A method for preparing chitosan-derived carbon dots, the derived carbon dots and their applications

Abstract

This application discloses a method for preparing chitosan-derived carbon dots, the derived carbon dots themselves, and their applications. These carbon dots are prepared by amidation covalent coupling of octyl-citric acid chitosan-based amphiphilic polymer carbon dots and 2,3-epoxypropyltrimethylammonium chloride-modified lignin-based carbon dots. The octyl hydrophobic chain disrupts the hydrogen bond shielding of chitosan, exposing active sites and enhancing reactivity. Quaternization modification of the lignin-based carbon dots improves compatibility and imparts targeting properties. This achieves a triple synergistic anti-aging effect, scavenging reactive oxygen species, upregulating LaminB1 expression, and inhibiting IL-6 / IL-8 secretion. Cell experiments show that these carbon dots significantly reduce UVA-induced oxidative damage, increase hyaluronic acid synthesis, and reduce cellular senescence levels, with no significant cytotoxicity within effective concentrations. They show promising application prospects in the fields of antioxidants, skin photoaging protection, and anti-aging cosmetics and medical repair materials.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, specifically to a method for preparing chitosan-derived carbon dots, the derived carbon dots, and their applications. Background Technology

[0002] With the increasing aging population and the growing demand for skin health and anti-aging solutions, research in the field of anti-aging has become a hot topic in biomaterials, cosmetics, and medical biomaterials. Skin aging is a direct manifestation of overall aging, and its main causes include ultraviolet radiation, inflammatory responses, and the accumulation of reactive oxygen species. Among these, photoaging caused by excessive reactive oxygen species generated from UVA radiation and inflammatory aging mediated by inflammatory factors are the core reasons for skin laxity, wrinkles, and impaired barrier function. Simultaneously, fibroblast aging and reduced hyaluronic acid synthesis further exacerbate the skin aging process. Therefore, developing novel anti-aging materials with antioxidant, anti-inflammatory, and low cytotoxic properties that can effectively delay cell aging and promote skin repair has significant theoretical and practical value.

[0003] Chitosan is a natural alkaline polysaccharide widely found in the shells of crustaceans such as shrimp and crabs, as well as in the cell walls of fungi. It boasts advantages such as abundant availability, low cost, good biocompatibility, biodegradability, and non-toxicity. Furthermore, its molecular structure contains numerous active groups such as amino and hydroxyl groups, making it easy to chemically modify and functionalize, and it has been widely applied in food, pharmaceuticals, and cosmetics. Carbon dots, as a novel nanomaterial, possess advantages such as small particle size, large specific surface area, abundant active sites, and good biocompatibility. Their unique conjugated structure and surface functional groups endow them with excellent free radical scavenging capabilities, leading to their widespread application in anti-oxidation and anti-aging fields. Combining chitosan with carbon dots allows the structural advantages of carbon dots to compensate for the lack of active sites in chitosan, while simultaneously enhancing the biocompatibility of chitosan and improving the biosafety of carbon dots.

[0004] Existing chitosan-carbon dot composite materials still have many shortcomings: On the one hand, most composite materials are prepared by physical mixing, resulting in poor binding stability and easy dissociation in vivo, which makes it difficult to maintain antioxidant activity and cannot effectively solve the problem of chitosan active site shielding; on the other hand, existing composite materials lack targeting and are still difficult to accurately reach deep tissues or specific organelles, making it impossible to achieve precise antioxidant effects, and cannot simultaneously achieve the synergistic anti-aging effects of clearing ROS, regulating LaminB1 expression, and inhibiting the secretion of inflammatory factors, which makes it difficult to meet the needs of practical applications.

[0005] Lignin, as a natural aromatic polymer, is abundant and inexpensive. Its molecular structure contains functional groups such as phenolic hydroxyl and methoxy groups, which possess excellent antioxidant activity. After hydrothermal carbonization, it can form lignin-based carbon dots, which not only retain the antioxidant sites of natural lignin but also possess the nanoscale properties of carbon dots, making it an ideal antioxidant modification material. However, lignin-based carbon dots lack targeting specificity and have poor compatibility with chitosan. Direct compounding makes it difficult to form stable composite materials, thus failing to fully exert their synergistic antioxidant effect.

