Sunscreen carbon quantum dots for clearing cfDNA, hydrogel dressing and preparation method and application thereof

Abstract

The application discloses a sunscreen carbon quantum dot for removing cfDNA, a hydrogel dressing and a preparation method and application thereof. The sunscreen carbon quantum dot with positive electricity is prepared by using chitosan as an amino donor through one-step hydrothermal method, is loaded in a sodium alginate / carboxymethyl chitosan hydrogel matrix together with astragalus polysaccharide, and is obtained through ionic crosslinking to obtain a multifunctional dressing. The application blocks ultraviolet damage from the source, simultaneously realizes ROS removal, cfDNA adsorption, inflammation inhibition and vascular regeneration promotion, completely breaks the malignant cycle of inflammation and aging induced by ultraviolet, restores the proliferation ability of fibroblasts and effectively reduces cell aging. The application solves the problem that traditional plant source carbon dots are negatively charged and difficult to adsorb cfDNA, and the positively charged carbon dots maintain good biocompatibility. Meanwhile, the composite hydrogel overcomes the defects of easy aggregation and cracking of the carbon dots, realizes long-acting protection, the preparation process is controllable, and the application has important application value in the fields of refractory wound treatment and skin photoaging protection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a sunscreen carbon quantum dot for clearing cfDNA, a hydrogel dressing, and its preparation method and application. Background Technology

[0002] The skin is the largest barrier organ in the human body, undertaking the core functions of resisting external physical, chemical and biological stimuli and maintaining the homeostasis of the internal environment. Ultraviolet (UV) radiation is one of the primary environmental causes of acute and chronic skin damage. Among them, UVA (315-400nm) has strong penetrating power and can reach the dermis and even subcutaneous tissue, while UVB (280-315nm) can directly cause cellular DNA damage. The combined effect of the two is the core culprit leading to delayed wound healing, photoaging of the skin, and even skin cancer.

[0003] When skin wounds are exposed to ultraviolet (UV) radiation, they directly damage surviving fibroblasts and keratinocytes, inducing apoptosis or senescence. Simultaneously, this triggers excessive local generation of reactive oxygen species (ROS), causing oxidative stress damage and inhibiting fibroblast proliferation and collagen synthesis. Damaged fibroblasts continuously release large amounts of cell-free DNA (cfDNA) into the wound microenvironment. Excessive accumulation of cfDNA, as a key damage-associated molecular pattern (DAMP), activates inflammatory signaling pathways in macrophages, amplifying the inflammatory response. This over-activated inflammatory microenvironment further exacerbates oxidative damage and senescence in fibroblasts, ultimately forming a vicious cycle of "ROS-induced oxidative stress - fibroblast damage and senescence - massive cfDNA release - intensified inflammation - aggravated cell senescence," resulting in non-healing wounds. Therefore, efficient repair of UV-induced non-healing wounds requires a multi-target synergistic intervention across the entire chain, from initial UV protection to breaking the "inflammatory-senescence" cycle.

[0004] Current technologies in this field suffer from the following core shortcomings: Traditional UV shielding agents (such as titanium dioxide and zinc oxide nanoparticles) can block UV rays, but they suffer from performance degradation due to photodegradation, and the photocatalytic production of ROS exacerbates skin damage. Furthermore, they lack repair capabilities for existing wounds and cannot intervene in the "inflammation-aging" cycle. Clinically used wound dressings (such as natural polymer hydrogels and synthetic polymer dressings) only provide basic barrier functions like moisturizing and preventing bacterial contamination, lacking the ability to target the core pathological mechanisms of UV-induced non-healing wounds. They cannot simultaneously clear ROS or block cfDNA-mediated inflammatory cascades, resulting in very limited therapeutic effects. Intervention technologies targeting cfDNA primarily rely on deoxyribonuclease (DNase), which can enzymatically degrade cfDNA, but suffer from poor in vivo stability, easy inactivation, short half-life, high production costs, and stringent storage and transportation conditions. Cationic polymer (such as polyethyleneimine) cfDNA scavengers generally have strong cfDNA adsorption capacity but also exhibit high cytotoxicity and hemolytic toxicity.

[0005] Carbon dots, as a novel type of carbon nanomaterial, have attracted widespread attention in the biomedical field due to their excellent optical properties, good biocompatibility, and ease of surface functionalization modification. However, in existing technologies, carbon dots derived from leaves or traditional Chinese medicine are limited by raw materials and preparation processes, and their surfaces are generally negatively charged. This leads to electrostatic repulsion between carbon dots and negatively charged cfDNA in the wound microenvironment, making it impossible to achieve efficient removal of cfDNA through electrostatic adsorption and thus difficult to block cfDNA-mediated inflammatory cascade reactions.

[0006] Currently, there are a few research reports on carbon dots derived from Ginkgo biloba leaves, but three major technical bottlenecks remain: First, no research has yet systematically integrated the UV shielding performance, ROS scavenging ability, and cfDNA targeted scavenging function of Ginkgo biloba leaf carbon dots, making it impossible to intervene in all pathological stages of UV-induced skin damage. Second, although Ginkgo biloba leaf carbon dots possess excellent UV shielding performance, their inherent defects, such as easy aggregation and film cracking after drying, lead to a rapid decline in their UV shielding effect over time, making it impossible to achieve long-term stable UV protection. Third, existing research has not functionally combined Ginkgo biloba leaf carbon dots with Astragalus polysaccharides, which have pro-angiogenic activity, to construct a full-chain intervention system covering damage blocking, microenvironment regulation, and tissue regeneration, and it is even more impossible to break the vicious cycle of "inflammation-aging" induced by UV radiation: "excessive ROS generation - fibroblast damage and aging - massive release of cfDNA - increased inflammation - aggravated cell aging."

