CN115887731A - Preparation method of beta lactoglobulin fiber-polyvinyl alcohol aerogel and application of beta lactoglobulin fiber-polyvinyl alcohol aerogel in preparation of skin dressing - Google Patents
Preparation method of beta lactoglobulin fiber-polyvinyl alcohol aerogel and application of beta lactoglobulin fiber-polyvinyl alcohol aerogel in preparation of skin dressing Download PDFInfo
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- CN115887731A CN115887731A CN202211544287.5A CN202211544287A CN115887731A CN 115887731 A CN115887731 A CN 115887731A CN 202211544287 A CN202211544287 A CN 202211544287A CN 115887731 A CN115887731 A CN 115887731A
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Abstract
The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of beta lactoglobulin fiber-polyvinyl alcohol aerogel and application of the beta lactoglobulin fiber-polyvinyl alcohol aerogel in preparation of skin dressing. The beta lactoglobulin fiber-polyvinyl alcohol aerogel prepared by the invention has the advantages of good biocompatibility and strong hydrophilicity. The aerogel can rapidly absorb exudate on skin wound surface, is converted into hydrogel to seal the wound surface and keep the wound surface moist, has good air permeability and promote the adhesion property of fiber cells, and can promote the healing of the wound surface. The beta lactoglobulin fiber-polyvinyl alcohol aerogel prepared by the invention has the advantages of spongy and hydrogel wound dressing, and is simple in preparation process, low in cost and easy to store and transport.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a preparation method of beta lactoglobulin fiber-polyvinyl alcohol aerogel and application of the beta lactoglobulin fiber-polyvinyl alcohol aerogel in preparation of skin dressing.
Background
The skin is an important organ of the human body, and plays an important role in maintaining homeostasis of the body and protecting the body from invasion of harmful substances and microorganisms. Because of direct contact with the outside, the skin is not easy to be injured to form a wound surface. The skin has certain repair capacity, and the healing of the wound surface mainly comprises four stages of hemostasis, inflammation, hyperplasia and tissue remodeling [1] . However, the whole healing process usually does not proceed perfectly orderly, especially in the presence of infection, ischemia, excessive inflammation, extensive burns, vascular diseases, diabetes and other factors, which easily make the wound surface difficult to heal [2] . Appropriate dressings are therefore required to achieve wound closure and accelerated healing.
From the clinical application point of view, the ideal dressing should have multiple properties [3-5] The method comprises the following steps: 1. the biocompatibility is good, and toxicity or inflammation is not caused; 2. has good absorption effect on blood and tissue fluid exuded from the wound surface, and can maintain the moist environment of the wound surface; 3. the material has enough mechanical strength, and avoids the material damage caused by the invasion of external bacteria; 4. can promote cell adhesion, proliferation and differentiation. Therefore, the traditional dressing such as gauze and bandage mainly made of cotton yarn is difficult to meet the clinical requirement. There are currently some sponge-like or hydrogel-like dressings on the market, however these dressings still have some limitations. The sponge dressing has the advantage of quickly absorbing the exudate, but the exudate is easy to leak back to the tissues of the wound surface, so that the secondary pollution of the wound surface is caused; the hydrogel dressing has strong exudate storage capacity, but the hydrogel dressing absorbs exudate slowly and has poor air permeability. Secondly, from the viewpoint of material components and preparation methods, the main components of the existing sponge or hydrogel are cellulose, alginate, chitosan, gelatin and the like, and in order to improve the bonding strength among the components, most dressings are formed by chemical crosslinking [6-8] . The addition of chemical cross-linking agents increases the complexity of the manufacturing process and also risks biological safety. These can adversely affect the cost, storage and transport of the dressing. Therefore, developThe novel dressing which has the advantages of good biocompatibility, simple process and non-chemical crosslinking and has the advantages of sponge and hydrogel simultaneously becomes a technical problem to be solved urgently.
Polyvinyl alcohol is a semi-crystalline high molecular polymer, and has the characteristics of high mechanical strength and good biocompatibility. The polyvinyl alcohol can obtain physically cross-linked spongy or hydrogel materials by a circulating freeze-thaw process method, and a chemical cross-linking agent is not required to be added in the process. Therefore, the polyvinyl alcohol has important application value in the field of biological materials [9-12] . However, polyvinyl alcohol sponges or hydrogels prepared by freeze-thaw methods have low porosity and poor hydrophilicity, resulting in poor absorption and retention of exudates [4] . In addition, the polyvinyl alcohol has weak cell adhesion capacity, and research shows that more than 45 freeze-thaw cycles are required to obtain certain cell adhesion capacity [10] Excessive freeze-thaw times increase the complexity and cost of the manufacturing process. Therefore, the pure polyvinyl alcohol is difficult to meet the requirements of clinical use and large-scale preparation of the wound dressing.
