Drug delivery system based on red-light carbon quantum dot composite nanoparticles and application thereof

Abstract

The application discloses a drug delivery system based on red-light carbon quantum dot composite nanoparticles and application thereof, and belongs to the field of medical treatment. The drug delivery system has tumor targeting and bioimaging functions and can be used for tumor treatment. In the application, dopamine and o-phenylenediamine are used as precursors to prepare red-light carbon quantum dots, and gallic acid, a polyphenol substance, is used as an organic ligand to prepare gallic acid-ferric ion nanoparticles by coordination with divalent iron ions. The red-light carbon quantum dots with bioimaging and PTT effects and the gallic acid-ferric ion nanoparticles with high Fenton reaction catalytic capacity are self-assembled into composite nanoparticles CGFH by using HA with tumor targeting to participate in the self-assembly. A broad-spectrum anticancer drug doxorubicin hydrochloride is used as a model drug to construct a DOX / CGFH drug delivery system, and the drug delivery system has a synergistic effect on the combined treatment of tumor chemotherapy / PTT / CDT.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a drug delivery system based on red light carbon quantum dot composite nanoparticles and its application. Specifically, it relates to the construction and application of a drug loading system consisting of red light carbon quantum dots coated with hyaluronic acid and gallic acid-ferrous nanoparticles. Background Technology

[0002] Drug delivery systems typically require a certain amount of time from administration to reaching the treatment site. Traditional drug delivery systems, lacking the ability to specifically identify the treatment site and trigger drug release upon arrival, can lead to premature drug release, resulting in low bioavailability and various toxic side effects.

[0003] To effectively increase drug concentration in lesion tissues, reduce dosage, and mitigate the toxic side effects of anticancer drugs on normal tissues and organs, stimulus-responsive drug delivery systems that utilize the specific physiological characteristics of target sites to induce drug release have become a hot topic in pharmaceutical formulation research. In recent years, stimulus-responsive drug delivery systems with properties such as reduction sensitivity, pH sensitivity, enzyme sensitivity, and photothermal sensitivity have been designed, with reduction sensitivity currently being the most widely used. Glutathione (GSH), an important intracellular reducing agent, typically has a concentration of only 2-20 μM in extracellular fluid and plasma, while the concentration in intracellular fluid can reach 1-10 mM. In some specific lesion tissues (such as tumor tissue), the GSH concentration can be more than three times that of normal tissues. Since complex nanoparticles are prone to breakage under high GSH concentrations, this can be used to design such a drug delivery system. Simultaneously, hyaluronic acid can be used to target the drug delivery system to tumor tissues. Tumor cells contain hyaluronidase, which enables drug release after reaching the target cells. Stimulus-responsive drug delivery systems can effectively control premature drug release, thereby reducing drug toxicity.

[0004] Treatment of lesions solely through medication remains limited and prone to problems such as drug resistance. Therefore, photothermal therapy (thermotherapy) has emerged as an adjunctive treatment method. Its principle primarily involves injecting materials or carriers with high photothermal conversion efficiency into the body. Using targeted recognition technology, these materials concentrate near the tumor tissue, and under the irradiation of an external light source (usually near-infrared light), the photothermal material efficiently converts light energy into heat energy, thus producing a therapeutic effect. Some denser lesions (such as tumor tissue) dissipate heat slowly and are more sensitive to thermal effects; therefore, photothermal therapy can raise the local temperature of the lesion tissue while having relatively little impact on normal cells. Near-infrared light has stronger tissue penetration than visible and ultraviolet light, making non-invasive photothermal therapy possible for tumor treatment. The advantages of photothermal therapy include: reduced patient pain; shorter treatment time (a few minutes); and significant therapeutic effects. The core of photothermal therapy is nanomaterials with ultra-high photothermal conversion efficiency. Currently, the photothermal conversion materials used in photothermal therapy are mainly inorganic nanoparticles such as gold nanorods, carbon nanotubes, and graphene, as well as organic fluorescent dyes. Carbon quantum dots, as a type of carbon nanomaterial, have been prepared through formulation screening to produce fluorescent carbon dots emitting red light, capable of emitting red fluorescence with maximum excitation and emission wavelengths of 570 nm and 622 nm, respectively. These carbon quantum dots also exhibit good stability and photothermal conversion capabilities, and their surface possesses modifiable amino groups.