[0007] In summary, current technologies cannot provide a solution that simultaneously blocks UV damage at its source, eliminates excess ROS to alleviate cell aging, removes cfDNA to block inflammatory cascade reactions, and promotes angiogenesis to accelerate wound healing, thereby effectively breaking the vicious cycle of UV-induced inflammation-aging. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention aims to solve the following core technical problems:

[0009] (1) Solve the inherent defects of Ginkgo biloba carbon dots being prone to agglomeration when dried and cracked when applied alone, which leads to rapid decay of UV shielding effect, and achieve long-term stable UV protection;

[0010] (2) Solve the technical bottleneck that the carbon dots of traditional leaf / Chinese medicine sources are generally negatively charged and cannot efficiently adsorb cfDNA, and ensure excellent biosafety while achieving efficient removal of cfDNA.

[0011] (3) Construct a full-chain intervention system of “damage blocking - microenvironment regulation - tissue regeneration” to completely break the vicious cycle of “inflammation-aging” induced by ultraviolet rays, solve the core pain point of existing technologies having single function and being unable to intervene in all pathological aspects of the wound, and meet the clinical treatment needs of ultraviolet-induced difficult-to-heal wounds.

[0012] (4) Solve the problems of uncontrollable quality of traditional Chinese medicine crude extracts and complex preparation process of existing functional dressings.

[0013] The primary objective of this invention is to provide a sunscreen carbon quantum dot for clearing cfDNA, its preparation method, and its application.

[0014] Another objective of this invention is to provide a hydrogel dressing for removing cfDNA, its preparation method, and its application.

[0015] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0016] A sunscreen carbon quantum dot for clearing cfDNA was prepared by hydrothermal method using ginkgo leaf powder as raw material and chitosan as amino donor. The surface has positively charged amino functional groups, the zeta potential is +15 mV to +40 mV, and it has characteristic absorption peaks in the 200 to 400 nm ultraviolet band.

[0017] The preferred Zeta potential is +18 mV to +38.5 mV.

[0018] The above-mentioned method for preparing sunscreen carbon quantum dots that remove cfDNA includes the following steps: fresh ginkgo leaves are blanched, dried, and pulverized. The resulting ginkgo leaf powder is mixed evenly with chitosan in a weakly acidic aqueous solution to obtain a chitosan-dissolved suspension. The suspension is subjected to a hydrothermal reaction at 160–200 °C for 4–8 h. After the reaction, the solid and liquid are separated. The supernatant is filtered through a 0.22 μm microfiltration membrane to remove large particulate impurities. The filtrate is dialyzed in deionized water using a dialysis bag with a molecular weight cutoff of 500 Da. The dialysate obtained from the dialysis is freeze-dried to obtain sunscreen carbon quantum dots that remove cfDNA.

[0019] The preferred conditions for blanching are treatment at 95–100 °C for 5–15 min; more preferably, treatment at 100 °C for 10 min.

[0020] The drying temperature is preferably 55-65 °C; more preferably 60 °C.

[0021] The degree of drying refers to drying to a constant weight.

[0022] The weakly acidic aqueous solution is an aqueous solution with pH = 3.0 to 5.0; preferably an acetic acid aqueous solution with pH = 3.5 to 4.5; more preferably an acetic acid aqueous solution with pH = 4.

[0023] The preferred conditions for the hydrothermal reaction are a constant temperature reaction at 180 °C for 6 h.

[0024] The preferred method for solid-liquid separation is centrifugation.

[0025] The centrifugation conditions are preferably 10,000 to 15,000 rpm for 5 to 15 minutes; more preferably 10,000 rpm for 10 minutes.

[0026] The preferred dialysis time is 24 hours; the deionized water is changed every 4 to 6 hours.

[0027] The above-mentioned sunscreen carbon quantum dots that remove cfDNA are used in the preparation of sunscreen skin care products or hydrogel excipients.

[0028] A composite hydrogel dressing for clearing cfDNA contains the aforementioned sunscreen carbon quantum dots; preferably, it includes a hydrogel matrix and functional active components dispersed in the hydrogel matrix; the functional active components include the aforementioned sunscreen carbon quantum dots (CD) and astragalus polysaccharide (APS).

[0029] The hydrogel matrix is ​​obtained by ion crosslinking sodium alginate and carboxymethyl chitosan; wherein the mass ratio of sodium alginate to carboxymethyl chitosan is 1:0.5-2; preferably 1:1-2.

[0030] The preferred ion is calcium ion.

[0031] The calcium ions are preferably derived from calcium chloride.

[0032] The calcium chloride content is based on a mass ratio of sodium alginate to calcium chloride of 6 to 12:1.

[0033] The sunscreen carbon quantum dots and the astragalus polysaccharide are preferably mixed in a mass ratio of 1-2:1-2; more preferably in a mass ratio of 1:1.

[0034] The preparation method of the above-mentioned composite hydrogel dressing for clearing cfDNA includes the following steps:

[0035] (1) Mix sodium alginate, carboxymethyl chitosan and water, and stir at 55-65°C until a transparent and uniform hydrogel precursor solution is obtained;

[0036] (2) Mix the above-mentioned sunscreen carbon quantum dots, astragalus polysaccharide and hydrogel precursor solution obtained in step (1), and stir at 55-65℃ until the astragalus polysaccharide is dissolved and the positively charged Ginkgo biloba carbon dots are evenly dispersed to obtain a mixed solution;

[0037] (3) Add calcium chloride solution to the mixed solution obtained in step (2), and let it stand after the addition is complete to obtain a composite hydrogel dressing that removes cfDNA.

[0038] The concentration of sodium alginate in the hydrogel precursor solution in step (1) is 1-5% (w / v, g / mL); preferably 1.5-3.0% (w / v, g / mL).

[0039] The concentration of carboxymethyl chitosan in step (1) in the hydrogel precursor solution is 1-5% (w / v, g / mL); preferably 1.5-3.0% (w / v, g / mL).

