Lipoic acid-based material as well as preparation method and application thereof

Abstract

The invention discloses a lipoic acid-based material as well as a preparation method and application thereof. The preparation method comprises the following steps: providing cuttlefish ink spherical melanin nanoparticles CINPs and nobiletin; the method comprises the following steps: dissolving lipoic acid LA and choline Ch in an organic solvent, and carrying out ring-opening polymerization reaction on LA and Ch to obtain an LA-Ch solution; the CINPs and the nobiletin are dissolved in the LA-Ch solution, and a precursor solution is obtained; and stirring the precursor solution until the precursor solution is viscous, so as to obtain CINPs at LA-Ch gel containing nobiletin, namely the lipoic acid-based material. After the dynamic disulfide bond contained in the zinc sulfate-based material is in contact with blood, the hydrophobic interaction force between the dynamic disulfide bond and the blood is remarkably enhanced, so that the stability of a gel network of the zinc sulfate-based material is enhanced, and the gel network of the zinc sulfate-based material is effectively prevented from being damaged due to high-pressure blood ejection; and the success rate of hemostasis is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a lipoic acid-based material and a preparation method and application thereof. Background Art

[0002] With the rapid development of society and the economy, people's quality of life has significantly improved, and their emphasis on life and health has also increased. However, despite the significant progress made in modern medical technology, the development of first aid materials, especially hemostatic materials for emergency situations, still lags behind, with many key technologies and materials remaining at the level of the last century. This situation stands in stark contrast to the overall development of modern medicine, highlighting the urgency and importance of first aid material research and development. Therefore, the development of new first aid materials for emergency medical situations not only supplements and improves modern medical technology but also is an inevitable requirement for meeting the needs of socioeconomic development and the health of the people.

[0003] In medical emergencies, such as war, traffic accidents, and industrial accidents, casualties are often devastating. Internal bleeding caused by puncture wounds with sharp objects is a leading cause of death and disability. These wounds are often characterized by rapid bleeding and high blood flow, rapidly depleting the victim's circulating blood volume and potentially exceeding the body's natural coagulation mechanisms. Furthermore, due to the deep wounds and relatively small wound surface, currently available hemostatic materials struggle to penetrate deeply into the human body to achieve rapid hemostasis. Therefore, developing emergency materials that can be easily and quickly injected, rapidly adhere to hemostasis, and prevent death and sequelae from internal bleeding is crucial for improving treatment efficiency and reducing mortality. Furthermore, these materials must effectively suppress inflammation and prevent organ failure caused by the inflammatory storm, ensuring that patients are protected within the critical rescue window and avoiding serious sequelae.

[0004] Lipoic acid (LA) is a small molecule widely found in the body. Its molecule contains multiple carboxyl groups, which can form hydrogen bonds with amino groups on tissue surfaces in moist environments, enabling strong adhesion to visceral tissues. Lipoic acid also possesses antioxidant and anti-inflammatory properties. Choline (Ch), an essential nutrient for the human body, has a trimethylamino group in its molecular structure that forms stable electrostatic interactions with the carboxyl groups of LA. This electrostatic interaction not only enhances the stability of LA but also reduces the bond energy of the disulfide bonds within the LA molecule through long-range electron effects, promoting ring opening and releasing a large number of sulfhydryl free radical sites. Long-range electron effects refer to the electronic influence of the quaternary ammonium group in choline on other distant disulfide groups within the molecule through indirect intramolecular bonding interactions (such as conjugation, hyperconjugation, and field effects). This can reduce the bond energy of the SS bonds and promote ring opening more rapidly. However, the combination of LA and choline still presents some challenges, particularly in gel preparation and application. Although choline accelerates the polymerization of lipoic acid while stabilizing the lipoic acid system to prevent its depolymerization to form lipoic acid monomers, current practice shows that this combination may cause the cohesion of the synthetic gel to be too weak during gel preparation, thereby affecting the mechanical properties and water stability of the gel.

[0005] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a lipoic acid-based material and a preparation method and application thereof, so as to solve the problem of poor stability of the existing gel prepared by using lipoic acid and choline.

[0007] The technical solutions adopted by the present invention to solve the above technical problems are as follows:

[0008] The first aspect of the present invention provides a method for preparing a lipoic acid-based material, the flow chart of which is shown in FIG. Figure 1 , the preparation method comprises the following steps:

[0009] Provides cuttlefish ink spherical melanin nanoparticles CINPs and nobiletin;

[0010] Dissolving lipoic acid LA and choline Ch in an organic solvent to allow LA and Ch to undergo a ring-opening polymerization reaction to obtain a LA-Ch solution;

[0011] dissolving the CINPs and nobiletin in the LA-Ch solution to obtain a precursor solution;

[0012] The precursor solution is stirred until it becomes viscous to obtain a CINPs@LA-Ch gel containing nobiletin, which is the lipoic acid-based material.

[0013] Preferably, the concentration of LA in the LA-Ch solution is 1-1.5 g / mL.