[0006] Therefore, given the limitations of existing technologies such as the limited number of active sites in natural chitosan, its susceptibility to shielding effects, insufficient ROS capture capacity, the inability of existing modification methods to achieve precise antioxidant effects, and the difficulty in synergistically regulating LaminB1 expression and inflammatory factor secretion, developing a chitosan-derived material with high-efficiency antioxidant properties, precise targeting, and synergistic anti-aging functions has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, such as the limited number of active sites in chitosan leading to insufficient ROS capture capacity due to shielding effects, and the lack of targeting capabilities in existing composite materials, which makes it difficult to achieve precise antioxidant effects and synergistic regulation of LaminB1 and inflammatory factors, this application proposes a method for preparing chitosan-derived carbon dots, the derived carbon dots themselves, and their applications.

[0008] This application provides a method for preparing chitosan-derived carbon dots, the method being as follows:

[0009] A: Preparation of carbon dots from octyl-citric acid chitosan-based amphiphilic polymers:

[0010] SA1: Mix 2.0g chitosan with 0.8g octanal, add 50mL methanol, and stir to obtain a mixed solution;

[0011] SA2: Dissolve potassium borohydride and add it dropwise to the mixed solution described in SA1 to react. Filter, wash, and freeze-dry to obtain octyl chitosan.

[0012] SA3: Dissolve 4.5g of citric acid in 50mL of deionized water, add 1g of the octyl chitosan described in SA2 and stir, add 0.02mol of EDC and 0.02mol of NHS and react in the dark, dialyze with deionized water, and freeze dry to obtain octyl-citric acid chitosan.

[0013] SA4: Mix 0.25g of the octyl-citric acid chitosan described in SA3 with 0.25g of anhydrous citric acid, 1mL of N-(2-hydroxyethyl)ethylenediamine and 8mL of deionized water, react, dialyze, and freeze-dry to obtain octyl-citric acid chitosan-based amphiphilic polymer carbon dots.

[0014] B: Preparation of modified lignin-based carbon dots:

[0015] SB1: Weigh 0.1g of alkali lignin and 0.1g of p-toluenesulfonic acid and dissolve them in 30mL of deionized water for hydrothermal reaction. Filter the solution through a membrane to obtain a carbon dot solution.

[0016] SB2: The carbon dot solution described in SB1 is concentrated, separated by chromatographic column, filtered, and freeze-dried to obtain lignin-based carbon dot powder;

[0017] SB3: Weigh 0.5g of the lignocellulosic carbon dot powder described in SB2, disperse it in 50mL of deionized water, adjust the pH, add 0.5g of 2,3-epoxypropyltrimethylammonium chloride and stir to react. After filtration, concentration and freeze-drying, the modified lignocellulosic carbon dots are obtained.

[0018] C: Preparation of chitosan-derived carbon dots:

[0019] Disperse 0.1 g of the octyl-citric acid chitosan-based amphiphilic polymer carbon dots from step SA4 in 100 mL of buffer solution, and add 0.01 mol of EDC. The chitosan-derived carbon dots were obtained by amidation activation with HCl and 0.012 mol NHS, covalent coupling with the modified lignin-based carbon dots from step SB3, dialysis, and freeze-drying.

[0020] Preferably, the washing method in step SA2 is to wash three times with a mixed solvent of water and methanol.

[0021] Preferably, the dialysis time in step SA4 is 72 hours.

[0022] Preferably, the hydrothermal reaction conditions in step SB1 are 180°C and 12 hours.

[0023] Preferably, the pore size of the filter membrane in step SB1 is 0.22 μm.

[0024] Preferably, the pH value adjusted in step SB3 is 8.6 ± 0.2.

[0025] Preferably, the stirring reaction in step SB3 is carried out under constant temperature stirring at 50°C.

[0026] Preferably, the buffer solution in step C is PBS buffer.

[0027] In another aspect, this application provides chitosan-derived carbon dots prepared by any of the preparation methods described above.