[0040] The stirring time in step (1) is preferably 25 to 35 minutes.

[0041] The content of the sunscreen carbon quantum dots in the mixed solution in step (2) is preferably 0.15-0.25% (w / v, g / mL); preferably 0.2% (w / v, g / mL).

[0042] The content of Astragalus polysaccharide in the mixed solution in step (2) is preferably 0.15-0.25% (w / v, g / mL); preferably 0.2% (w / v, g / mL).

[0043] The stirring time in step (2) is preferably 15 to 25 minutes.

[0044] The concentration of the calcium chloride solution in step (3) is preferably 1.0 to 1.5% (w / v, g / mL); preferably 1.25% (w / v, g / mL).

[0045] The above-mentioned composite hydrogel dressing for clearing cfDNA is used in the preparation of medical preparations or skin care products.

[0046] The medical preparation is preferably a preparation for treating UV-induced, refractory skin wounds; it possesses at least one function among UV shielding, ROS scavenging, cfDNA adsorption and scavenging, inflammation inhibition, and angiogenesis promotion; or it has the function of preventing UV-induced precancerous skin lesions. The skin care product is preferably a facial mask; it has the function of preventing or reversing photoaging of the skin.

[0047] Compared with the prior art, the present invention has the following outstanding substantive features and significant progress:

[0048] (1) This invention achieves a balance between efficient cfDNA removal and excellent biocompatibility. Using chitosan as the amino donor, this invention prepares positively charged Ginkgo biloba leaf carbon dots via a one-step hydrothermal method, overcoming the technical bottleneck of traditional leaf-derived carbon dots being unable to adsorb cfDNA due to their negative surface charge. Simultaneously, it exhibits high safety. Under the premise that the carbon dots of this invention have essentially the same zeta potential and comparable cfDNA adsorption efficiency as the commercially available cationic polymer PEI, the survival rate of human skin fibroblasts is increased from 32.35% for PEI to over 90%, demonstrating high biocompatibility.

[0049] (2) This invention solves the inherent application defects of carbon dots from Ginkgo biloba leaves, achieving long-term stable UV protection. This invention utilizes a three-dimensional hydrophilic network of sodium alginate / carboxymethyl chitosan hydrogel to achieve uniform distribution of carbon dots, fundamentally solving the inherent defects of rapid loss of UV shielding performance caused by drying agglomeration and film cracking when Ginkgo biloba leaf carbon dots are used alone. Experimental verification shows that after 48 hours of continuous UVA+UVB irradiation, the transmittance in the 200–400 nm UV band of the composite hydrogel of this invention remains below 20%, stably blocking more than 85% of UVA radiation and more than 98% of UVB radiation, achieving long-term stable UV protection and filling a gap in existing technologies.

[0050] (3) This invention constructs a full-chain synergistic intervention system, completely breaking the vicious cycle of "inflammation-aging" induced by ultraviolet radiation. This invention combines positively charged Ginkgo biloba carbon dots with Astragalus polysaccharides, relying on the long-acting sustained-release system of hydrogel to achieve synergistic effects: ① Long-acting ultraviolet shielding at the source, reducing direct damage to cells and abnormal production of cfDNA by ultraviolet radiation; ② Synergistically clearing ROS, alleviating oxidative damage and aging of fibroblasts; ③ Targeted adsorption and clearing of cfDNA, blocking the inflammatory cascade reaction mediated by it; ④ Sustained release of Astragalus polysaccharides, promoting angiogenesis in the wound. The four functions are interconnected, completely breaking the vicious cycle of "inflammation-aging" induced by ultraviolet radiation. In vivo experiments have confirmed that the composite hydrogel of this invention, in the treatment of UV-induced refractory wounds, compared with the PBS group under UV irradiation alone, showed a relative healing rate of 57.18%±14.04% in the CD treatment group, 49.5%±6.55% in the APS treatment group, and 131.91% in the GCA treatment group after 12 days. The healing rate was significantly better than that of the carbon dot group and the astragalus polysaccharide group alone, and higher than the sum of the healing rates of the two groups, demonstrating a synergistic effect.

[0051] (4) The preparation process is simple and controllable, and has both clinical value and industrialization prospects.

[0052] The raw materials used in this invention are all commercially available natural polymer materials and medicinal herbs that are both food and medicine. No toxic or harmful reagents are added. The core preparation process is only a one-step hydrothermal method and mild calcium ion crosslinking. The reaction conditions are mild, the operation steps are simple, and the batch stability is good. This solves the problems of complex composition and uncontrollable quality of traditional Chinese medicine crude extracts, and it is very easy to achieve large-scale mass production. At the same time, the dressing of this invention can be widely used in multiple scenarios such as the treatment of refractory wounds, protection against photoaging of the skin, and treatment of chronic inflammatory skin diseases, and has both high clinical value and industrialization prospects. Attached Figure Description

[0053] Figure 1 Figure 1 shows the physicochemical characterization results of the synthesized Ginkgo biloba leaf carbon dots; where A is a photograph of the carbon dots under white light and 365nm ultraviolet light; B is the XRD pattern of the carbon dots; C is the XPS pattern of the C1s carbon dots; D is the XPS pattern of the O1s carbon dots; E is the XPS pattern of the N1s carbon dots; and F is the full XPS spectrum of the carbon dots.

[0054] Figure 2 Figure 1 shows the characterization results of the functional properties of carbon dots in Ginkgo biloba leaves; where A is the UV absorption curve of the carbon dot solution; B is the transmittance curve of the carbon dot solution; and C is the reflectance curve of the carbon dot solution.

[0055] Figure 3 The image shows the morphology of carbon dots in Ginkgo biloba leaves; where A is a TEM image of the carbon dots and B is the average particle size of the carbon dots.