The beta lactoglobulin is one of the main proteins in the fresh milk of cattle and sheep, and has the advantages of wide source, low extraction cost, good biological safety and the like [13] . However, beta lactoglobulin is a globular protein having a diameter of only about 7nm under natural conditions, and it is difficult to satisfy the conditions for forming a gel. Although studies have shown that heating under acidic conditions of pH =2.0 promotes the polymerization of beta lactoglobulin to form beta lactoglobulin fibers in a fibrous state [14] However, the beta lactoglobulin fiber obtained by the method is difficult to apply to the field of biological materials, particularly to the preparation of skin dressings. The main reasons are three points:
first, beta lactoglobulin fiber is prepared and stored in a solution (pH = 2.0) having strong acidity, which causes discomfort to the skin, and the process of adjusting the pH of the solution from acidity to neutrality destroys the stability of beta lactoglobulin fiber, easily causing the fiber to be shortened or decomposed [15] 。
Secondly, the existing methods for preparing beta lactoglobulin fibers as a gel mainly rely on chemical cross-linking,chemical agents such as ethanol, ca/Na salt, urease, surfactant and the like are required to be added to promote winding and crosslinking among beta lactoglobulin fibers [16] . The addition of chemical cross-linking agents increases the complexity of the preparation process and also has the risk of biological safety, and these forming methods are not suitable for the preparation of wound dressings.
Thirdly, although the polyvinyl alcohol can realize physical crosslinking under circulating freeze-thaw, the freeze-thaw forming of the polyvinyl alcohol is premised on the need of improving the crystallinity of the system [10] However, the beta lactoglobulin fiber will destroy the crystallinity of the system, and further affect the forming process. And the stability of the beta lactoglobulin fiber is easily influenced by the temperature and pH value of the external environment [17] . Therefore, the influence of the preparation process flow and parameters on the physicochemical properties of the beta lactoglobulin fiber-polyvinyl alcohol aerogel and the influence of the property changes on the final wound repair effect are unknown.
In the invention, the beta lactoglobulin fiber and the polyvinyl alcohol are hoped to be used for preparing the aerogel which has good biocompatibility, simple process, good water absorption and physical crosslinking, and the aerogel can be converted into hydrogel after absorbing exudates and is beneficial to cell adhesion so as to meet the design requirement of the wound dressing with the advantages of sponge and hydrogel.
Reference:
[1]Zhu J,Li F,Wang X,et al.Hyaluronic Acid and Polyethylene Glycol Hybrid Hydrogel Encapsulating Nanogel with Hemostasis and Sustainable Antibacterial Property for Wound Healing.ACS Appl Mater Interfaces.2018；10(16):13304-13316.
[2]Liang Y,He J,Guo B.Functional Hydrogels as Wound Dressing to Enhance Wound Healing.ACS Nano.2021；15(8):12687-12722.
[3]Zou F,Wang Y,Tang T,et al.Synergistic strategy constructed hydrogel-aerogel biphase gel(HAB-gel)with self-negative-pressure exudate absorption,M2 macrophage-polarized and antibacterial for chronic wound treatment.Chem Eng J.2023；451(P4):138952.
[4]Varshney N,Sahi AK,Poddar S,et al.Freeze–Thaw-Induced Physically Cross-linkedSuperabsorbent Polyvinyl Alcohol/Soy Protein Isolate Hydrogels for Skin Wound Dressing:In Vitro and In Vivo Characterization.ACS Appl Mater Interfaces.2022；14(12):14033-14048.
[5]Nuutila K,Eriksson E.Moist Wound Healing with Commonly Available Dressings.Adv Wound Care.2021；10(12):685-698.
[6]Hu Y,Li N,Yue P,et al.Highly antibacterial hydrogels prepared from amino cellulose,dialdehyde xylan,and Ag nanoparticles by a green reduction method.Cellulose.2022；29(2):1055-1067.
[7]Yin M,Wan S,Ren X,et al.Development of Inherently Antibacterial,Biodegradable,and Biologically Active Chitosan/Pseudo-Protein Hybrid Hydrogels as Biofunctional Wound Dressings.ACS Appl Mater Interfaces.2021；13(12):14688-14699.
[8]Nazarnezhada S,Abbaszadeh-Goudarzi G,Samadian H,et al.Alginate hydrogel containing hydrogen sulfide as the functional wound dressing material:In vitro and in vivo study.Int J Biol Macromol.2020；164:3323-3331.
[9]Dou X,Li P,
H.Three-Dimensional Microstructured Poly(vinyl alcohol)Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids.Biomacromolecules.2018；19(1):158-166.
[10]Gupta S,Webster TJ,Sinha A.Evolution of PVA gels prepared without crosslinking agents as a cell adhesive surface.J Mater Sci Mater Med.2011；22(7):1763-1772.
[11]Wang LY,Wang MJ.Removal of Heavy Metal Ions by Poly(vinyl alcohol)and Carboxymethyl Cellulose Composite Hydrogels Prepared by a Freeze-Thaw Method.ACS Sustain Chem Eng.2016；4(5):2830-2837.
[12]Kenawy ER,Kamoun EA,Mohy Eldin MS,et al.Physically crosslinked poly(viny lalcohol)-hydroxyethyl starch blend hydrogel membranes:Synthesis and characterization for biomedical applications.Arab J Chem.2014；7(3):372-380.
[13]Jain S,Dongave SM,Date T,et al.Succinylatedβ-Lactoglobuline-Functionalized Multiwalled Carbon Nanotubes with Improved Colloidal Stability and Biocompatibility.ACS Biomater Sci Eng.2019；5(7):3361-3372.
[14]
G,Roder L,Fernández-ronco MP,et al.Amyloid Templated Organic–Inorganic Hybrid Aerogels.Adv Funct Mater.2017；1703609:1-11.
[15]Jay G,Osvaldo C,Owen GJ.Electrostatic Stabilization ofβ-lactoglobulin Fibrils at Increased pH with Cationic Polymers.Biomacromolecules,2014；15(8):3119-3127.