[0028] This application also provides chitosan-derived carbon dots prepared by any of the preparation methods described above, and the application of the chitosan-derived carbon dots in the preparation of anti-aging cosmetics.

[0029] The chitosan-derived carbon dots in this application are synthesized by a composite of octyl-citric acid chitosan-based amphiphilic polymer carbon dots and modified lignin-based carbon dots via amidation covalent coupling.

[0030] First, addressing the issue of chitosan's active sites being easily shielded by intramolecular hydrogen bonds, this application introduces octyl and citrate groups onto the chitosan backbone to form an amphiphilic polymer structure. The hydrophobic chain of the octyl group can disrupt the intermolecular and intramolecular hydrogen bond network of chitosan, exposing shielded active sites such as amino and hydroxyl groups; the citrate group provides abundant carboxyl groups, creating conditions for subsequent covalent coupling. After hydrothermal carbonization, this amphiphilic polymer forms carbon dots, possessing the small size, high specific surface area, and amphiphilic properties of carbon dots, which is beneficial for penetrating deep layers of the skin and transmembrane transport.

[0031] Secondly, addressing the poor compatibility between lignin-based carbon dots and chitosan, this application pre-modifies the lignin-based carbon dots with amino groups on their surface. The modified lignin-based carbon dots then form stable covalent bonds with the aforementioned amphiphilic polymer carbon dots through an amidation reaction, avoiding the defects of easy dissociation in physical mixing. This covalent coupling not only improves the structural stability of the composite material but also allows the antioxidant functional groups such as phenolic hydroxyl and methoxy groups on the lignin-based carbon dots to complement the amino and hydroxyl groups on the chitosan carbon dots, synergistically enhancing the ability to capture various reactive oxygen species and effectively overcoming the problem of insufficient antioxidant activity of single chitosan carbon dots.

[0032] Furthermore, addressing the challenge of existing materials lacking targeting and failing to achieve precise antioxidant effects, the chitosan-derived carbon dots of this application utilize the self-assembly behavior induced by their amphiphilic structure to accumulate around inflamed or senescent cells. Simultaneously, their nanoscale size and positively charged surface facilitate binding to mitochondrial or nuclear membranes via electrostatic interactions, achieving passive targeting to mitochondrial and nuclear membrane regions. This targeting capability enables the carbon dots to scavenger excess ROS in senescent cells, thereby reducing the oxidative degradation of nuclear laminin LaminB1 by ROS, upregulating LaminB1 protein expression levels, stabilizing nuclear membrane structure, and subsequently inhibiting the secretion of downstream inflammatory factors IL-6 and IL-8. This achieves a synergistic anti-aging effect through ROS scavenging, LaminB1 protection, and inflammation suppression.

[0033] The beneficial effects of the embodiments in this application are as follows:

[0034] (1) By using amidation covalent coupling to combine two carbon dots, the chemical bonds are stabilized instead of the physical mixing, thus avoiding the dissociation of the composite material from the structure, so that the antioxidant functional group can exist stably for a long time and ensure the continuous performance of the effect.

[0035] (2) Chitosan is modified by octyl hydrophobic chain, which breaks the intramolecular / intermolecular hydrogen bond, removes the shielding effect of active site, and provides a large number of carboxyl groups in combination with citric acid, which significantly increases the number of active sites and ROS capture efficiency, and solves the problem of insufficient activity of traditional chitosan.

[0036] (3) After being modified with quaternary ammonium salt, the surface of the lignin-based carbon dots is positively charged, which greatly improves their compatibility with chitosan carbon dots. At the same time, by taking advantage of amphiphilicity and nano-size, passive targeted enrichment can be achieved, which can accurately bind to mitochondria and nuclear membranes, remove excess ROS in situ, and improve antioxidant utilization.

[0037] (4) By clearing ROS, LaminB1 is protected to inhibit inflammatory factors, reduce cell aging, and promote hyaluronic acid synthesis, thereby achieving a triple synergistic anti-aging effect of anti-oxidation, anti-photoaging, and anti-inflammatory repair.