[0005] Glutathione (GSH) is a natural tripeptide widely found in animal and plant cells. It is widely distributed and present in high concentrations. In cancer cells, it maintains redox balance by scavenging reactive oxygen species (ROS), protecting cancer cells from damage and promoting tumor growth. Intracellular glutathione levels in tumor cells can reach 2-10 mmol / L. -1 It is approximately 100–1000 times that of the extracellular matrix (2–20 μmol·L⁻¹). -1 The levels of reactive oxygen species (ROS) in these kinetic therapies, including photodynamic therapy (PDT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT), are more than three times higher than in normal tissues. ROS are the primary cell death inducers in these kinetic therapies. Although different reactive oxygen species are generated during these treatments, excess GSH in tumor cells directly counteracts the generated ROS, thus weakening the therapeutic effect of various kinetic therapies. To avoid unnecessary ROS loss and thus improve anti-tumor efficacy, reducing intracellular GSH during these kinetic therapies is reasonable and necessary. Therefore, depleting intracellular GSH is considered one of the effective methods to enhance cancer treatment efficacy. This carbon quantum dot has a certain glutathione-depleting capacity. Gallic acid-ferrous iron nanoparticles are used in chemodynamic therapy to generate excess ROS within tumor cells, thereby killing the tumor cells. Summary of the Invention

[0006] The purpose of this invention is to provide a drug delivery system based on red light carbon quantum dot composite nanoparticles and its application. This drug delivery system has near-infrared photothermal conversion capability to achieve drug targeting to tumor tissue. It also enhances the therapeutic effect and reduces toxic side effects through the synergistic effect of stimulus-responsive release in the target tissue and chemotherapy, photothermal therapy, and chemokinetic therapy. At the same time, it has bioimaging function to track the drug delivery process.

[0007] This invention uses hyaluronic acid to complex carbon quantum dots and gallic acid-ferrous nanoparticles to prepare larger composite nanoparticles via precipitation, thereby constructing a pH-responsive gated system with near-infrared photothermal conversion capability for tumor targeting, and releasing hyaluronidase and high-concentration GSH-responsive drugs from the tumor tissue environment.

[0008] The drug delivery system described in this invention has pH-responsive drug release capability and synergistic effects with photothermal therapy under near-infrared irradiation and chemokinetics.

[0009] This invention prepares photothermal-sensitive red-light carbon quantum dots and utilizes the chemical kinetics of gallic acid-ferrous nanoparticles to create a nanomedicine delivery system with thermotherapy capabilities under near-infrared light irradiation and pH-responsive drug release capabilities.

[0010] The technical solution adopted in this invention is as follows:

[0011] 1) Preparation of photothermally sensitive red carbon quantum dots:

[0012] Red carbon quantum dots with good photothermal conversion ability were prepared by hydrothermal method using 50-150 mg o-phenylenediamine and 100-200 mg dopamine as carbon and nitrogen sources, respectively.

[0013] 2) Preparation of gallic acid-ferrous divalent nanoparticles (GF):

[0014] Polyvinylpyrrolidone K30 (PVP) and ferrous chloride were dispersed in ultrapure water and reacted with gallic acid (GA) under nitrogen protection to prepare gallic acid-ferrous divalent nanoparticles (GF). Unreacted ferrous chloride and gallic acid were then removed by dialysis.

