CN116251224B

CN116251224B - Lactoferrin gel dressing and application thereof

Info

Publication number: CN116251224B
Application number: CN202310549629.0A
Authority: CN
Inventors: 常天安; 王曙蒙
Original assignee: Jiangsu Zhengda Tianchuang Biological Engineering Co ltd
Current assignee: Jiangsu Zhengda Tianchuang Biological Engineering Co ltd
Priority date: 2023-05-16
Filing date: 2023-05-16
Publication date: 2023-09-12
Anticipated expiration: 2043-05-16
Also published as: CN116251224A

Abstract

The invention discloses a lactoferrin gel dressing and application thereof, wherein the dressing comprises an outer layer and an inner layer, the outer layer is a polyurethane film, and the inner layer is betaine modified hyaluronic acid and lactoferrin composite gel; the polyurethane outer layer has good elasticity and ductility, isolates bacteria and can exchange water vapor at the same time; the gel inner layer not only provides a moist healing environment, absorbs exudates and is completely absorbed and degraded, but also plays a role in synergistic antibacterial effect of quaternary ammonium and lactoferrin, and the durability of the dressing can be greatly improved by combining the two layers.

Description

Lactoferrin gel dressing and application thereof

Technical Field

The invention belongs to the technical field of synthetic biomedical materials, and particularly relates to a lactoferrin gel dressing and application thereof.

Background

Lactoferrin (LF) is a natural active iron-binding glycoprotein with multiple physiological functions, mainly derived from milk of mammals, and is not only involved in the transportation of iron in organisms and promoting the growth and differentiation of bone cells, but also has the functions of resisting microorganisms, viruses, oxidation, cancers and the like. Among the many biological functions, its antibacterial activity is the most attractive. A large number of researches show that the lactoferrin has good inhibition capability to various gram-negative bacteria, gram-positive bacteria and some fungi, has no toxic or side effect to human bodies, is recognized as a safe food preservative auxiliary agent, and is widely applied to the field of foods.

Currently, lactoferrin is rarely reported to be applied to wound dressings, wang reports that lactoferrin/lithium magnesium silicate are compounded by ionic bonds to form injectable hydrogel wound dressings, the hydrogel dressings are powdery after the moisture volatilizes, cannot permanently act on wounds, and are easy to cause secondary infection when being difficult to clean or washed (Wang. Preparation of lactoferrin/lithium magnesium silicate injectable hydrogels and research on promoting wound healing [ D ] university of Lanzhou, 2022). Chinese patent CN 107496971B reports a bacterial cellulose loaded lactoferrin dressing that is not adhesive and is easily dropped on the skin.

Disclosure of Invention

Aiming at the defects of the prior art of wound dressing containing lactoferrin, the invention provides a lactoferrin gel dressing, which comprises an outer layer and an inner layer, wherein the outer layer is a polyurethane film, and the inner layer is a betaine modified hyaluronic acid and lactoferrin composite gel.

The outer layer provides elasticity and ductility, can resist damage by external force and prevent excessive quick volatilization of moisture; the inner layer has the sticky and durable and stable adhered skin surface, has strong moisture retention, can absorb wound secretion, and provides moist environment for wound surface to promote healing.

The thickness of the polyurethane film is 0.8mm.

The preparation method of the betaine modified hyaluronic acid is further as follows:

heating and stirring hyaluronic acid and 1, 2-epoxy-3- (diethylamino) propane under alkaline condition to react and obtain amino grafted hyaluronic acid; and (3) heating the amino grafted hyaluronic acid and 1, 3-Propane Sultone (PS) in tetrahydrofuran for reaction, purifying and drying to obtain betaine modified hyaluronic acid.

The molecular weight of the hyaluronic acid is 10-80 ten thousand.

The mass ratio of the hyaluronic acid to the 1, 2-epoxy-3- (diethylamino) propane is 1:0.4.

The mass ratio of the amino grafted hyaluronic acid to the 1, 3-propane sultone is 1:0.4.

Further mixing the betaine modified hyaluronic acid solution with lactoferrin solution to obtain a sol solution, wherein the mixing ratio of the betaine modified hyaluronic acid solution to the lactoferrin solution is 1:0.5 to 0.8.

The betaine modified hyaluronic acid solution is 1g/mL, and the solvent is Tris-HCl protein buffer solution; the lactoferrin solution is 0.2g/mL, and the solvent is Tris-HCl protein buffer solution.

The gel is further adhered to the polyurethane film by a smearing method, and the method specifically comprises the following steps: spin-coating the sol solution on a polyurethane film, and drying for 3-4 hours at normal temperature under vacuum to obtain the dressing.

The thickness of the gel layer is controlled to be 0.5-1 mm.

The dressing provided by the invention comprises a polyurethane outer layer and a gel inner layer, wherein the polyurethane outer layer has good elasticity and ductility, isolates bacteria and can exchange water vapor; the gel inner layer can provide a moist healing environment, can be completely degraded and absorbed on the surface of the skin, and can greatly improve the durability of the dressing by combining the two.

