Amino acid-porous carbon dot composite material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of carbon nanomaterials, and particularly relates to an amino acid-porous carbon dot composite material as well as a preparation method and application thereof. The amino acid-porous carbon dot composite material comprises porous carbon dots and amino acid loaded on the porous carbon dots, the amino acid is connected to a pore structure of the porous carbon dot. The prepared porous carbon dots have a porous structure, a closed space is provided for encapsulation of amino acid, the conjugated structure of the porous carbon dots is destroyed by doping of the amino acid in nanopores, and characteristic fluorescence of the porous carbon dots is converted from yellow emission to cyan emission of an amino acid-porous carbon dot composite material. Acidic conditions can trigger amino acid to be released from the porous carbon dots, transfer and release of amino acid can be tracked in real time through fluorescence color changes, and real-time fluorescence monitoring is achieved. And the amino acid-porous carbon dot composite material can promote wound healing.

Description

Technical Field

[0001] This invention belongs to the field of carbon nanomaterials technology, and specifically relates to an amino acid-porous carbon dot composite material, its preparation method, and its application. Background Technology

[0002] Currently, fluorescence color changes in carbon dot-based smart drug delivery systems typically originate from energy transfer between the carbon dot and the fluorophore-containing drug. However, most clinically used drugs lack fluorescence properties and cannot participate in the energy transfer process between the carbon dot and the drug, thus limiting drug release monitoring.

[0003] Carbon dots (CDs) are novel carbon-based nanomaterials with dimensions below 10 nanometers. Their excellent biocompatibility, non-toxicity, and tunable fluorescence properties have spurred extensive research into their application in biofluorescence imaging, making them a versatile drug delivery platform for biomedical applications. Intelligent drug delivery systems based on carbon dots can achieve real-time visualization of drug pharmacokinetics and pharmacodynamics through dynamic imaging of spatial distribution and intracellular metabolic processes. However, how to reflect the effective loading and release dynamics of non-fluorescent drugs through structural changes in the carrier, particularly by developing stimulus-responsive fluorescent variants capable of structural and optical modulation under external triggering conditions, still requires further exploration. Summary of the Invention

[0004] The present invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial alternative. Specifically, the present invention provides an amino acid-porous carbon dot composite material that can achieve pH-sensitive fluorescence color switching, thereby enabling real-time tracking of drug release at the cellular level, and also promoting wound healing.

[0005] The inventive concept of this invention is as follows: The amino acid-porous carbon dot composite material of this invention comprises porous carbon dots and amino acids loaded on the porous carbon dots; the amino acids are connected to the pore structure of the porous carbon dots. The surface of the porous carbon dots of this invention has a molecular-level pore structure, and amino acids are adsorbed on the pore surface. This provides a closed space for the encapsulation of amino acids. The incorporation of amino acids into the nanopores disrupts the conjugated structure of the porous carbon dots, causing the characteristic fluorescence of the porous carbon dots to change from the original yellow emission to the cyan emission of the amino acid-porous carbon dot composite material. Under acidic conditions, basic amino acids can be triggered to be released from the porous carbon dots, and the fluorescence color changes from cyan to yellow, realizing pH-sensitive fluorescence color switching, thereby enabling real-time tracking of drug release at the cellular level. In addition, the amino acid-porous carbon dot composite material prepared by this invention can significantly accelerate wound healing and has the effect of promoting wound healing.

[0006] Therefore, a first aspect of the present invention provides an amino acid-porous carbon dot composite material.

[0007] Specifically, the amino acid-porous carbon dot composite material includes porous carbon dots and amino acids loaded on the porous carbon dots; the amino acids are connected to the pore structure of the porous carbon dots.

[0008] Preferably, the surface of the porous carbon dot structure is connected to amino acids via hydrogen bonds.

[0009] Preferably, the porous carbon dots have a carbon core with a graphene-like lattice.

[0010] Preferably, the porous carbon dots have a particle size of 2-10 nm and a pore size of 0.4-0.8 nm.

[0011] More preferably, the particle size of the porous carbon dots is 3-4 nm, and the pore size of the porous carbon dots is 0.5-0.6 nm.

[0012] Preferably, the amino acids include basic amino acids.

[0013] More preferably, the basic amino acid includes at least one of lysine, arginine, and histidine; even more preferably, the basic amino acid includes at least one of lysine and arginine.

[0014] Specifically, the basic amino acid refers to an amino acid having two amino groups and one carboxyl group, and having a net positive charge at neutral pH.

[0015] Preferably, the mass ratio of the porous carbon dots to amino acids is 1:(0.1-11); more preferably, the mass ratio of the porous carbon dots to amino acids is 1:(0.1-10).

[0016] Preferably, at pH < 6, the amino acid release rate in the amino acid-porous carbon dot composite material is ≥ 80%; more preferably, at pH < 5, the amino acid release rate in the amino acid-porous carbon dot composite material is ≥ 90%.

