Multifunctional oil gel wound dressing and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a multifunctional oil gel wound dressing and a preparation method thereof. The raw material of the multifunctional oleogel wound dressing comprises oleogel; the preparation method of the oleogel comprises the following steps: preheating oil, adding anti-solvent beta-sitosterol and candelilla wax, and uniformly mixing to obtain a mixture; and adding curcumin into the mixture, uniformly mixing, and cooling to obtain the oleogel. The anti-solvent beta-sitosterol and candelilla wax are utilized to prepare the curcumin lipid carrier (mixture) with good stability and smearing property, and the stability of curcumin is improved. The prepared oleogel has the capability of promoting healing of chronic wounds of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, in particular to a multifunctional oil gel wound dressing and a preparation method thereof. Background Art

[0002] As one of the most common chronic diseases in the world, the prevalence of diabetes continues to rise. According to statistics in 2021, the number of diabetes patients worldwide has reached 537 million, and is expected to surge to 783 million in 2045. Among this large patient population, approximately 15%-25% of patients suffer from chronic wounds and face the risk of amputation or even death. Chronic diabetic wounds are characterized by persistent inflammation and susceptibility to bacterial infection, which significantly hinder the wound healing process. At the same time, the microenvironment of diabetic wounds is complex, involving multiple local pathological changes such as oxidative stress, immune cell dysfunction, and chronic proinflammatory state. In response to this complex microenvironment, oil gels containing bioactive ingredients have gradually become an alternative option for the treatment of chronic diabetic wounds.

[0003] Among numerous gelling agents, β-sitosterol has attracted considerable attention due to its significant anti-inflammatory, anti-diabetic, antioxidant, and anti-cardiovascular effects. β-sitosterol can often combine with other compounds to form stable oil gel structures. However, its high melting point, water insolubility, and poor oil dispersibility have significantly limited its application in biomedical materials and the delivery of bioactive substances. Summary of the Invention

[0004] Based on the above content, the present invention provides a multifunctional oil gel wound dressing and a preparation method thereof; a novel oil gel system using an antisolvent β-sitosterol (AS-Sit) and candelilla wax (Cw) is used for transdermal delivery of curcumin, which has a good effect on promoting wound healing in diabetic mice.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for preparing an oil gel, comprising the following steps:

[0007] After preheating the oil, anti-solvent β-sitosterol and candelilla wax are added and mixed to obtain a mixture;

[0008] adding curcumin to the mixture, mixing evenly, and then cooling to obtain the oil gel;

[0009] The oil includes at least one of soybean oil, olive oil, linseed oil, and chia seed oil.

[0010] The second technical solution of the present invention is an oil gel prepared according to the above preparation method.

[0011] The third technical solution of the present invention is a multifunctional oil gel wound dressing, the raw materials of which include the above-mentioned oil gel.

[0012] A fourth technical solution of the present invention is a drug delivery carrier, the raw materials of which include the above-mentioned oil gel.

[0013] The present invention discloses the following technical effects:

[0014] The present invention utilizes anti-solvent β-sitosterol and candelilla wax to prepare a curcumin lipid carrier (mixture) with good stability and spreadability, thereby improving the stability of curcumin. The prepared oil gel has the ability to promote the healing of chronic diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is the appearance of the oil gel prepared in Examples 1-5.

[0017] Figure 2 The antibacterial activity of the oil gels prepared in Examples 1-5.

[0018] Figure 3 Effects of the oil gels prepared in Examples 1-5 on the in vitro migration of fibroblasts.

[0019] Figure 4 The ROS scavenging ability of the oil gels prepared in Examples 1-5.

[0020] Figure 5 Effects of the oil gels prepared in Examples 1-5 on macrophage polarization.

[0021] Figure 6 The cell compatibility of the oil gels prepared in Examples 1-5.

[0022] Figure 7 Blood compatibility of the oil gels prepared in Examples 1-5.