[0014] Preferably, in the LA-Ch solution, the concentration of Ch is 0.3-0.5 g / mL.

[0015] Preferably, the organic solvent is selected from ethanol, ethyl acetate and acetone.

[0016] Preferably, after the step of dissolving lipoic acid LA and choline Ch in an organic solvent, the method further comprises the step of stirring the mixed solution obtained after dissolution.

[0017] Preferably, the stirring speed is 300-500 rpm, and the stirring time is 60-180 min.

[0018] Preferably, the concentration of CINPs in the precursor solution is 5-20 mg / mL.

[0019] Preferably, the concentration of nobiletin in the precursor solution is 0.1-0.2 g / mL.

[0020] The second aspect of the present invention provides a lipoic acid-based material, which is prepared using the above-mentioned preparation method.

[0021] The third aspect of the present invention provides the use of the above-mentioned lipoic acid-based material, wherein the lipoic acid-based material can be used as a hemostatic material and / or an antibacterial material.

[0022] Beneficial effects:

[0023] The present invention discloses a thioctic acid-based material and its preparation method and application. The thioctic acid-based material disclosed in the present invention is a hemostatic material (CINPs, nobiletin, LA and Ch) composed of natural ingredients, with excellent biocompatibility and biological activity. In addition, the zinc sulfate-based material disclosed in the present invention has dynamic disulfide bonds and hydrogen bonds, and its unique injectability is derived from the dynamic disulfide bonds and hydrogen bonds in the zinc sulfate-based material, so that the zinc sulfate-based material can be directly injected into the visceral bleeding site caused by sharp puncture wounds. After the dynamic disulfide bonds in the zinc sulfate-based material come into contact with blood, the hydrophobic interaction force between the dynamic disulfide bonds and the blood is significantly enhanced, which not only enhances the stability of the zinc sulfate-based material gel network, but also effectively avoids the destruction of the zinc sulfate-based material gel network caused by high-pressure blood ejection, thereby improving the success rate of hemostasis.

[0024] Furthermore, the hydroxyl, catechol hydroxyl, and carboxyl groups in zinc sulfate-based materials can form electrostatic interactions or hydrogen bonds with corresponding groups in human tissue, enabling rapid adhesion and improving stability. The addition of nobiletin, through its antimicrobial properties, further reduces the risk of wound infection and creates a more favorable environment for wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The flowchart of the preparation method of lipoic acid-based materials in a preferred embodiment of the present invention is shown in FIG.

[0026] Figure 2 Middle: A is spherical melanin nanoparticles (CINPs) derived from cuttlefish ink; B is the UV-visible absorption spectrum of CINPs at a concentration of 1 mg / mL; C is the hydrodynamic diameter distribution and zeta potential of CINPs; D and E are SEM images and TEM images of CINPs; F is the temperature change of CINPs at different concentrations (0.5, 1, 2 mg / mL) under NIR irradiation (808 nm).

[0027] Figure 3 Middle: A is an image of LA-Ch and CINPs@LA-Ch hydrogel containing nobiletin; B is the temperature change of free CINPs and CINPs@LA-Ch containing nobiletin under near-infrared light irradiation (808 nm) at different concentrations; C is the thermal image of free CINPs and CINPs@LA-Ch containing nobiletin recorded by an infrared camera; D is the Raman spectra of LA powder, LA-Ch, and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; E is the FT-IR spectra of LA powder, choline, LA-Ch, and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs.

[0028] Figure 4 Middle: AC are XRD patterns of LA powder, choline, LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; D is the frequency sweep viscosity results of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; E is the dynamic scanning rheological test results of CINPs@LA-Ch containing nobiletin with 0.1% CINPs in the injection state; F is the dynamic scanning rheological test results of CINPs@LA-Ch containing nobiletin with 0.1% CINPs after solidification in water; G is the image of CINPs@LA-Ch hydrogel containing nobiletin with LA-Ch and different concentrations of CINPs hardening in water; H is the thermal remodeling of CINPs@LA-Ch containing nobiletin with 0.1% CINPs under 808 nm light irradiation.

[0029] Figure 5 Middle: A is a photo of LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs attached to pig skin after stretching, compression, twisting and bending; B is the adhesion test between fingers of LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs; C shows that LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs can be injected; D is an image of LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs injected into water; E is a lap shear test; F is the average shear strength of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs on pig skin (n=3); G is the interfacial toughness of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs on pig skin (n=3); H is a water pipe constructed using CINPs@LA-Ch hydrogel containing nobiletin containing 0.1% CINPs; I is a schematic diagram of the bursting pressure test and a schematic diagram of the bursting pressure test; J is a summary of the bursting pressures of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs (n=3).