[0017] Preferably, the porous carbon dots have a fluorescence emission peak with a wavelength of 500-600 nm under an excitation light source of 400-500 nm;

[0018] More preferably, the porous carbon dots have a fluorescence emission peak with a wavelength of 550-600 nm under an excitation light source of 450-500 nm.

[0019] Preferably, the amino acid-porous carbon dot composite material has a fluorescence emission peak with a wavelength of 450-500 nm under an excitation light source of 350-450 nm.

[0020] More preferably, the amino acid-porous carbon dot composite material has a fluorescence emission peak with a wavelength of 470-500 nm under an excitation light source of 400-450 nm.

[0021] Specifically, the steric hindrance introduced by amino acids disrupted the original porous carbon dots sp. 2 The π-π interactions between fragments lead to the formation of sp in amino acid-porous carbon dot composites. 2 The conjugated π-π stacking between fragments is less than that of the original porous carbon dots. The porous carbon dots exhibit fluorescence emission peaks of 500-600 nm under an excitation source of 400-500 nm, while the amino acid-porous carbon dot composite material exhibits fluorescence emission peaks of 450-500 nm under an excitation source of 350-450 nm, corresponding to the original porous carbon dot sp... 2 The π-π interactions between fragments and the conjugated π-π stacking of amino acid-porous carbon dot composites are disrupted.

[0022] A second aspect of the present invention provides a method for preparing the amino acid-porous carbon dot composite material described in the first aspect of the present invention.

[0023] Specifically, the preparation method of the amino acid-porous carbon dot composite material includes the following steps:

[0024] (1) Mix polycyclic aromatic hydrocarbons and amide compounds containing carboxylic acids and heat them to obtain porous carbon dots;

[0025] (2) The porous carbon dots and amino acids are mixed in a solvent and heated for incubation to obtain the amino acid-porous carbon dot composite material.

[0026] Specifically, the porous carbon dots in this invention are synthesized from carboxylic acid-containing polycyclic aromatic hydrocarbons and solvent amide compounds via a free radical-assisted solvothermal method. In this process, the carboxyl groups in the carboxylic acid-containing polycyclic aromatic hydrocarbons undergo amidation with the amino groups in the amide compounds, while the hydrogen on the amide undergoes dehydrogenation with the hydrogen on the polycyclic aromatic hydrocarbon, forming a cyclic structure. The inventors discovered that these carbon dots possess a graphene-like carbon core and a molecular-level porous structure on their surface. The hydrogen on the amide bonds at the pores is more reactive than the hydrogen on the aromatic rings, making it easier to form intermolecular hydrogen bonds with amino acid molecules. Therefore, the pore surface can adsorb amino acids through hydrogen bonds. This provides a closed space for the encapsulation of amino acids. The incorporation of amino acids into the nanopores disrupts the conjugated structure of the porous carbon dots, causing the characteristic fluorescence of the porous carbon dots to change from the original yellow emission to the cyan emission of the amino acid-porous carbon dot composite material.

[0027] Preferably, in step (1), the carboxylic acid-containing polycyclic aromatic hydrocarbon includes at least one of 3,9-perylenedicarboxylic acid, 2,7-pyrenedicarboxylic acid, 2-indolecarboxylic acid, fluorene-9-carboxylic acid, 5-acenaphthenic acid, and 2,6-naphthalenedicarboxylic acid; more preferably, the carboxylic acid-containing polycyclic aromatic hydrocarbon includes 3,9-perylenedicarboxylic acid.

[0028] Preferably, in step (1), the amide compound includes at least one of formamide and N,N-dimethylformamide.

[0029] Preferably, in step (1), the mixing further includes adding a catalyst, wherein the catalyst includes at least one of potassium persulfate, ammonium persulfate, and sodium persulfate.

[0030] Preferably, in step (1), the mass ratio of the carboxylic acid-containing polycyclic aromatic hydrocarbon to the catalyst is 1:(1-10); more preferably, the mass ratio of the carboxylic acid-containing polycyclic aromatic hydrocarbon to the catalyst is 1:(1-5); even more preferably, the mass ratio of the carboxylic acid-containing polycyclic aromatic hydrocarbon to the catalyst is 1:(1-2).

[0031] Preferably, in step (1), the amount of the amide compound used is 5-30 mL; more preferably, the amount of the amide compound used is 10-20 mL; even more preferably, the amount of the amide compound used is 10-15 mL.

[0032] Preferably, in step (1), the heating temperature is 50-90℃ and the heating time is 10-120 min; more preferably, the heating temperature is 70-90℃ and the heating time is 10-60 min; for example, the heating temperature is 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, etc.; the heating time is 10-120 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.

[0033] Preferably, in step (1), after heating, the process further includes removing the catalyst and amide compounds.

[0034] Preferably, the step of removing the catalyst and amide compounds is as follows: dialyzing for 11-100 hours using a dialysis bag with a molecular weight cutoff of 450-2200 Da.

[0035] More preferably, the step of removing the catalyst and amide compound is as follows: dialyzing for 12-96 hours using a dialysis bag with a molecular weight cutoff of 500-2000 Da.

