Lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel, preparation method and application

Abstract

The invention discloses a lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel (LA-MPC deep eutectic gel) as well as a preparation method and application thereof, and belongs to the field of medical biological materials. According to the LA-MPC deep eutectic gel, appropriate raw materials are screened, the characteristics of an LA hydrogen bond donor and an MPC hydrogen bond acceptor are combined, the deep eutectic effect is introduced, and the deep eutectic gel formed by co-melting two kinds of powder is prepared under the simple conditions of no solvent and 40 DEG C water bath heating. The preparation method disclosed by the invention is simple and convenient to operate, mild in reaction condition and low in cost, does not need complex chemical reaction and tedious raw material input, and effectively avoids the safety problems of cytotoxicity and the like caused by introduction of an organic solvent, a cross-linking agent and other solutions. The deep eutectic gel provided by the invention has good mechanical properties and self-healing ability, can be used as a novel anti-inflammatory skin dressing, and can effectively relieve inflammatory response of a wound part and promote wound healing in a full-thickness skin injury model of a rat.

Description

Technical Field

[0001] The invention belongs to the field of medical biomaterials and relates to a lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel, a preparation method and an application thereof. Background Art

[0002] As the largest organ in the human body, the skin, with its integrated multilayered structure (including the epidermis, dermis, and subcutaneous tissue), performs a crucial barrier function. However, epidemiological data show that over 30 million new cases of chronic, non-healing wounds occur worldwide each year, with infection rates exceeding 60% for diabetic foot ulcers. This clinical dilemma stems from the skin's continuous exposure to multiple threats, including mechanical injury, pathogens, and oxidative stress, which hinder wound healing. Wound healing is a dynamic and complex biological process encompassing four interconnected phases: hemostasis, inflammation, proliferation, and remodeling. The hemostatic phase occurs 0-2 hours after injury, characterized by platelet aggregation to form a fibrin clot, initiating the healing cascade. The inflammatory phase, occurring 2-5 days after injury, is characterized by pathogen clearance by neutrophils and the initiation of the inflammatory response by M1 macrophages. Over the next 5-14 days, the wound enters a proliferative phase, characterized by fibroblast migration, angiogenesis, and collagen deposition. Finally, the wound enters a prolonged remodeling phase, during which collagen cross-linking and scar maturation occur.

[0003] A key issue affecting wound healing is that over 85% of chronic wounds remain stuck in the inflammatory phase due to persistently high concentrations of pro-inflammatory factors (such as TNF-α and IL-6). Currently, there is no effective topical treatment available clinically to alleviate the inflammatory response at the wound site. Furthermore, surgical sutures are widely used as the gold standard for closing wounds clinically, but this method struggles to cope with the challenges of irregular wounds and wound microenvironments. Compared to traditional materials, hydrogels are expected to become the next generation of wound closure materials due to their biomimetic ECM structure, dynamic mechanical properties that match skin mechanics, and a load-release system to maintain local drug concentrations. Therefore, the development of hydrogel dressings that can cope with the skin's inflammatory microenvironment is of great significance.

[0004] Lipoic acid (LA) is an endogenous small molecule coenzyme with excellent anti-inflammatory and antioxidant properties due to its unique five-membered disulfide heterocycle structure. Its mechanisms of action include biphasic antioxidant activity and inhibition of the NF-κB pathway. Currently, researchers are attempting to prepare LA into hydrogels for medical applications. For example, Professor Liu Wenguang's team at Tianjin University has designed a hydrogel composed of LA and methacrylate-modified gelatin. The hydrogel exhibits wettable adhesion and excellent antioxidant activity, making it suitable for the treatment of periodontitis. However, existing synthesis strategies often rely on free radical polymerization or the use of double-bond crosslinkers, which not only complicate the reaction steps but also pose the risk of chemical toxicity. Therefore, the development of green preparation processes is urgently needed. Summary of the Invention

[0005] In order to overcome the shortcomings of the above technologies, the purpose of the present invention is to avoid the introduction of tedious chemical reactions and harmful reagents, prepare a new anti-inflammatory skin dressing that is simple, easy to synthesize and has good use effects, and provide a preparation method and application of LA-MPC deep eutectic gel.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] The LA-MPC deep eutectic gel of the present invention is prepared by selecting suitable raw materials, combining the characteristics of LA hydrogen bond donors and MPC hydrogen bond acceptors, and introducing a deep eutectic effect. This deep eutectic gel is formed by co-melting the two powders in a simple, solvent-free, 40°C water bath heating condition. The preparation method of the present invention is simple to operate, has mild reaction conditions, and is low-cost. It does not require complex chemical reactions or cumbersome raw material inputs, and effectively avoids safety issues such as cytotoxicity caused by the introduction of organic solvents, cross-linking agents, and other solutions.

[0009] The LA-MPC deep eutectic gel of the present invention has good biological tissue adhesion and can be fixed on the wound surface; it has good self-healing properties and can heal itself after being broken and deformed under the action of external force; it has good antioxidant activity and can promote wound healing.