[0030] Figure 6 Middle: A, B are hemolytic activity detection and hemolysis rate diagrams of CINPs@LA-Ch hydrogel containing nobiletin (200 μg / mL) with different concentrations of CINPs, PBS (negative control) and water (positive control) (n=5); C is the cell survival rate in CINPs@LA-Ch containing nobiletin with 0.1% CINPs; D is a representative live / dead staining image of HUVCE cells after incubation with LA-Ch and CINPs@LA-Ch hydrogel extracts containing nobiletin with different concentrations of CINPs for different times (n=5, scale bar: 300 μm); E is the representative live / dead staining image of HUVCE cells after incubation with LA-Ch and different Figure 3 is the OD value change curve of HUVEC cells on the 1st, 3rd and 5th days in CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; F is the photo of bacteria growing on agar plates treated with LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs and those not treated with hydrogel; G is the interfacial toughness of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs on pig skin (n=3); G is the coagulation behavior of the LA-Ch and CINPs@LA-Ch treatment groups containing nobiletin with different concentrations of CINPs over time. DETAILED DESCRIPTION

[0031] The present invention provides a lipoic acid-based material and its preparation method and application. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0032] Combining lipoic acid and choline to form a gel (LA-Ch system) presents several challenges, particularly in gel preparation and application. Specifically, these challenges include the following: 1. Weak gel cohesion: When lipoic acid and choline are combined, the interaction between them can loosen the gel's internal structure and reduce its cohesion. This can cause the gel to easily break or deform when subjected to external forces, impacting its effectiveness and stability. 2. Poor mechanical properties: Gels with weak cohesion often exhibit poor mechanical properties, such as tensile strength and compressive strength. These deficiencies limit their application in environments requiring resistance to external forces or pressure. 3. Poor water stability: Lipoic acid has the ability to form hydrogen bonds with tissue surfaces in moist environments. However, this property can lead to water molecules competing with the carboxyl groups of zinc sulfate for hydrogen bonding sites during gel preparation, disrupting the existing hydrogen bond network and reducing stability.

[0033] Based on this, an embodiment of the present invention provides a method for preparing a lipoic acid-based material, the preparation method comprising the following steps:

[0034] Provides cuttlefish ink spherical melanin nanoparticles CINPs and nobiletin;

[0035] Dissolving lipoic acid LA and choline Ch in an organic solvent to allow LA and Ch to undergo a ring-opening polymerization reaction to obtain a LA-Ch solution;

[0036] dissolving the CINPs and nobiletin in the LA-Ch solution to obtain a precursor solution;

[0037] The precursor solution is stirred until it becomes viscous to obtain a CINPs@LA-Ch gel containing nobiletin, which is the lipoic acid-based material.

[0038] As can be seen from the above, the LA-Ch system suffers from poor stability. Based on this, this example introduces CINPs, spherical melanin nanoparticles derived from cuttlefish ink, into the LA-Ch system. The catechol groups in CINPs can undergo polymerization reactions with certain radicals, such as thiol or carbon radicals. Therefore, CINPs act as free radical-initiated polymerization sites, enhancing the cohesion and stability of the gel network. Furthermore, as reinforcement and adhesion sites within the gel network, they can rapidly respond to wounds and promote hemostasis and tissue repair.

[0039] In addition, the catechol groups rich in the surface of CINPs can form a stable bond with the sulfur free radicals released after the ring opening of LA. At the same time, the catechol groups on CINPs also have the effect of scavenging free radicals and anti-inflammatory, which can further enhance the anti-inflammatory effect of LA.

[0040] This embodiment also introduces nobiletin into the LA-Ch system. The unique molecular structure of nobiletin enables it to further enhance the hydrophobic interaction with the LA-Ch system through hydrophobic interaction, π-π stacking, etc., thereby stabilizing the LA-Ch system. In addition, nobiletin has significant antibacterial and anti-inflammatory activity, and its antibacterial and anti-inflammatory activity produces a synergistic effect with the LA-Ch system, further enhancing the antibacterial and anti-inflammatory effect of the entire system. The LA-Ch system exerts its antibacterial effect by destroying bacterial cell membranes, interfering with bacterial metabolism, and other means. Nobiletin may be antibacterial by inhibiting bacterial cell wall synthesis, blocking bacterial DNA replication, or interfering with bacterial signal transduction. When the two are used in combination, they can act on different targets of bacteria at the same time, forming multiple antibacterial mechanisms, thereby significantly improving the antibacterial effect and exerting a synergistic effect.

[0041] This synergistic effect not only enhances the ability of lipoic acid-based materials to inhibit and kill pathogenic microorganisms but also reduces inflammatory responses, providing new insights into the treatment of infectious and inflammatory diseases. Furthermore, as a small molecule drug, nobiletin offers other advantages. For example, it can more easily penetrate cell membranes and exert its effects within cells. Furthermore, its smaller molecular weight facilitates its distribution and metabolism within the body, thereby enhancing the bioavailability and therapeutic efficacy of zinc sulfate-based materials.

[0042] In summary, the introduction of nobiletin not only enhanced the antibacterial and anti-inflammatory effects of the LA-Ch system, but also improved the stability of the LA-Ch system and exerted other advantages of small molecule drugs, providing a broader prospect for the application of the LA-Ch system in the biomedical field.