[0023] Figure 8 This is the effect of the oil gel prepared in Example 3 on wound healing in diabetic mice.

[0024] Figure 9 Histopathological analysis of wounds in diabetic mice.

[0025] Figure 10Comparing the appearance and smearing property of the oil gel prepared in Example 3 and Comparative Example 1 (without anti-solvent β-sitosterol addition). DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present application will now be described in detail, without intending to be limited thereto, and are to be understood as being merely illustrative of certain aspects, features and embodiments of the present application.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the ranges is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the limits are also included.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0029] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the specific embodiments described herein. Those skilled in the art will readily devise their own

[0030] As used herein, the terms "comprise", "comprising", "include", "including", "contain", "containing", "have", "having" and the like are open-ended terms that are intended to mean including, but not limited to.

[0031] The first aspect of the present application provides a method for preparing an oil gel, comprising the following steps:

[0032] After preheating the oil, adding anti-solvent β-sitosterol and candelilla wax to mix to obtain a mixture;

[0033] After adding curcumin to the mixture and mixing, cooling to obtain the oil gel;

[0034] The oil comprises at least one of soybean oil, olive oil, flaxseed oil, and chia seed oil.

[0035] In a preferred embodiment of the present invention, the preheating temperature is 90°C.

[0036] In a preferred embodiment of the present invention, the mass concentration of the anti-solvent β-sitosterol in the mixture is 1% to 10%.

[0037] In a preferred embodiment of the present invention, the method for preparing the anti-solvent β-sitosterol comprises the following steps:

[0038] dissolving β-sitosterol in anhydrous ethanol to obtain an anhydrous ethanol solution containing β-sitosterol;

[0039] The anhydrous ethanol solution containing β-sitosterol and the PBS solution are mixed in a volume ratio of 5:1 to 1:5, and then the ethanol is removed by rotary evaporation and freeze-dried to obtain the anti-solvent β-sitosterol.

[0040] In a preferred embodiment of the present invention, the mass concentration of the candelilla wax in the mixture is 1% to 9%.

[0041] In a preferred embodiment of the present invention, the concentration of curcumin in the mixture is 0.5-2.5 mg / mL.

[0042] In a preferred embodiment of the present invention, the mixing method is stirring, the stirring speed is 1,000 rpm, and the stirring time is 5 to 30 minutes.

[0043] The second aspect of the present invention provides an oil gel prepared according to the above preparation method.

[0044] A third aspect of the present invention provides a multifunctional oil gel wound dressing, the raw materials of which include the above-mentioned oil gel.

[0045] The multifunctional oil gel wound dressing can be applied to wounds with inflammation, bacterial infection or oxidative stress.

[0046] A fourth aspect of the present invention provides a drug delivery carrier, the raw materials of which include the above-mentioned oil gel.

[0047] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0048] The soybean oil used in the examples of the present invention was purchased from Xing'an Supermarket, Jinan University, Tianhe District, Guangzhou.

[0049] The preparation method of the anti-solvent β-sitosterol used in the embodiment of the present invention comprises the following steps:

[0050] β-sitosterol was mixed with absolute ethanol in a ratio of 1 : 100 (w / v). To ensure complete dissolution, the mixture was sonicated until all the large particles were completely gone. Subsequently, the solution was heated in a constant temperature water bath at 45 °C for 5 min to ensure complete dissolution of β-sitosterol, thus obtaining a solution of β-sitosterol in absolute ethanol. After the solution was cooled to room temperature, the solution of β-sitosterol in absolute ethanol (organic phase) was rapidly added to the aqueous phase (PBS, pH = 7) within 2 min using a T25 high-speed homogenizer under vigorous stirring at 8000 rpm. During this process, the volume ratio of the organic phase to the aqueous phase (anti-solvent ratio) was set to 1 : 1. After mixing well, the homogenization was continued for 2 min to ensure the uniformity of the solution. Finally, the entire ethanol and part of the water in the solution were removed using a rotary evaporator, thus obtaining a water dispersion. The water dispersion was subjected to freeze-drying for 72 h, and finally the anti-solvent β-sitosterol microparticles were obtained.