[0056] Figure 4Figure 1 shows the performance characterization results of the GCA composite hydrogel; where A is a photograph of the hydrogel before and after gelation; B is the swelling curve of G and GCA hydrogels; C is the degradation curve of G and GCA hydrogels; D is the sustained-release curve of carbon dots in GCA hydrogel; and E is the sustained-release curve of astragalus polysaccharide in GCA hydrogel.

[0057] Figure 5 The figure shows the results of the antioxidant performance verification of GCA composite hydrogel; where A represents the H2O2 scavenging efficiency of different groups; and B represents the DPPH free radical scavenging efficiency of different groups.

[0058] Figure 6 Figure 1 shows the UV shielding performance characterization results of GCA composite hydrogel; where A is the UVA shielding rate of different groups as a function of irradiation time; and B is the UVB shielding rate of different groups as a function of irradiation time.

[0059] Figure 7 The image shows the therapeutic effect of GCA composite hydrogel on UV-induced refractory wounds; where A is a schematic diagram of wound modeling and drug administration; B is a photograph of mouse wounds at different time points; and C is the wound healing rate of different treatment groups on day 12.

[0060] Figure 8 The figure shows the effects of GCA composite hydrogel on cell damage, cfDNA clearance and cell senescence. Among them, A is the γ-H2AX immunofluorescence image of wound tissue in different treatment groups; B is the cfDNA level in wound exudate after 24 hours of ultraviolet irradiation; and C is the expression level of the senescence marker Lamin B1 in wound tissue in different treatment groups.

[0061] Figure 9 The image shows the effect of GCA composite hydrogel on downregulating inflammatory factors in wounds; where A represents the expression level of TNF-α in wound tissue and B represents the expression level of IL-6 in wound tissue.

[0062] Figure 10 CD31 immunofluorescence images of wound tissue (angiogenesis status) in different treatment groups. Detailed Implementation

[0063] To make the objectives, technical solutions, and beneficial effects of this invention clearer and to facilitate better understanding and implementation of the technical solutions in this application by those skilled in the art, the technical solutions of this invention are clearly and completely described below in conjunction with specific embodiments. It should be particularly noted that the embodiments described below are only some embodiments of this invention, not all embodiments, and are only used to explain and exemplify this invention, and are not intended to limit the scope of protection of this invention; all other embodiments obtained by those skilled in the art based on the core technical concepts disclosed above, without substantial innovation, through non-essential improvements, parameter adjustments, and equivalent substitutions, should fall within the scope of protection claimed by this invention.

[0064] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0065] There are no particular restrictions on the purity of any raw materials used in this invention. However, it is preferred to use analytical grade materials or materials that meet medical purity standards or standards for carbon dot preparation.

[0066] Example 1: Preparation of positively charged carbon dots from Ginkgo biloba leaves with different Zeta potentials

[0067] This embodiment prepared positively charged Ginkgo biloba leaf carbon dots with different Zeta potentials by adjusting the amount of chitosan added, and verified the effect of potential on cfDNA adsorption efficiency and biocompatibility. The specific steps are as follows:

[0068] Fresh ginkgo leaves were blanched at 100℃ for 10 minutes, dried at 60℃, and then pulverized to obtain ginkgo leaf powder. Four 2.0 g portions of ginkgo leaf powder were placed in 50 mL of acidic deionized water (pH=4, adjusted with glacial acetic acid) and stirred at room temperature for 30 minutes to form a homogeneous suspension. 0 g, 0.2 g, 0.5 g, and 1.0 g of chitosan powder were added respectively, and stirred at room temperature until completely dissolved, resulting in four mixtures with corresponding ginkgo leaf powder to chitosan powder mass ratios of ginkgo leaf alone, 10:1, 4:1, and 2:1. The four mixtures were transferred to 100 mL polytetrafluoroethylene-lined hydrothermal reactors and reacted at 180℃ for 6 hours. After the reaction, the mixtures were allowed to cool naturally to room temperature. The reaction product was centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microfiltration membrane to remove large particulate impurities. The filtrate was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 500 Da, with deionized water replaced every 6 h to remove unreacted small molecule impurities. After dialysis, the dialysate was freeze-dried for 48 h to obtain four groups of Ginkgo biloba carbon dot powders, labeled as CD1, CD2, CD3, and CD4, respectively.

[0069] The physicochemical properties and biocompatibility of the four groups of carbon dot and cationic polymers PEI were characterized, and the results are shown in Table 1 below.

[0070] Table 1 Core performance parameters of carbon dots from Ginkgo leaves

[0071]

[0072] Experimental results confirmed that within a potential range of +15mV to +40mV, as the positive charge of the carbon dots increased, the survival rate of human skin fibroblasts remained above 90% (final concentration of 1 mg / mL), demonstrating high safety and cfDNA clearance rate. Commercially available cationic polymers exhibited high charge and cfDNA clearance rate, but lower cell survival efficiency. Considering synthesis yield, electronegativity, and safety, the optimal Zeta potential for positively charged Ginkgo biloba carbon dots was +18mV to +38.5mV, with the best being approximately +18 to +19mV ​​(CD3 group).