[0043] In some embodiments, in the lipoic acid-based material (CINPs@LA-Ch gel of nobiletin) provided by the present invention, the mass ratio of nobiletin, CINPs, LA and Ch is 0.1-0.2:1-1.5:0.3-0.5:5-20.

[0044] By adjusting the concentration, the gel adhesion properties and antibacterial function can be achieved in different situations.

[0045] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them, and are intended only to illustrate the present invention and in no way limit the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0046] Example 1

[0047] A preparation method of a lipoic acid-based material comprises the following steps:

[0048] (1) Extraction of CINPs: CINPs were extracted from commercial cuttlefish ink powder by centrifugation at 2000, 3000, and 4000 rpm for 10 min to remove large particles, and then centrifuged at 12000 rpm for 30 min at 4°C to obtain CINPs. Finally, the CINPs were washed three times with deionized water (DI water) and freeze-dried in a vacuum to obtain black powder with concentrations of 0.5, 1, and 2 mg / mL for subsequent use.

[0049] Particle size and zeta potential were assessed by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS): UV-Vis-NIR absorption spectra of the extracted CINPs (1 mg / mL) were measured using a Shimadzu UV-2600 spectrometer. The temperature of the CINPs aqueous solution was recorded under continuous laser irradiation (808 nm) until the solution reached a steady-state temperature after several minutes.

[0050] (2) Preparation of LA-Ch solution: Dissolve lipoic acid and choline in 5 mL of ethanol at a molar ratio of 1:0.6. Place the solution in a glass container equipped with a magnetic stirrer and stir thoroughly at 25°C for 30 minutes to allow the two to react fully. After the reaction, LA-Ch solution is obtained.

[0051] (3) Preparation of lipoic acid-based materials (CINPs@LA-Ch containing nobiletin): First, CINPs were dispersed in an ethanol medium at a concentration of 10 mg / mL by ultrasonic treatment to obtain a CINPs suspension. Subsequently, the CINPs suspension was slowly added to the LA-Ch solution prepared above at a mass ratio of 0.05%, while gently stirring to ensure that the two were evenly mixed. Then, 0.2 g / mL of nobiletin was added to obtain a precursor solution. After stirring the precursor solution at room temperature for several hours, the viscosity of the precursor solution gradually increased, and finally CINPs@LA-Ch containing nobiletin (wherein the concentration of CINPs was 0.05%) was formed.

[0052] Example 2

[0053] Preparation of a lipoic acid-based material. The method of this embodiment is basically the same as that of Example 1, except that the concentration of CINPs in the final formed CINPs@LA-Ch containing nobiletin is 0.1%.

[0054] Example 3

[0055] Preparation of a lipoic acid-based material. The method of this embodiment is basically the same as that of Example 1, except that the concentration of CINPs in the final formed CINPs@LA-Ch containing nobiletin is 0.2%.

[0056] Performance testing experiment

[0057] Characterization

[0058] The phase structure was examined by X-ray diffraction (XRD, Homelab, Rigaku). The morphology and elemental distribution of the lipoic acid-based materials were observed using a field emission scanning electron microscope (NovaNanoSem450, FEI). The valence distribution and composition of the elements were evaluated by X-ray photoelectron spectroscopy (XPS, ESCALab 250Xi, Thermo Scientific) and Fourier transform infrared (FT-IR) spectroscopy (BrukerVertex 70v, Bruker). Raman measurements were performed using a laser micro-Raman spectrometer (LabRAM HR Evolution, HORIBA Scientific) with an excitation wavelength of 532 nm. Frequency sweep viscosity measurements were performed on an AntonPaar MCR702 (AntonPaar, Graz, Austria) using a solution mode rotor at 0.1 to 100 s -1 Dynamic rheological measurements were performed at 25°C in all cases using an oscillatory mode with a time sweep to determine the storage modulus (G') and loss modulus (G") of the lipoic acid-based materials.

[0059] Tissue adhesion properties

[0060] A piece of pig skin was excised and washed with PBS. Then, 500 μL of LA-Ch and the nobiletin-containing CINPs@LA-Ch hydrogel were injected onto the pig skin at 25°C. After 10 minutes, the adhesion properties of the hydrogel on the skin were examined using stress tests such as tension, compression, twisting, and bending.

[0061] Lap shear test: First, a 50×20 mm rectangular piece of pigskin tissue was moistened, and then about 500 μL of the CINPs@LA-Ch hydrogel sample containing nobiletin was applied to one end. Next, another piece of pigskin of the same size was pressed against the hydrogel side, ensuring a contact area of 20×20 mm. The fitted pigskin was pressed for 30 seconds. The test was performed using a universal testing system (INSTRON 68TM-5) at a constant shear rate of 10 mm / min. The shear bond strength was calculated by dividing the maximum stress by the area.

[0062] 180° peel test: Porcine skin tissue was cut into 50×20 mm rectangles and wetted, then coated with approximately 500 μL of a CINPs@LA-Ch hydrogel sample containing nobiletin. Another piece of pig skin of the same size was then pressed against the hydrogel side, maintaining a 20×20 mm bonding area. After the bonded sample was pressed for 30 seconds, peel measurements were performed using a universal testing system at a constant speed of 10 mm / min. Interfacial toughness was calculated using the following formula, where Fplateau is the plateau tension and W is the width of the bonded surface.

