Crinis carbonisatus / castor oil-lecithin oil gel as well as preparation and application thereof

Abstract

The invention relates to the technical field of medical dressings, in particular to carbonized hair / castor oil-lecithin oil gel as well as preparation and application thereof. The preparation method comprises the following steps: firstly, calcining hair, then grinding to obtain carbonized hair, then carrying out post-treatment on the carbonized hair, and then carrying out dialysis treatment to obtain the carbonized hair carbon quantum dots; uniformly mixing lecithin and castor oil to obtain an oil gel mixture; and adding the carbonized hair carbon quantum dots into the oleogel mixture, heating and uniformly mixing, and treating to obtain the carbonized hair / castor oil-lecithin oleogel. The carbonized hair / castor oil-lecithin oil gel disclosed by the invention can be used for quickly stopping bleeding, effectively resisting breeding and reproduction of bacteria and regulating oxidative stress and inflammation microenvironment around a wound, so that wound healing is quickly promoted; the preparation method is simple in process flow and low in cost, and is a biomedical material with good application value.

Description

Technical Field

[0001] The invention relates to the technical field of medical dressings, in particular to a charcoal / castor oil-lecithin oil gel and the preparation and application thereof. Background Art

[0002] Wounds following trauma and surgery often present complex healing challenges, with uncontrolled bleeding and bacterial colonization exacerbating the challenge. A treatment approach that works with the body’s natural wound healing cascade is critical to successful wound healing. The typical process of wound repair consists of four interdependent phases: hemostasis, inflammation, proliferation, and tissue remodeling. Therefore, a typical wound dressing should not only be biocompatible and cost-effective, but also easy to use, while synergistically promoting each phase of wound healing.

[0003] The importance of safe, convenient and effective hemostasis during the complex stages of wound healing is often underestimated. Traditional hemostatic agents such as sponges, gauze and powders are insufficient due to poor adhesion and ineffectiveness for different wound types. In addition, complex manufacturing processes and challenges in ensuring biocompatibility and biosafety have hindered the application of new hemostatic materials. A new generation of wound dressings not only promotes hemostasis but also meets higher functional requirements. Wound rupture causes the local tissue to lose skin barrier protection, paving the way for pathogen invasion and subsequent persistent infection around the wound. This ultimately leads to oxidative stress, pH changes and inflammatory responses around the wound, forming a microenvironment that is not conducive to wound healing. In this harsh environment, newly formed endothelial cells are easily damaged, impairing local angiogenesis, which in turn affects nutrient supply and immune efficacy, causing the wound to fall into a chronic and non-healing state. Therefore, it is crucial to design a biocompatible material that can maintain a high level of safety while meeting the different requirements of different wound healing stages.

[0004] Wound repair has problems such as easy infection, slow repair speed and high medical costs. So far, there are a wide variety of new dressings on the market. Several major new medical dressings include foam dressings, hydrocolloid dressings, alginate dressings, etc. However, they all have some shortcomings.

[0005] ① Foam dressing: It has poor adhesion and requires an outer dressing to fix it; it is opaque and the wound condition cannot be directly observed; its raw material source is complex and the cost is relatively high.

[0006] ② Hydrogel dressings: Hydrogel dressings do not have the ability to quickly stop bleeding from wounds; they are easily confused for infection after dissolving, have an odor, and leave brown residual glue when removed. When the hydrogel dressing is replaced, it affects the fragile skin around it; hydrogel dressings are not suitable for wounds with a lot of exudate and can easily soak the skin.

[0007] ③ Alginate dressings: Alginate dressings require a second layer of dressings; alginate dressings cannot be used for wounds with a small amount of exudate and dry eschar; for wounds with a lot of exudate and deep fistulas, it is also difficult to remove residual dressings; the cost of alginate dressings is relatively high and it is not easy to promote them on a large scale.