[16]Hoppenreijs LJG,Fitzner L,Ruhmlieb T,et al.Engineering amyloid and amyloid-like morphologies ofβ-lactoglobulin.Food Hydrocoll.2022；124(PA):107301.
[17]Heyn TR,Garamus VM,Neumann HR,et al.Influence of the polydispersity of pH 2and pH 3.5beta-lactoglobulin amyloid fibril solutions on analytical methods.Eur Polym J.2019；120(May):109211.
disclosure of Invention
In view of the above technical problems, the present invention provides a method for preparing a beta lactoglobulin fiber-polyvinyl alcohol aerogel. The aerogel realizes physical crosslinking through a circulating freeze-thawing method, does not need to add a chemical crosslinking agent, can be quickly converted into a hydrogel state after absorbing exudates, has good air permeability and moisture retention, can create a microenvironment beneficial to proliferation and adhesion of fibroblasts, and is suitable for promoting healing of wound surfaces.
The invention also aims to provide application of the aerogel prepared by the method.
The preparation method of the beta lactoglobulin fiber-polyvinyl alcohol aerogel comprises the following steps:
step 1: preparing a beta lactoglobulin solution with the concentration of 3-4 wt% by using ultrapure water, adjusting the pH to 1.5-2.5 by using hydrochloric acid, and stirring and heating for 3-7 hours under the water bath condition of 80-90 ℃ at the rotating speed of 200-500 rpm; cooling the prepared beta lactoglobulin fiber solution to 0-4 ℃, and then adjusting the pH value to 7.0-7.5 by using 8-12M NaOH;
step 2: adding polyvinyl alcohol solid particles into ultrapure water at 85-100 ℃, heating until the polyvinyl alcohol solid particles are completely dissolved to obtain a polyvinyl alcohol solution with the concentration of 10-15 wt%, and cooling to 0-25 ℃ for storage; the alcoholysis degree of the polyvinyl alcohol 1799 is 98-99%;
and step 3: adding the beta lactoglobulin fiber solution obtained in the step 1 and the polyvinyl alcohol solution obtained in the step 2 into ultrapure water at 15-25 ℃ according to a proportion, so that the sum of the mass fractions of the beta lactoglobulin fiber and the polyvinyl alcohol in the solution is 3.5-8.5 wt%, wherein the mass fraction of the beta lactoglobulin fiber is 1-3.5 wt%, and the mass fraction of the polyvinyl alcohol is 2.5-5 wt%; after being uniformly mixed, the mixture is subjected to cyclic freeze thawing for 3 to 4 times, each cycle is carried out for 21 to 24 hours at a temperature of between 20 ℃ below zero and 80 ℃ below zero, and the cycle is carried out for 3 to 6 hours at a temperature of between 15 and 25 ℃; and (3) performing vacuum freeze drying on the sample obtained by freeze thawing to obtain the beta lactoglobulin fiber-polyvinyl alcohol aerogel, wherein the polyvinyl alcohol is polyvinyl alcohol 1799.
Among the above-mentioned methods, preferred are: the sum of the mass fractions of the beta lactoglobulin fibers and the polyvinyl alcohol in the solution is 6wt%;
among the above-mentioned methods, preferred are: the mass fraction of the beta lactoglobulin fiber is 2-3.5 wt%;
among the above-mentioned methods, preferred are: the beta lactoglobulin is derived from cow milk, and the dry weight purity of the beta lactoglobulin is more than or equal to 93wt%;
the temperature of the vacuum freeze drying in the step 3 is-50 to-80 ℃, and the time is 24 to 48 hours;
the concentrations of the beta lactoglobulin fiber and the polyvinyl alcohol in the solution are both 3wt%;
the protection scope of the present invention also includes the application of the beta lactoglobulin fiber-polyvinyl alcohol aerogel prepared by the method in preparing skin dressing, and the application includes but is not limited to: the aerogel is directly prepared into a skin wound dressing, or is combined with other effective substances to prepare the skin wound dressing; or can be prepared into a carrier of a skin-care facial mask and the like.
Compared with the prior art, the invention has the following advantages:
(1) The beta lactoglobulin fiber-polyvinyl alcohol aerogel realizes physical crosslinking through a circulating freeze-thaw method, does not need to add a chemical crosslinking agent, and has good biocompatibility. The method is simple to operate, mild in reaction condition and easy for large-scale preparation.
(2) The beta lactoglobulin fiber-polyvinyl alcohol aerogel has the advantages of high porosity, high water absorption rate and high hydrophilicity, can quickly absorb exudate of a wound surface, and increases the adhesion area of cells.
(3) The beta lactoglobulin fiber-polyvinyl alcohol aerogel can be converted into a hydrogel state after absorbing exudates, has good air permeability and moisture retention, can maintain a proper humid environment, and can prevent the formation of dry scabs.
(4) The beta lactoglobulin is rich in highly repetitive beta sheet structures, and can promote cell adhesion. The beta lactoglobulin fiber-polyvinyl alcohol aerogel can promote the adhesion of fibroblasts only by 3-4 times of circulating freeze thawing, and the cell adhesion is very important for the healing of wound surfaces. Pure-phase polyvinyl alcohol requires more than 45 freeze-thaw cycles to obtain a certain cell adhesion promoting capacity.