[0073] Example 2 Characterization and UV shielding properties of positively charged carbon dots in Ginkgo biloba leaves

[0074] Further analysis of the properties of the preferred CD3 was conducted, through... Figure 1 A in the study found that the synthesized Ginkgo biloba carbon dots exhibited a brownish-yellow color and emitted blue fluorescence under 365 nm ultraviolet light. Analysis of the freeze-dried carbon dot powder revealed a broad peak in the X-ray diffraction (XRD) pattern, centered at approximately 20°, indicating the presence of carbon defects in amorphous carbon caused by heteroatom doping or sp2 (see [reference needed]). Figure 1 (B in the image). XRD results indicate that the carbon dots contain no obvious inorganic impurity phases (such as metal oxides, salts, etc.), indicating high purity and a predominantly carbon-based structure. The XPS spectra of C1s, N1s, and O1s are shown in the image. Figure 1 CF in the C1s spectrum. For the C1s spectrum (see...) Figure 1 The peak at 284.80 eV (C in the graphite) is attributed to the C=C bond, indicating the presence of a graphitic carbon structure; the peak at 286.23 eV corresponds to the CN / CO bond, indicating that nitrogen is incorporated into the carbon structure; the peak at 287.92 eV is related to the C=O bond, which may suggest the presence of carbonyl or imine groups on the carbon dot surface; the peak at 292.69 eV may be related to the π-π* transition in the aromatic system. For the O1s spectrum (see...), see... Figure 1 The peak at 530.86 eV (in the D spectrum) is related to C=O, while the peak at 532.18 eV (C-OH / C-O-C) is attributed to the hydroxyl or ether oxygen group. In the N1s spectrum (see...), Figure 1In the E group, the binding energies are 399.56 eV and 401.48 eV, corresponding to NH / NH2 (amino / imino) and quaternary nitrogen (NQ), respectively. The main peak at 399.56 eV indicates that amino is the main nitrogen-containing functional group on the carbon dot surface, endowing the carbon dots with excellent water solubility and surface reactivity. The weak peak at 401.48 eV confirms that the carbon dots sp 2 Graphitic nitrogen is present in the hybrid carbon framework. These oxygen-containing functional groups play an important role in the surface chemistry and properties of carbon dots. Figure 1 F in the figure represents the XPS full spectrum of CD, showing that CD is composed of C, N and O atoms, with weight percentages of 62.35%, 4.24% and 33.41%, respectively.

[0075] A 0.5 mg / mL aqueous solution of Ginkgo biloba carbon dots was prepared, and the absorption characteristics of the sample were detected using a UV absorption spectrometer. The UV absorption curve results are shown below. Figure 2 As shown in Figure A, the absorption peak of CD is close to λ=210 nm, which is attributed to the π-π* transition containing the C=C bond. The absorption peak near λ=280 nm is attributed to the n-π* transition containing the C=O bond. Simultaneously, the transmittance and reflectance of the 2 mg / mL carbon dot solution were measured using a spectrometer. CD exhibits good low transmittance in the ultraviolet region (see Figure A). Figure 2 CD (type B) has the ability to absorb ultraviolet light. Its transmittance gradually saturates and stabilizes at λ>700 nm, reaching a maximum value (74.7%). At a concentration of 2.0 mg / mL, the transmittance in the ultraviolet region (λ<400 nm) is 1.8%, at which point CD absorbs 98.2% of the ultraviolet light. Similarly, CD also exhibits excellent ultraviolet shielding performance in refractive index measurements (see...). Figure 2 (C in the text). Further morphological observation of CDs using transmission electron microscopy revealed that CDs exhibited a uniform particle size in the range of 2–4 nm (see C). Figure 3 )

[0076] Example 3: Preparation of the preferred formulation of the GCA composite hydrogel dressing of the present invention

[0077] Preparation of hydrogel precursor solution: Different masses of sodium alginate (Aladdin, S100128) and different masses of carboxymethyl chitosan (Aladdin, C304738) were weighed and added to 100 mL of deionized water. The mixture was stirred at 60℃±5℃ for 30 min until completely dissolved, resulting in a uniform and transparent hydrogel precursor solution. 10 mL of the above hydrogel precursor solution was taken, and 20 mg of the carbon dot powder (CD3) prepared in Example 1 and 20 mg of astragalus polysaccharide powder (Solebo, SA9790) were added. The mixture was stirred at 60℃±5℃ for 20 min until completely dissolved, with each carbon dot and astragalus polysaccharide having a mass fraction of 0.2%. 2.0 mL of a 1.25% (w / v) calcium chloride solution was slowly added dropwise to the solution. After the addition was complete, the mixture was allowed to stand at room temperature to complete cross-linking and gel formation, resulting in the GCA composite hydrogel dressing.

[0078] Table 2 Different GCA composite hydrogel ratios

[0079]

[0080] Experimental results confirm that when the mass ratio of sodium alginate to carboxymethyl chitosan is 1:1 and both have a mass-volume fraction of 3% (w / v), they successfully crosslink into a gel with good formability. That is, the hydrogel GCA2 not only forms a gel but also has good transparency.

[0081] Characterization of the physicochemical properties of 4 GCA composite hydrogels

[0082] 4.1 Physicochemical characterization of GCA multifunctional composite hydrogel

[0083] Hydrogel G was prepared to evaluate the performance of hydrogel GCA2. The composition of hydrogel G is as follows: 3g of sodium alginate and 3g of carboxymethyl chitosan sodium alginate were added to 100mL of deionized water and stirred at 60℃±5℃ for 30 min until completely dissolved, yielding a homogeneous and transparent hydrogel precursor solution. 10mL of the above hydrogel precursor solution was slowly added dropwise to 2mL of a 1.25% (w / v) calcium chloride solution, and the mixture was placed in a circular mold and allowed to stand at room temperature to form a gel, thus obtaining hydrogel G.