[0008] In summary, the wound dressings available on the market have defects such as poor adhesion, opacity, inability to quickly stop bleeding, single function and high cost. Summary of the invention

[0009] In order to solve the above problems, the purpose of the present invention is to provide a charcoal / castor oil-lecithin oil gel and its preparation and application. The present invention prepares an oil gel wound dressing loaded with multifunctional charcoal carbon quantum dots by simple self-assembly, which can quickly stop bleeding while effectively resisting the growth and reproduction of bacteria, regulating the oxidative stress and inflammatory microenvironment around the wound, thereby quickly promoting wound healing; its preparation process is simple and low in cost, and it is a biomedical material with good application value.

[0010] The purpose of the present invention can be achieved by the following technical solutions:

[0011] The first object of the present invention is to provide a method for preparing a castor oil-lecithin oleogel, comprising the steps of:

[0012] (S1) calcining hair and then grinding it to obtain CrCi-carbon, and then post-treating the CrCi-carbon and performing dialysis treatment to obtain CrCi-carbon quantum dots (abbreviated as "CrCi-CDs");

[0013] Mixing lecithin and castor oil to obtain an oleogel mixture (abbreviated as "OG");

[0014] (S2) adding the CrCi-CDs / OG prepared in step (S1) to the oil gel mixture, heating and mixing, and then post-treating to obtain CrCi-CDs / OG / castor oil-lecithin oil gel (abbreviated as "CrCi-CDs / OG").

[0015] In one embodiment of the present invention, in step (S1), the hair is hair that has been washed to remove oil and fat and dried.

[0016] In one embodiment of the present invention, the hair is washed with distilled water and carbonate buffer solution to remove oil;

[0017] During the drying process, the temperature was 70°C and the time was 24 hours.

[0018] In one embodiment of the present invention, in step (S1), during the calcination process, the temperature is 300-400° C. and the time is 1-3 hours.

[0019] In one embodiment of the present invention, during the calcination process, the temperature is 350° C. and the time is 2 hours.

[0020] In one embodiment of the present invention, before dialysis, the blood charcoal is soaked in 70° C. alcohol for 1 hour and then filtered through a 0.22 μm cellulose acetate membrane.

[0021] In one embodiment of the present invention, in step (S1), during the dialysis treatment, the molecular weight cutoff of the dialysis membrane is 900-1100 Da, and the dialysis time is 64-80 h.

[0022] In one embodiment of the present invention, in step (S1), the ratio of lecithin to castor oil is 0.15 g to 0.25 g: 1 mL (when the amount of lecithin added is too little, the subsequent gel cannot be formed; when the amount of lecithin added is too much, the subsequent gel hardness is too high and is not convenient for further application);

[0023] When lecithin and castor oil are mixed, the temperature is 80-100°C.

[0024] In one embodiment of the present invention, in step (S2), the amount ratio of castor oil in the mixture of carbon quantum dots and oil gel is 4-6 mg: 1 mL.

[0025] In one embodiment of the present invention, in step (S2), during the heating and stirring process, the temperature is 80-100° C. and the time is 20-40 min.

[0026] In one embodiment of the present invention, in step (S2), the post-treatment is a room temperature cooling treatment.

[0027] The second object of the present invention is to provide a kind of charcoal / castor oil-lecithin oil gel prepared by the above method.

[0028] The third object of the present invention is to provide a use of charcoal / castor oil-lecithin oil gel in the preparation of wound dressings.

[0029] Carbon quantum dots: Quantum dots (QuantμM Dots) are extremely small-scale materials with a particle size of less than 10 nanometers. They exhibit quantum size effects and have optical and electrochemical properties that are completely different from macroscopic materials. They are a milestone in the field of artificial materials and have achieved qualitative breakthroughs in the fields of bioimaging, lighting and displays, communication technology, and photoelectrocatalysis. Professors Bawendi, Brus, and Ekimov from the United States won the 2023 Nobel Prize in Chemistry for their contributions to the "discovery and synthesis of quantum dots." Carbon quantum dots (abbreviated as carbon dots) are a new type of nanoparticle material made from carbon as raw material. In addition to the characteristics of quantum dots, they also have many advantages such as good water solubility, low toxicity, good biocompatibility, wide source of raw materials, and low cost. They show broad application prospects in the biomedical field.