[0038] (5) The carbon dots of this application have small particle size, good dispersibility and strong amphiphilicity, which makes them easy to be absorbed through the skin and transported across the membrane. Cell experiments have confirmed that they have excellent biocompatibility, no obvious toxicity within the effective dose, are safe and degradable, and are suitable for cosmetics and medical repair fields. Attached Figure Description

[0039] Figure 1 Line graph showing IL-6 data at different concentrations of anti-inflammatory factors in Example 3 of this application.

[0040] Figure 2 Line graph showing IL-8 data for different concentrations of anti-inflammatory factors in Example 3 of this application. Detailed Implementation

[0041] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0042] Example 1

[0043] Octyl-citric acid chitosan-based amphiphilic polymer carbon dots

[0044] (1) Preparation of octyl chitosan: 2.0 g of chitosan (Guoyao) and 0.8 g of octaldehyde (Guoyao, which is 0.6 to 0.8 times the amount of chitosan amino group) were added to a 250 mL three-necked flask, 50 mL of methanol was added, and the mixture was stirred for 12 h under nitrogen protection. 1.5 times the amount of chitosan amino group potassium borohydride was dissolved in 50 mL of water and added dropwise to the above mixed solution. The reaction was continued for 12 h. After the reaction was completed, the mixture was vacuum filtered, washed three times with a mixture of water and methanol, and freeze-dried to obtain the product. The octyl grafting rate was calculated by elemental analysis.

[0045] (2) Synthesis of octyl-citric acid chitosan: 4.5 g of citric acid was dissolved in 50 mL of deionized water, 1 g of octyl-chitosan was added and stirred for 12 h, then 0.02 mol of EDC was added, and after stirring for 10 min, 0.02 mol of NHS was added. The reaction was carried out in the dark for 48 h, and the resulting product was dialyzed against deionized water for 72 h (M). WCO =8000u), and then freeze-dried to obtain the target product;

[0046] (3) Preparation of carbon dots in octyl-citric acid chitosan-based amphiphilic polymers: 0.25 g of octyl-citric acid chitosan was placed in a hydrothermal reactor, and 0.25 g of anhydrous citric acid, 1 mL of N-(2-hydroxyethyl)ethylenediamine, and 8 mL of deionized water were added sequentially and mixed thoroughly. The mixture was reacted at 180 °C for 3 h. After the reaction, the mixture was dialyzed with deionized water for 72 h. (M WCO =8000u), the obtained product was freeze-dried: pre-frozen at -50℃ for 8h until completely frozen; cold trap temperature ≤-60℃, vacuum degree 5~15Pa, shelf temperature -30℃, maintained for 18~20h to remove free water; vacuum degree reduced to ≤5Pa, shelf temperature raised to 25℃, maintained for 10~12h to remove bound water; sample temperature consistent with shelf temperature, vacuum degree stable for 30min, drying ended. The quantum yield of carbon dots of octyl-citric acid chitosan-based amphiphilic polymer was determined using quinine sulfate (0.05mol / L H2SO4 solution, quantum yield 0.54 under 360nm excitation light) as standard, calculated according to the following formula: Φ x Where Φ is the quantum yield, F is the measured integral emission intensity, A is the optical density, η is the refractive index of the solvent, the subscript "s" refers to a reference with a known quantum yield, and "x" represents the measured carbon dots of the octyl-citric acid chitosan-based amphiphilic polymer; the calculated quantum yield is 43.1%.

[0047] Example 2

[0048] Preparation of modified lignin-based carbon dots

[0049] (1) Weigh 0.1g of alkali lignin (Solebo) and 0.1g of p-toluenesulfonic acid (Guoyao) and dissolve them in 30mL of deionized water. Stir the reaction at 90℃ for 1h and filter it through a 0.22μm filter membrane after cooling.

[0050] (2) Transfer the filtered solution to a 50 mL polytetrafluoroethylene reactor and perform a hydrothermal reaction in an oven at 180 °C for 12 h. After the reaction is completed, allow the reactor to cool naturally to room temperature and filter through a 0.22 μm filter membrane to obtain a carbon dot solution.

[0051] (3) The carbon dot solution was concentrated to about 5 mL using a rotary evaporator and then added to a silica gel column. A mixed solvent of ethyl acetate and methanol in a volume ratio of 1:3 was used as the mobile phase to separate the carbon dot solution by column chromatography.