[0084] Figure 4 Figure A shows the state of the hydrogel before and after gelation. To test the swelling properties of the hydrogels, the formed hydrogels G and GCA (10 mm in diameter, 2 mm in thickness) were freeze-dried to constant weight, and the initial weight was recorded as W0. The G and GCA gels were then immersed in 10 mL of PBS (pH 7.4, 0.01 mol·L⁻¹). -1 The samples were placed in a 37℃ constant temperature incubator for incubation. Samples were removed at preset time points (0, 0.5, 1, 2, 6, 8, 12, 24h), the surface free moisture was blotted dry with filter paper, and the samples were weighed. The mass W after swelling was recorded. tThis continues until swelling equilibrium is reached. Swelling rate SR t =[(W t -W0) / W0]×100%, plot the swelling kinetics curve with time as the x-axis (e.g., Figure 4 (B in the text). The results showed that both G and GCA hydrogels could rapidly absorb water and swell within 2 hours, reaching swelling equilibrium within 6 hours, with a swelling rate ≥1400%. They possessed excellent rapid water absorption capacity, efficiently absorbing wound exudate and maintaining a moist healing environment, fully meeting the application requirements of medical wound dressings. Further testing of the hydrogel's solubility involved immersing the gel in PBS at 37°C. After reaching swelling equilibrium, its initial weight was measured as W0. Samples were removed at preset time points (0, 12, 24, 36, and 48 hours), and the remaining weight was measured as W0. t The solubility is calculated using the formula DR%=[(W t -W0) / W0]×100%, plot the swelling kinetics curve with time as the x-axis (e.g., Figure 4 (C in the text). Swelling and dissolution curves indicate that the hydrogel surface has a strong water absorption capacity. The hydrogel was further immersed in PBS solution at pH 7.4, shaken at 100 rpm at 37°C, and the extract was collected at preset time points (0, 1, 3, 6, 12, 24, 36, 48 h). The sustained-release effect of CD was detected by UV absorption (e.g., C). Figure 4 (D in the original text). Further analysis was conducted using the phenol-sulfuric acid method to determine the content of Astragalus polysaccharides in the extract (e.g., ...). Figure 4 (E in the text). Figure 4 Further analysis of D and E in the study confirms that GCA possesses excellent drug sustained-release capabilities. GCA hydrogel exhibits superior long-term sustained-release capabilities for both carbon dots and astragalus polysaccharides, with no burst release within 48 hours. The cumulative release rate of carbon dots over 48 hours is 87%, and the cumulative release rate of astragalus polysaccharides is 79.27%. This enables continuous and stable release of active ingredients locally on the wound surface, overcoming the core shortcomings of existing technologies such as easy loss of astragalus polysaccharides, easy inactivation of DNase, and short window of action, thus meeting the intervention needs throughout the entire wound healing cycle.

[0085] 4.2 Characterization of the therapeutic properties of GCA multifunctional composite hydrogel

[0086] Ginkgo leaves are a traditional Chinese medicinal herb used in both food and medicine. Their active ingredients possess antioxidant, anti-inflammatory, and tissue-repair-promoting properties. However, current applications primarily utilize crude extracts of the herb, and ginkgo leaf extracts suffer from several core issues, including complex composition, poor batch-to-batch quality control, low bioavailability, and high price. In contrast, carbon dots derived from ginkgo leaves offer high bioavailability, simple synthesis, and possess the ROS scavenging and antioxidant effects associated with ginkgo leaf extracts.

[0087] The Solarbio reagent kit (BC3595) was used for detection: H2O2 (30 mM) was mixed with G, CD (2 mg / mL, this concentration is the final concentration in the detection system, the same below), APS (2 mg / mL), and GCA (CDs content 2 mg / mL, APS 2 mg / mL), respectively, and incubated for 30 min. Titanium sulfate working solution was then added and incubated for 5 min. The amount of titanium peroxide complex formed was measured at 415 nm. The scavenging efficiency of hydrogen peroxide for different groups was calculated. Hydrogen peroxide scavenging efficiency = (S0 - S...) S ) / S0×100%, where S0 is the initial hydrogen peroxide concentration, S S This represents the hydrogen peroxide concentration after the reaction. The results are as follows: Figure 5 As shown in Figure A, CD, APS, and GCA all exhibit H2O2 scavenging capabilities, with GCA hydrogel showing the best scavenging efficiency. Conversely, hydrogel G alone showed no therapeutic effect. The DPPH scavenging abilities of G, CD, APS, and GCA were assessed using the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) method. A DPPH stock solution (0.1 mmol·L⁻¹) was prepared. -1 ) and G, CD (2 mg·mL) respectively -1 ), APS (2 mg·mL) -1 ) and GCA (CDs content of 2 mg·mL) -1 APS 2 mg·mL -1 Mix the samples and incubate at room temperature for 30 min. Measure the absorbance at 515 nm. Calculate the DPPH scavenging efficiency of different groups. Scavenging efficiency = (S0 - S...) S ) / S0×100% (where S0 is the initial OD value, S S (OD value after reaction). Results are as follows: Figure 5 As shown in B, gel G represents antioxidant capacity. CD, APS, and GCA all have antioxidant capacity, with GCA hydrogel showing the best effect.

[0088] 4.3 UV shielding function of GCA multifunctional composite hydrogel

[0089] Carbon dots, rich in aromatic conjugated structures and surface functional groups, exhibit strong and broad absorption characteristics in the 200-400 nm range, laying the foundation for their UV-resistant applications. Introducing carbon dots into a hydrogel network allows them to synergistically construct a dense UV barrier, effectively delaying carbon dot aggregation and cracking, thus significantly enhancing UV shielding. Hydrogels were formed on quartz glass slides using 1 mL of G and 1 mL of GCA solution, respectively, and 1 mL of CD aqueous solution (1 mg·mL⁻¹) was added. -1 ) and 1 mL of APS aqueous solution (1 mg·mL) -1The samples were evenly laid flat on a quartz glass slide (75 mm × 25 mm × 1 mm) and exposed to a UV-LED (UV-LED, Longpro LTD., Guangzhou, China) emitting UVA340 and UVB313. The light intensity beneath the quartz slide was monitored at predetermined intervals using UVA and UVB power meters. The UV shielding efficiency was calculated using the following formula: UV shielding efficiency (%) = (J0 - J) / J × 100%, where J0 represents the light power measured through a clean quartz slide, and J represents the light power measured through a quartz slide coated with various samples. Figure 6 As shown in Figures A and B, initially, group CD exhibited excellent UVA and UVB blocking performance, but over time, it began to agglomerate and develop cracks, resulting in a decrease in its shielding effectiveness. In contrast, group GCA consistently blocked over 85% of UVA radiation and 98% of UVB radiation.