(5) The beta lactoglobulin is derived from milk, and has the advantages of low cost, easy extraction and good biological safety. The beta lactoglobulin fiber formed by the method has good biocompatibility, the preparation method is simple, and the shape of the product is easy to control.
(6) The applicant finds that the pH value of the acidic beta lactoglobulin fiber is adjusted step by step in the earlier research process, so that the obtained sample has poor effect, and the beta lactoglobulin fiber is easy to decompose. The invention quickly adjusts the beta lactoglobulin fiber through the high-concentration sodium hydroxide solution, overcomes the problem and enables the beta lactoglobulin fiber to be formed into gel with polyvinyl alcohol under the condition of physiological pH.
Drawings
FIG. 1 is a photograph of different types and concentrations of polyvinyl alcohol solutions before and after cyclic freeze-thawing;
in the figure, a is a photograph before freeze thawing, b is a photograph after freeze thawing, and each photograph is respectively 1750 (2.5 wt%), 1750 (10 wt%), 1788 (2.5 wt%), 1788 (10 wt%), 1799 (2.5 wt%), 1750 (10 wt%) from left to right.
Fig. 2 is an Atomic Force Microscope (AFM) picture of beta lactoglobulin monomers and beta lactoglobulin fibers at pH = 7.4;
wherein a is a picture of a beta lactoglobulin monomer, and b is a picture of a beta lactoglobulin fiber after being placed for one week.
FIG. 3 shows the results of mixing beta-lactoglobulin fibers with polyvinyl alcohol at different ratios and subjecting the resulting mixture to cyclic freeze-thawing, wherein beta-lactoglobulin fibers are abbreviated as BLGF and polyvinyl alcohol is abbreviated as PVA;
wherein a is a statistical chart of the content of beta lactoglobulin fibers and polyvinyl alcohol in each group of samples and whether gel is formed, and b is a photograph after freeze thawing.
FIG. 4 is a photograph of a polyvinyl alcohol aerogel and a beta lactoglobulin fiber-polyvinyl alcohol aerogel;
PVA represents polyvinyl alcohol aerogel, and BLGF-1/PVA-5, BLGF-2/PVA-4 and BLGF-3/PVA-3 represent beta lactoglobulin fiber-polyvinyl alcohol aerogel with different proportions.
Fig. 5 is a Scanning Electron Microscope (SEM) Image of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel and the results of pore size distribution using Image J software statistics.
Fig. 6 is a statistical result of porosities of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel.
FIG. 7 shows the results of the contact angle characterization of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel;
contact angle photographic image where a is 10 s; b is the contact angle statistic.
Fig. 8 is a graph showing the statistical results of the water absorption rate changes of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel.
Fig. 9 is a statistical result of water vapor transmission rates of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel.
Fig. 10 is a graph of the leachate cytotoxicity evaluation of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel. Control represents a complete medium group, PVA represents a polyvinyl alcohol aerogel group, BLGF-1/PVA-5, BLGF-2/PVA-4 and BLGF-3/PVA-3 represent beta lactoglobulin fiber-polyvinyl alcohol aerogel groups with different proportions, and NS represents no statistical significance compared with the Control group.
FIG. 11 shows the nuclear and cytoskeletal staining results of cell adhesion experiments for polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel;
in the figure, a is the result after 24 hours of culture, and b is the result after 72 hours of culture. PVA represents polyvinyl alcohol aerogel group, and BLGF-1/PVA-5, BLGF-2/PVA-4 and BLGF-3/PVA-3 represent beta lactoglobulin fiber-polyvinyl alcohol aerogel group with different proportions.
FIG. 12 shows the results of animal experiments with beta lactoglobulin fiber-polyvinyl alcohol aerogel (BLGF-3/PVA-3) dressings.
The specific implementation mode is as follows:
in order to illustrate the embodiments of the present invention more clearly, the preparation method, material characterization and application fields of the beta lactoglobulin fiber-polyvinyl alcohol aerogel provided by the present invention will be described by specific examples, but the present invention is not limited to these examples, and those skilled in the art who make non-essential improvements or modifications under the core teaching of the present invention still belong to the protection scope of the present invention.
Example 1:
screening of beta lactoglobulin fiber-polyvinyl alcohol aerogel formation conditions:
1) Preparation of component materials
Preparation of polyvinyl alcohol hydrogel:
polyvinyl alcohol can realize physical crosslinking under circulating freeze-thaw, but beta lactoglobulin fibers can damage the crystallinity of a system, thereby influencing the forming process. In order to find out a polyvinyl alcohol material with a good gel effect and explore preparation process conditions, polyvinyl alcohol 1750, polyvinyl alcohol 1788 and polyvinyl alcohol 1799 are added into ultrapure water with the temperature of 95 ℃, and water bath is carried out in boiling water until the polyvinyl alcohol 1750, the polyvinyl alcohol 1788 and the polyvinyl alcohol 1799 solution with the concentration of 2.5wt% and 10wt% are completely dissolved, respectively. Cooling to 25 deg.c, freezing and thawing for 3-4 times at-80 deg.c for 21 hr and at 25 deg.c for 3 hr.
As shown in FIG. 1, polyvinyl alcohol 1750, 1788 did not gel at concentrations of 2.5wt% and 10wt%, while 1799 did gel at concentrations of 2.5wt% and 10 wt%. Thus, all of the polyvinyl alcohols described in the subsequent experiments were polyvinyl alcohol 1799.