[0051] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0052] Example 1

[0053] First, an appropriate amount of soybean oil was preheated to 90 °C, and then 5% (w / v) of anti-solvent β-sitosterol and candelilla wax (concentration 7% w / v) were added. The mixture was stirred at a speed of 1,000 rpm for 30 min to ensure uniform mixing.

[0054] Next, curcumin (concentration 0.5 mg / mL) was added to the mixture, and the stirring was continued for 5 min until the curcumin was completely dissolved.

[0055] After that, the mixture was cooled to room temperature to form a stable oil gel. To reduce the degradation of curcumin, the entire preparation process was carried out in the dark.

[0056] Example 2

[0057] The difference between Example 1 and Example 2 is only that the concentration of curcumin in the mixture is 1.0 mg / mL.

[0058] Example 3

[0059] The difference between Example 1 and Example 3 is only that the concentration of curcumin in the mixture is 1.5 mg / mL.

[0060] Example 4

[0061] The difference between Example 1 and Example 4 is only that the concentration of curcumin in the mixture is 2.0 mg / mL.

[0062] Example 5

[0063] The only difference from Example 1 is that the concentration of curcumin in the mixture is 2.5 mg / mL.

[0064] Figure 1 The appearance of the oil gel prepared in Example 1-5 is shown below. Figure 1 It can be seen that all five groups of oil gels have good gel properties. However, unencapsulated free curcumin was present in Examples 4 and 5, which may have affected the functional properties of the oil gel.

[0065] Figure 2 The antibacterial activity of the oil gel prepared in Examples 1-5. Test method: 100 μL of bacteria (10 5 CFU / mL, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) were added to the oil gel surface and incubated at 37°C for 12 hours. The bacteria were then washed off the oil gel surface with 300 μL of PBS solution, and 20 μL of the solution was spread on an agar plate and incubated at 37°C for another 12 hours. The surviving bacterial colonies were observed. Figure 2 It can be seen that the antibacterial activity of the oil gel is significantly improved with the increase of curcumin addition.

[0066] Figure 3 The effect of the oil gel prepared in Example 1-5 on the migration of fibroblasts. Test method: 1×10 6 NIH3T3 cells were seeded into each well of a 12-well plate and co-cultured with LPS (lipopolysaccharide) and glucose for 12 hours. When the cell confluence reached >90%, a vertical line was formed between the cells using the tip of a 100 μL pipette to form an artificial gap. The cells were then rinsed three times with PBS to remove any floating cells. Subsequently, 500 μL of serum-free culture medium and 500 μL of oil gel extract (the sterilized oil gel sample was immersed in the corresponding culture medium containing 1% dimethyl sulfoxide and allowed to stand in a cell culture incubator for 48 hours to prepare the extract. The extract was then centrifuged and filtered to obtain the final extract for cell culture; the same below) were added to the 12-well plate. The cells were observed under a microscope after 0 and 24 hours. Figure 3 It can be seen that under normal conditions and high-glucose oxidative damage conditions, the prepared oil gel can significantly promote the migration of fibroblasts in vitro, among which Example 3 shows the best migration-promoting ability.

[0067] Figure 4 The ROS scavenging ability of the oil gel prepared in Examples 1-5. Test method: 1×10 6RAW264.7 cells were cultured for 12 hours until they adhered to the wall of the culture flask. To simulate and differentiate the oxidative stress microenvironment of normal wounds and diabetic wounds, RAW 264.7 cells were co-cultured with LPS and LPS+glucose for 12 hours. Then, the solution was discarded and the cells were co-cultured with 500 μL of different oil gel extracts. After 24 hours of co-culture, the cells were covered with DCFH-DA (1:1000) for 30 minutes. The results were obtained using an inverted fluorescence microscope. Figure 4 It can be seen that the prepared oil gel effectively scavenged ROS generated in cells under normal conditions and high glucose oxidative damage conditions.