[0036] Preferably, the process of removing the catalyst and amide compounds further includes a freeze-drying process.

[0037] Preferably, in step (2), the mass ratio of the porous carbon dots to the amino acids is 1:(0.1-11).

[0038] More preferably, in step (2), the mass ratio of the porous carbon dots to the amino acids is 1:(0.1-10).

[0039] Preferably, in step (2), the solvent includes at least one of water, culture medium, and phosphate buffer.

[0040] Preferably, the culture medium includes a minimum essential culture medium (MEM medium).

[0041] Preferably, in step (2), the concentration of the porous carbon dots after mixing is 0.08-0.25 mg / mL; more preferably, the concentration of the porous carbon dots after mixing is 0.09-0.11 mg / mL; even more preferably, the concentration of the porous carbon dots after mixing is 0.1 mg / mL.

[0042] Preferably, in step (2), the heating and incubation temperature is 35-65℃ and the heating and incubation time is 9-65 min; more preferably, the heating and incubation temperature is 40-60℃ and the heating and incubation time is 10-60 min; for example, the heating and incubation temperature is 40℃, 45℃, 50℃, 55℃, 60℃, etc.; for example, the heating and incubation time is 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min.

[0043] Preferably, in step (2), the heating and incubation are followed by cooling to obtain an amino acid-porous carbon dot composite material.

[0044] A third aspect of the present invention provides the application of the amino acid-porous carbon dot composite material described in the first aspect of the present invention in the preparation of cell fluorescence imaging reagents and skin repair drug formulations.

[0045] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0046] (1) The porous carbon dots prepared in this invention have a graphene-like carbon core and a molecular-level porous structure on the surface. Amino acids can be adsorbed through hydrogen bonds on the pore surface. The porous structure provides a sealed space for the encapsulation of amino acids. The incorporation of amino acids into the nanopores disrupts the conjugated structure of the porous carbon dots, causing the characteristic fluorescence of the porous carbon dots to change from the original yellow emission to the cyan emission of the amino acid-porous carbon dot composite material. Under acidic conditions, the amino acid-porous carbon dot composite material can trigger the release of basic amino acids from the porous carbon dots, changing the fluorescence color from cyan to yellow. This enables pH-sensitive fluorescence color switching, allowing real-time tracking of amino acid delivery and release, achieving real-time fluorescence monitoring and tracking of drug release at the cellular level. Furthermore, the amino acid-porous carbon dot composite material in this invention significantly accelerates the healing of burn wounds in mice by promoting macrophage activation and fibroblast proliferation, demonstrating excellent skin repair effects.

[0047] (2) The amino acid-porous carbon dot composite material of the present invention is prepared by using polycyclic aromatic hydrocarbons containing carboxylic acids (such as at least one of 3,9-perylenedicarboxylic acid, 2,7-pyrenedicarboxylic acid, 2-indolecarboxylic acid, fluorene-9-carboxylic acid, 5-acenaphthenic acid, and 2,6-naphthalenedicarboxylic acid) and amide compounds as raw materials, and then reacting them with amino acids in an aqueous solution by heating and incubation. It is used for the transfer and release of amino acids. The preparation process is simple and easy to industrialize. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the synthesis mechanism of porous carbon dots in Example 1 of the present invention;

[0049] Figure 2 The images shown are transmission electron microscope (TEM) images and high-resolution TEM images of the porous carbon dots prepared in Example 1 of this invention.

[0050] Figure 3 An atomic force microscope image of the porous carbon dots prepared in Example 1 of this invention;

[0051] Figure 4 The optical properties test diagrams of the porous carbon dots and amino acid-porous carbon dot composite materials prepared in Example 1 of this invention are shown.

[0052] Figure 5 The density functional theory (DFT) plot shows the correlation between the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy level and fluorescence emission.

[0053] Figure 6 The image shows the cytotoxicity of the porous carbon dots and amino acid-porous carbon dot composite materials prepared in Example 11 of this invention.

[0054] Figure 7This is a cellular uptake diagram of the amino acid-porous carbon dot composite material prepared in Example 12 of the present invention;

[0055] Figure 8 This is a lysosomal colocalization diagram of the amino acid-porous carbon dot composite material prepared in Example 12 of the present invention;

[0056] Figure 9 Metabolic diagram of the amino acid-porous carbon dot composite material prepared in Example 13 of this invention in major organs of mice;

[0057] Figure 10 This is a comparison diagram of the porous carbon dots and amino acid-porous carbon dot composite materials prepared in Example 13 of the present invention during the healing process of a mouse burn model. Detailed Implementation

[0058] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0059] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0060] The MEM culture medium used in this embodiment of the invention is sourced from Gibco 11095080.

[0061] Example 1

[0062] A method for preparing an amino acid-porous carbon dot composite material includes the following steps:

[0063] (1) Mix 3,9-perylenedicarboxylic acid (0.1g), potassium persulfate (0.2g) and formamide (10mL) in an open beaker and heat in a 90℃ constant temperature oven for 60min. After the reaction, a dark red liquid sample is obtained in the beaker, which is the carbon dot solution. Dialyze the carbon dot solution with a dialysis bag with a molecular weight cutoff of 500Da for 48h to remove formamide and potassium persulfate. After the dialysis is completed, collect the solution in the dialysis bag and freeze-dry it to obtain an orange-red carbon dot solid powder, which is the porous carbon dot.