[0030] Blood charcoal: Blood charcoal is a traditional Chinese medicine, mainly made from human hair that has been washed, treated with alkaline water, rinsed with clean water, dried, and then simmered and calcined into carbonized material. In traditional Chinese medicine, blood charcoal is believed to have the effects of astringent hemostasis, blood stasis removal, and diuresis. It can be used to treat a variety of bleeding symptoms (such as vomiting blood, hemoptysis, hematuria, blood in the stool, metrorrhagia, etc.) and urinary problems. The main components of blood charcoal include elements such as protein, fat, nitrogen, and sulfur, as well as minerals such as calcium, potassium, zinc, copper, iron, manganese, and arsenic. Blood charcoal has an effect on the blood system, can shorten bleeding and coagulation time, and also has antibacterial effects, inhibits certain bacteria, and has a diuretic effect.

[0031] Oil gel: Oil gel is a mixture of oil and gelling agent, and its characteristics are mainly reflected in the following aspects: 1) High consistency: Oil gel has high viscosity and fluidity, and can maintain stable shape and not easy to lose when applied or coated; 2) Good transparency: Oil gel has good transparency, which enables the user to clearly observe the condition of the object being applied; 3) Not easy to volatilize: Oil gel has low volatility and can keep the coated object moist for a long time; 4) Soft texture: Oil gel has a soft touch, which can make the user feel a comfortable application experience.

[0032] The present invention first obtains hemostatic carbon quantum dots (with good biological functions) by carbonization and purification, and then loads the carbon quantum dots onto castor oil-lecithin oil gel (the oil gel itself has anti-inflammatory and antioxidant capabilities) to obtain a multifunctional wound dressing with the capabilities of hemostasis, antibacterial, anti-inflammatory, antioxidant, and angiogenesis promotion.

[0033] The CrCi-CDs / OG prepared by the present invention can form a physical barrier at the wound site due to its high viscosity, promoting rapid hemostasis; moreover, in the wound healing stage, CrCi-CDs / OG can inhibit bacterial growth and biofilm formation, reduce oxidative stress and inflammatory cell activation, thereby regulating the wound microenvironment to promote healing; in addition, the present invention finds that CrCi-CD has strong angiogenesis properties, which can significantly enhance blood vessel growth around the wound and accelerate the wound healing process. Therefore, the CrCi-CDs / OG of the present invention can be further applied to the preparation of wound dressings.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention takes the poor adhesion of current wound dressings as its starting point, uses oil gel as the main body of the dressing, and fully utilizes the high viscosity and fluidity of the oil gel to ensure that the shape remains stable and is not easily lost during application or coating.

[0036] (2) The present invention takes the opacity of current wound dressings as a starting point and utilizes the good transparency of oil gel to ensure that the recovery of the wound can be clearly observed during use.

[0037] (3) The present invention takes the fact that current wound dressings cannot bleed quickly as a starting point, and loads the carbon quantum dots with excellent hemostatic ability onto the oil gel to achieve the purpose of sealing the wound and quickly stopping bleeding.

[0038] (4) Based on the single function of current wound dressings, the present invention prepares a castor oil-lecithin multifunctional oil gel loaded with carbon quantum dots, which can quickly stop bleeding from ruptured wounds, effectively resist the growth and reproduction of bacteria, and regulate the oxidative stress and inflammatory microenvironment around the wound, thereby effectively promoting wound healing.

[0039] (5) Based on the high cost of current wound dressings, the present invention uses hair from natural sources as raw material, carbonizes and purifies to obtain blood carbon quantum dots, and uses castor oil and lecithin as precursors to obtain the oil gel body through simple self-assembly. The materials used are simple in source and low in cost, and the preparation process is simple and has low equipment requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the preparation process of the mixture of carbon quantum dots and oil gel in Example 1;

[0041] Figure 2 This is a schematic diagram of the action of CrCi-CDs / OG prepared in Example 1;

[0042] Figure 3 This is a graph showing the hemolysis test results of CrCi-CDs / OG prepared in Example 1;

