CN111714285A

CN111714285A - Layer-by-layer self-assembled film and preparation method thereof

Info

Publication number: CN111714285A
Application number: CN202010615765.1A
Authority: CN
Inventors: 叶梦琦; 何华成; 吴疆; 廖雨萍; 董炜飞
Original assignee: Wenzhou University
Current assignee: Wenzhou University
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2020-09-29
Anticipated expiration: 2040-06-29
Also published as: CN111714285B

Abstract

A layer-by-layer self-assembled film and a preparation method thereof relate to the field of dressing. The preparation method of the layer-by-layer self-assembled film comprises the following steps: and (3) sequentially soaking the base film in a weakly acidic polyvinylpyrrolidone aqueous solution and a weakly acidic tannic acid aqueous solution to form an assembly period, and drying after the assembly period of preset times is finished. The preparation method is simple to operate, the materials are easy to obtain, the utilization rate is high, and the load can be adjusted by changing the concentration of the tannic acid aqueous solution and the preset times of the assembly period; the layer-by-layer self-assembled film prepared by the method has good stability, can be stored and used for a long time, and can be used for slowly releasing tannic acid in a wound surface environment to promote wound healing.

Description

Layer-by-layer self-assembled film and preparation method thereof

Technical Field

The application relates to the field of dressings, in particular to a layer-by-layer self-assembled film and a preparation method thereof.

Background

In recent years, antimicrobial wound dressings have become increasingly popular and most commercial suppliers now offer silver-plated or silver nanoparticle impregnated dressings. Although the broad spectrum antimicrobial effect of this dressing has been demonstrated, there have also been studies reporting the cytotoxicity of silver to mammalian cells and the negative effects on the wound healing process. Therefore, we have sought an alternative antibacterial agent.

Tannic Acid (TA) is a weakly acidic polyphenol compound, which is widely present in nature. TA is known as an antioxidant, an antibacterial agent, an anti-inflammatory agent, etc., and it has good biocompatibility. But the TA solution is directly used on the wound surface, the TA solution is easy to run off, and the antibacterial effect is poor.

In view of this, the present application is hereby presented.

Disclosure of Invention

The present application provides a layer-by-layer self-assembled film and a method for manufacturing the same, so as to improve or alleviate the above technical problems.

The preparation method of the layer-by-layer self-assembled film according to the embodiment of the first aspect of the application comprises the following steps:

and (3) sequentially soaking the base film in a weakly acidic polyvinylpyrrolidone aqueous solution and a weakly acidic tannic acid aqueous solution to form an assembly period, and drying after the assembly period of preset times is finished.

According to the preparation method of the layer-by-layer self-assembled film, the basement membrane is sequentially soaked in the weakly acidic polyvinylpyrrolidone aqueous solution and the weakly acidic tannic acid aqueous solution, so that the alternating weakly acidic polyvinylpyrrolidone layer and the weakly acidic tannic acid layer are formed on the surface of the basement membrane, the tannic acid layer is stably loaded on the basement membrane through the polyvinylpyrrolidone layer, and meanwhile, the weakly acidic condition is more favorable for bacteriostasis.

Because polyvinylpyrrolidone and tannic acid are mainly connected by hydrogen bonds which are reversible in nature, in aqueous solution, the layer-by-layer self-assembled film can be gradually decomposed, so that tannic acid is released into a medium from the layer-by-layer self-assembled film, when the layer-by-layer self-assembled film is applied to a wound surface as a wound surface dressing (a base film is positioned on one side far away from the wound surface), the wound surface is a moist microenvironment, and the layer-by-layer self-assembled film can be gradually disintegrated to realize the slow release of tannic acid. And the polyvinylpyrrolidone has high biocompatibility and has no adverse effect on wound surfaces.