The gel principle of the betaine modified hyaluronic acid solution and lactoferrin solution after mixing is as follows: lactoferrin has a positive point, whereas betaine contains a sulfonic acid group (SO ₃ ^- ) And quaternary ammonium (N) ⁺ ) After the lactoferrin and the sulfonic acid are mixed, the lactoferrin and the sulfonic acid are electrostatically attracted, so that the lactoferrin becomes a crosslinking agent of the modified hyaluronic acid to form a net-shaped crosslinking object; because of the higher water solubility of both, the mixture is in a sol state when the concentration of the mixture in water is low, and a gel is formed as the water content is lower. Furthermore, the free quaternary ammonium in betaine has good antibacterial effect due to negative charge, so that lactoferrin and quaternary ammonium have synergistic antibacterial effect. Lactoferrin has strong iron binding capacity, can be competitively chelated with iron ions required by bacterial reproduction, reduces the combination of bacteria and the iron ions, reduces the nutrition absorption required in the bacterial growth process, and inhibits the growth of the bacteria; and the quaternary ammonium is positively charged, so that the negative electricity on the surface of a bacterial cell membrane can be destroyed, and the cell membrane is broken, thereby playing a role in sterilization.

Advantageous effects

The inner layer of the dressing provided by the invention has the effects of moisturizing, absorbing exudates, inhibiting bacteria and diminishing inflammation, can be completely degraded and absorbed by skin, and has good safety.

The dressing provided by the invention has good elasticity and ductility on the outer layer and synergistic antibacterial factors on the inner layer, so that the durability of the dressing is greatly prolonged.

Drawings

FIG. 1 is a graph showing the results of a biotoxicity experiment-the morphology of the cells.

FIG. 2 is an infrared spectrum of lactoferrin, betaine-modified hyaluronic acid, gel sample 4.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Example 1

Dissolving 10g of hyaluronic acid in 100mL of isopropanol solution, adding 5mL of NaOH solution with the mass concentration of 20%, then placing the solution into a water bath kettle with the room temperature, magnetically stirring for 2 hours, adding 4g of 1, 2-epoxy-3- (diethylamino) propane, stirring uniformly, adjusting the pH to be 8, continuously stirring at 60 ℃ for 10 hours, pouring the obtained solution into methanol solution with the mass concentration of 20% of HCl after the reaction is finished, precipitating, filtering to obtain a precipitate, washing the precipitate with ethanol, filtering, and drying under reduced pressure to obtain the amino grafted hyaluronic acid.

Dissolving amino grafted hyaluronic acid and 1, 3-Propane Sultone (PS) in tetrahydrofuran, loading into a round bottom flask, heating and stirring in a water bath at 50 ℃ for 12 hours, volatilizing the solvent, dissolving the obtained crude product with water, dialyzing in 10000Da dialysis bag for 3 days, and drying to obtain betaine modified hyaluronic acid. The feeding mass ratio of the amino grafted hyaluronic acid to the 1, 3-propane sultone is 1:0.4.

The molecular weight of the hyaluronic acid is respectively 10-20 ten thousand, 20-40 ten thousand and 40-80 ten thousand, and the obtained betaine modified hyaluronic acid is respectively named as HA-1, HA-2 and HA-3. Hyaluronic acid was purchased from Shanghai Meilin Biochemical technologies Co.

Example 2

Hydrogels were prepared.

Preparing Tris-HCl protein buffer solution: 50mL of 0.1M Tris solution and 42mL of 0.1M HCl solution are mixed, the volume is fixed to 100mL (pH 7.4), then steam sterilization is carried out for 30min under the pressure of 0.08-0.1 MPa, and the protein buffer is obtained through refrigeration.

1g/mL of betaine-modified hyaluronic acid solution and 0.2g/mL of lactoferrin solution were prepared, respectively.

The lactoferrin was purchased from baichuan biotechnology limited.

The gels prepared with different betaine-modified hyaluronic acid and different mixing ratios are shown in Table 1.

TABLE 1

Example 3

Cutting a polyurethane film into a sample piece with the thickness of 3 multiplied by 3cm, dipping the sol liquid obtained in the embodiment 2 by using a brush, coating one surface of the sample piece, drying for 3-4 hours at normal temperature under vacuum, and repeating the coating and drying until the thickness of a gel layer is between 0.5 and 1mm.

The polyurethane film is purchased from Shanghai Xin Suo environmental protection technology Co., ltd and has a thickness of 0.8mm.

Example 4

The tackiness of the dressing was tested.

The tackiness of the film was characterized by testing the peel force after bonding between the dressing and a smooth glass sheet (sulfuric acid surface treatment). Cutting the film into strip-shaped sample strips with the thickness of 10mm multiplied by 60mm, attaching one surface of the sample strip to a smooth glass sheet, applying certain pressure to enable the dressing to be tightly attached to the glass sheet, and then placing the glass sheet into a baking oven at 37 ℃ for standing for 2 hours. After the sample strip is taken out, the force required for 180 DEG stripping the sample strip is tested by a pulling machine, and the stripping rate is 50mm/min.