[0015] 3) Preparation of drug-loaded CGFH system

[0016] Weigh the prepared red carbon quantum dots and GF dispersed in distilled water, sonicate in a water bath, add the drug to the above solution, continue sonicating in a water bath, weigh the hyaluronic acid dispersed in distilled water, stir and disperse evenly, add it to the above solution, sonicate in a water bath, centrifuge, and obtain drug-loaded CGFH nanoparticles.

[0017] In step 2), GA and Fe2+ Under nitrogen protection, GF nanoparticles are polymerized through coordination. The specific ratio is: 15-25 mg FeCl2, 70-90 mg polyvinylpyrrolidone (PVP), and 10-15 mg GA.

[0018] In step 3), the drug is preferably doxorubicin; hyaluronic acid self-assembly is used, and the positively charged doxorubicin interacts with the negatively charged hyaluronic acid and GA. At the same time, due to the presence of anthraquinone ring structure, doxorubicin can form a drug-loaded system with GA through non-covalent interactions of π-π stacking.

[0019] In step 3), 10-30 mg of the drug is dissolved in 2-5 mL of distilled water and dispersed evenly under ultrasonication in a water bath for 10-15 min. 10-30 mg of the prepared red-light carbon quantum dots and 10-30 mg of GF are weighed and dispersed in 5-10 mL of distilled water, and ultrasonicated in a water bath for 10-20 min. The dispersed drug solution is added to the above solution, and ultrasonication continues for 10-20 min. 5-10 mg of hyaluronic acid is weighed and dispersed in 5-10 mL of distilled water, stirred for 10-20 min until evenly dispersed, and then added to the above liquid. The mixture is ultrasonicated in a water bath for 15-20 min. The above system is centrifuged at 12000-40000 rpm for 6-30 min to obtain the drug-loaded system. The drug adsorbed on the surface is removed by washing with PBS (pH 7.4), and the system is vacuum dried for 10-20 h to remove the distilled water, yielding the drug-loaded nanoparticles. Positively charged doxorubicin is preferred as the model drug to improve the antitumor effect of the drug-loaded system. The CGFH system consists of red carbon quantum dots, GF, and hyaluronic acid. CGFH refers to hyaluronic acid complex nanoparticles without drug loading. The ratio of drug to CGFH system is between 2:1 and 1:10 by mass.

[0020] This invention utilizes hyaluronic acid-encapsulated carbon quantum dots, gallic acid-ferrous nanoparticles, and drugs to achieve synergistic effects with target tissue stimulation-responsive drug release, chemotherapy, photothermal therapy, and chemokinetics, while reducing toxic side effects. The drugs mentioned are doxorubicin and paclitaxel.

[0021] Take doxorubicin (DOX) as an example:

[0022] Doxorubicin was added to the CGFH system to prepare a DOX / CGFH nanodelivery system.

[0023] The specific preparation method of DOX / CGFH includes the following steps:

[0024] Dissolve 10-30 mg DOX in 2-5 mL of distilled water and disperse evenly under ultrasonication in a water bath for 10 min. Weigh 10 mg of the prepared red carbon quantum dots and 20 mg of GF and disperse them in 5-10 mL of distilled water. Ultrasonicate in a water bath for 20 min. Add the dispersed doxorubicin to the above solution and continue ultrasonication in a water bath for 10 min. Weigh 5-10 mg of hyaluronic acid and disperse it in 10 mL of distilled water. Stir for 10 min to disperse evenly and then add it to the above liquid. Ultrasonicate in a water bath for 20 min. Centrifuge the above system at 12000 rpm for 6 min to obtain the drug-loaded system. Wash with PBS at pH 7.4 to remove DOX adsorbed on the surface of DOX / CGFH. Vacuum dry for 10-20 h to remove distilled water to obtain drug-loaded nanoparticles.