[0064] (2) The porous carbon dot powder (0.25 mg) obtained in step (1) is mixed with L-arginine (0.025, 0.05, 0.125, 0.25, 0.5, 1.25, 2.5 mg) in 2.5 mL of aqueous solution at 40 °C. The mixture is heated and incubated for 20 min, and then cooled to room temperature to obtain a bright green solution, which is the amino acid-porous carbon dot composite material.

[0065] Example 1: Schematic diagram of the synthesis mechanism of porous carbon dots. Figure 1As shown, potassium persulfate can achieve free radical catalysis, promoting the activation of the carboxyl group and aromatic hydrogen. Subsequently, the amino group in formamide undergoes an amidation reaction with the carboxyl group in the activated 3,9-perylene dicarboxylic acid molecule to form a monomer. Then, two such monomers form a dimer through the dehydration reaction of the amide and aromatic hydrogen. This dimer can further polymerize to form a trimer with a molecular ring diameter of approximately 0.6 nm. This π-conjugated molecular ring further grows as a building unit and assembles through π-π interactions to form porous carbon dots with yellow fluorescence.

[0066] Example 2

[0067] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Embodiment 1 is that the mass of potassium persulfate in Embodiment 2 is 0.1g.

[0068] Example 3

[0069] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Embodiment 1 is that the mass of potassium persulfate in Embodiment 3 is 0.3g.

[0070] Example 4

[0071] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that the solvothermal temperature for free radical-assisted porous carbon dot synthesis in Example 4 is 80°C, that is, heating in an 80°C constant temperature oven for 60 minutes.

[0072] Example 5

[0073] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that the solvothermal temperature for free radical-assisted porous carbon dot synthesis in Example 5 is 85°C, that is, heating in an 85°C constant temperature oven for 60 minutes.

[0074] Example 6

[0075] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that the solvothermal heating time for free radical-assisted porous carbon dot synthesis in Example 6 is 40 min, that is, heating in a 90°C constant temperature oven for 40 min.

[0076] Example 7

[0077] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Embodiment 1 is that the solvothermal heating time for free radical-assisted porous carbon dot synthesis in Embodiment 7 is 50 min, that is, heating in a 90°C constant temperature oven for 50 min.

[0078] Example 8

[0079] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Embodiment 1 is that in Embodiment 8, the porous carbon dots and amino acids are heated and incubated in an aqueous solution at a temperature of 35°C, that is, they are mixed and heated in an aqueous solution at 35°C for 20 minutes.

[0080] Example 9

[0081] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Embodiment 1 is that in Embodiment 9, the porous carbon dots and amino acids are heated and incubated in an aqueous solution for 30 minutes, that is, they are mixed in an aqueous solution at 40°C and heated and incubated for 30 minutes.

[0082] Example 10

[0083] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that in Example 10, the porous carbon dots and amino acids are heated and incubated in an aqueous solution for 40 minutes, that is, they are mixed in an aqueous solution at 40°C and heated and incubated for 40 minutes.

[0084] Example 11

[0085] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that in step (2), porous carbon dot powder (2.5 mg) and L-arginine (25 mg) are mixed in MEM medium at 40°C and 10 mL. The rest is the same as in Example 1.

[0086] Example 12

[0087] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that in step (2), porous carbon dot powder (1 mg) and L-arginine (10 mg) are mixed in 10 mL of MEM medium at 40 °C, i.e., the concentration of porous carbon dots is 100 μg / mL. -1 The mass ratio of porous carbon dots to L-arginine was 1:10, and other aspects were the same as in Example 1.

[0088] Example 13

[0089] This embodiment provides a method for preparing an amino acid-porous carbon dot composite material. The only difference from Example 1 is that in step (2), porous carbon dot powder (1 mg) and L-arginine (10 mg) are mixed in 10 mL of phosphate buffered saline (PBS) at 40 °C, i.e., the concentration of porous carbon dots is 100 μg / mL. -1 The mass ratio of porous carbon dots to L-arginine was 1:10, and other aspects were the same as in Example 1.

[0090] Comparative Example 1

[0091] Comparative Example 1 is the carbon dot-amino acid composite reagent prepared in Example 1 of the prior art CN119055784A.

[0092] Transmission electron microscopy (TEM) image of the carbon dot-amino acid composite reagent prepared in Comparative Example 1 is shown in CN119055784A. Figure 1 As shown, Figure 1 The carbon dots shown do not have a porous structure. After adsorbing dextrorotatory arginine (D-Arg), the emission peak at 650 nm shows no shift, as in CN119055784A. Figure 3 As shown, it is impossible to reflect the adsorption / desorption process in real time through fluorescence changes. In Example 1 of this invention, the porous carbon dots exhibit a significant blue shift in emission peak position after adsorbing amino acids, as indicated by the present invention. Figure 4 Figures b and d in the paper show that the amino acid-porous carbon dot composite material of this application can monitor the amino acid release process.