The preparation method of the layer-by-layer self-assembled film is simple to operate, the raw materials of polyvinylpyrrolidone and tannic acid are easy to obtain, the long-acting slow release of tannic acid can be realized by changing the preset times of the assembly period, the tannic acid load can be adjusted by changing the concentration of the tannic acid aqueous solution and the preset times, and the finally obtained layer-by-layer self-assembled film shows good antibacterial activity and good cell behavior and can promote better healing of wounds.

In addition, the preparation method of the layer-by-layer self-assembled film according to the embodiment of the application has the following additional technical characteristics:

in one possible embodiment, the sequential immersion in an aqueous solution of weakly acidic polyvinylpyrrolidone and an aqueous solution of weakly acidic tannic acid comprises: soaking in a weakly acidic polyvinylpyrrolidone aqueous solution for 5-10 min, washing, then soaking in a weakly acidic tannic acid aqueous solution for 5-10 min, and washing.

In the implementation process, redundant polyvinylpyrrolidone and tannic acid on the surface are respectively removed by washing after soaking every time, the stability of connection between the finally formed tannic acid layer and the polyvinylpyrrolidone layer is ensured, and the long-acting slow-release effect is realized.

The acidic fibroblast growth factor (aFGF) can effectively promote cell proliferation and tissue regeneration, further promote wound repair and improve wound healing quality, but the aFGF is used on the wound surface independently, and is easy to lose and inactivate. Meanwhile, the weak-acid polyvinylpyrrolidone aqueous solution is a high polymer solution with good biocompatibility, the applicant finds that aFGF can be well wrapped in PVP high polymer chain and can also keep the activity of the PVP high polymer chain to the maximum, and then PVP wrapping the aFGF forms a layer-by-layer self-assembly film with TA through hydrogen bond self-assembly.

Therefore, in a possible embodiment, the weakly acidic aqueous polyvinylpyrrolidone solution contains acidic fibroblast growth factor, and the concentration of the acidic fibroblast growth factor in the weakly acidic aqueous polyvinylpyrrolidone solution is 0.5-100 ug/ml.

In the implementation process, the sustained release of the TA and the aFGF is realized through the arrangement, so that the TA and the aFGF can achieve the aims of sterilization and cell proliferation at different stages of wound healing, and further, the layer-by-layer self-assembled film can show good antibacterial activity and good cell behavior, angiogenesis and tissue regeneration are promoted, and the wound healing is promoted better.

In one possible embodiment, each assembly cycle is carried out at a temperature of 2 to 8 ℃.

In the implementation process, the activity of TA and aFGF is kept in a better range through proper temperature selection, so that the layer-by-layer self-assembled film has good antibacterial activity and good cell behavior, and the wound can be promoted to heal better.

In one possible embodiment, the pH value of the weakly acidic polyvinylpyrrolidone aqueous solution is 5-6.

In the implementation process, because the most suitable pH value of the aFGF is 5-6, the aFGF can keep better activity by the pH value, the wound environment can be improved, and the bacteriostatic effect of the layer-by-layer self-assembled film is improved.

Optionally, the concentration of polyvinylpyrrolidone in the weakly acidic polyvinylpyrrolidone aqueous solution is 0.5-10 mg/mL.

In one possible embodiment, the pH value of the weakly acidic aqueous tannic acid solution is 5-6.

Optionally, the concentration of tannic acid in the weakly acidic aqueous tannic acid solution is 0.5-10 mg/mL.

Because the surfaces of the base film which completes the assembly period of the preset times are the weakly acidic polyvinylpyrrolidone layer and the tannic acid layer, in order to avoid influencing the distribution uniformity of the polyvinylpyrrolidone layer and the tannic acid layer and ensure better activity of the tannic acid, in one possible embodiment, the base film is hung and dried at 2-8 ℃ after the assembly period of the preset times is completed.

In the implementation process, the drying effect is good, the structural integrity of the layer-by-layer self-assembled film is guaranteed, and the effect of promoting wound healing is good.

Optionally, the base membrane is a hydrophobic membrane.

In the implementation process, the wound is prevented from being infected due to the fact that external water is adsorbed to the wound, the TA slow release effect is guaranteed, and after the TA slow release film is used, the basement membrane is easy to tear and does not adhere to the wound.