[0025] This invention utilizes self-assembly technology and red-light carbon quantum dots to achieve photothermal therapy and bioimaging functions. Then, GF nanoparticles with highly efficient Fenton catalysis and doxorubicin are self-assembled to prepare a drug delivery system, forming a multi-stimulus-responsive nanodrug delivery system with pH / redox / HAase / NIR properties. When the delivery system targets the lesion tissue, near-infrared light is used to irradiate the lesion. The carbon quantum dots efficiently convert light energy into heat, raising the local temperature and increasing the uptake of the target carrier by cells. Simultaneously, the sensitivity of the delivery system to pH and GSH release within the cells increases, accelerating drug release and achieving a synergistic effect between chemotherapy and photothermal therapy. Simultaneously, hyaluronidase degrades hyaluronic acid, releasing carbon quantum dots and GF nanoparticles, increasing the concentration of reactive oxygen species within tumor cells, thus achieving chemokinetic therapy. The synergistic effect of these three therapies further reduces the toxic side effects of chemotherapy drugs. The drug delivery system described in this invention can serve as a stimulus-responsive drug delivery system combining traditional chemotherapy with novel photothermal and chemokinetic therapies. Attached Figure Description

[0026] Figure 1 Transmission electron microscopy image of the R-CDs prepared in Example 1;

[0027] Figure 2 Atomic force microscopy (AFM) images of the R-CDs prepared in Example 1; (A) AFM three-dimensional image; (B) AFM topography; (C) Height profile along line (B);

[0028] Figure 3 XRD data of R-CDs prepared in Example 1;

[0029] Figure 4 XPS data for the R-CDs prepared in Example 1;

[0030] Figure 5(A) Excitation and emission spectra of R-CDs prepared in Example 1 at different excitation wavelengths; (B) FL emission spectra;

[0031] Figure 6 The emission spectrum of sodium fluorescein and R-CDs in Example 1 is shown.

[0032] Figure 7 Transmission electron microscopy image of GF prepared in Example 3;

[0033] Figure 8 The particle size results for GF prepared in Example 3;

[0034] Figure 9 (A) XPS data and (B) Fe 2p spectrum of GF prepared in Example 3;

[0035] Figure 10 Data for detecting hydroxyl radicals generated by GF in MB;

[0036] Figure 11 The drug release curves of DOX / CGFH prepared in Example 5 under NIR-free (A) and NIR-exposed (B) conditions over 24 hours are shown.

[0037] Figure 12 The potential of DOX / CGFH prepared in Example 10;

[0038] Figure 13 The results of cytotoxicity in Example 11;

[0039] Figure 14 This is a graph showing the results of mitochondrial membrane potential changes in Example 12;

[0040] Figure 15 The results show the stability of the system in Example 13;

[0041] Figure 16 The image shows the effect of ROS generation in Example 14. Detailed Implementation

[0042] Example 1

[0043] Weigh 108 mg of o-phenylenediamine (O-PDA) and 184 mg of dopamine (DA) into a 50 mL Erlenmeyer flask, add 19 mL of ultrapure water and 1 mL of concentrated sulfuric acid, and sonicate in a water bath for 10 min to disperse them evenly. Place the reaction mixture in a muffle furnace and heat at 200 °C for 8 h at a rate of 5 °C / min. After the reaction is complete, allow it to cool naturally to room temperature to obtain a blue-brown suspension, which is the crude product, red-light carbon quantum dots (R-CDs). Centrifuge the suspension (11000 rpm, 10 min) to remove large particles. Place the supernatant in a dialysis bag and dialyze it in pure water for 24 h, changing the pure water every 4 h. Collect the solution in the dialysis bag, freeze-dry it, and obtain the R-CDs.

[0044] Figure 1 Transmission electron microscopy image of the R-CDs prepared in Example 1; Figure 2 Atomic force microscopy (AFM) images of R-CDs; (A) 3D AFM image, (B) AFM topography, (C) height profile along line (B); Figure 3 XRD data for R-CDs; Figure 4 XPS data for R-CDs; Figure 5 (A) Excitation and emission spectra of R-CDs at different excitation wavelengths, and (B) FL emission spectra; Figure 6 The emission spectra of sodium fluorescein and R-CDs are shown.