[0043] Figure 4 This is a graph showing the CCK8 experimental results of CrCi-CDs / OG prepared in Example 1 in vascular endothelial cells (HUVEC);

[0044] Figure 5 This is a graph showing the CCK8 experimental results of CrCi-CDs / OG prepared in Example 1 in RAW264.7 cells;

[0045] Figure 6 This is a graph showing the results of a live-dead staining experiment of CrCi-CDs / OG prepared in Example 1 on vascular endothelial cells (HUVEC);

[0046] Figure 7 This is a graph showing the results of a live-dead staining experiment of RAW264.7 cells using CrCi-CDs / OG prepared in Example 1;

[0047] Figure 8 This is a schematic diagram of the pathological sections of the heart, liver, spleen, lung and kidney of SD rats treated with CrCi-CDs / OG prepared in Example 1;

[0048] Fig. 9 This is a graph showing the BCI coagulation index results of CrCi-CDs / OG prepared in Example 1;

[0049] Fig.10 This is a schematic diagram of the therapeutic effect of CrCi-CDs / OG prepared in Example 1 on liver hemorrhage in New Zealand white rabbits;

[0050] Fig.11 This is the live-dead staining result of MRSA and E.coli under the stimulation of CrCi-CDs / OG prepared in Example 1;

[0051] Fig.12 This is a schematic diagram of the agar plate culture counting of MRSA and E.coli under the stimulation of CrCi-CDs / OG prepared in Example 1;

[0052] Fig.13 This is a schematic diagram of the inhibitory effect of CrCi-CDs / OG prepared in Example 1 on LPS-induced inflammation in RAW264.7 cells;

[0053] Fig.14 Schematic diagram of the inhibitory effect of CrCi-CDs / OG prepared in Example 1 on the production of ROS by RAW264.7 induced by hydrogen peroxide;

[0054] Fig.15 This is a schematic diagram showing that the CrCi-CDs / OG prepared in Example 1 promotes the proliferation of vascular endothelial cells (HUVEC);

[0055] Fig.16 This is a schematic diagram of the CrCi-CDs / OG prepared in Example 1 promoting the scratch healing of vascular endothelial cells (HUVEC);

[0056] Fig.17 This is a schematic diagram of the CrCi-CDs / OG prepared in Example 1 promoting the migration of vascular endothelial cells (HUVEC);

[0057] Fig.18 This is a schematic diagram of the CrCi-CDs / OG prepared in Example 1 promoting angiogenesis of vascular endothelial cells (HUVEC). DETAILED DESCRIPTION

[0058] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0059] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0060] Example 1

[0061] This embodiment provides a method for preparing a charcoal / castor oil-lecithin oil gel, comprising the following steps:

[0062] (S1) Hair (obtained from young healthy volunteers with no history of hair treatment such as dyeing or perming) was first washed with distilled water and carbonate buffer solution to remove any oil, and then dried at 70°C for 24 hours. Subsequently, the dried hair was calcined in a muffle furnace at 350°C for 2 hours. Once cooled to room temperature, it was pulverized using a micromill to obtain the charcoal; the charcoal fine powder was then soaked in an alcohol solution and kept in a constant temperature water bath at 70°C for 1 hour, and then filtered through a 0.22 μm cellulose acetate membrane, and then dialyzed in finished water for 72 hours using a dialysis membrane with a molecular weight cutoff of 1000 Da to obtain charcoal carbon quantum dots: CrCi-CDs ( Figure 1 A);

[0063] 0.25g, 0.50g, 0.75g, 1.00g and 1.25g of lecithin were mixed with 5ml of castor oil to obtain oleogel mixtures with concentrations of 5%, 10%, 15%, 20% and 25% (w / w), respectively, and then stirred and heated at 300rpm for 30 minutes at 90°C with a magnetic stirrer to obtain a clear and uniform solution; cooled at room temperature for 24 hours until gelation. The critical gel concentration of castor oil-lecithin oleogel was found to be 15% using a tilt test.