The layer-by-layer self-assembled film according to the embodiment of the second aspect of the present application is prepared by the layer-by-layer self-assembled film preparation method according to the embodiment of the first aspect of the present application.

The layer-by-layer self-assembly film is simple in structure, low in cost, good in antibacterial activity and cell behavior and capable of effectively promoting wound healing.

The layer-by-layer self-assembled film according to the third aspect of the present application includes a base film and at least one functional layer formed on the base film.

Each functional layer comprises a weakly acidic polyvinylpyrrolidone layer and a weakly acidic tannic acid layer which is connected with one side of the polyvinylpyrrolidone layer away from the base film through hydrogen bonds.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is a graph showing the antibacterial effect of various samples in test example 1;

FIG. 2 is a statistical chart of wound surface areas of the mouse wound surface in different periods of time in experimental example 2;

FIG. 3 is a comparative graph of agar-coated plates of exudate from infected wounds of experimental example 3 mice.

Detailed Description

Reference will now be made in detail to the embodiments of the present application, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application.

The application provides a layer-by-layer self-assembled film, which is prepared by the following preparation method:

s1, sequentially soaking a basement membrane in a weakly acidic polyvinylpyrrolidone aqueous solution and a weakly acidic tannic acid aqueous solution to form an assembly period.

Alternatively, the soaking in the weakly acidic aqueous solution of polyvinylpyrrolidone and the weakly acidic aqueous solution of tannic acid in this order comprises: soaking in a weakly acidic polyvinylpyrrolidone aqueous solution for 5-10 min, washing, then soaking in a weakly acidic tannic acid aqueous solution for 5-10 min, and washing.

Specifically, washing is carried out for 1-2 s by adopting a washing liquid. Wherein, the flushing liquid is water, the flushing effect is good and no new substance is introduced.

The basement membrane is a hydrophobic membrane, so that the wound is prevented from being infected, and the basement membrane is easy to tear and does not adhere to the wound after being used.

Specifically, the base film includes, but is not limited to, a polydimethylsiloxane film, and may also be made of one of polylactic acid and polycaprolactone mixed with chitosan, which is not limited herein.

Optionally, the base film is a polydimethylsiloxane film.

Optionally, the pH of the weakly acidic aqueous solution of polyvinylpyrrolidone is 5-6, for example, the pH of the weakly acidic aqueous solution of polyvinylpyrrolidone is 5, 5.5 or 6.

Alternatively, the concentration of polyvinylpyrrolidone in the weakly acidic aqueous polyvinylpyrrolidone solution is 0.5 to 10mg/mL, for example, the concentration of polyvinylpyrrolidone in the aqueous polyvinylpyrrolidone solution is 0.5mg/mL, 1mg/mL, 3mg/mL, 5mg/mL, 7mg/mL, 10mg/mL, or the like.

Optionally, the pH of the weakly acidic aqueous tannic acid is 5 to 6, for example, the pH of the weakly acidic aqueous tannic acid is 5, 5.5, or 6.

Optionally, the concentration of tannic acid in the weakly acidic aqueous tannic acid solution is 0.5-10 mg/mL. For example, the concentration of tannic acid in the weakly acidic aqueous tannic acid solution is 0.5mg/mL, 1mg/mL, 3mg/mL, 5mg/mL, 7mg/mL, 10mg/mL, or the like.

It should be noted that the concentrations of the weakly acidic polyvinylpyrrolidone aqueous solution and the weakly acidic tannic acid aqueous solution used in each assembly cycle may be the same or different, and may be changed, such as increased or decreased, with the increase of the assembly cycle, which is not limited herein.

Optionally, the weakly acidic polyvinylpyrrolidone aqueous solution contains acidic fibroblast growth factor, and the concentration of the acidic fibroblast growth factor in the weakly acidic polyvinylpyrrolidone aqueous solution is 0.5-100 ug/ml, such as 0.1ug/ml, 1ug/ml, 10ug/ml, 20ug/ml, 30ug/ml, 50ug/ml, 55ug/ml, 70ug/ml, 80ug/ml, 100ug/ml, etc.