Example 5

The gels were tested for in vitro antimicrobial properties.

Antibacterial activity test: the colibacillus and staphylococcus aureus are used as test strains, and the bacterial liquid is diluted to 1.4x10 respectively by PBS buffer solution ⁴ CFU/mL (E.coli) and 1X 10 ⁶ CFU/mL (Staphylococcus aureus). Sterilizing the dressing, adding into 75mL of bacterial solution, shaking and contacting for 12h in a constant temperature shaker, and then taking out a proper amount of bacterial solution for plating culture according to growthThe bacterial colony number of (2) calculates the bacterial liquid concentration and the bacteriostasis rate, and the bacteriostasis rate has the following calculation formula:

wherein W is _t Is the concentration of the bacterial liquid of the blank control group; q (Q) _t Is the concentration of the bacterial liquid after the sample is contacted with the bacterial liquid.

The test results of example 4 and example 5 are shown in Table 2.

TABLE 2

From the results, the dressing provided by the invention has higher antibacterial property on escherichia coli and staphylococcus aureus, and after the betaine modified hyaluronic acid solution and lactoferrin are crosslinked into gel, the electronegativity of the gel is not reduced excessively due to the existence of free quaternary ammonium in the betaine, so that the antibacterial property of the gel is kept at a higher level. On the other hand, the gel plays a synergistic antibacterial role of quaternary ammonium and lactoferrin, so that the gel has higher antibacterial activity. From the test results, it is observed that the gel has better viscosity, and the viscosity is related to the molecular weight of the hyaluronic acid and the mixing proportion of the two solutions.

Example 6

The dressing was subjected to a biotoxicity experiment.

A suspension of mouse epithelial fibroblasts L929 (2 mL) was added to the vessel and at 37℃CO ₂ CO at a concentration of 5% ₂ Culturing in an incubator. Fresh RPMI 1640 medium was added to each vessel. The sterilized dressing (sample 4) was gently placed on the cell layer in the center of each vessel and the culture was continued for 24 hours to observe the cell morphology using an optical microscope. LDPE film is used as negative control and PVC film containing organotin stabilizer is used as positive control. The control group membranes were purchased from Shandong Dexuda geotechnical materials Co. The cell morphology is shown in figure 1.

Example 7

The gel (sample 4), HA-1 and lactoferrin were subjected to infrared broad spectrum analysis, the spectrum of which is shown in FIG. 2.

As can be seen from the figure, the mixed gel has a quaternary ammonium (N ⁺ ) Sulfonic acid group (SO) ₃ ^- ) The characteristic peaks of (2) are displaced to different extents due to the change in polarity of the bonds. Wherein the characteristic peaks of the two sulfonic acid groups are 1190cm respectively ^-1 (telescoping vibration of s=o) and 1040cm ^-1 （S-O ^- Antisymmetric telescopic vibration).

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The lactoferrin gel dressing is characterized by comprising an outer layer and an inner layer, wherein the outer layer is a polyurethane film, and the inner layer is a composite gel of betaine modified hyaluronic acid and lactoferrin;

the preparation method of the betaine modified hyaluronic acid comprises the following steps:

heating and stirring hyaluronic acid and 1, 2-epoxy-3- (diethylamino) propane under alkaline condition to react and obtain amino grafted hyaluronic acid; the amino grafted hyaluronic acid and 1, 3-Propane Sultone (PS) react in tetrahydrofuran by heating, and betaine modified hyaluronic acid is obtained after purification and drying;

the molecular weight of the hyaluronic acid is 10-80 ten thousand;

the compound gel is prepared by mixing a betaine modified hyaluronic acid solution and a lactoferrin solution, wherein the solvent is Tris-HCl protein buffer solution, the concentration of the betaine modified hyaluronic acid solution is 1g/mL, the concentration of the lactoferrin solution is 0.2g/mL, and the mixing volume ratio of the betaine modified hyaluronic acid solution to the lactoferrin solution is 1:0.5 to 0.8.

2. The lactoferrin gel dressing of claim 1, wherein the mass ratio of hyaluronic acid to 1, 2-epoxy-3- (diethylamino) propane is 1:0.4.

3. The lactoferrin gel dressing of claim 1, wherein the mass ratio of the amino grafted hyaluronic acid to the 1, 3-propane sultone is 1:0.4.

4. A method of preparing a lactoferrin gel dressing as claimed in claim 1, comprising the steps of:

preparing betaine modified hyaluronic acid solution and lactoferrin solution respectively, wherein the solvent is Tris-HCl protein buffer;

mixing the two materials in proportion to obtain sol solution;

spin-coating the sol solution on a polyurethane film, and drying for 3-4 hours at normal temperature under vacuum to obtain the dressing.

5. The method for preparing a lactoferrin gel dressing as claimed in claim 4, characterized in that the thickness of the polyurethane film is 0.8mm, and the thickness of the gel layer is 0.5-1 mm.

6. The use of a lactoferrin gel dressing as claimed in any one of claims 1 to 5, wherein the dressing is applied to a human skin wound.