[0090] This invention utilizes the three-dimensional hydrophilic network of GCA hydrogel to achieve nanoscale uniform dispersion and spatial confinement of carbon dots, fundamentally solving the inherent defects of drying and agglomeration and rapid decay of UV shielding performance when Ginkgo leaf carbon dots are used alone. It achieves long-term stable UV protection and fills the gap in the existing technology.

[0091] Example 5: Assessment of the ability of multifunctional composite hydrogel GCA to treat UV-induced delayed wound healing

[0092] SPF-grade ICR mice (6 weeks old, weighing 20-25 g) were purchased from Ruige Biotechnology Co., Ltd. [SCXK(Yue)2023-00592026595270] and housed in the SPF-grade Animal Experiment Department of Southern Medical University [SYXK(Yue)2021-0167]. The housing environment was maintained at a temperature of 24±2°C and a relative humidity of 50±10%, following a 12-hour light-dark cycle. All animal experiments were conducted according to the protocols approved by the Animal Management and Use Committee of Southern Medical University (Animal Experiment Approval No.: SMUL202603087). A UV-induced delayed healing model was established. During the experiments, the mice were anesthetized with 2% isoflurane, and their dorsal fur was shaved. The skin was disinfected alternately with 70% alcohol and povidone-iodine to minimize the risk of infection. A full-thickness excision wound (1.2 cm in diameter) was created in the center of the back using a sterile circular biopsy punch, ensuring the incision depth reached the subcutaneous tissue. After wound establishment, patients were divided into four groups: Con (control group, no treatment or UV irradiation), PBS+UV (50 μL sterile PBS applied to the wound), G+UV (50 μL G base hydrogel applied to the wound), CD+UV (50 μL 2 mg / mL carbon dot solution applied to the wound), APS+UV (50 μL 2 mg / mL APS solution applied to the wound), and GCA+UV treatment group (50 μL Ginkgo biloba carbon dot composite hydrogel GCA applied to the wound). The medication was changed every two days, followed by UV irradiation. All treatment groups except Con received daily UVA (64.8 kJ·m²). -2 ) and UVB (11.76 kJ·m -2 Exposure is used to simulate the wound healing environment under ultraviolet radiation. A single exposure lasts 20 minutes (see [link to relevant documentation]). Figure 7 A). Wound healing curve record, such as Figure 7 As shown in Figure B, the PBS group and the G hydrogel group showed significant delayed wound healing due to UV interference. GCA significantly reversed the UV-induced healing delay and accelerated wound healing. ImageJ software was used to analyze the wound area. Using the wound healing rate of the UV irradiation model control group (PBS) as a baseline, the healing improvement rate of the experimental group was calculated using the formula: Relative healing promotion rate (%) = (S0 - S) / S × 100%, where S0 represents the absolute wound healing rate of the experimental group at 12 days, and S represents the absolute wound healing rate of the PBS group at 12 days. Wound size and time curves were also plotted. Figure 7As shown in Figure C, the healing rates of the CD and APS treatment groups under UV interference were 57.18%±14.04 and 49.5%±6.55% higher than those of the PBS group, respectively. The healing rate of the GCA composite hydrogel after treatment on day 12 was 131.91%±13.66% higher than that of the PBS group, significantly superior to both the CD and APS groups, and far exceeding the expected additive effect of either group acting alone. This confirms the unexpected synergistic effect of positively charged Ginkgo biloba carbon dots and Astragalus polysaccharides in the hydrogel system of this invention. The successful promotion of wound healing under UV interference by GCA further illustrates the significant delay in wound healing under UV intervention.

[0093] Frozen sections were taken from the wound sites of mice treated with Con, PBS, G, CD, APS, and GCA. The sections were then subjected to immunofluorescence staining with γ-H2AX (the procedure is described in the reference "Acedera JD, et al. BTG3-dependent VCP / p97 nuclear translocation is required for efficient repair of UV-induced DNA lesions Nucleic Acids Research. 2025(53), Issue 13") to assess the extent of UV damage. Figure 8 In the A group, the high expression of γ-H2AX in the tissues of the PBS and G treatment groups was observed due to the lack of UV shielding, indicating severe cell damage caused by PBS and G. The cfDNA content in the exudate from the wound site after wound modeling (24 hours after UV irradiation) was measured using an ELISA kit. Figure 8 The results in Figure B showed that CD and GCA significantly downregulated the cfDNA content in wound exudate, with GCA hydrogel dressing showing the best effect. Conversely, the cfDNA content in wound exudate in the PBS and G treatment groups was significantly higher than that in the Con control group. The aging marker Lamin B1 in wound tissue was detected using a Lamin B1 assay kit (SEF548Mi, Cloud-Clone Corp.), and the results are shown in Figure B. Figure 8 In the control group, the levels of Lamin B1 in the C, PBS, G, CD, and APS treatment groups were significantly downregulated compared to the Con group, with the most significant reduction observed in PBS and G. There was no significant difference between the GCA treatment group and the Con control group, indicating that the PBS, G, CD, and APS treatment groups experienced varying degrees of cellular senescence under UV irradiation, while GCA multifunctional hydrogel treatment effectively alleviated cellular senescence caused by UV irradiation.

[0094] Skin tissue was collected from the wound and lysed with RIPA lysis buffer containing a protease inhibitor. The homogenate was homogenized on ice and centrifuged at 12000 g for 15 min at 4°C. The supernatant was collected, and protein concentration was determined using the BCA method. The levels of TNF-α and IL-6 inflammatory factors in the tissue homogenate supernatant were detected using an ELISA kit. Figure 9 As shown in A and B, the GCA composite hydrogel exhibits a strong ability to downregulate inflammation and effectively reduces the levels of TNF-α and IL-6 in the wound site, indicating that the GCA hydrogel effectively reduces the level of inflammation in the wound site.