Optionally, each assembly cycle is performed at a temperature of 2-8 ℃, e.g., 2 ℃, 4 ℃, 5 ℃, 7 ℃, or 8 ℃.

And S2, drying after the assembly period of preset times is finished.

The preset times are one or more times, and the skilled person can select the times according to actual requirements.

Alternatively, the preset number is multiple, specifically, for example, 2 times, 10 times, 30 times, 50 times, etc., and one skilled in the art can limit the preset number according to actual needs.

Wherein, hang the drying at 2 ~ 8 ℃ after accomplishing the equipment cycle of predetermineeing the number of times, for example hang the drying at 2 ℃, 4 ℃, 5 ℃, 7 ℃, or 8 ℃, wherein hang and include: clamping at least one edge of the layer-by-layer self-assembled film to make it suspended.

The preparation method selects polyvinylpyrrolidone (PVP) and Tannic Acid (TA) to form the layer-by-layer self-assembled film through hydrogen bond self-assembly, is not limited by the size and the shape of the basement membrane, is easy to control the thickness of the layer-by-layer self-assembled film, the TA and the aFGF load capacity, and has good drug slow-release effect.

The application provides a layer-by-layer self-assembled film, which comprises a base film and at least one functional layer formed on the base film.

Each functional layer comprises a weakly acidic polyvinylpyrrolidone layer and a weakly acidic tannic acid layer which is connected with one side of the polyvinylpyrrolidone layer away from the base film through hydrogen bonds. Wherein, at least one layer is, for example, two, five or thirty layers, etc., which is not limited herein.

The layer-by-layer self-assembled film and the preparation method thereof are further described in detail with reference to the following examples.

Example 1

A layer-by-layer self-assembly film for slowly releasing TA is prepared by the following steps:

the method comprises the following steps: obtaining a Polydimethylsiloxane (PDMS) film, washing the PDMS film with deionized water, and drying.

Step two: respectively preparing a 5mg/mL PVP (MW 8000) aqueous solution and a 5mg/mL TA aqueous solution, and respectively adjusting the pH values of the PVP aqueous solution and the TA aqueous solution to 5-6 by using 0.1M hydrochloric acid and 0.1M NaOH solution to obtain a weakly acidic PVP aqueous solution and a weakly acidic TA aqueous solution.

Step three: and (3) soaking the dried PDMS film in a weak acid PVP (polyvinyl pyrrolidone) aqueous solution for 5min, cleaning for 1-2 s in deionized water, soaking in a weak acid TA (TA) solution for 5min, taking out and cleaning for 1-2 s, and completing an assembly period to form a functional layer on the PDMS film, wherein the functional layer comprises a weak acid polyvinylpyrrolidone layer and a weak acid tannic acid layer connected with one side, far away from the PDMS film, of the polyvinylpyrrolidone layer through a hydrogen bond.

Step four: and repeating the third step until 10 functional layers are obtained, and obtaining the film body. Wherein, the second to the fourth steps are all carried out at 4 ℃.

Step five: and (4) hanging the film body obtained in the fourth step at the temperature of 4 ℃ until the water is completely evaporated to obtain the layer-by-layer self-assembled film.

Example 2

Step four: and repeating the third step until 30 functional layers are obtained, and obtaining the film body. Wherein, the second to the fourth steps are all carried out at 4 ℃.

Example 3

Step four: and repeating the third step until 50 functional layers are obtained, and obtaining the film body. Wherein, the second to the fourth steps are all carried out at 4 ℃.