[0068] Figure 5 The effect of the oil gel prepared in Example 1-5 on macrophage polarization. Test method: RAW264.7 cells were cultured with glucose (200 μm) and LPS (10 μg / mL) for 12 hours. To study macrophage polarization, the above RAW 264.7 cells were co-cultured with the oil gel extract in a cell culture incubator for 24 hours. The polarized cells were then collected and resuspended in PBS solution. The resuspended cells were plated at 1×10 6 The concentration of cells / 100 μL was transferred to a medical PE tube. FITC-conjugated anti-mouse CD86 antibody and PE-conjugated anti-mouse CD206 (MMR) antibody were added to the tube at appropriate concentrations. Flow cytometry and immunofluorescence staining were then performed. Figure 5 It can be seen that the prepared oil gel can promote the M2 polarization of macrophages.

[0069] Figure 6 and Figure 7 The biocompatibility of the oil gels prepared in Examples 1-5 is shown in Figure 2. Test method: NIH3T3, RAW264.7 and HUVEC cells were cultured in the corresponding culture medium at a concentration of 1×10 5 Next, 100 μL of cell suspension was inoculated into a 96-well plate and incubated in a cell culture incubator for 12 h to promote cell adhesion. Subsequently, 100 μL of oil gel extract was added and incubated for another 24 h. Then, 100 μL of 10% CCK-8 solution was added to each control group and sample group. The cells were then placed in a cell culture incubator and incubated for 3 h. The optical density (OD) value was measured at 450 nm using a microplate reader. Figure 6 It can be seen that a large number of dead cells appeared in the cells cultured in Examples 4 and 5. Figure 7 It also shows that the hemolysis rate of Example 5 is greater than 5%, which does not meet the standards for biomedical materials. Therefore, the maximum addition amount of curcumin in the oil gel is 1.5 mg / mL.

[0070] Figure 8Effect of the oil gel prepared in Example 3 on wound healing of diabetic mice. Test method: Under a sterile operating environment, type I diabetic C57BL / 6 mice were anesthetized to ensure animal welfare and experimental accuracy during the experiment. Subsequently, the mouse back hair was carefully treated with professional instruments to fully expose the back skin, providing a clear view for subsequent operations. A 1 cm diameter skin sampler was used to accurately mark the skin defect modeling site on the fixed position of the mouse back. Then, using surgical scissors and tweezers, the marked line was accurately operated to successfully construct a mouse full-thickness skin defect model, simulating a clinical wound scenario. According to the experimental grouping (PBS group as blank control group, oil gel as experimental group, RMC group as commercial gel control group), 50 mg of oil gel or RMC gel was weighed and evenly applied to the skin defect site of different groups of mice. After the gel flowability decreased, 3M biological semi-permeable membrane dressing was used for coverage and fixation to maintain the local drug concentration and prevent external pollution. In order to ensure the comfort of the mice during the gel dressing change process, isoflurane was used for gas anesthesia every other day. At key time points such as 0, 3, 7, and 14 days after the operation, the dressing was changed and the wound image was taken using a digital camera, and the image was accurately analyzed using Image J software. (Commercial gel purchased from Jingdong Mall, main parameters: Rui Mei Chuang RMC chitosan medical biological gel) Figure 8 It can be seen that compared with the control group and the commercial gel group, Example 3 has the best effect on promoting wound healing of diabetic mice. At 14 days, the wound is almost completely healed.