[0052] (4) The yellow-green carbon dot solution was eluted from the chromatographic column, and the separated solution was evaporated to dryness and dissolved in water. The obtained aqueous solution was filtered through a 0.22 μm filter membrane to obtain the purified lignin-based carbon dot solution. Finally, it was freeze-dried to obtain lignin-based carbon dot powder.

[0053] (5) Weigh 0.5g of lignin-based carbon dots powder, disperse it in 50mL of deionized water, sonicate for 15min until uniformly dispersed, adjust the pH of the system to 8.6, add 0.5g of 2,3-epoxypropyltrimethylammonium chloride, and stir the reaction at 50℃ for 5h. After the reaction is completed, filter through a filter membrane, concentrate by rotary evaporation, and then freeze dry at -50℃ and 10Pa for 24h. Freeze dry to obtain modified lignin-based carbon dots;

[0054] (6) Characterization (TEM): The modified lignin-based carbon dot solution was dropped onto a carbon support film and air-dried naturally. The morphology was observed using a transmission electron microscope. The analysis showed that the modified lignin-based carbon dot solution had good dispersibility, an average particle size of 2.23 nm, and the carbon core had a graphite-like structure.

[0055] Example 3

[0056] Preparation of chitosan-derived carbon dots

[0057] (1) Pretreatment: Take 0.1g of the octyl-citric acid chitosan-based amphiphilic polymer carbon dots prepared in Example 1, disperse them in 100mL of PBS buffer (concentration 0.01mol / L) with pH=6.0, and sonicate for 20min to ensure uniform dispersion and no obvious agglomerates;

[0058] (2) Amide activation: Add 0.01 mol EDC·HCl and 0.012 mol NHS to the above dispersion, and stir for 30 min under room temperature and light protection to fully activate the carboxyl groups on the carbon dots of the octyl-citric acid chitosan amphiphilic polymer, in preparation for subsequent covalent coupling.

[0059] (3) Covalent coupling reaction: According to the mass ratio of octyl-citric acid chitosan-based amphiphilic polymer carbon dots to modified lignin-based carbon dots prepared in Example 2 of 1:1.2, the modified lignin-based carbon dots were added to the activated dispersion, and the reaction was carried out under the conditions of avoiding light and room temperature for 12 hours with magnetic stirring.

[0060] (4) Purification: Transfer the reaction mixture to a dialysis bag (M WCO =8000u), and purified by dialysis with deionized water. The deionized water was replaced every 12 hours and dialysis was continued for 72 hours to completely remove unreacted small molecule impurities such as EDC·HCl and NHS, as well as uncoupled free carbon points.

[0061] (5) Product preparation: The purified solution after dialysis was placed in a freeze dryer and freeze-dried according to the following parameters: pre-freezing at -50℃ for 8 hours until completely frozen, cold trap temperature ≤ -60℃, vacuum degree 5~10Pa, shelf temperature -30℃ maintained for 19 hours to remove free water; then the vacuum degree was reduced to ≤5Pa, the shelf temperature was slowly raised to 25℃ and maintained for 11 hours to remove bound water. After the sample temperature was consistent with the shelf temperature and the vacuum degree was stable for 30 minutes, the drying was stopped and a light yellow loose chitosan-derived carbon dot powder was obtained.

[0062] (6) Performance verification: The quantum yield of the chitosan-derived carbon dots was determined using the same method as in Example 1. The quantum yield was 48.3%, which is significantly higher than that of the single carbon dots in Example 1. At the same time, TEM characterization showed that the derived carbon dots had good dispersion, with an average particle size of 2.58 nm and no obvious agglomeration.

[0063] Example 4

[0064] Preparation of chitosan-derived carbon dots

[0065] (1) Pretreatment: Take 0.1g of the octyl-citric acid chitosan-based amphiphilic polymer carbon dots prepared in Example 1, disperse them in 100mL of PBS buffer (concentration 0.01mol / L) with pH=6.0, and sonicate for 10min to ensure uniform dispersion and no obvious agglomerates;

[0066] (2) Amide activation: Add 0.01 mol EDC·HCl and 0.012 mol NHS to the above dispersion, and stir for 30 min at room temperature and in the dark to fully activate the carboxyl groups on the surface of the carbon dots of the octyl-citric acid chitosan amphiphilic polymer, in preparation for subsequent covalent coupling.