[0093] Performance testing

[0094] 1. Microscopic morphological characterization

[0095] The morphology of the porous carbon dots prepared in Example 1 was observed using transmission electron microscopy and high-resolution transmission electron microscopy, respectively. The specific test results are as follows: Figure 2 As shown. Among them, Figure 2 Figure a in the image is a transmission electron microscope (TEM) image of the porous carbon dots prepared in Example 1. Figure 2 Figures b and c in the figure are high-resolution transmission electron microscopy (HRTEM) images of the porous carbon dots circled in figure a at different magnifications.

[0096] Depend on Figure 2 As shown in Figure a, the porous carbon dots are uniformly distributed with a particle size of 4-5 nm; Figure 2 As shown in Figure b, the porous carbon dots exhibit graphene-like lattice fringes with a lattice spacing of 0.21 nm; Figure 2 As shown in Figure c, the porous carbon dots have a porous structure with a pore size of 0.6 nm.

[0097] The porous carbon dots obtained in Example 1 were tested using atomic force microscopy (AFM), and the test results are as follows: Figure 3 As shown. Among them, Figure 3 The left image in the image is an atomic force microscope image of porous carbon dots; Figure 3 The right-hand image in the figure is a statistical chart of the height of the scribbled area in the porous carbon dot atomic force microscope image.

[0098] Depend on Figure 3It can be seen that the porous carbon dots have a uniform height distribution, with an average height of about 4 nm.

[0099] 2. Optical performance testing

[0100] The porous carbon dots prepared in Example 1 were placed in water at a mass fraction of 1 mg / mL to obtain an aqueous solution of porous carbon dots, which was then characterized by spectroscopic analysis. The amino acid-porous carbon dot composite material prepared in Example 1 (where the mass ratio of porous carbon dots to L-arginine was 1:10) was also characterized by spectroscopic analysis. The optical performance test results are as follows: Figure 4 As shown.

[0101] in, Figure 4 Figure a shows the absorption spectra of the raw material 3,9-perylenedicarboxylic acid and porous carbon dots; Figure 4 Figure b in the figure is a three-dimensional excitation-emission mapping of the porous carbon dot aqueous solution in Example 1; Figure 4 Figure c in the figure is the absorption spectrum of the amino acid-porous carbon dot composite material in Example 1 (since the amino acid-porous carbon dot composite material is prepared by porous carbon dot powder and L-arginine in an aqueous solution, the amino acid-porous carbon dot composite material is tested in an aqueous solution state). Figure 4 Figure d in the figure is the excitation-emission three-dimensional mapping of the amino acid-porous carbon dot composite material prepared in Example 1; Figure 4 Figure e in the figure shows the absorption spectra of the amino acid-porous carbon dot composite material prepared in Example 1 at different pH values; Figure 4 Figure f in Example 1 shows the fluorescence intensity of the amino acid-porous carbon dot composite material under different pH values ​​at excitation wavelengths of 480 nm and 570 nm, where Ex and Em represent the excitation wavelength and emission wavelength, respectively.

[0102] Depend on Figure 4 As shown in Figure a, the absorption peak of the porous carbon dots exhibits a red shift, with the absorption peak located at 510 nm. From... Figure 4 As shown in Figure b, the porous carbon dots are located at the emission peak at 580 nm, corresponding to the sp of the carbonized core. 2 The emission spectrum of light emitted by π-π stacking between fragments. Figure 4 As shown in Figures c and d, the steric hindrance introduced by the amino acids disrupted the original porous carbon dots sp. 2 The π-π interactions between fragments lead to the formation of sp in amino acid-porous carbon dot composites. 2 The less conjugated π-π stacking between fragments compared to the original porous carbon dots results in a blue shift of the absorption and emission peaks in the amino acid-porous carbon dot composite material compared to the original porous carbon dots. Figure 4 As shown in Figures e and f, acidic conditions trigger the release of basic amino acids from the amino acid-porous carbon dot composite material, and the fluorescence color changes from cyan to yellow.

[0103] Furthermore, density functional theory (DFT) was used to simulate the changes in the carbon point band gap before and after the disruption of π-π stacking, establishing four molecular models. The DFT calculation diagram of the correlation between HOMO-LUMO energy levels and fluorescence emission is shown below. Figure 5 As shown. Among them, Figure 5 Figure a shows the four molecular structure models of 3,9-perylenedicarboxylic acid monomer, dimer, trimer, and pentamer in the formamide reaction; Figure 5 Figure b shows the distribution of the lowest unoccupied molecular orbitals and the highest occupied molecular orbitals of the four molecular structures in a vacuum.

