin glacial acetic acid solution with the mass volume concentration of 15-25% to obtain a gelatin giant salamander mixed spinning solution with the volume concentration of 1-5%.

4. The method according to claim 3, wherein the electrospinning in the step (2) is performed by using a syringe of 12-18 mL, the voltage is 50-70 kv, the solution flow rate is 0.8-2 mL/h, the needle receiver is a nonwoven fabric, the distance from the needle tip of the syringe to the receiver is 15-19 cm, the indoor temperature is 22-28 ℃ relative humidity is 40-60%, and the spinning time is 20-30 min.

5. The method of claim 4, wherein the diameter of the polylactic acid nanofiber membrane and the gelatin giant salamander mixed nanofiber membrane is 345-405 nm.

6. The method of claim 1, wherein the crosslinking in step (3) is performed using glutaraldehyde solution: the volume concentration of the glutaraldehyde solution is 45-55%.

7. The method of claim 1, wherein the hot pressing in step (4) is at a temperature of 90 to 110 ℃; the pressure of the hot pressing is 0.4-0.6 MPa.

8. An asymmetric nanofiber membrane containing skin secretions of giant salamanders obtained according to the preparation method of any one of claims 1 to 7.

9. Use of an asymmetric nanofiber membrane containing skin secretions of giant salamander according to claim 8 as a wound dressing.