[0045] Example 2

[0046] Weigh 100 mg O-PDA and 170 mg DA into a 50 mL Erlenmeyer flask, add 19 mL ultrapure water and 1 mL concentrated sulfuric acid, and sonicate in a water bath for 10 min to disperse them evenly. Place the reaction mixture in a muffle furnace and heat at 200 °C for 8 h at a rate of 5 °C / min. After the reaction is complete, allow it to cool naturally to room temperature to obtain a blue-brown suspension, which is the crude product R-CDs. Centrifuge the suspension (11000 rpm, 10 min) to remove large particles. Place the supernatant in a dialysis bag and dialyze against pure water for 24 h, changing the pure water every 4 h. Collect the solution in the dialysis bag, freeze-dry it, and obtain R-CDs.

[0047] Example 3

[0048] 23 mg of FeCl2 and 80 mg of polyvinylpyrrolidone (PVP) were weighed and mixed into 4 mL of ultrapure water, and stirred at room temperature for 5 min. Then, under nitrogen protection, 1 mL of GA (10 mg / mL) aqueous solution was added dropwise to the above solution, and stirring continued for 24 h. Finally, the resulting deep purple gallic acid-ferrous nanoparticles (GF) were concentrated by ultrafiltration using an ultrafiltration tube with a half-molecule cutoff of 10 kDa and stored at 4 °C for later use.

[0049] Figure 7 Transmission electron microscopy image of GF prepared in Example 3; Figure 8 The particle size results for GF; Figure 9 XPS data for GF; Figure 10 Data for detecting hydroxyl radicals generated by GF in MB.

[0050] Example 4

[0051] 18 mg of FeCl2 and 80 mg of polyvinylpyrrolidone (PVP) were weighed and mixed, then added to 4 mL of ultrapure water and stirred at room temperature for 5 min. Subsequently, under nitrogen protection, 1 mL of GA (10 mg / mL) aqueous solution was added dropwise to the above solution and stirring was continued for 24 h. Finally, the resulting deep purple gallic acid-ferrous divalent nanoparticles (GF) were concentrated by ultrafiltration using an ultrafiltration tube with a half-molecule cutoff of 10 kDa and stored at 4 °C for later use.

[0052] Example 5

[0053] Accurately weigh 10 mg of doxorubicin and dissolve it in 2 mL of distilled water. Disperse it evenly under ultrasonic conditions in a water bath for 10 min. Weigh 10 mg of the prepared red carbon quantum dots and 20 mg of GF and disperse them in 10 mL of distilled water. Sonicate in a water bath for 20 min. Add the dispersed doxorubicin to the above solution and continue to sonicate in a water bath for 10 min. Weigh 5 mg of hyaluronic acid and disperse it in 10 mL of distilled water. Stir for 10 min to disperse it evenly and then add it to the above liquid. Sonicate in a water bath for 20 min. Centrifuge the above system at 12000 rpm for 6 min to obtain the drug-loaded system. Wash with PBS at pH 7.4 to remove DOX adsorbed on the surface of DOX / CGFH. Vacuum dry for 10 h to remove distilled water to obtain drug-loaded nanoparticles (DOX / CGFH).

[0054] Figure 11 The drug release behavior of DOX / CGFH prepared in Example 5 was investigated. It can be seen that the constructed DOX / CGFH drug delivery system has multiple stimulation response characteristics of pH / redox / HAase / NIR light, which synergistically promotes drug release and achieves better anti-tumor therapeutic effect.