Preparation of beta lactoglobulin fibers:
preparing a beta lactoglobulin solution with the concentration of 4wt% by using ultrapure water, adjusting the pH to 2.0 by using hydrochloric acid, stirring and heating for denaturation for 5 hours under the condition of a water bath at 90 ℃, and obtaining the beta lactoglobulin fiber solution with the stirring speed of 300rpm. In order to make beta-lactoglobulin fibers more suitable as a dressing material, it is necessary to obtain a solution of beta-lactoglobulin fibers at pH 7.4 (physiological conditions). However, according to the report (biomacromolecules.2014, 15, 3119-3127), the isoelectric point of beta lactoglobulin is around pH 5.0, so the process of adjusting the pH of the solution from 2.0 to 7.4 through the isoelectric point (around 5.0) destroys the stability of the beta lactoglobulin fiber, and easily causes the fiber to be shortened or decomposed.
In this application, the inventors used a single addition of a moderate amount of high concentration NaOH solution (10M concentration) to rapidly adjust the pH of the beta lactoglobulin fiber solution to 7.4. Under the method, the beta lactoglobulin fiber can keep better stability (as shown in figure 2), and the sample still presents obvious fiber shape after one week.
The beta lactoglobulin is derived from cow milk, and the dry weight purity of the beta lactoglobulin is more than or equal to 93wt%.
2) Beta lactoglobulin fiber-polyvinyl alcohol gel forming condition screening
The beta lactoglobulin fiber solution with pH =7.4 and the polyvinyl alcohol solution were mixed in different proportions, and the mixture ratio of the two solutions in the obtained mixed solution is shown in fig. 3, where the beta lactoglobulin fiber is abbreviated as BLGF and the polyvinyl alcohol is abbreviated as PVA. The mixed solution is stirred uniformly and then is subjected to cyclic freeze thawing for 4 times, wherein each cycle is carried out at-80 ℃ for 21 hours and at 25 ℃ for 3 hours.
As shown in fig. 3, the ratio of the β lactoglobulin fibers to the polyvinyl alcohol in the mixed solution is important for gel formation after freezing and melting. On one hand, the crystallinity of the polyvinyl alcohol can be damaged by the beta lactoglobulin fibers, and the gel forming process is further influenced, so that the increase of the content of the polyvinyl alcohol is beneficial to the gel forming of the sample. On the other hand, too much polyvinyl alcohol may cause a decrease in the performance of the resulting gel dressing, such as: small porosity, poor hydrophilicity, low water absorption and retention, and lack of sufficient cell adhesion promoting ability, etc. The final experimental results are: when the mass fraction of the beta lactoglobulin fiber in the mixed solution is 1-3.5 wt% and the mass fraction of the polyvinyl alcohol is 2.5-5 wt%, gel forming can be carried out.
Example 2:
preparation of beta lactoglobulin fiber-polyvinyl alcohol aerogel
Step 1: preparing a beta lactoglobulin solution with the concentration of 4wt% by using ultrapure water, adjusting the pH to 2 by using hydrochloric acid, and stirring and heating the solution for 5 hours at the rotation speed of 300rpm under the condition of a water bath at the temperature of 90 ℃. Beta lactoglobulin is formed into beta lactoglobulin fibers through thermal denaturation, and the prepared beta lactoglobulin fiber solution is cooled to 4 ℃ and then is adjusted to pH 7.4 by using a proper amount of 10M NaOH solution.
And 2, step: adding solid particles of polyvinyl alcohol 1799 into ultrapure water at 95 ℃, heating for 10min to obtain a polyvinyl alcohol solution with the concentration of 12wt%, and cooling to 25 ℃ for storage.
And step 3: adding the beta lactoglobulin fiber solution in the step 1 and the polyvinyl alcohol solution in the step 2 into ultrapure water at 25 ℃ according to the proportion. In the mixed solution, the mass fraction ratios of beta lactoglobulin fibers (abbreviated as BLGF) and polyvinyl alcohol are respectively as follows: 5wt% for 1wt%, 4wt% for 2wt%, and 3wt% for 3wt%, which are labeled as BLGF-1/PVA-5, BLGF-2/PVA-4, and BLGF-3/PVA-3 samples in this order. After being uniformly mixed, the mixture is subjected to cyclic freeze thawing for 3 to 4 times, each cycle is carried out for 21 hours at minus 80 ℃, and the cycle is carried out for 3 hours at 25 ℃. And (3) carrying out vacuum freeze drying on the sample obtained by freeze thawing to obtain the beta lactoglobulin fiber-polyvinyl alcohol aerogel.
Obtaining of polyvinyl alcohol aerogels
To demonstrate the important effect of the addition of beta lactoglobulin fibers on the properties of the dressing, a polyvinyl alcohol aerogel without beta lactoglobulin fibers was used as a control. The preparation method comprises the following steps: adding solid particles of polyvinyl alcohol 1799 into ultrapure water at 95 ℃ until the solid particles are dissolved to obtain a polyvinyl alcohol solution with the concentration of 12wt%, cooling to 25 ℃, and diluting with ultrapure water at 25 ℃ to 6wt%. After being uniformly mixed, the mixture is subjected to cyclic freeze thawing for 3 to 4 times, each cycle is carried out for 21 hours at minus 80 ℃, and the cycle is carried out for 3 hours at 25 ℃. And (4) carrying out vacuum freeze drying on the sample obtained by freeze thawing to obtain the polyvinyl alcohol aerogel.