[0095] The degree of angiogenesis at the wound site indicates the level of wound healing. Skin tissue from the wounds of mice in each group was collected after treatment on day 12, embedded and sectioned, and then subjected to CD31 immunofluorescence (the procedure is referenced in "Xu SH, et al. Magnesium Silicate Composite Patch With Neurovascular Regenerative Properties Promotes Diabetic Wound Healing in Mice Interdisciplinary Materials. 2025(4): Issue 5"). CD31 fluorescence was observed in the sections, with higher fluorescence intensity in APS and GCA hydrogels, followed by CD and Con treatment groups, and almost no red fluorescence of CD31 in PBS and G treatment groups. This indicates that APS can effectively promote angiogenesis at the wound site, and GCA multifunctional hydrogel can also effectively promote angiogenesis by releasing APS at the wound site. Figure 10 ).

[0096] This invention overcomes the limitation that the nucleic acid adsorption capacity and biocompatibility of cationic materials in this field cannot be simultaneously achieved, and solves the inherent defect of rapid decay of UV shielding performance when Ginkgo biloba carbon dots are used alone. It realizes a full-chain synergistic intervention of UV protection, ROS removal, cfDNA targeted removal, inflammation inhibition, and angiogenesis promotion, providing a new solution with high efficiency, low toxicity, and excellent biocompatibility for UV-induced refractory wounds, filling the technological gap in related fields.

[0097] Finally, it should be noted that the above descriptions are merely embodiments of the present invention, used only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A sunscreen carbon quantum dot for clearing cfDNA, characterized in that: It was prepared by hydrothermal method using ginkgo leaf powder as raw material and chitosan as amino donor. It has positively charged amino functional groups on the surface, a zeta potential of +15 mV to +40 mV, and characteristic absorption peaks in the 200 to 400 nm ultraviolet band.

2. The method for preparing sunscreen carbon quantum dots for clearing cfDNA as described in claim 1, characterized in that... The process includes the following steps: fresh ginkgo leaves are blanched, dried, and pulverized. The resulting ginkgo leaf powder is mixed evenly with chitosan in a weakly acidic aqueous solution to obtain a chitosan-dissolved suspension. The suspension is subjected to a hydrothermal reaction at 160–200 °C for 4–8 h. After the reaction, the solid and liquid are separated. The supernatant is filtered through a 0.22 μm microfiltration membrane to remove large particulate impurities. The filtrate is dialyzed in deionized water using a dialysis bag with a molecular weight cutoff of 500 Da. The dialysate obtained from the dialysis is freeze-dried to obtain sunscreen carbon quantum dots that remove cfDNA.

3. The application of the sunscreen carbon quantum dots for clearing cfDNA as described in claim 1 in the preparation of sunscreen skin care products or hydrogel excipients.

4. A composite hydrogel dressing for clearing cfDNA, characterized in that: It includes a hydrogel matrix and functional active components dispersed in the hydrogel matrix; the functional active components include the sunscreen carbon quantum dots and astragalus polysaccharide for clearing cfDNA as described in claim 1.

5. The composite sunscreen hydrogel dressing for clearing cfDNA according to claim 4, characterized in that: The hydrogel matrix is ​​obtained by ion crosslinking sodium alginate and carboxymethyl chitosan; wherein the mass ratio of sodium alginate to carboxymethyl chitosan is 1:0.5-2. The ion mentioned is a calcium ion; The sunscreen carbon quantum dots that remove cfDNA and the astragalus polysaccharide are mixed in a mass ratio of 1-2:1-2.

6. The composite hydrogel dressing for clearing cfDNA according to claim 5, characterized in that: The mass ratio of sodium alginate to carboxymethyl chitosan is 1:1 to 2; The calcium ions mentioned are derived from calcium chloride; The calcium chloride content is based on a mass ratio of sodium alginate to calcium chloride of 6 to 12:

1.

7. The method for preparing the composite hydrogel dressing for clearing cfDNA according to any one of claims 4 to 6, characterized in that... Includes the following steps: (1) Mix sodium alginate, carboxymethyl chitosan and water, and stir at 55-65°C until a transparent and uniform hydrogel precursor solution is obtained; (2) Mix the sunscreen carbon quantum dots for clearing cfDNA as described in claim 1, astragalus polysaccharide and the hydrogel precursor solution obtained in step (1), and stir at 55-65°C until the astragalus polysaccharide is dissolved and the positively charged Ginkgo biloba carbon dots are evenly dispersed to obtain a mixed solution. (3) Add calcium chloride solution to the mixed solution obtained in step (2), and let it stand after the addition is complete to obtain a composite sunscreen hydrogel dressing that removes cfDNA.

8. The method for preparing the composite hydrogel dressing for clearing cfDNA according to claim 7, characterized in that: The concentration of sodium alginate in the hydrogel precursor solution in step (1) is 1-5% w / v; The concentration of carboxymethyl chitosan in step (1) in the hydrogel precursor solution is 1-5% w / v; The content of the sunscreen carbon quantum dots for clearing cfDNA mentioned in step (2) in the mixed solution is 0.15-0.25% w / v; The content of Astragalus polysaccharide in the mixed solution mentioned in step (2) is 0.15-0.25% w / v; The concentration of the calcium chloride solution mentioned in step (3) is 1.0 to 1.5% w / v.

9. The use of the composite hydrogel dressing for clearing cfDNA as described in any one of claims 4 to 6 in the preparation of medical preparations or skin care products.

10. The application according to claim 9, characterized in that: The medical preparation described is a medical preparation for ultraviolet-induced refractory skin wounds; The skincare product mentioned is a face mask.

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