[0055] Example 6

[0056] Accurately weigh 15 mg of doxorubicin and dissolve it in 3 mL of distilled water. Disperse it evenly under ultrasonic conditions in a water bath for 10 min. Weigh 10 mg of the prepared red carbon quantum dots and 20 mg of GF and disperse them in 5 mL of distilled water. Sonicate in a water bath for 20 min. Add the dispersed doxorubicin to the above solution and continue to sonicate in a water bath for 10 min. Weigh 6 mg of hyaluronic acid and disperse it in 10 mL of distilled water. Stir for 10 min to disperse it evenly and then add it to the above liquid. Sonicate in a water bath for 20 min. Centrifuge the above system at 12000 rpm for 6 min to obtain the drug-loaded system. Wash with PBS at pH 7.4 to remove DOX adsorbed on the surface of DOX / CGFH. Vacuum dry for 20 h to remove distilled water to obtain drug-loaded nanoparticles (DOX / CGFH).

[0057] Example 7

[0058] Accurately weigh 20 mg of doxorubicin and dissolve it in 4 mL of distilled water. Disperse it evenly under ultrasonic conditions in a water bath for 10 min. Weigh 10 mg of the prepared red carbon quantum dots and 20 mg of GF and disperse them in 5 mL of distilled water. Sonicate in a water bath for 20 min. Add the dispersed doxorubicin to the above solution and continue to sonicate in a water bath for 10 min. Weigh 7 mg of hyaluronic acid and disperse it in 10 mL of distilled water. Stir for 10 min to disperse it evenly and then add it to the above liquid. Sonicate in a water bath for 20 min. Centrifuge the above system at 12000 rpm for 6 min to obtain the drug-loaded system. Wash with PBS at pH 7.4 to remove DOX adsorbed on the surface of DOX / CGFH. Vacuum dry for 10 h to remove distilled water to obtain drug-loaded nanoparticles (DOX / CGFH).

[0059] Example 8

[0060] Accurately weigh 25 mg of doxorubicin and dissolve it in 5 mL of distilled water. Disperse it evenly under ultrasonic conditions in a water bath for 10 min. Weigh 10 mg of the prepared red carbon quantum dots and 20 mg of GF and disperse them in 5 mL of distilled water. Sonicate in a water bath for 20 min. Add the dispersed doxorubicin to the above solution and continue to sonicate in a water bath for 10 min. Weigh 8 mg of hyaluronic acid and disperse it in 10 mL of distilled water. Stir for 10 min to disperse it evenly and then add it to the above liquid. Sonicate in a water bath for 20 min. Centrifuge the above system at 12000 rpm for 6 min to obtain the drug-loaded system. Wash with PBS at pH 7.4 to remove DOX adsorbed on the surface of DOX / CGFH. Vacuum dry for 20 h to remove distilled water to obtain drug-loaded nanoparticles (DOX / CGFH).

[0061] Example 9

[0062] Accurately weigh 30 mg of doxorubicin and dissolve it in 5 mL of distilled water. Disperse it evenly under ultrasonic conditions in a water bath for 10 min. Weigh 10 mg of the prepared red carbon quantum dots and 20 mg of GF and disperse them in 10 mL of distilled water. Sonicate in a water bath for 20 min. Add the dispersed doxorubicin to the above solution and continue to sonicate in a water bath for 10 min. Weigh 8 mg of hyaluronic acid and disperse it in 10 mL of distilled water. Stir for 10 min to disperse it evenly and then add it to the above liquid. Sonicate in a water bath for 20 min. Centrifuge the above system at 12000 rpm for 6 min to obtain the drug-loaded system. Wash with PBS at pH 7.4 to remove DOX adsorbed on the surface of DOX / CGFH. Vacuum dry for 20 h to remove distilled water to obtain drug-loaded nanoparticles (DOX / CGFH).

[0063] Example 10

[0064] The DOX / CGFH prepared in Example 7 was dispersed in distilled water and ultrasonically dispersed for 5 minutes. A small amount of the test solution was then taken into a particle size cup and a potential cup to determine the particle size and potential of the nanoparticles (n=3).