Aerogel performance characterization and detection
1) Aerogel morphology analysis
As shown in fig. 4, the beta lactoglobulin fiber-polyvinyl alcohol aerogel is lower in density and larger in volume than the polyvinyl alcohol aerogel. And as the content of the beta lactoglobulin fiber increases, the beta lactoglobulin fiber-polyvinyl alcohol aerogel is more fluffy.
2) Scanning Electron Microscope (SEM) characterization analysis
And detecting the shapes of the polyvinyl alcohol aerogel and the beta lactoglobulin fiber-polyvinyl alcohol aerogel by adopting a scanning electron microscope. The results are shown in fig. 5, where the polyvinyl alcohol aerogel is tightly polymerized without significant pore size and network scaffolding. The beta lactoglobulin fiber-polyvinyl alcohol aerogel has a porous structure which is mutually communicated, and the pore diameter is gradually increased along with the increase of the content of the beta lactoglobulin fiber, so that the beta lactoglobulin fiber-polyvinyl alcohol aerogel has an obvious network scaffold structure.
3) Porosity analysis
To characterize the porosity of polyvinyl alcohol aerogels and beta lactoglobulin fiber-polyvinyl alcohol aerogels, the weight of each aerogel was recorded as W 0 And immersed in an ethanol solution, and then all samples were placed in a vacuum desiccator and repeatedly evacuated until the aerogel was completely filled with ethanol. The surface of the aerogel was wiped clean with ethanol and the saturated aerogel was weighed as W 1 The experiment was repeated three times. The porosity calculation formula is as follows:
wherein V and ρ represent the apparent volume of the beta lactoglobulin fiber-polyvinyl alcohol aerogel and the density of ethanol (0.789 g/cm), respectively 3 ). As a result, as shown in fig. 6, the porosity of the β lactoglobulin fiber-polyvinyl alcohol aerogel is significantly higher than that of the polyvinyl alcohol aerogel, and the porosity of the β lactoglobulin fiber-polyvinyl alcohol aerogel gradually increases as the content of the β lactoglobulin fiber increases. Porosity can affect cell viability, angiogenesis, stability and degradation in biological fluids, and cell migration and distribution, and high porosity wound dressings can improve the ability to absorb wound exudate.
4) Surface wetting Performance analysis
To evaluate the surface wetting properties of polyvinyl alcohol aerogels and beta lactoglobulin fiber-polyvinyl alcohol aerogels, the contact angles of the aerogels were measured at room temperature. 10 mu L of ultrapure water is dropped on the surface of the aerogel, and an image after standing for 10s is recorded by a digital camera. As shown in fig. 7, the contact angle of the beta lactoglobulin fiber-polyvinyl alcohol aerogel is significantly smaller than that of the polyvinyl alcohol aerogel, and the contact angle gradually decreases as the content of the beta lactoglobulin fiber increases. The beta lactoglobulin fiber can obviously improve the hydrophilicity of the aerogel.
5) Analysis of Water absorption
To explore the water absorption of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel at room temperature. Weigh each aerogel as W 0 The aerogel was soaked in 30mL of PBS (pH 7.4), the water-absorbed aerogel was periodically removed from the PBS, and the liquid on the surface of the aerogel was wiped with filter paper. The aerogel weighed at each time point was recorded as W S . The water absorption was calculated from the following formula
As shown in fig. 8, the water absorption of the β lactoglobulin fiber-polyvinyl alcohol aerogel is significantly higher than that of the polyvinyl alcohol aerogel, and the water absorption of the β lactoglobulin fiber-polyvinyl alcohol aerogel gradually increases as the content of the β lactoglobulin fiber increases. The higher water absorption rate can quickly absorb tissue fluid or blood exuded from the wound surface, and is helpful for hemostasis and sealing of the wound surface. When the concentrations of the beta lactoglobulin fiber and the polyvinyl alcohol in the mixed solution are both 3wt%, the prepared beta lactoglobulin fiber-polyvinyl alcohol aerogel is the dressing with the best effect.
Example 3:
application of beta lactoglobulin fiber-polyvinyl alcohol aerogel in promoting wound healing
1) Water vapor transmission rate analysis
To evaluate the water vapor permeability of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel. First, 6mL of ultrapure water was added to 10mL of a centrifuge tube, the diameter of the bottle mouth was 1.4cm, the bottle mouth was covered with an aerogel, and the tube mouth was sealed with a sealing film. The whole apparatus (aerogel + centrifuge tube + water + sealing film) was weighed and recorded as W i And placing in an incubator with the temperature of 37 ℃ and the relative humidity of 60 +/-15% for 24h. Finally, the entire apparatus is removed from the incubator and weighed as W t . In addition, the blank Control group (Control group) was a centrifuge tube filled with water, and the surface of the entire apparatus was not covered with any material and was placed under the same environmental conditions as the experimental group to simulate the state of natural evaporation of water. The calculation formula of the water vapor transmission rate of the aerogel is as follows:
wherein A and T are the areas (m) of the centrifugal tube openings respectively 2 ) And the time the device was in the incubator (day).