[0104] The calculated HOMO-LUMO band gaps for the four structures were 2.91 eV, 2.74 eV, 2.78 eV, and 2.66 eV, respectively. The results indicate that the formation of the 3,9-perylenedicarboxylic acid-derived porous structure has no significant effect on the band gap, and the luminescence is similar to that of the 3,9-perylenedicarboxylic acid unit. According to theoretical calculations, the presence of conjugated interlaminar π-π layering typically leads to a narrowing of the band gap. These simulation results can explain the luminescence in sp... 2 Yellow luminescence was observed in pure carbon dots with tight π-π stacking interactions between their domains. Once L-arginine is inserted into the molecular pores of the porous carbon dots, sp... 2 Increased interlayer distance between structural domains disrupts π-π stacking, resulting in a blue shift in fluorescence from yellow to cyan.

[0105] 3. Performance testing of amino acid-porous carbon dot composite materials for cell fluorescence imaging

[0106] The cytotoxicity of the porous carbon dots and amino acid-porous carbon dot composite materials obtained in Example 11 was evaluated. The cytotoxicity of the porous carbon dots and amino acid-porous carbon dot composite materials in different cell lines, such as HeLa cell line (HeLa), rat cardiomyocyte cell line (H9C2), mouse breast cancer cells (4T1), and mouse melanoma cells (B16), was studied using the following methods:

[0107] Cytotoxicity was assessed using the CCK-8 assay. Specifically, approximately 10,000 cells per well were seeded into 96-well plates and incubated at 37°C with 5% (v / v) CO2 for 24 hours. The complete culture medium (89% DMEM (Gibco 11965092) + 10% fetal bovine serum (FBS, Gibco A5670701) + 1% penicillin-streptomycin (Gibco 15140122)) was replaced with 100 μL of MEM medium containing 250 ppm, 50 ppm, 10 ppm, and 0 ppm of porous carbon dots (CDs) and amino acid-porous carbon dot composites (Arg@CDs). After 24 hours of incubation, the CDs and Arg@CDs in the wells were washed three times with PBS to avoid interference with sample fluorescence. Cells were then treated with the CCK-8 reagent for 2 hours, and absorbance was measured at 450 nm using a microplate reader.

[0108] The cytotoxicity results of the porous carbon dots and amino acid-porous carbon dot composite materials prepared in Example 11 of this invention are as follows: Figure 6 As shown. Among them, Figure 6 Figure a shows the cytotoxicity of porous carbon dots in different cell lines; Figure 6 Figure b in the diagram shows the cytotoxicity of the amino acid-porous carbon dot composite material in different cell lines.

[0109] Figure 6 The concentration in the text represents the concentration of porous carbon dots; that is, when the porous carbon dot concentration is 250 ppm, the amino acid concentration is 2500 ppm. The preparation steps for porous carbon dots of different concentrations are as follows: Weigh 2.5 mg of porous carbon dot powder and dissolve it in 10 mL of MEM medium, then filter it through a 0.22 μm sterile enzyme-free filter to obtain a solution with a porous carbon dot concentration of 250 ppm. Then, dilute it stepwise with MEM medium to the target concentration. The preparation steps for amino acid-porous carbon dot composite materials of different concentrations are as follows: Heat the carbon dot powder and amino acid powder in MEM medium, adjusting the concentration as needed. For example, weigh 2.5 mg of porous carbon dot powder and 25 mg of amino acid powder and dissolve them in 10 mL of MEM medium.

[0110] Depend on Figure 6 It can be seen that at 250 μg mL -1 At the specified concentration, porous carbon dots exhibited a slight inhibitory effect on cell viability. In contrast, the amino acid-porous carbon dot composite material did not show a significant inhibitory effect on cell viability, indicating that L-arginine loading resulted in low or no cytotoxicity.

[0111] The cell fluorescence imaging capability of the porous carbon dot and amino acid-porous carbon dot composite materials prepared in Example 12 was evaluated. The cell uptake map of the porous carbon dot and amino acid-porous carbon dot composite materials is shown in the figure. Figure 7As shown, Figure 7 The carbon dots in the text represent porous carbon dots, and Arg@CDs represents amino acid-porous carbon dot composite materials.

[0112] The specific testing process is as follows: porous carbon dots (100 μg mL) were used respectively. -1 The concentration of porous carbon dots after dissolving them in MEM medium, and the amino acid-porous carbon dot composite material prepared in Example 12 (porous carbon dot concentration of 100 μg / mL). -1 The porous carbon dots (with a mass ratio of 1:10 to L-arginine) were named Arg@CDs. HeLa cells were incubated with these CDs for 30 min, and their fluorescence was evaluated under excitation at 405 nm and 488 nm, respectively. The results are as follows: Figure 7 As shown in Figure a, and with the same scale bar for each figure, it can be seen that under 405 nm excitation, Arg@CDs-treated cells exhibit clear cyan fluorescence in the cytoplasm, while porous carbon dot-treated cells only show weak yellow fluorescence under 488 nm excitation; indicating that L-arginine loading on porous carbon dots can promote the intracellular uptake of porous carbon dots.