[0063] Interface toughness (J / m 2 )="2Fplateau" / "W"

[0064] Burst pressure test

[0065] There is a 1cm 2 The porcine skin with holes was placed in a pressure bursting device connected to a syringe pump and pressure monitor. LA-Ch and CINPs@LA-Ch containing nobiletin were then deposited onto the holes, forming a hydrogel in situ. After the hydrogel formed, water was gradually injected into the pressure bursting device to apply pressure to the sealed holes. The bursting pressure was considered the critical pressure at which the seal would fail.

[0066] Hemolytic activity assay

[0067] Hemolytic activity assay: Different samples (water, PBS, LA-Ch, and CINPs@LA-Ch containing nobiletin) were added to 1 mL of erythrocyte suspension (5% in PBS, v / v). After incubation at 37°C for 4 hours, the samples were centrifuged at 2000 rpm for 10 minutes. Water and PBS were used as positive and negative controls, respectively. The absorbance of the resulting supernatant at 540 nm was read using a microplate reader to calculate the hemolysis rate.

[0068] In vitro blood coagulation test

[0069] Sodium citrate whole blood was collected from healthy rats and mixed with 0.1M calcium chloride solution at a volume ratio of 5:1. The mixture was vortexed for 3 seconds and then set aside. A 50μL volume of CINPs@LA-Ch hydrogel containing nobiletin was placed at the bottom of a 96-well plate, and 50μL of the blood sample was added. At predetermined time points, unclotted blood was gently washed with PBS. The time it took for a blood clot to form was recorded as the blood clotting time.

[0070] Cytotoxicity assessment

[0071] First, CINPs@LA-Ch hydrogel (40 mg) containing nobiletin was placed in 2 mL of DMEM medium and incubated at 37 ° C for 48 hours to prepare the hydrogel extract. The extract was sterilized by filtering through a 0.22 μm filter and then supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. HUVEC cells were seeded in 96-well plates at a density of 1000 cells per well and incubated for 24 hours. The culture medium was then replaced with the hydrogel extract prepared above and incubated for another 1, 3, and 5 days. Complete original DMEM was used as a control. Cell viability was assessed using a cell counting kit-8 (CCK-8) according to the manufacturer's instructions.

[0072] Similarly, HUVCE cells were seeded in 96-well plates, and the culture medium was replaced with the hydrogel extract prepared above. The cells were incubated for 1, 3, and 5 days, respectively. Cells were stained for viability using a Calcein / PI cell staining kit, and cell viability was assessed by laser confocal microscopy.

[0073] In vitro antibacterial activity

[0074] To investigate the antibacterial activity of LA-Ch and nobiletin-containing CINPs@LA-Ch hydrogels, hydrogel extracts were co-cultured with Staphylococcus aureus and Escherichia coli. The bacterial culture was extracted and diluted 10,000-fold. Subsequently, 100 μL of the diluted bacterial solution was evenly spread onto a Luria-Bertani (LB) plate using a cell spreader. Optical images were captured after overnight incubation at 37°C.

[0075] The above test results are as follows:

[0076] Figure 2 Middle: A is spherical melanin nanoparticles (CINPs) derived from cuttlefish ink; B is the UV-visible absorption spectrum of CINPs at a concentration of 1 mg / mL; C is the hydrodynamic diameter distribution and zeta potential of CINPs; D and E are SEM images and TEM images of CINPs; F is the temperature change of CINPs at different concentrations (0.5, 1, 2 mg / mL) under NIR irradiation (808 nm).

[0077] Transmission electron microscopy (TEM) results showed that CINPs had uniform particle size ( Figure 2 Dynamic light scattering (DLS) further confirmed that the hydrated particle size of CINPs dispersed in water was approximately 190 nm ( Figure 2 C). Such as scanning electron microscope ( Figure 2 Middle D), transmission electron microscopy ( Figure 2 As shown in Figure E), CINPs appear as spherical particle aggregates with an average diameter of 130 nm and a rough surface, with good dispersibility and uniform particle size. At the same time, the zeta potential of CINPs was detected to be -17 mV, and the absolute value of the zeta potential was large, which means that CINPs have good dispersibility. Ultraviolet-visible spectrophotometer (UV) can reflect the absorption behavior of NPs (nanoparticles) at different wavelengths. CINPs show broad spectrum absorption (UV-visible) spectroscopy in the range of 200 to 900 nm. Figure 2 The photothermal properties of CINPs in aqueous solution were evaluated by NIR laser (808 nm, 0.5-1 W / cm 2 ) were irradiated in PBS solutions of different concentrations for 15 minutes. During the irradiation test, the temperature of PBS did not increase significantly. The CINPs solution showed a concentration- and super-irradiation intensity-dependent temperature rise under near-infrared irradiation, ranging from ≈30-80°C, with a maximum of ≈80°C at 2 mg / mL. Figure 2 Figure (F) shows that CINPs have good photothermal transduction properties. Among them, the laser power determines the photothermal effect of CINPs. With the increase of laser power, the temperature of CINPs increases significantly. For CINPs of different concentrations, the laser intensity is 1W / cm 2 It showed the best photothermal properties, so 1W / cm2 was used uniformly in the future. 2 The photothermal experiment was carried out with the laser power of 100 nm.