[0067] (3) Covalent coupling reaction: According to the mass ratio of octyl-citric acid chitosan-based amphiphilic polymer carbon dots to modified lignin-based carbon dots prepared in Example 2 of 1:1.2, the modified lignin-based carbon dots were added to the activated dispersion, and the reaction was carried out under the conditions of avoiding light and room temperature for 24 hours with magnetic stirring.

[0068] (4) Purification: Transfer the reaction mixture to a dialysis bag (M WCO =8000u), and purified by dialysis with deionized water. The deionized water was replaced every 12 hours and dialysis was continued for 72 hours to completely remove unreacted small molecule impurities such as EDC·HCl and NHS, as well as uncoupled free carbon points.

[0069] (5) Product preparation: The purified solution after dialysis was placed in a freeze dryer and freeze-dried according to the following parameters: pre-freezing at -50℃ for 8 hours until completely frozen, cold trap temperature ≤ -60℃, vacuum degree 10Pa, shelf temperature -30℃ maintained for 19 hours to remove free water; then the vacuum degree was reduced to ≤5Pa, the shelf temperature was slowly raised to 25℃ and maintained for 11 hours to remove bound water. After the sample temperature was consistent with the shelf temperature and the vacuum degree was stable for 30 minutes, the drying was stopped, and a light yellow loose chitosan-derived carbon dot powder was obtained.

[0070] (6) Performance verification: The quantum yield of the chitosan-derived carbon dots was determined using the same method as in Example 1. The quantum yield was 47.5%, which is significantly higher than that of the single carbon dots in Example 1. At the same time, TEM characterization showed that the derived carbon dots had good dispersion, with an average particle size of 2.62 nm and no obvious agglomeration.

[0071] Test case

[0072] Performance test

[0073] (1) Experimental objectives: Antioxidant capacity: to measure the level of reactive oxygen species (ROS) induced by UVA irradiation; Dermal matrix repair: to measure the level of hyaluronic acid (HA) secretion; Anti-aging: to detect the activity of aging-related β-galactosidase (SA-β-Gal), the expression of LaminB1 protein, and the secretion of inflammatory factors IL-6 and IL-8.

[0074] (2) Experimental materials and reagents: P3 generation human dermal fibroblasts (HSF) were purchased from Bio-Tech, chitosan-derived carbon dots (CTS-CDs) in Example 3 and Example 4, DCFH-DA reactive oxygen species detection kit was purchased from Beyotime, hyaluronic acid (HA) ELISA kit was purchased from Beyotime, SA-β-Gal staining kit was purchased from Beyotime, LaminB1 antibody (1:1000 dilution) was purchased from CST, and IL-6 / IL-8 ELISA kit was purchased from Beyotime.

[0075] (3) Grouping: In order to evaluate the efficacy of CTS-CDs, the following groups (each group has 3 replicate wells) will be set up as shown in Table 1.

[0076] Table 1 Experimental Groups

[0077]

[0078] (4) Experimental steps:

[0079] 1) Cell culture and treatment:

[0080] HSF cells were seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells; Culture: Incubate at 37°C in a 5% CO2 incubator for 24 hours to ensure cell adhesion.

[0081] 2) UVA irradiation treatment (for UVA group and UVA+CTS-CDs group):

[0082] a. Rinsing: Gently discard the old culture medium containing 10% serum and rinse the cells once with PBS (to remove residual serum).

[0083] b. Irradiation: The orifice plate was placed in a UVA lamp box (365nm), and the irradiation dose was set to 0.5 J / cm². 2 (10 minutes).

[0084] c. Recovery: Immediately after irradiation, replace with complete culture medium containing 10% serum and allow to recover for 2 hours.

[0085] 3) CTS-CDs processing (for CTS-CDs group and UVA+CTS-CDs group):

[0086] a. Rinsing: Gently discard the old culture medium again and rinse the cells once with PBS to ensure no serum protein interference.