[0113] After 1 hour of incubation, the extracellular amino acid-porous carbon dot composite material was washed away, and cell metabolism was further studied. The trends of cyan and yellow fluorescence changes over 48 hours are as follows: Figure 7 As shown in Figure b, and with the same scale bars in all figures, the fluorescence intensity variation curves are as follows. Figure 7 As shown in the last image of Figure b, it can be seen that the cyan fluorescence in the cytoplasm gradually decreases with time, and obvious yellow fluorescent spots gradually appear after 12 hours. After 48 hours, the cyan fluorescence disappears completely, and only a small number of yellow fluorescent spots are detected.

[0114] Considering that the change in fluorescence color may be due to the release of L-arginine in an acidic environment, these yellow fluorescent spots may originate from the more acidic lysosomes (pH 4.5-5.0). Therefore, the inventors used a commercial lysosomal dye, LysoTracker Red DND-99 (LTR), to confirm the lysosomal localization characteristics of Arg@CDs. The specific test method was as follows: 100 μg mL -1 Porous carbon dots (concentration after dissolving porous carbon dots in MEM medium) were incubated in HeLa cells at 37°C and 5% (volume fraction) CO2 for 4 hours, washed with PBS, and then incubated with 100 nM LTR working solution for 30 min. After incubation, the cells were washed with PBS and the fluorescence of the 488 nm and 561 nm channels was observed using a confocal microscope.

[0115] Lysosomal colocalization diagram of amino acid-porous carbon dot composite material is shown below. Figure 8 As shown. Among them, Figure 8 The image in the upper left corner shows the yellow fluorescence of Arg@CDs-treated HeLa cells in the 488nm channel; Figure 8 The lower left image shows the red fluorescence of LTR probe-labeled lysosomes in the 561nm channel; Figure 8 The image in the upper right corner is a merged channel fluorescence image, and the scale bars of each image are the same; Figure 8 The lower right figure is a statistical graph of fluorescence intensity.

[0116] Depend on Figure 8 The results showed that the yellow fluorescence (488 nm channel) of Arg@CDs-treated cells after 24 hours exhibited good overlap with the red fluorescence (561 nm channel) of LTR-labeled lysosomes, with a Pearson correlation coefficient of 0.86. These results confirm that the amino acid-porous carbon dot composite material is first endocytosed into the cytoplasm and then enters the lysosome, where L-arginine is released in an acidic environment, changing the fluorescence color from cyan to yellow. This phenomenon indicates that the amino acid-porous carbon dot composite material can not only serve as a carrier for L-arginine delivery but also as a novel intracellular pH-responsive fluorescent probe.

[0117] 4. Performance testing of amino acid-porous carbon dot composite materials for skin repair

[0118] The biocompatibility and biodistribution of the amino acid-porous carbon dot composite material obtained in Example 13 were evaluated in mice.

[0119] The amino acid-porous carbon dot composite material obtained in Example 13 was administered intravenously (porous carbon dot concentration was 100 μg / mL). -1 The mass ratio of porous carbon dots to L-arginine was 1:10, and the solvent was PBS. Fluorescence imaging was performed on the major organs (heart, liver, spleen, lung, and kidney) of mice. The metabolic map of the amino acid-porous carbon dot composite material in the major organs of mice is shown below. Figure 9 As shown.

[0120] Depend on Figure 9 It can be seen that the fluorescence signal of the amino acid-porous carbon dot composite material mainly appears in the liver and kidneys, with the maximum fluorescence intensity appearing at 8h and 2h, respectively. The signal disappears after 48h, indicating that the amino acid-porous carbon dot composite material can be rapidly cleared by the liver and kidneys.

[0121] In addition, the inventors used a mouse skin scald wound model with second-degree burns to study the efficacy of the amino acid-porous carbon dot composite material in Example 13 in promoting skin repair. The specific test methods are as follows:

[0122] Four groups of mice with 10mm diameter burn wounds were intravenously injected with 0.1mL PBS (control group) and L-arginine (1000μg / mL) on days 1, 3, 5, 7, and 9 post-injury. -1 Example 13 Porous carbon dots (100 μg mL) -1 The concentration of porous carbon dots dissolved in PBS) and the amino acid-porous carbon dot composite material of Example 13 (porous carbon dot concentration of 100 μg / mL) -1 The mass ratio of porous carbon dots to L-arginine was 1:10, referred to as Arg@CDs. The skin repair in mice was then observed. A comparison of the effects of porous carbon dots and the amino acid-porous carbon dot composite material on the healing process in a mouse burn model is shown in the figure below. Figure 10 As shown. Among them, Figure 10 Figure a in the figure shows a comparison of burn repair in mice from different test groups, with the same scale bar. Figure 10 Figure b in the figure shows a comparison of the wound area of ​​the scalded lesions in mice after different test groups were treated. Figure 10 Figure c in the figure shows a comparison of scar size at the site of burn wound healing in mice on day 30, with the same scale bar.