[0065] According to Malvern's measurements, the average particle size of DOX / CGFH is approximately 63 nm, and the PDI is 0.172, indicating that the prepared DOX / CGFH has good dispersibility. The potential changes of CGFH and DOX / CGFH were also compared, and the results are as follows... Figure 12 As shown, the potential of CGFH is -25.93mV, and the potential of DOX / CGFH is -13.76mV, indicating that DOX self-assembles into the drug delivery system.

[0066] Example 11

[0067] The cytotoxicity of DOX, CGFH, and DOX / CGFH to 4T1 cells was assessed using the MTT assay. (2 × 10⁻⁶ cells) 4 Cell / well density: 4 T1 cells were seeded into 96-well plates. 100 μL of sterile PBS was added to the edge wells. After incubation for 24 h, the old culture medium was discarded, and serum-free medium was used to prepare sample test solutions with gradient concentrations (equivalent to DOX concentrations of 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL). Wells containing cell-containing serum-free medium served as the control group. Test samples were added to the corresponding wells (n=5). After administration, the cells were incubated for a total of 24 h. For the NIR irradiation group, 1.5 W / cm² was applied 10 h after administration. 2The samples were irradiated with NIR light for 3 min. After incubation, 50 μL of 2 mg / mL MTT solution was added to each well, and incubation was continued at 37 °C for 4 h. The liquid in the old plate was aspirated, and 150 μL of DMSO was added to each well. The plates were shaken in the dark for 15 min, and the absorbance of each well was measured at a wavelength of 570 nm. The IC50 of each group was calculated. 50 The values ​​were calculated, and the survival rate of each group of cells was determined.

[0068] The results showed that when 4T1 cells were co-incubated with gradient concentrations of CGFH blank carrier, the cell survival rate was less than 80% at a CGFH concentration of 5 μg / mL. The carrier itself, without NIR light irradiation, exhibited cytotoxicity in the CDT efficacy of CGFH. Under NIR light irradiation, the cell survival rate of the CGFH preparation decreased with increasing carrier concentration, reaching 50.24% at a concentration of 10 μg / mL. This is attributed to the strong photothermal conversion ability and cytotoxicity of CGFH. When the DOX concentration in the DOX / CGFH mixture reached 10 μg / mL, the cell survival rate was only 27.29% under NIR light irradiation, showing significant cytotoxicity compared to the chemotherapy, PTT, CDT, and free DOX groups. This was mainly attributed to the synergistic effect of chemotherapy, CDT, and PTT. Figure 13 ).

[0069] Example 12

[0070] 4T1 cells were seeded in 6-well plates and cultured for 24 h. DOX / CGFH was added and the cells were co-incubated for 2 h. Rh123 staining was then performed. Ten h after drug administration, cells were treated with 1.5 W / cm² of DOX / CGFH. 2 The samples were irradiated with NIR light for 3 min, and the fluorescence intensity of Rh123 in the cells was detected by FCM. Changes in mitochondrial membrane potential indicated by Rh123 staining were observed using CLSM. The specific steps were as follows: 4T1 cells in logarithmic growth phase were seeded into 24-well plates pre-coated with sterile cell spreaders and cultured in an incubator. After cell adhesion, DOX / CGFH was added and co-incubated with the cells for 2 h. Rh123 staining was then added. Ten h after drug administration, 1.5 W / cm² of DOX / CGFH was used. 2 The sample was irradiated with NIR light for 3 min, and the fluorescence intensity of Rh123 in the cells was detected by FCM.

[0071] The results are as follows Figure 14 As shown, compared with the control group without drugs, the mitochondrial membrane potential of DOX / CGFH at the NIR was significantly reduced. This indicates that the mitochondria were severely damaged, resulting in the highest degree of apoptosis.

[0072] Example 13

[0073] The lyophilized CGFH and DOX / CGFH injectable nano-formulations were dispersed in 2 mL of ultrapure water, and after being ultrasonically dispersed and allowed to stand, appropriate samples were taken at given times (0 h, 4 h, 8 h, 12 h and 24 h). The samples were diluted to appropriate concentrations and the particle size and PDI (n = 3) of the two nano-formulations were determined.