[0010] The deep eutectic gel of the present invention has suitable mechanical properties and self-healing ability, can adhere to the skin and serve as a new type of skin dressing. In a rat full-thickness skin injury model, it can effectively reduce the inflammatory response at the wound site and increase the speed of wound closure and skin tissue reconstruction. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Photographs of gel macromorphology;

[0012] Figure 2 The microstructure of the gel under an electron microscope;

[0013] Figure 3 Gel strain sweep rheology test

[0014] Figure 4 Gel time scan rheology test;

[0015] Figure 5 Gel viscosity scanning rheology test;

[0016] Figure 6 Scanning rheological test of gel self-healing;

[0017] Figure 7 Gel swelling capacity test;

[0018] Figure 8 Gel adhesion ability test;

[0019] Figure 9 Image of the full-thickness skin injury model in SD rats;

[0020] Figure 10 H&E and Masson staining images of wound tissue; DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0022] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0023] The present invention is described in further detail below with reference to the accompanying drawings:

[0024] Example 1

[0025] Weigh a certain amount of LA and MPC and place them in an EP tube. Vortex for 1 minute to thoroughly mix the two powders, and heat in a water bath at 40°C for 30 minutes to obtain a transparent yellow gel. By trying various ratios, it was found that when the mass ratio of LA to MPC was less than 1.5:1, MPC did not melt fully, and a large amount of MPC solids could be observed in the gel; by increasing the LA content, when the mass ratio reached 1.5:1, the melting point of the LA-MPC blend dropped to about 40°C, and both powders melted completely; by further increasing the LA content, when the mass ratio was 2:1, both powders melted completely, but the gel appeared solid and lost fluidity, which was not conducive to application on the skin surface. Therefore, LA:MPC=1.5:1 was selected as the final preparation process. The state of the deep eutectic gel under various ratios is shown as follows. Figure 1 shown.

[0026] 1. Hydrogel morphology characteristics experiment

[0027] The injectable hydrogel sample prepared in Example 1 was placed in a vacuum freeze dryer for freeze drying. The dried gel sample was taken out and cut in liquid nitrogen. The cross section of the gel was fixed with conductive glue and sprayed with gold. The microscopic morphology of the cross section of the sample was then observed using a scanning electron microscope. Figure 2 As shown, from Figure 2 The hydrogel can be observed to have a relatively regular three-dimensional mesh pore structure, which provides good mechanical strength and elasticity. Without destroying its overall structure, the hydrogel can withstand certain mechanical stresses and deformations, which is beneficial for bearing certain mechanical loads in areas with high mobility. The formation of this three-dimensional network structure may be due to the hydrogen bonding between LA and MPC, forming a loose network structure, which is why the three-dimensional mesh pore structure is observed under a scanning electron microscope.

[0028] 2. Mechanical properties experiment of injectable hydrogel

[0029] Rheological tests of LA-MPC Gel hydrogels were performed using an Anton Paar rheometer (MCR 302, Austria). All rheological measurements were performed on a cone-plate with a diameter of 25 mm and the temperature was set to 37°C. In the strain sweep test, the dynamic storage modulus (G') and loss modulus (G") were measured for each sample at a frequency of 1 Hz, with a strain range of 1% to 2000%. The dynamic time sweep experiment was performed at a strain value of 1% and a frequency of 1 Hz, with a time range of 0 to 200 s. At a constant frequency of 1 Hz, three step strain sweeps (100 s per cycle) were performed with alternating switching between high shear strain (500%) and low shear strain (0.5%). The time from 1 to 1000 s was recorded.-1 Viscosity-shear rate curve. Figure 3 As shown in the strain sweep, when the strain range is from 1% to 1500%, the storage modulus G' is greater than the loss modulus G', indicating that the gel has a relatively stable cross-linked structure. The stability of the LA-MPC deep eutectic gel was evaluated by time sweep, as shown in Figure 4 As shown in Figure 2, the G' and G' values ​​of the gel remained stable during the test time (0–300 s). Figure 5 As shown in the figure, the viscosity curve shows that the LA-MPC deep eutectic gel has a high viscosity, which means it is difficult to flow during application, thus being able to remain in the wound site for a long time. In addition, the LA-MPC deep eutectic gel also has significant shear thinning phenomenon, which has the potential for injection.

[0030] 3. Self-healing experiment

[0031] The LA-MPC deep eutectic gel was tested by rheological method. Under the conditions of frequency of 1 Hz, low strain of 5%, high strain of 500%, low strain and high strain were changed alternately, each strain interval was 100 s, and the cycle was repeated 3 times. Step strain test was performed ( Figure 6 When the gel was subjected to a large stress (500%), G' and G'' decreased rapidly, indicating the collapse of the hydrogel network structure. However, when the stress was restored to 0.1%, the G' and G'' values ​​quickly returned to their initial levels, demonstrating the efficient and rapid self-healing function of the LA-MPC deep eutectic gel.