[0087] b. Add culture medium: Add 200 μL of culture medium containing CTS-CDs to each well (add the corresponding concentration of CDs working solution or an equal volume of PBS according to the group); "add" here means replacing the original culture medium and allowing the cells to incubate in an environment containing CDs.

[0088] 4) Joint incubation

[0089] Incubation: All treated well plates were returned to a 37°C, 5% CO2 incubator for 24 hours. During this period, CTS-CDs will be phagocytosed or adsorbed by the cell membrane; cellular damage processes such as oxidative stress and inflammatory responses caused by UVA irradiation will be manifested or inhibited during this time.

[0090] (5) Performance verification test

[0091] 1) Use CCK The effects of chitosan-derived carbon dots on healthy human dermal fibroblasts were detected using an 8-method approach. The results showed that the cell viability of the carbon dots prepared in Example 3 at concentrations of 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL was 99.8%, 99.1%, and 98.5%, respectively; and the cell viability of the carbon dots prepared in Example 4 at concentrations of 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL was 99.6%, 98.7%, and 98.1%, respectively. The results indicate that the chitosan-derived carbon dots prepared in this application have no obvious cytotoxicity, and low and medium concentrations do not inhibit cell proliferation but have a slight promoting effect. This indicates that the carbon dots are safe and do not damage healthy cells within the tested dosage range and can be used for subsequent anti-aging efficacy studies.

[0092] 2) Antioxidant capacity test (ROS inhibition rate):

[0093] a. Fluorescent staining: After treatment, remove the culture medium, add 10µM DCFH-DA working solution (containing PBS), and incubate at 37℃ in the dark for 30 min.

[0094] b. Washing: Wash away any unabsorbed dye and add PBS to maintain.

[0095] c. Detection: The fluorescence intensity of 488nm excitation and 525nm emission was detected using a fluorescence microplate reader.

[0096] d. Calculation: ROS inhibition rate = (fluorescence intensity of UVA group - fluorescence intensity of UVA + CDs group) / fluorescence intensity of UVA group × 100%.

[0097] 3) Dermal matrix repair assessment (hyaluronic acid (HA) production)

[0098] a. Sampling: After 24 hours of treatment, collect the cell culture supernatant and centrifuge to remove the residue.

[0099] b. Detection: Follow the instructions in the ELISA kit (sample addition, incubation, washing, substrate addition, and reaction termination).

[0100] c. Reading: Use an ELISA reader to read the OD value at a wavelength of 450nm.

[0101] d. Calculation: Calculate the HA concentration for each sample based on the standard curve.

[0102] 4) Anti-aging marker detection (β-galactosidase & LaminB1)

[0103] A. SA-β-Gal staining (for detecting cell senescence rate):

[0104] a. Fixation: After treatment, fix with 0.5% PFA for 15 min.

[0105] b. Staining: Add staining solution containing X-gal and incubate overnight in a water bath at 37°C (pH 6.0).

[0106] c. Observation and counting: Observe the blue-stained cells under a microscope; randomly select 5 fields of view and calculate the percentage (%) of blue-positive cells in the total number of cells.

[0107] B.LaminB1 protein expression:

[0108] a. Lysis: Collect cells, add RIPA lysis buffer, and extract total protein.

[0109] b. Electrophoresis: Proteins are separated by SDS-PAGE electrophoresis.

[0110] c. Transfer: Transfer the protein to a PVDF membrane.

[0111] d. Incubate the antibody: Antibody: Anti-LaminB1, Internal control: β-actin.

[0112] e. Development: Detection using chemiluminescence immunoassay (ECL).

[0113] 5) Anti-inflammatory factor detection (IL-6 & IL-8)

[0114] a. Sampling: Collect the cell culture supernatant 24 hours after treatment.

[0115] b. Detection: Perform ELISA tests according to the kit instructions.

[0116] c. Reading: OD value is measured at a wavelength of 450nm.