Example 4

A layer-by-layer self-assembled film for slowly releasing TA and aFGF is prepared by the following steps:

Step two: a 5mg/mL PVP (MW 8000) aqueous solution and a 5mg/mL TA aqueous solution were prepared, respectively, the pH of the PVP aqueous solution and the TA aqueous solution was adjusted to 5 to 6 with 0.1M hydrochloric acid and 0.1M NaOH solution, respectively, and aFGF was added to the PVP aqueous solution adjusted to 5 to 6, to obtain a weakly acidic PVP aqueous solution and a weakly acidic TA aqueous solution containing 100ug/mL aFGF.

Step three: and (3) soaking the dried PDMS film in a weak acid PVP (polyvinyl pyrrolidone) aqueous solution for 5min, cleaning for 1-2 s in deionized water, soaking in a weak acid TA (polyethylene glycol) solution for 5min, taking out and cleaning for 1-2 s, and completing an assembly period to form a functional layer on the PDMS film, wherein the functional layer comprises a weak acid polyvinylpyrrolidone layer containing aFGF (alpha-FGF) and a weak acid tannic acid layer connected with one side, far away from the PDMS film, of the polyvinylpyrrolidone layer through a hydrogen bond.

Test example 1

Control McLeod 0.5 tube (1.5 × 10)⁸CFU/mL), LB broth with staphylococcus aureus was diluted to similar turbidity with normal saline, giving bacteria of order 10⁸A suspension of (a). Sucking 100 μ L of the suspension, and diluting to 1mL to obtain bacteria with 10 orders of magnitude⁷A suspension of (a). 200. mu.L of the above bacteria of the order of 10 were added to each 96-well plate⁷For the bacterial suspension, 75uL of the leachate (the single PDMS films with uniform size, and the self-assembled films layer by layer prepared in 1, 2, and 3 in the examples) of the corresponding PDMS film and the self-assembled films layer by layer with different layers were added to each 96-well plate, and the plates were immersed in 1mL of PBS buffer solution with pH of 7.4 for 4 days, and each 96-well plate was placed in a 37 ℃ incubator for 5 hours. After taking out, the plate was shaken on a shaker for 20 seconds, 20. mu.L of the bacterial suspension in each well plate was diluted in 980. mu.L of PBS, and 70. mu.L of the diluted bacterial suspension was added dropwise to a nutrient agar plate and spread uniformly. All agar plates were placed in a 37 ℃ incubator for 12 hours.

The results are shown in FIG. 1. Wherein, in figure 1, white dots are staphylococcus aureus. As can be seen from FIG. 1, the layer-by-layer self-assembled film in example 3 has a significant antibacterial effect.

Test example 2

Four groups of wound surfaces with the same size and quantity are set for a mouse to serve as a blank control group, a control group 1, a test group 1 and a test group 2, the wound surfaces of the groups are infected by staphylococcus aureus with the same quantity and then are administrated, the blank control group is not treated, the control group 1 is coated with weak acid TA aqueous solution containing aFGF, the test group 1 covers the layer-by-layer self-assembled film obtained in the embodiment 3, the test group 2 covers the layer-by-layer self-assembled film prepared in the embodiment 4, the test group 1 and the test group 2 have the same layer-by-layer self-assembled film size, and the weak acid TA aqueous solution containing aFGF is prepared by the following method: adding aFGF into a 5mg/mL TA aqueous solution with the pH value of 5-6 to obtain a weakly acidic TA aqueous solution containing 100ug/mL aFGF. The amount of aFGF and TA used in control 1 was substantially the same as the amount of aFGF and TA released from the layered self-assembled film in test 2.

The wounds of mice from control 1, test 2 were evaluated as controls to blank control 7, 10, 14, 17 days after wound administration, and the statistics are shown in fig. 2.

According to fig. 2, the wound healing speed of the layer-by-layer self-assembled film prepared in example 4 is relatively fast.

Test example 3

Wound healing was described on day 17 after administration of the wound in mice of control group 1, test group 1, and test group 2 in test example 2, and the results are shown in table 1.