[0123] Depend on Figure 10 As shown in Figure a, compared with the control group, the L-arginine group, and the porous carbon dot group of Example 12, the Arg@CDs group exhibited faster wound healing. Figure 10 As shown in Figure b, on day 7, the wound area difference between the Arg@CDs treatment group and other groups was statistically significant, and on day 18, the scabs in the Arg@CDs-treated mice completely fell off, and the skin completely healed. Figure 10 As shown in Figure c, on day 30, the scars at the healing sites in the Arg@CDs group were smaller and lighter than those in other groups. This study indicates that the amino acid-porous carbon dot composite material obtained in Example 12 has the ability to promote skin repair.

[0124] Tests showed that the amino acid-porous carbon dot composite materials prepared in Examples 1-13 all had essentially the same properties.

[0125] In summary, a porous carbon dot exhibiting yellow fluorescence was prepared using a mild free radical-assisted synthesis method under ambient pressure and low temperature conditions. Structurally, the sub-nanometer-sized porous carbon dots (approximately 0.6 nm in diameter) provided a closed space for L-arginine encapsulation. The incorporation of L-arginine into the nanopores disrupted the conjugated structure of the porous carbon dots, causing the characteristic fluorescence of the porous carbon dots to change from the original yellow emission to the cyan emission of the amino acid-porous carbon dot composite material. Acidic conditions triggered the release of L-arginine from the porous carbon dots, and the release of L-arginine could be tracked in real time by observing changes in fluorescence color. Furthermore, the study found that the amino acid-porous carbon dot composite material significantly accelerated the healing of burn wounds in mice.

[0126] Furthermore, the preparation process and conditions of the amino acid-porous carbon dot composite material in this invention are simple, low in cost, and easy to promote and apply. As a novel intelligent drug delivery platform, it can be applied to the delivery and release of amino acids, realize real-time fluorescence monitoring, and promote wound healing.

[0127] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An amino acid-porous carbon dot composite material, characterized in that, It includes porous carbon dots and amino acids loaded on the porous carbon dots; the amino acids are attached to the pore structure of the porous carbon dots.

2. The amino acid-porous carbon dot composite material according to claim 1, characterized in that, The porous structure of the carbon dots has amino acids bonded to its surface via hydrogen bonds.

3. The amino acid-porous carbon dot composite material according to claim 1, characterized in that, The porous carbon dots have a carbon core with a graphene-like lattice; and / or, the particle size of the porous carbon dots is 2-10 nm, and the pore size of the porous carbon dots is 0.4-0.8 nm; and / or, the amino acids include basic amino acids; and / or, the mass ratio of the porous carbon dots to the amino acids is 1:(0.1-11).

4. The amino acid-porous carbon dot composite material according to claim 3, characterized in that, The basic amino acid includes at least one of lysine, arginine, and histidine; and / or, at pH < 6, the release rate of the amino acid in the amino acid-porous carbon dot composite material is ≥ 80%.

5. The amino acid-porous carbon dot composite material according to any one of claims 1-4, characterized in that, The porous carbon dots exhibit a fluorescence emission peak with a wavelength of 500-600 nm under an excitation light source of 400-500 nm. And / or, the amino acid-porous carbon dot composite material has a fluorescence emission peak with a wavelength of 450-500 nm under an excitation light source of 350-450 nm.

6. The method for preparing the amino acid-porous carbon dot composite material according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Mix polycyclic aromatic hydrocarbons and amide compounds containing carboxylic acids and heat them to obtain porous carbon dots; (2) The porous carbon dots and amino acids are mixed in a solvent and heated for incubation to obtain the amino acid-porous carbon dot composite material.

7. The preparation method according to claim 6, characterized in that, In step (1), the carboxylic acid-containing polycyclic aromatic hydrocarbon includes at least one of 3,9-perylene dicarboxylic acid, 2,7-pyrene dicarboxylic acid, 2-indole carboxylic acid, fluorene-9-carboxylic acid, 5-acenaphthenic acid, and 2,6-naphthalenedicarboxylic acid; and / or, the amide compound includes at least one of formamide and N,N-dimethylformamide; and / or, the mixing further includes the addition of a catalyst, the catalyst including at least one of potassium persulfate, ammonium persulfate, and sodium persulfate.

8. The preparation method according to claim 7, characterized in that, In step (1), the mass ratio of the carboxylic acid-containing polycyclic aromatic hydrocarbon to the catalyst is 1:(1-10); and / or, the amount of the amide compound used is 5-30 mL; And / or, the heating temperature is 50-90°C, and the heating time is 10-120 min; And / or, the heating process further includes a process for removing the catalyst and amide compounds.

9. The preparation method according to claim 6, characterized in that, In step (2), the mass ratio of the porous carbon dots to the amino acids is 1:(0.1-11); And / or, the solvent includes at least one of water, culture medium, and phosphate buffer; And / or, after mixing, the concentration of the porous carbon dots is 0.08-0.25 mg / mL; And / or, the temperature of the heating incubation is 35-65°C, and the heating incubation time is 9-65 min.

10. The use of the amino acid-porous carbon dot composite material according to any one of claims 1-5 in the preparation of cell fluorescence imaging reagents and skin repair drug formulations.

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