[0117] (6) Test Results: Performance test data are shown in Table 2. IL-6 data for Example 3 are shown in Table 2. Figure 1 As shown; IL-8 data for Example 3 are as follows Figure 2 As shown:

[0118] Table 2 Performance test data

[0119]

[0120] Through Table 2 and Figure 1 , Figure 2 Compared with the group irradiated by UVA alone, the secretion of hyaluronic acid (HA) in human dermal fibroblasts significantly increased after treatment with chitosan-derived carbon dots, showing a clear dose-dependent increase. Specifically, the HA content in the high-concentration group of Example 3 reached 91.2 ng / mL, and in the high-concentration group of Example 4 it reached 88.5 ng / mL, both close to the level of the blank control group. These results indicate that the chitosan-derived carbon dots of this application can effectively improve UVA-induced dermal matrix synthesis disorders, significantly promote hyaluronic acid production, enhance skin's water retention and repair capabilities, and have a positive effect on delaying skin aging and improving skin laxity and dryness.

[0121] In summary, the experimental results show that the chitosan-derived carbon dots prepared in this application have no significant toxicity to human dermal fibroblasts in the concentration range of 0.01–0.1 mg / mL, demonstrating good biocompatibility. They can significantly scavenge UVA-induced reactive oxygen species, inhibit oxidative stress damage, upregulate LaminB1 protein expression, reduce aging-related β-galactosidase activity, effectively inhibit the secretion of inflammatory factors IL-6 and IL-8, and promote hyaluronic acid synthesis. They significantly improve UVA-induced photoaging of skin cells, exhibiting highly efficient, stable, and safe anti-aging properties, and can be used as a novel anti-aging active ingredient in cosmetics and medical repair materials.

[0122] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A method for preparing chitosan-derived carbon dots, characterized in that, include: A: Preparation of octyl-citric acid chitosan-based amphiphilic polymer carbon dots: SA1: Mix 2.0g chitosan, 0.8g octanal and methanol to obtain a mixed solution; SA2: Dissolve potassium borohydride and add it dropwise to the mixed solution, then wash to obtain octyl chitosan; SA3: Dissolve 4.5g of citric acid, add the octyl chitosan, and add EDC and NHS to obtain octyl-citric acid chitosan; SA4: The octyl-citric acid chitosan was mixed with anhydrous citric acid, N-(2-hydroxyethyl)ethylenediamine and deionized water, and dialyzed to obtain octyl-citric acid chitosan-based amphiphilic polymer carbon dots; B: Preparation of modified lignocellulosic carbon dots: SB1: Dissolve 0.1g of alkali lignin and 0.1g of p-toluenesulfonic acid in water and carry out a hydrothermal reaction. Filter the solution through a membrane to obtain a carbon dot solution. SB2: The carbon dot solution is concentrated, separated, and filtered to obtain lignin-based carbon dot powder; SB3: Dissolve 0.5g of the lignin-based carbon dot powder, adjust the pH, add 0.5g of 2,3-epoxypropyltrimethylammonium chloride and stir to obtain modified lignin-based carbon dots; C: Carbon-dot the octyl-citric acid chitosan-based amphiphilic polymer from step SA4 in the buffer solution, and add EDC. Chitosan-derived carbon dots were obtained by amidation activation with HCl and NHS, covalent coupling with the modified lignin-based carbon dots from step SB3, and dialysis.

2. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, The washing method in step SA2 is to wash three times with a mixed solvent of water and methanol.

3. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, The dialysis time in step SA4 is 72 hours.

4. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, The hydrothermal reaction conditions in step SB1 are 180℃ and 12h.

5. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, The pore size of the filter membrane in step SB1 is 0.22 μm.

6. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, In step SB3, the pH value is adjusted to 8.6 ± 0.

2.

7. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, The stirring reaction in step SB3 is carried out under constant temperature stirring at 50°C.

8. The method for preparing chitosan-derived carbon dots as described in claim 1, characterized in that, The buffer solution used in step C is PBS buffer.

9. Chitosan-derived carbon dots prepared by the preparation method according to any one of claims 1-8.

10. The application of chitosan-derived carbon dots prepared by the preparation method according to any one of claims 1-8, and the chitosan-derived carbon dots according to claim 9, in the preparation of anti-aging cosmetics.

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