[0064] Therefore, a 15% concentration of oleogel mixture was used in subsequent experiments: OG (0.75 g lecithin + 5 ml castor oil) ( Figure 1 B).

[0065] (S2) Add 5 mg of CrCi-CDs to 15% castor oil-lecithin gel, heat and stir at 90°C for 30 min, and cool at room temperature for 24 h to obtain CrCi-CDs / OG; the schematic diagram of its action is shown in Figure 2 shown.

[0066] Performance Analysis:

[0067] 1) Hemolysis test (results such as Figure 3 shown):

[0068] The red blood cell suspension (2% v / v) was prepared by washing the red blood cells of New Zealand rabbits with fresh PBS. A negative control group (200 μL red blood cell suspension + 800 μL PBS), a positive control group (200 μL red blood cell suspension + 800 μL DW (distilled water)), an OG group (200 μL red blood cell suspension + 800 μL OG), a CrCi-CDs group (200 μL red blood cell suspension + 800 μL CrCi-CDs) and a CrCi-CDs / OG group (200 μL red blood cell suspension + 800 μL CrCi-CDs / OG) were prepared, and after incubation at room temperature for 1 hour, the mixture was centrifuged at 3000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate was calculated as follows:

[0069] Hemolysis rate (%) = (A s -A n ) / (A p -A n )×100

[0070] Among them A s , A n and A p Represents the absorbance of the experimental groups (OG group, CrCi-CDs group and CrCi-CDs / OG group), the negative control group and the positive control group.

[0071] pass Figure 3 It can be found that the CrCi-CDs / OG prepared in this example has good hemolytic performance.

[0072] 2) CCK8 cytotoxicity assay (results as Figure 4 and Figure 5 (shown)

[0073] 100 μL of cell suspension (5×10 3 Cells / mL) were inoculated in a single well of a 96-well plate and inoculated at 37°C, 5% CO2 for 12 hours; then, 1mg / ml OG, 1mg / ml CrCi-CDs and 1mg / mlCrCi-CDs / OG were co-cultured with HUVEC cells and RAW264.7 cells under the same conditions (the culture medium was DMEM medium containing 5% serum, 37°C, 5% CO2) for 1, 3 or 5 days, and the culture medium was replaced every 3 days. Subsequently, the culture medium was removed at each time point, 100 μL of 10% CCK8 solution was added to each well and cultured for 2 hours. Then, the absorbance at 450nm was measured, and the relative cell viability was calculated, and the calculation formula is as follows:

[0074] Relative Cell Viability=(A s -A b ) / (A c -A b )

[0075] Among them A s , A c and A b Represent the absorbance of the experimental group (10% CCK8 solution containing cells and OG, CrCi-CDs, CrCi-CDs / OG, respectively), the control group (10% CCK8 solution containing cells but no sample) and the blank group (10% CCK8 solution).

[0076] pass Figure 4 and Figure 5 It can be found that the CrCi-CDs / OG prepared in this example has no toxic effect on HUVEC cells and RAW264.7 cells.

[0077] 3) Cell live-death staining experiment (results such as Figure 6 and Figure 7 (shown)

[0078] To further evaluate the cytocompatibility of the hydrogel, the viability of HUVEC and RAW264.7 cells was detected by live / dead staining. Using the same method as the CCK8 cytotoxicity test, 1 mg / ml OG, 1 mg / ml CrCi-CDs, and 1 mg / ml CrCi-CDs / OG were co-cultured with cells (HUVEC cells or RAW264.7 cells, respectively) under the same conditions (the culture medium was DMEM culture medium containing 5% serum, 37°C, 5% CO2) for 1, 3, or 5 days, and the culture medium was replaced every 3 days. After the culture, the cells were washed with PBS, and then calcein AM and PI solutions were added, incubated in the dark at 37°C for 30 minutes, and immediately observed under a fluorescence microscope. Among them, the blank group was used without adding any material (marked as "Blank" in the figure).

[0079] pass Figure 6 and Figure 7 It can be found that the CrCi-CDs / OG prepared in this example does not affect the viability of HUVEC and RAW264.7 cells.