[0074] The stability of nanoformulations is crucial for clinical applications. Results are as follows... Figure 15 The figure shows the particle size and PDI results of the DOX / CGFH formulation after reconstitution. The average particle size of the reconstituted nanoparticles did not change significantly during the 24-hour standing period, and the PDI values ​​remained around 0.2, indicating good dispersion stability.

[0075] Example 14

[0076] 4T1 cells in logarithmic growth phase were seeded into 24-well plates pre-coated with sterile cell spreaders and cultured in an incubator. After cell attachment, 10 μg / mL CGFH suspension was added, and the cells were co-incubated for 2 h. Then, 25 μM MDCFH-DA was added, and the cells were co-incubated for 20 min. For the NIR irradiation group, 808 nm NIR light (1.50 W / cm²) was used. 2 Irradiate for 3 min. After incubation, gently wash the cells with PBS, add 200 μL of 4% paraformaldehyde solution, and continue incubation for 20 min. Then, remove the cell slide, invert it onto a glass slide with mounting solution, and observe the fluorescence changes of dichlorofluorescein (DCF) under CLSM.

[0077] The results are as follows Figure 16 As shown, the blank control group without CGFH exhibited very little green fluorescence, presumably due to spontaneously generated ROS in the cells. In contrast, the CGFH group showed a slightly stronger green fluorescence intensity, indicating that CGFH can generate ROS in the tumor microenvironment, thus demonstrating the therapeutic effect of CGFH in CDT. Under 808nm NIR laser irradiation, the CGFH group showed an even stronger green fluorescence intensity, mainly attributed to the increased cell temperature after NIR irradiation, which accelerated ROS generation in the CDT reaction. These experimental results demonstrate that the prepared CGFH possesses the ability of Fenton catalysis, capable of generating a large amount of ROS in tumor cells to kill them, further proving the potential of CGFH in PTT combined with CDT for tumor treatment.

Claims

1. A drug delivery system based on red-light carbon quantum dot composite nanoparticles, characterized in that, The drug delivery system utilizes red-light carbon quantum dots and gallic acid-ferrous nanoparticles, along with hyaluronic acid self-assembly to form a drug loading system for loading the antitumor drug doxorubicin. The red-light carbon quantum dots possess bio-imaging capabilities and glutathione consumption capabilities. They are prepared using dopamine and o-phenylenediamine as precursors via a solvothermal method.

2. A method for preparing a drug delivery system based on red-light carbon quantum dot composite nanoparticles as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of gallic acid-ferrous nanoparticles; (2) Preparation of self-assembled drug loading system.

3. The preparation method according to claim 2, characterized in that, In step (1), polyvinylpyrrolidone K30 is added.

4. The preparation method according to claim 3, characterized in that, In step (1), gallic acid and Fe 2+ Gallic acid-ferrous iron nanoparticles were polymerized through coordination under nitrogen protection.

5. The preparation method according to claim 3, characterized in that, In step (2), hyaluronic acid self-assembly is used, and the drug is doxorubicin. The positively charged doxorubicin interacts with the negatively charged hyaluronic acid and gallic acid. At the same time, due to the presence of anthraquinone ring structure, doxorubicin can form a drug-loaded system with gallic acid through non-covalent interaction of π-π stacking.

6. The preparation method according to claim 5, characterized in that, Doxorubicin is loaded into the drug delivery system of step (2), and the ratio of drug to drug delivery system is between 2:1 and 1:10 by mass. The drug delivery system is composed of hyaluronic acid, gallic acid-ferrous nanoparticles and red carbon quantum dots.

7. The application of the drug delivery system based on red-light carbon quantum dot composite nanoparticles as described in claim 1 in the preparation of combined therapeutic drugs for chemotherapy, photothermal therapy, and chemodynamic therapy, characterized in that, The drug contains doxorubicin.

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