[0071] Figure 9 Histopathological analysis of wound tissue of diabetic mice. The wound tissue of diabetic mice in each treatment group was subjected to pathological analysis, and the test method was as follows: H&E staining steps included washing, fixing, dehydrating, antigen repairing, staining, re-staining, mounting, and observation. First, the skin tissue of the mouse back at 3, 7, and 14 days was taken, and the skin tissue was washed with water and fixed with 4% paraformaldehyde. Alcohol gradient dehydration was performed according to 75%, 85%, 90% alcohol, anhydrous ethanol, and xylene. Then, liquid paraffin was poured into the embedding box for embedding, and after embedding, the section was cut to a thickness of 3 μm and placed in an oven for drying. The section was placed in xylene for two times of deparaffinization, and then rehydrated in anhydrous ethanol, 95%, 85%, and 70% alcohol. The section was placed in hematoxylin staining solution for 4-5 min, differentiated with hydrochloric acid ethanol, and stained in eosin staining solution for 1 min. Then the section was dehydrated in 85% and 95% alcohol, respectively. Finally, after xylene permeation, the section was mounted for microscopic examination.

[0072] Mason staining: Dorsal skin samples were obtained from mice at 3, 7, and 14 days of age. Tissue embedding and sectioning procedures were the same as above. Sections were immersed in ethylene glycol ether acetate and then rehydrated in a gradient of ethanol. Sections were stained with Weigert's iron hematoxylin solution, differentiated with acidic ethanol solution, and washed. Subsequently, sections were blued with Masson's bluing solution, stained with Ponceau fuchsin, and then stained with aniline blue solution, followed by washing. Finally, sections were dehydrated, permeabilized, mounted, and observed.

[0073] Depend on Figure 9 It can be seen that compared with the control group and the commercial gel group, the inflammatory cells in the wound tissue of the mice in Example 3 group were significantly reduced, and collagen deposition was greater, which promoted wound healing.

[0074] Comparative Example 1

[0075] The only difference from Example 3 is that the addition of the anti-solvent β-sitosterol is omitted.

[0076] Figure 10 Comparison of the appearance and spreadability of the oil gel prepared in Example 3 and Comparative Example 1 (without the addition of anti-solvent β-sitosterol); Figure 10 It can be seen that compared with Example 3, the oil gel prepared in Comparative Example 1 has poor mechanical properties and has obvious granularity after application, and is not suitable for transdermal administration of curcumin.

[0077] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing an oil gel, characterized in that: The following steps are involved: After preheating the oil, anti-solvent β-sitosterol and candelilla wax are added and mixed to obtain a mixture; adding curcumin to the mixture, mixing evenly, and then cooling to obtain the oil gel; The oil includes at least one of soybean oil, olive oil, linseed oil, and chia seed oil.

2. The method for preparing the oil gel according to claim 1, wherein The preheating temperature is 90°C.

3. The method for preparing the oil gel according to claim 1, wherein The mass concentration of the anti-solvent β-sitosterol in the mixture is 1% to 10%.

4. The method for preparing the oil gel according to claim 1, wherein The preparation method of the anti-solvent β-sitosterol comprises the following steps: dissolving β-sitosterol in anhydrous ethanol to obtain an anhydrous ethanol solution containing β-sitosterol; The anhydrous ethanol solution containing β-sitosterol and the PBS solution are mixed in a volume ratio of 5:1 to 1:5, and then the ethanol is removed by rotary evaporation and freeze-dried to obtain the anti-solvent β-sitosterol.

5. The method for preparing the oil gel according to claim 1, wherein The mass concentration of the candelilla wax in the mixture is 1% to 9%.

6. The method for preparing the oil gel according to claim 1, wherein The concentration of the curcumin in the mixture is 0.5 to 2.5 mg / mL.

7. The oil gel prepared according to the preparation method according to any one of claims 1 to 6.

8. A multifunctional oil gel wound dressing, characterized in that: The raw material comprises the oil gel according to claim 7.

9. A drug delivery carrier, characterized in that The raw material comprises the oil gel according to claim 7.

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