[0080] pass Figures 3 to 7 It can be found that the CrCi-CDs / OG prepared in this example has good biocompatibility.

[0081] 4) SD rats were treated with CrCi-CDs / OG prepared in this example (the results are shown in Table 2). Figure 8 (shown)

[0082] A back wound defect model was established in SD rats to evaluate the effect of materials (OG, CrCi-CDs, CrCi-CDs / OG) in promoting wound healing in vivo. After SD rats (weight 200-250g) were anesthetized with sutane, two circular full-thickness skin defects with a diameter of 10mm were cut on the back of SD rats. On the 1st, 3rd and 5th days after surgery, each wound was treated with 100μL 1mg / mL OG, 100μL 1mg / mL CrCi-CDs and 100μL 1mg / mL CrCi-CDs / OG, respectively; at the same time, wounds treated with sterile gauze were used as controls. On the 14th day after surgery, the heart, liver, spleen, lungs and kidneys were collected and HE staining was performed to evaluate the in vivo biocompatibility of the materials.

[0083] pass Figure 8 It can be found that the CrCi-CDs / OG prepared in this example has a strong ability to promote wound healing in vivo.

[0084] 5) BCI in vitro coagulation test and in vivo coagulation test (results such as Fig. 9 and Fig.10 (shown)

[0085] The BCI coagulation test was used to evaluate the in vitro anticoagulant ability of the materials (OG, CrCi-CDs, CrCi-CDs / OG). First, 1 mg OG, 1 mg CrCi-CDs, and 1 mg CrCi-CDs / OG were placed in a preheated 48-well plate and placed at 37°C for 10 minutes. Subsequently, 200 μL of fresh citrate anticoagulated rat whole blood was dropped on the surface of the above materials, and then 20 μL CaCl2 (0.25 M) solution was added, and then incubated at 37°C for another 5 minutes. Next, 10 mL of deionized water was added to each well and incubated at 37°C for another 5 minutes to collect the supernatant to collect free red blood cells. Finally, the absorbance of each supernatant was measured at 540 nm, and the coagulation of whole blood on cotton gauze and gelatin sponge was compared. Among them, 200 μL of blood and 20 μL of CaCl2 solution were directly added to the well as a negative control group (marked as "Blank" in the figure). The BCI is calculated as follows:

[0086] BCI(%)=A s / A b ×100%

[0087] Among them, A s and A b is the absorbance of the experimental groups (OG group, CrCi-CDs group, CrCi-CDs / OG group) and the negative control group.

[0088] Subsequently, the in vivo hemostatic ability of the material was evaluated by establishing a liver bleeding model in New Zealand white rabbits. Male New Zealand white rabbits aged 12 weeks were anesthetized with 1% sodium pentobarbital, and then nearly half of the liver was exposed, and a blank, clean filter paper was placed under the liver. A 2 cm long and 0.5 cm deep wound was cut at the same position in the liver of each New Zealand white rabbit with a scalpel to obtain a liver injury model. Subsequently, the wounds were immediately treated with 100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs, and 100 μL 1 mg / mL CrCi-CDs / OG, respectively, and wounds without any treatment were used as the control group. The hemostasis time was recorded, and the filter paper weight was weighed before and after hemostasis to obtain the amount of blood loss.

[0089] pass Fig. 9 It can be found that the CrCi-CDs / OG prepared in this example has a strong in vitro hemostatic ability; Fig.10 It can be found that the CrCi-CDs / OG prepared in this example has excellent in vivo hemostatic ability.

[0090] 6) Bacterial live and dead staining test and agar plate culture count (results such as Fig.11 and Fig.12 (shown)

[0091] The antibacterial ability was evaluated using a bacterial LIVE / DEAD staining kit. 100 μL of PBS, 100 μL of 1 mg / mL OG, 100 μL of 1 mg / mL CrCi-CDs, and 100 μL of 1 mg / mL CrCi-CDs / OG were incubated with 25 mL of a bacterial suspension in the logarithmic growth phase (methicillin-resistant Staphylococcus aureus (MRSA, Xen29) and Escherichia coli (E. coli, ATCC25922)) at room temperature in the dark for 15 minutes. 5 μL of the stained bacterial suspension was transferred to a glass slide with a pipette and covered with a coverslip for fluorescence microscopy observation.