[0078] Figure 3 Middle: A is an image of LA-Ch and CINPs@LA-Ch hydrogel containing nobiletin; B is the temperature change of free CINPs and CINPs@LA-Ch containing nobiletin under near-infrared light irradiation (808 nm) at different concentrations; C is the thermal image of free CINPs and CINPs@LA-Ch containing nobiletin recorded by an infrared camera; D is the Raman spectra of LA powder, LA-Ch, and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; E is the FT-IR spectra of LA powder, choline, LA-Ch, and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs.

[0079] Different ratios of CINPs were introduced into the mixture of lipoic acid and choline to obtain CINPs@LA-Ch composite gels containing nobiletin with CINPs proportions of 0.05%, 0.1% and 0.2% ( Figure 3 Middle A).

[0080] Verification of the photothermal properties of CINPs@LA-Ch gels containing nobiletin at different ratios ( Figure 3 B and C), during the irradiation measurement (1W / cm 2 ), while the temperature of PBS did not significantly increase. The CINPs@LA-Ch gel containing nobiletin also produced a photothermal effect, primarily due to the CINPs. CINPs-containing PBS solutions (0.5, 1, and 2 mg / mL, respectively) were tested at the same concentration as the gel group as a control. As the CINPs concentration in LA-Ch increased, the temperature increased accordingly. The temperature rose rapidly within 5 minutes of laser irradiation, after which the temperature trend decreased.

[0081] After synthesis, the CINPs@LA-Ch gel containing nobiletin can be heated to 75°C by infrared heating to form a fluid, injectable gel. It can also remain injectable for a month after cooling to room temperature. This excellent injectability makes it easier to use and inject into wounds of varying shapes.

[0082] like Figure 3 Shear rate-viscosity analysis of 0, 0.05, 0.1, and 0.2% nobiletin-containing CINPs@LA-Ch glues (D) highlights their excellent shear-thinning behavior. The viscosity of the gels decreases with increasing shear rate, demonstrating injectability and shape adaptability. This property is particularly beneficial for covering irregularly shaped wounds, highlighting the versatility and potential of nobiletin-containing CINPs@LA-Ch glues in biomedical applications.

[0083] Due to the long-range electronic effect and nucleophilic carboxylic acid, deprotonating LA to promote ring-opening polymerization is an efficient and simple method. Choline (Ch) is added to a lipoic acid ethanol solution, where the optimal mass ratio of LA and Ch is 1:0.6. The two form an LA-Ch ionic liquid, which can promote ring-opening polymerization more quickly at room temperature. FT-IR was further used to verify the changes in chemical bonds after the combination, such as Figure 3 As shown in E, the stretching vibration of the C=O bond in the -COOH group is from 1694 cm -1 Transformed to 1580cm -1 The asymmetric stretching vibration and 1380cm -1This change confirms the deprotonation of LA by choline.

[0084] The changes in the ring-opening polymerization of LA's cyclic disulfide cyclopentanes can be detected by Raman spectroscopy. By comparing the SS bond peaks in the Raman spectra of CINPs@LA-Ch containing nobiletin at different ratios of 0, 0.05, 0.1, and 0.2% and LA, the reactivity of the SS bond in the samples is reflected. The SS peak of LA-Ch shifts from 508 to 503 and 524 cm -1 This indicates that the cyclic disulfide ring opens after deprotonation. In addition, with the increase of CINPs ratio, the change rate of SS bond peak decreases, which indicates that the addition of CINPs may form a chemical bond with the opened sulfur ring.

[0085] Figure 4 Middle: AC are XRD patterns of LA powder, choline, LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; D is the frequency sweep viscosity results of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; E is the dynamic scanning rheological test results of CINPs@LA-Ch containing nobiletin with 0.1% CINPs in the injection state; F is the dynamic scanning rheological test results of CINPs@LA-Ch containing nobiletin with 0.1% CINPs after solidification in water; G is the image of CINPs@LA-Ch hydrogel containing nobiletin with LA-Ch and different concentrations of CINPs hardening in water; H is the thermal remodeling of CINPs@LA-Ch containing nobiletin with 0.1% CINPs under 808 nm light irradiation.

[0086] X-ray diffraction (XRD) was used to verify the structural changes. Figure 4 Figures A and B show the sharp crystallization peaks of LA monomer and Ch monomer. After adding Ch to LA, the crystallization peaks of the original LA and Ch completely disappeared ( Figure 4 (C) This suggests that the post-polymerization of LA and Ch stabilizes LA and inhibits its depolymerization. CINPs@LA-Ch containing nobiletin at varying ratios also exhibited a disappearance of the crystalline diffraction peak after the addition of CHIPs. This indicates that the amorphous phase dominates the ternary system of CHIPs, LA, and Ch.