TABLE 1 wound healing results

Meanwhile, when the wound surfaces of the mice of the blank control group, the control group 1, the test group 1 and the test group 2 are administrated for 2, 7 and 10 days, the secretion on the wound surface of each group of mice is wiped by a sterile cotton swab, diluted by normal saline and coated on an agar plate, and the result is shown in fig. 3.

As can be seen from table 1 and fig. 3, the bacterial load on the wound surface of test group 1 and test group 2 decreased all the time, while the bacterial load on the wound surface of control group 1 decreased greatly at the beginning, but increased again at the later stage.

In the actual operation process, the applicant tries to assemble the membrane by a one-step method, that is, firstly, a weakly acidic polyvinylpyrrolidone aqueous solution and a weakly acidic tannic acid aqueous solution are mixed, or firstly, the polyvinylpyrrolidone and the tannic acid are mixed and then added with water, then the pH is adjusted, and then the PDMS is soaked in the mixed solution.

In conclusion, the preparation method provided by the application is simple to operate, the materials are easy to obtain, the utilization rate is high, and the load capacity can be adjusted by changing the concentration of the TA solution and the preset times of the assembly period; the film prepared by the method has good stability and can be stored and used for a long time.

The TA can be slowly released in the wound surface environment by the layer-by-layer self-assembled film, so that the wound healing is promoted; and TA has high biocompatibility and low cytotoxicity, and when the wound healing wound contains aFGF, TA and aFGF are slowly released through the layer-by-layer self-assembled film, so that the aims of sterilizing and promoting cell proliferation are fulfilled at different stages of wound healing.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a layer-by-layer self-assembled film is characterized by comprising the following steps:

and sequentially soaking the base film in a weakly acidic polyvinylpyrrolidone aqueous solution and a weakly acidic tannic acid aqueous solution to form an assembly period, and drying after the assembly period of preset times is finished.

2. The method for preparing a layer-by-layer self-assembled film according to claim 1, wherein the sequentially immersing in the weakly acidic aqueous solution of polyvinylpyrrolidone and the weakly acidic aqueous solution of tannic acid comprises: soaking in the weakly acidic polyvinylpyrrolidone aqueous solution for 5-10 min, washing, and then soaking in the weakly acidic tannic acid aqueous solution for 5-10 min, and washing.

3. The method for preparing a layer-by-layer self-assembled film according to claim 1, wherein the weakly acidic polyvinylpyrrolidone aqueous solution contains acidic fibroblast growth factor, and the concentration of the acidic fibroblast growth factor in the weakly acidic polyvinylpyrrolidone aqueous solution is 0.5-100 ug/ml.

4. The method for preparing a layer-by-layer self-assembled film according to any one of claims 1 to 3, wherein each assembling cycle is carried out at a temperature of 2 to 8 ℃.

5. The method for preparing a layer-by-layer self-assembled film according to any one of claims 1 to 3, wherein the pH value of the weakly acidic polyvinylpyrrolidone aqueous solution is 5 to 6;

6. The method for preparing a layer-by-layer self-assembled film according to any one of claims 1 to 3, wherein the pH value of the weakly acidic aqueous solution of tannic acid is 5 to 6;

optionally, the concentration of tannic acid in the weakly acidic tannic acid aqueous solution is 0.5-10 mg/mL.

7. The method of producing a layer-by-layer self-assembled film according to any one of claims 1 to 3, wherein the suspension drying is performed at 2 to 8 ℃ after the completion of the predetermined number of assembly cycles.

8. The method for preparing a layer-by-layer self-assembled film according to any one of claims 1 to 3, wherein the base film is a hydrophobic film.

9. A layer-by-layer self-assembled film, which is produced by the method for producing a layer-by-layer self-assembled film according to any one of claims 1 to 8.

10. The layer-by-layer self-assembled film is characterized by comprising a base film and at least one functional layer formed on the base film, wherein each functional layer comprises a weakly acidic polyvinylpyrrolidone layer and a weakly acidic tannic acid layer which is connected with one side, far away from the base film, of the polyvinylpyrrolidone layer through a hydrogen bond.