As a result, as shown in FIG. 9, the water vapor transmission rate of the polyvinyl alcohol aerogel was 526. + -.27 g m -2 day -1 The water vapor transmission rate of each group of beta lactoglobulin fiber-polyvinyl alcohol aerogel is greater than 526 +/-27 g m -2 day -1 . With the increase of the content of the beta lactoglobulin fiber, the beta lactoglobulin fiber-polyvinyl alcoholThe water vapor transmission rate of the aerogel is obviously increased (1149 +/-170, 1467 +/-151 and 1632 +/-92 g m) -2 day -1 ). Under the same conditions, the water vapor transmission rate of the blank group is 2190 +/-23 g m - 2 day -1 . It is believed that excessive accumulation of exudate may occur when the water vapor transmission rate is too low, which increases the risk of bacterial growth, while excessive water vapor transmission rate may result in excessive drying of the wound surface and scarring.
2) Leaching solution cytotoxicity assay
To evaluate the biocompatibility of the cells, the CCK8 method was used to determine the viability of the cells. And (3) placing the beta lactoglobulin fiber-polyvinyl alcohol aerogel under ultraviolet light for 1h for sterilization. And putting the sample into a complete culture medium for incubation to obtain a leaching liquor. Mouse L929 fibroblasts were seeded in 96-well plates in complete medium containing minimal essential medium, 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin solution, placed at 37 ℃ in 5% CO 2 And (5) in the incubator, after culturing for 24 hours, replacing a complete culture medium with the leaching liquor, and continuously culturing the fibroblasts. After 12h and 24h of culture, the leaching solution was replaced by complete medium. CCK8 reagent was added to each well for incubation. Finally, the absorbance was measured at 450nm with a microplate reader. Fibroblasts cultured by a complete culture medium alone are used as a Control group (Control group), and fibroblasts cultured by a leaching liquor of polyvinyl alcohol aerogel and beta lactoglobulin fiber-polyvinyl alcohol aerogel are used as an experimental group.
As shown in fig. 10, when the culture is carried out for 12 hours, the influence of the leaching solutions of the polyvinyl alcohol aerogel and the beta lactoglobulin fiber-polyvinyl alcohol aerogel on cell proliferation is not significantly different; compared with a blank control group, the cell survival rate of each group is more than 90%, and no obvious difference exists among the groups. When the cells are cultured for 24 hours after the culture, the cells cultured by the polyvinyl alcohol aerogel and the beta lactoglobulin fiber-polyvinyl alcohol aerogel leaching liquor show a proliferation tendency, but the proliferation capacity of the cells in the polyvinyl alcohol aerogel group is weaker than that in the blank control group. Compared with the polyvinyl alcohol aerogel group, the cells of the beta lactoglobulin fiber-polyvinyl alcohol aerogel group show good proliferation capacity, which is close to or even exceeds that of the control group, and the beta lactoglobulin fiber can improve the cell compatibility of the aerogel.
3) Cell adhesion assay
To evaluate the effect of the dressing on cell morphology and adhesion, groups of samples were sterilized by exposure to UV light for 1h and then preincubated overnight in culture medium. Absorbing the culture medium, dripping the cell suspension on the sample, and placing in CO 2 Culturing in an incubator. After 24h and 72h of culture, taking out and fixing with 4% paraformaldehyde, and washing with PBS for three times; adding Triton X-100 for permeation, and washing with PBS for three times; the cytoskeleton and nucleus were then stained with rhodamine-labeled phalloidin and DAPI, respectively, and images were taken with a fluorescence microscope. The method takes the culture of fibroblasts by polyvinyl alcohol aerogel as a control group and the culture of the fibroblasts by beta lactoglobulin fiber-polyvinyl alcohol aerogel as an experimental group.
As shown in a of fig. 11, after 24h of culture, the number of cells adhered to the surface of each beta lactoglobulin fiber-polyvinyl alcohol aerogel group was significantly increased compared to the polyvinyl alcohol aerogel group, showing good cell adhesion ability, and as the content of beta lactoglobulin fibers was increased, the cells showed a tendency to proliferate and form cell spheres, and the number was significantly increased.
As shown in b of fig. 11, after 72h of culture, the surface cell attachment number of the beta lactoglobulin fiber-polyvinyl alcohol aerogel group was significantly greater than that of the polyvinyl alcohol aerogel group, and the growth characteristics of multicellular spheroids were exhibited. As the beta lactoglobulin fiber content increases, a tendency is shown that the number of cell adhesions increases and that multicellular spheroids tend to increase in volume. We believe that the main reason for this phenomenon is the high pore structure and the larger specific surface area of the beta lactoglobulin fiber-polyvinyl alcohol gel, and these specific structures can transfer more nutrients into the cells, promoting cell growth and adhesion. In addition, beta lactoglobulin fibers are rich in beta sheets and have the property of promoting cell adhesion. Compared with beta lactoglobulin fiber-polyvinyl alcohol aerogel, the polyvinyl alcohol aerogel has a compact surface, poor hydrophilicity and no pore structure, so that the cell adhesion and growth are not facilitated.
4) Animal experiments
Is prepared by WistarA full-thickness skin defect animal model is constructed by mice (6-8 weeks old and male) so as to further evaluate the wound healing performance of the beta lactoglobulin fiber-polyvinyl alcohol aerogel as a wound dressing. After anesthetizing the Wistar rats, the hairs were cut from the dorsal side and the skin surface was cleaned with 75% alcohol solution and iodophor; subsequently, a skin biopsy sampler (diameter 10mm, area about 78.5 mm) was used 2 ) And sterile surgical scissors were used to create a wound model on the back of the mouse. The wound was covered with beta lactoglobulin fiber-polyvinyl alcohol aerogel (BLGF-3/PVA-3) with a diameter of 12mm and the healing of the wound surface was recorded until the dressing fell off. Meanwhile, the wound surface covered with gauze was used as a control group.