[0032] 4. Swelling rate experiment

[0033] Weigh 3 portions of LA-MPC Gel, 0.15 g each, and place them in 50 mL EP tubes. Add 40 mL of ultrapure water to each tube and shake them in a constant temperature water bath at 37°C and 120 rpm / min for 24 h. Afterwards, weigh the tubes. Figure 7 As shown, the swelling rate of LA-MPC Gel is extremely low, reaching 20% ​​at equilibrium. This is due to the strong hydrophobicity of the lipoic acid in the gel, which makes it difficult to bind to water molecules, thus maintaining the gel swelling rate at a low level. The low swelling rate prevents the LA-MPC deep eutectic gel from losing its adhesion in a wet environment. At the same time, the gel structure is not easily destroyed by wound exudate, maintaining continuous adhesion to the wound site.

[0034] 5. Adhesion strength test

[0035] LA-MPC deep eutectic gel was sandwiched between two layers of pigskin, glass, wood, and metal surfaces, with a gel area of ​​4 cm 2 , use a universal testing machine to stretch and test the maximum adhesion strength of the gel on the surface of each material. Figure 8 As shown in the figure, the gel has a maximum adhesion strength of about 60, 80 and 300 kPa on glass, wood and metal surfaces, respectively, and a maximum adhesion strength of about 16 kPa on the surface of fresh pig skin. This strong adhesion ability should be attributed to the rich hydrogen bonds of LA and the electrostatic interaction provided by MPC, which enables the LA-MPC deep eutectic gel to adhere firmly to the surface of skin tissue and not easily fall off.

[0036] Example 2

[0037] 1. Evaluation of the therapeutic effect of LA-MPC deep eutectic gel on SD rat skin injury model

[0038] Twelve male SD rats weighing 250-300 g were randomly divided into three groups: a 3-day group, a 7-day group, and a 14-day group. After anesthesia, the rats were skin-prepared. Three wounds were created on similar sites on the rats' backs using a 10 mm diameter punch. These wounds were divided into the model group, the 3M membrane group, and the gel group. The 3M membrane group was treated with a 10 mm diameter 3M membrane, and the gel group was treated with 0.1 mL of gel. All wounds were sutured with bandages to prevent contamination.

[0039] Images of the wounds were taken on days 0, 3, 7, and 14, and the wound area healing rate was calculated.

[0040] The wound healing trajectory heat map and wound healing rate quantification showed that the wound in the model group was not completely closed at 14 days, with a closure rate of 80%; the wound closure rate in the 3M group was 85% at 14 days, slightly faster than that of the model group; the wound in the gel group was completely closed at 14 days, and hair regeneration was observed at the wound site, indicating that the wound healed well.

[0041] Example 3

[0042] 1. Tissue H&E staining and Masson staining

[0043] The rats were euthanized, and the skin tissues at the wound sites were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin and sliced. The pathological changes of the skin tissues were observed under an optical microscope after HE staining. Figure 10 It can be seen that obvious tissue defects and inflammatory reactions were observed in the model group at all time points, and the wound had not yet healed completely; the wound in the 3M membrane group had healed at 14 days, but there was still obvious inflammatory reaction, and the new connective tissue was irregular; the wound in the gel group was significantly smaller than that in the model group and the 3M membrane group at 7 days, and the wound was completely healed at 14 days without obvious inflammatory reaction. New hair follicles and structurally orderly new connective tissue could be observed.

[0044] The experimental data of the present invention were processed using SPSS15.0 statistical software package, and the test results were expressed as mean plus or minus standard deviation. The quantitative data were tested using Student's T test; the means between groups were compared using analysis of variance (ANOVA).

[0045] The above experimental results comprehensively show that the LA-MPC deep eutectic gel prepared in the present invention has a good therapeutic effect in the rat full-thickness skin injury model without obvious organ toxicity and side effects, providing a new idea for the development of new skin injury dressings.

[0046] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel, comprising the following steps: 1) Weigh a certain amount of lipoic acid (LA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) so that the mass ratio is 1.5:

1. 2) Vortex for 1 min to thoroughly mix the two powders. 3) Heating in a water bath at 40°C for 30 min yielded a lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel.

2. The method for preparing the lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel according to claim 1, characterized in that: The eutectic point of the mixture to form a gel is 40°C.

3. The method for preparing the lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel according to claim 1, characterized in that: The mass ratio of LA to MPC is 1.5:

1.

4. The method for preparing the lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel according to claim 1, characterized in that: The molecular weight of the lipoic acid is 206.

32.

5. The method for preparing the lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel according to claim 1, characterized in that: The molecular weight of the 2-methacryloyloxyethyl phosphorylcholine is 295.

27.

6. The lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel according to any one of claims 1 to 5, characterized in that: It is a gel formed by lowering the melting point after mixing LA and MPC solids.

7. A use of the lipoic acid-2-methacryloyloxyethyl phosphorylcholine deep eutectic gel according to claim 6, characterized in that: As a new type of anti-inflammatory skin dressing, it reduces inflammatory response and promotes wound healing.

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