[0087] Figure 4Step strain measurements in Figures E and F reveal the self-healing ability of the nobiletin-containing CINPs@LA-Ch glue before and after injection into water. Under alternating cycles of low (1%) and high (300%) strain, the glue exhibited distinct behaviors, namely, those of an elastic solid and a viscous liquid, respectively, before immersion in water. While the G' and G" values dropped sharply upon transitioning from low to high strain, the glue quickly recovered its original modulus upon abruptly removing the high strain. This ability to alternate between elastic solid and viscous liquid behavior under varying strain conditions demonstrates the self-healing properties of the nobiletin-containing CINPs@LA-Ch glue, which is crucial for wound healing applications where hydrogels may encounter mechanical stress. The self-healing properties were also verified after injection into water for curing. Testing revealed that the glue exhibited the same self-healing properties after curing in water, but with a significant increase in the G' and G" values. This indicates that the mechanical strength of the glue increases upon exposure to water, while still exhibiting some modulus variation under alternating cycles of low and high strain. This may be because although the CINPs@LA-Ch glue containing nobiletin is enhanced after contact with water due to the interaction between the glue and water molecules, there are many reversible chemical bonds between LA-Ch molecules, so the glue network still exhibits self-healing properties.

[0088] like Figure 4 As shown in G, 0, 0.05, 0.1 and 0.2% CINPs@LA-Ch glue containing nobiletin was significantly whitened compared to the original sample after curing in water. This phenomenon may be caused by the interaction between the glue and water molecules. In order to more intuitively verify the situation of CINPs@LA-Ch glue containing nobiletin after NIR irradiation, 0.1% CINPs@LA-Ch glue containing nobiletin was squeezed into a glass beaker and irradiated with NIR laser (808nm, 1W / cm 2 ) irradiation and observation ( Figure 4 In the H), it was found that the glue transformed from a solid state to a fluid gel, which was due to the high temperature destroying the polymerization degree of LA and reducing the viscosity of the glue.

[0089] Figure 5Middle: A is a photo of LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs attached to pig skin after stretching, compression, twisting and bending; B is the adhesion test between fingers of LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs; C shows that LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs can be injected; D is an image of LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs injected into water; E is a lap shear test; F is the average shear strength of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs on pig skin (n=3); G is the interfacial toughness of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs on pig skin (n=3); H is a water pipe constructed using CINPs@LA-Ch hydrogel containing nobiletin containing 0.1% CINPs; I is a schematic diagram of the bursting pressure test and a schematic diagram of the bursting pressure test; J is a summary of the bursting pressures of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs (n=3).

[0090] First, the sample is subjected to a torsion test, such as Figure 5 As shown in Figure A. CINPs@LA-Ch hydrogel containing nobiletin can firmly adhere to wet pig skin without falling off and maintain its original shape under different external stresses (stretching, bending and twisting), indicating that a strong interfacial adhesion force has been formed and the gel has sufficient deformation ability to withstand the activities of internal organs. Next, the glove adhesion test was carried out ( Figure 5 In Figure B), the four groups of CINPs@LA-Ch hydrogel containing nobiletin, 0%, 0.05%, 0.1% and 0.2%, all showed excellent adhesion properties, indicating that the CINPs@LA-Ch hydrogel material containing nobiletin itself has good adhesion properties. In particular, when two fingers were stretched apart, the most obvious stringing phenomenon was observed in the 0.1% CINPs@LA-Ch hydrogel group containing nobiletin. The strings were thick and continuous, which further highlighted the superior adhesion performance of the CINPs@LA-Ch hydrogel containing nobiletin at this concentration. At the same time, although the groups with other concentrations also showed good adhesion, the stringing phenomenon did not reach the significant level of the 0.1% group. This phenomenon indicates that the 0.1% CINPs@LA-Ch hydrogel containing nobiletin may have reached a certain optimized state in terms of intermolecular interaction, material toughness or adhesion mechanism, allowing more stable and thicker gel filaments to be formed during the stretching process.

[0091] The injectability of the gel is crucial for its infusion into human tissue. Therefore, injectability experiments were conducted on 0%, 0.05%, 0.1%, and 0.2% CINPs@LA-Ch hydrogels containing nobiletin. When the 0%, 0.05%, 0.1%, and 0.2% CINPs@LA-Ch hydrogels were drawn into a 1 mL cylindrical syringe, they could all be easily extruded from the needle to achieve the desired shape and morphology, demonstrating good injectability.

[0092] Biocompatibility is a fundamental prerequisite for the clinical translation of adhesives. In vitro tests were performed to evaluate the biocompatibility and biodegradability of 0.1% nobiletin-containing CINPs@LA-Ch hydrogels. During 1, 3, and 5 days of culture, CCK-8 and live / dead staining assays revealed that HUVEC viability reached its maximum at 100 μg / mL, with almost no dead cells (red fluorescence) detected, confirming the cytocompatibility of the adhesive ( Figure 5 C and D).