[0092] The effect on the survival of methicillin-resistant Staphylococcus aureus and Escherichia coli was evaluated by plate count. Specifically, the bacteria were diluted to 5 × 5 in PBS. 10 CFU / mL concentration. Subsequently, 100 μL of 1 mg / mL OG, 100 μL of 1 mg / mL CrCi-CDs, and 100 μL of 1 mg / mL CrCi-CDs / OG were placed in a 24-well plate containing 1.5 mL PBS, mixed with 500 μL of bacterial suspensions in the logarithmic growth phase (methicillin-resistant Staphylococcus aureus (MRSA, Xen29) and Escherichia coli (E. coli, ATCC25922), respectively), and incubated at 37 ° C for 12 hours. After incubation, 200 μL of the mixture was evenly spread on the agar plate and further incubated at 37 ° C for 24 hours before counting the colonies.

[0093] pass Fig.11 and Fig.12 It can be found that the CrCi-CDs / OG prepared in this example has an inhibitory effect on MRSA (methicillin-resistant Staphylococcus aureus) and E. coli (Escherichia coli), and has a strong antibacterial ability.

[0094] 7) Anti-inflammatory test (results such as Fig.13 (shown)

[0095] Inflammation was induced in RAW264.7 cells by lipopolysaccharide (LPS), and quantitative real-time PCR was used to evaluate the expression of relevant inflammatory factors, including TNF-α, IL-1β, IL-6, and iNOS.

[0096] RAW264.7 cells (300,000 / well) were cultured with LPS (10 ng / mL) for 24 hours in 100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs, and 100 μL 1 mg / mL CrCi-CDs / OG treatments, respectively. Total RNA was then extracted from RAW264.7 cells using the Axygen RNA Miniprep Kit (Axygen, Union City, CA, USA). 1 mg of total RNA was reverse transcribed using the PrimeScript RT Kit (Takara Bio, Tokyo, Japan), and complementary DNA (cDNA) was used as a template for qRT-PCR assay using the TB Green Premix Ex-Taq Kit (Takara Bio) (the primers used are shown in Table 1). All reactions were performed in triplicate and normalized to the expression of the internal reference gene GAPDH. By Fig.13 It can be found that the CrCi-CDs / OG prepared in this example has strong anti-inflammatory ability.

[0097] Table 1 Primer summary

[0098]

[0099] 8) Antioxidant test (results such as Fig.14 (shown)

[0100] The generation of intracellular reactive oxygen species (ROS) was assessed using the cell-permeable fluorescent dye 5,6-carboxy 2′,7′-dichlorofluorescein diacetate (DCF-DA) in a reactive oxygen species assay kit (Beyotime, Shanghai, China).

[0101] In order to detect the fluorescence level of ROS, RAW264.7 was stimulated with hydrogen peroxide (final concentration of 10 μmol / L) to induce oxidative stress, and simultaneously placed in 100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs and 100 μL 1 mg / mL CrCi-CDs / OG, with the blank group as the control (no substance added). Subsequently, it was incubated with DCF-DA (final concentration of 10 μM) at 37°C for 15 minutes, washed twice with PBS, and fixed with 4% paraformaldehyde (pH 7.4) for 20 minutes; after washing twice more with PBS, the fluorescence of the stained cells was examined and imaged using a confocal laser microscope. Fig.14 It can be found that the CrCi-CDs / OG prepared in this example has excellent antioxidant ability.

[0102] 9) Angiogenesis assay (results as Figures 15 to 18 (shown)

[0103] Using BeyoClick TM The proliferation ability of HUVEC cells was evaluated by EdU-488 cell proliferation detection kit (Beyotime, Shanghai, China). First, HUVEC cells (300,000 per well) were co-cultured (37°C, 5% CO2) with materials (100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs, and 100 μL 1 mg / mL CrCi-CDs / OG, respectively) for 24 h, with the blank group as the control (no substance added); then cultured with 2 μM EdU working solution for 2 hours. After washing three times with PBS, it was fixed with paraformaldehyde for 15 minutes; then the cell nucleus was stained with Hoechst for 10 minutes, and finally observed under a fluorescence microscope.