The result is shown in fig. 12, the dressing of beta lactoglobulin fiber-polyvinyl alcohol aerogel group can be well attached on the wound, and can be detached only at the 5 th day. Compared with a control group, the dressing of the beta lactoglobulin fiber-polyvinyl alcohol aerogel group forms obvious scabs on the 2 nd day, and the wound healing speed is promoted. On days 3 and 4, the wound surface shrinkage degree of the beta lactoglobulin fiber-polyvinyl alcohol aerogel group was significantly enhanced and the scab area was smaller compared to the control group. On day 5, the scab on the wound surface of the experimental group fell off together with the dressing, and it was observed that the new tissue was significantly larger than that of the control group. The area of the wound surface is counted by using software, and the unhealed area of the wound surface of the experimental group is found to be about 30.08mm 2 Comparison of the initial area of the wound surface (78.5 mm) 2 ) The healing rate exceeds 61.68 percent. However, the area of the control group where the wound surface did not heal was about 47.06mm 2 Comparison of the initial area of the wound surface (78.5 mm) 2 ) The healing rate was only 40.05%.
Animal experiment results show that the beta lactoglobulin fiber-polyvinyl alcohol aerogel has the capability of promoting wound healing, mainly because the beta lactoglobulin fiber-polyvinyl alcohol aerogel has a good intercommunicating pore form, the pore diameter is less than 100 mu m, the porosity is more than 85 percent, and the beta lactoglobulin fiber-polyvinyl alcohol aerogel is suitable for the growth of cells and tissues. On the other hand, the beta lactoglobulin fiber-polyvinyl alcohol aerogel turns into a hydrogel state after absorbing liquid, and the beta lactoglobulin fiber-polyvinyl alcohol aerogel can be used as a barrier to prevent further bleeding and bacterial infection of wounds and guide a more effective healing process.
Claims (9)
1. The preparation method of the beta lactoglobulin fiber-polyvinyl alcohol aerogel comprises the following steps:
step 1: preparing a beta lactoglobulin solution with the concentration of 3-4 wt% by using ultrapure water, adjusting the pH to 1.5-2.5 by using hydrochloric acid, stirring and heating for 3-7 h under the water bath condition of 80-90 ℃ and at the rotating speed of 200-500 rpm; cooling the prepared beta-lactoglobulin fiber solution to 0-4 ℃, and then adjusting the pH value to 7.0-7.5 by using 8-12M NaOH;
and 2, step: adding the polyvinyl alcohol solid particles into ultrapure water at 85-100 ℃, heating until the polyvinyl alcohol solid particles are completely dissolved to obtain a polyvinyl alcohol solution with the concentration of 10-15 wt%, and cooling to 0-25 ℃ for later use; the alcoholysis degree of the polyvinyl alcohol 1799 is 98-99%;
and step 3: adding the beta-lactoglobulin fiber solution in the step 1 and the polyvinyl alcohol solution in the step 2 into ultrapure water at 15-25 ℃ according to a proportion, so that the sum of the mass fractions of the beta-lactoglobulin fiber and the polyvinyl alcohol in the solution is 3.5-8.5 wt%, wherein the mass fraction of the beta-lactoglobulin fiber is 1-3.5 wt%, and the mass fraction of the polyvinyl alcohol is 2.5-5 wt%; after being mixed evenly, the mixture is subjected to cyclic freeze thawing for 3 to 4 times, the temperature is circulated from-20 to-80 ℃ for 21 to 24 hours each time, and the temperature is circulated from 15 to 25 ℃ for 3 to 6 hours; carrying out vacuum freeze drying on the sample obtained by freeze thawing to obtain the freeze-dried powder;
the polyvinyl alcohol is polyvinyl alcohol 1799.
2. The method according to claim 1, wherein the sum of the mass fractions of the beta lactoglobulin fiber and the polyvinyl alcohol in the solution in step 3 is 6wt%.
3. The preparation method according to claim 1, wherein the mass fraction of the beta-lactoglobulin fibers in the step 3 is 2 to 3.5 wt%.
4. The method according to claim 1, wherein the beta lactoglobulin of step 3 is derived from cow's milk and has a purity of 93wt% or more on a dry weight basis.
5. The method for preparing the compound of claim 1, wherein the temperature of vacuum freeze drying in step 3 is-50 to-80 ℃ and the time is 24 to 48 hours.
6. The method of claim 1, wherein the solution of step 3 has a concentration of 3wt% of both the beta lactoglobulin fiber and the polyvinyl alcohol.
7. Use of the beta lactoglobulin fiber-polyvinyl alcohol aerogel prepared by the method of claim 1 in the preparation of a skin dressing.
8. The use of claim 7, wherein the skin dressing is a dermoad dressing.
9. The use of claim 7, wherein the skin dressing is a skin care mask carrier.
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| CN202211544287.5A CN115887731B (en) | 2022-12-04 | Preparation method of beta-lactoglobulin fiber-polyvinyl alcohol aerogel and application of beta-lactoglobulin fiber-polyvinyl alcohol aerogel in preparation of skin dressing |
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