[0093] Figure 6 Middle: A, B are hemolytic activity detection and hemolysis rate diagrams of CINPs@LA-Ch hydrogel containing nobiletin (200 μg / mL) with different concentrations of CINPs, PBS (negative control) and water (positive control) (n=5); C is the cell survival rate in CINPs@LA-Ch containing nobiletin with 0.1% CINPs; D is a representative live / dead staining image of HUVCE cells after incubation with LA-Ch and CINPs@LA-Ch hydrogel extracts containing nobiletin with different concentrations of CINPs for different times (n=5, scale bar: 300 μm); E is the representative live / dead staining image of HUVCE cells after incubation with LA-Ch and different Figure 3 is the OD value change curve of HUVEC cells on the 1st, 3rd and 5th days in CINPs@LA-Ch containing nobiletin with different concentrations of CINPs; F is the photo of bacteria growing on agar plates treated with LA-Ch and CINPs@LA-Ch hydrogels containing nobiletin with different concentrations of CINPs and those not treated with hydrogel; G is the interfacial toughness of LA-Ch and CINPs@LA-Ch containing nobiletin with different concentrations of CINPs on pig skin (n=3); G is the coagulation behavior of the LA-Ch and CINPs@LA-Ch treatment groups containing nobiletin with different concentrations of CINPs over time.

[0094] The hemolytic activity test of CINPs@LA-Ch hydrogels containing nobiletin at different ratios showed good blood compatibility, and the hemolysis rate was less than 2% ( Figure 6A and B). An ideal first aid medical glue should protect wounds from bacterial infection. Therefore, bacteria (Escherichia coli and Staphylococcus aureus) were incubated with 100 μg / mL of the above-mentioned 0.1% CINPs@LA-Ch hydrogel extract containing nobiletin to evaluate the in vitro antibacterial activity. After 12 hours of incubation, the antibacterial rates measured for all tested microorganisms were higher than those of LC-Ch ( Figure 6 F). These results reveal the anti-infective ability of the glue against Gram-positive and Gram-negative bacteria. Low cytotoxicity and good blood compatibility are important prerequisites before the application of hydrogels as hemostatic materials in vivo. Effective sealing hemostatic gels must immediately induce blood coagulation and minimize blood cell damage. To evaluate the coagulation ability, the coagulation time of whole blood was determined by adding it to 96-well plates coated with different samples ( Figure 6 (G) Blood coagulation in wells coated with 0%, 0.05%, 0.1%, and 0.2% nobiletin-containing CINPs@LA-Ch hydrogels took 0.25 minutes, respectively, significantly faster than the 6 minutes in uncoated wells (control). The superior coagulation ability of the nobiletin-containing CINPs@LA-Ch hydrogels may be attributed to the interaction between the SS bonds on LA and the catechol hydroxyl groups on CINPs and ions on negatively charged blood cells and proteins, thereby forming flocculation to accelerate the blood coagulation process.

[0095] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a lipoic acid-based material, characterized in that: The preparation method comprises the following steps: Provides cuttlefish ink spherical melanin nanoparticles CINPs and nobiletin; Dissolving lipoic acid LA and choline Ch in an organic solvent to allow LA and Ch to undergo a ring-opening polymerization reaction to obtain a LA-Ch solution; dissolving the CINPs and nobiletin in the LA-Ch solution to obtain a precursor solution; The precursor solution is stirred until it becomes viscous to obtain a CINPs@LA-Ch gel containing nobiletin, which is the lipoic acid-based material.

2. The preparation method of lipoic acid-based material according to claim 1, wherein In the LA-Ch solution, the concentration of LA is 1-1.5 g / mL.

3. The preparation method of lipoic acid-based material according to claim 1, wherein In the LA-Ch solution, the concentration of Ch is 0.3-0.5 g / mL.

4. The preparation method of lipoic acid-based material according to claim 1, wherein The organic solvent is selected from one of ethanol, ethyl acetate and acetone.

5. The preparation method of lipoic acid-based material according to claim 1, wherein After the step of dissolving lipoic acid LA and choline Ch in an organic solvent, the method further comprises the step of stirring the mixed solution obtained after the dissolution.

6. The method for preparing a lipoic acid-based material according to claim 5, wherein The stirring speed is 300-500 rpm, and the stirring time is 60-180 min.

7. The preparation method of lipoic acid-based material according to claim 1, wherein In the precursor solution, the concentration of CINPs is 5-20 mg / mL.

8. The preparation method of lipoic acid-based material according to claim 1, wherein In the precursor solution, the concentration of nobiletin is 0.1-0.2 g / mL.

9. A lipoic acid-based material, characterized in that The lipoic acid-based material is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the lipoic acid-based material according to claim 9, characterized in that The lipoic acid-based material can be used as a hemostatic material and / or an antibacterial material.

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