[0104] The horizontal migration of HUVECs was evaluated using a wound healing assay. HUVECs were added to a 24-well plate until they reached 90% confluence; a straight, parallel, cell-free wound was formed in each well using a 200 μL pipette tip. Next, HUVEC cells were grown to 90% of the plate density and placed in the materials (100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs, and 100 μL 1 mg / mL CrCi-CDs / OG, respectively) for culture (37°C, 5% CO2), with the blank group as the control (no substance was added); and photographed after calcein AM staining at 0, 12, and 24 hours.

[0105] Transwell chambers equipped with polycarbonate filters (8 mm pore size) (Merck Millipore, MA, USA) were used to evaluate the migration of HUVEC in the vertical direction. HUVEC cells (300,000 per well) were placed in materials (100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs, and 100 μL 1 mg / mL CrCi-CDs / OG) for culture (37 ° C, 5% CO2) for 24 h, with the blank group as the control (no substance added); the cells were then transplanted into the upper Transwell chamber. DMEM supplemented with 10% FBS was added to the lower chamber, while serum-free DMEM was applied to the upper chamber. After 24 hours, the cells at the bottom of the Transwell chamber were fixed in 4% paraformaldehyde at 4 ° C for 15 minutes and stained with crystal violet for 15 minutes. Finally, each chamber was randomly sampled using an optical microscope.

[0106] For the tube formation experiment, 50 μL of Matrigel (Corning, USA) was added to each well of a 96-well plate and polymerized at 37°C for half an hour. Next, HUVEC cells (5×10 4 ) were inoculated on Matrigel and cultured in materials (100 μL 1 mg / mL OG, 100 μL 1 mg / mL CrCi-CDs and 100 μL 1 mg / mL CrCi-CDs / OG) (37°C, 5% CO2) for 24 h, with the blank group as the control (no substance added); after 8 hours, HUVEC cells were stained with Calcein AM and imaged using a fluorescent inverted microscope.

[0107] pass Figures 15 to 18 It can be found that the CrCi-CDs / OG prepared in this example has the ability to promote the proliferation, migration and angiogenesis of vascular endothelial cells (HUVEC).

[0108] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these examples and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a castor oil-lecithin oleogel, characterized in that: The steps include: (S1) calcining the hair and then grinding it to obtain blood charcoal, and then post-treating the blood charcoal and performing dialysis treatment to obtain blood charcoal carbon quantum dots; Mixing lecithin and castor oil to obtain an oil gel mixture; (S2) adding the castor oil carbon quantum dots prepared in step (S1) to the oil gel mixture, heating and mixing, and then post-treating to obtain castor oil / castor oil-lecithin oil gel.

2. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S1), the hair is washed to remove oil and then dried.

3. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S1), during the calcination process, the temperature is 300-400° C. and the time is 1-3 hours.

4. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S1), during the dialysis treatment, the molecular weight cutoff of the dialysis membrane is 900-1100 Da, and the dialysis time is 64-80 h.

5. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S1), the usage ratio of lecithin and castor oil is 0.15 g to 0.25 g: 1 mL; When lecithin and castor oil are mixed, the temperature is 80-100°C.

6. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S2), the ratio of castor oil in the mixture of carbon quantum dots and oil gel is 4-6 mg: 1 mL.

7. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S2), during the heating and stirring process, the temperature is 80-100° C. and the time is 20-40 min.

8. The method for preparing the charcoal / castor oil-lecithin oil gel according to claim 1, characterized in that: In step (S2), the post-treatment is a room temperature cooling treatment.

9. A charcoal / castor oil-lecithin oil gel prepared by the method according to any one of claims 1 to 8.

10. Use of the blood charcoal / castor oil-lecithin oil gel as claimed in claim 9 in the preparation of wound dressings.

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