A degradable carbon dot composite system for enhancing tumor penetration and a preparation method and application thereof

Abstract

This invention discloses a depolymerizable carbon dot composite system for enhancing tumor penetration, its preparation method, and its applications, belonging to the field of pharmaceutical technology. Specifically, it relates to the preparation of a carbon dot composite system for enhancing tumor penetration and its application in tumor cascade catalytic therapy and photothermal therapy. The carbon dot composite system consists of iron-doped red-light nanozyme carbon dots, glucose oxidase, and hyaluronic acid to reduce the system's immunogenicity. Through the targeted action of hyaluronic acid on the tumor site, depolymerization occurs in the tumor microenvironment under the influence of hyaluronidase and photothermal heating. Glucose oxidase consumes glucose at the tumor site to produce hydrogen peroxide, thereby generating more hydroxyl radicals and simultaneously performing starvation therapy. This invention's carbon dot composite system for enhancing tumor penetration combines cascade catalytic therapy, photothermal therapy, and starvation therapy to achieve highly efficient tumor growth inhibition, providing an important reference for multimodal tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation of a decomposable carbon dot composite system that enhances tumor penetration, and its application in tumor cascade catalytic therapy, photothermal therapy, and starvation therapy. Background Technology

[0002] Cancer is one of the major diseases threatening human health, causing severe damage due to the uncontrolled proliferation, invasion, and destruction of surrounding healthy tissues by abnormal cells. Compared to traditional therapies, nanomedicines have been extensively studied due to their targeting of tumor sites, higher safety profile, and potential as a multifunctional platform. Limited penetration at the tumor site is a major obstacle to nanoplatform therapy for solid tumors, caused by tumor vascular system disturbances, increased tumor stroma pressure, and the dense extracellular matrix in the solid tumor microenvironment. To increase the penetration depth of nanomedicines into tumors, researchers have mainly developed two strategies: penetrating peptide modification and particle size control. Ultrasmall nanocarriers are beneficial for tumor penetration because diffusion capacity is inversely correlated with particle size. However, ultrasmall nanoparticles are often rapidly cleared in systemic circulation. To address this issue, nanoparticles with larger initial particle sizes and longer half-lives have been developed, which can rapidly decompose into ultrasmall nanoparticles within tumor tissue, thereby enhancing intratumoral diffusion. Simultaneously, researchers have also developed various peptides with different tumor targeting / penetration functions to increase tumor penetration depth. Overcoming the limited penetration depth within solid tumors is crucial for achieving efficient tumor treatment.

[0003] Carbon dots (CDs), as a novel type of ultrasmall carbon nanomaterials with luminescent properties, have attracted worldwide attention due to their excellent luminescent characteristics, biocompatibility, water dispersibility, nanozyme activity, abundant surface functional groups, and ease of modification and preparation. Compared with traditional carbon dots emitting blue and green light, red carbon dots can significantly avoid interference from the background fluorescence of some tissue proteins, thus achieving better tracking effects. It has been reported that certain carbon dots, due to their special edge functional groups, can enhance the uptake of carbon dots and their complexes by cells (Soft Matter, 2022, 18, 2776).

[0004] Glucose oxidase (GOx), a natural enzyme, can consume glucose and produce hydrogen peroxide, thereby enhancing oxidative stress and starvation therapy at the tumor site. However, due to the barrier of biological macromolecules, tumor cells have difficulty effectively taking up glucose oxidase.

[0005] Based on this, we designed and synthesized carbon dots with amino acid-like edge structures. After complexing with GOx, we utilized the large neutral amino acid transporters LAT1 / ASCT2 to enhance cellular uptake of the complex. Simultaneously, hyaluronic acid coated the system surface, allowing the system to depolymerize into smaller nanoparticles under hyaluronidase / photothermal heating, exposing the edge structures of the carbon dots and thus enhancing nanoparticle penetration into tumor sites. This has significant implications for the delivery of large molecules to tumors and clinical cancer treatment. Summary of the Invention

[0006] The primary objective of this invention is to construct a depolymerizable carbon dot composite system that enhances tumor penetration. Hyaluronic acid encapsulation reduces the system's immunogenicity, allowing for long-term circulation while simultaneously enabling depolymerization by hyaluronidase overexpressed at the tumor site into smaller nanocomposites, thus enhancing penetration. Simultaneously, iron-doped red-light carbon dots (carbon dot nanozymes) generate ROS to enhance oxidative stress at the tumor site, thereby achieving a synergistic anti-tumor effect through cascade catalytic therapy / photothermal therapy / starvation therapy.

[0007] The technical solution adopted in this invention is as follows:

[0008] The aforementioned depolymerizable carbon dot composite system that enhances tumor penetration possesses hyaluronidase-responsive depolymerization capabilities and synergistic effects with photothermal therapy, cascade catalytic therapy, and starvation therapy under near-infrared irradiation.

[0009] Preferably, the tumor-penetrating depolymerizable carbon dot composite system comprises photothermal responsive carbon dot nanozymes, glucose oxidase (GOx), and hyaluronic acid. Preferably, the composite system consists of photothermal responsive carbon dot nanozymes and glucose oxidase, with hyaluronic acid coating the outer layer of the composite.

[0010] Preferably, the photothermal responsive carbon nanoparticle nanozyme is formed by carbonization polymerization of citric acid, urea, disodium ethylenediaminetetraacetate, and an iron ion precursor under high temperature and high pressure, wherein the iron ion is Fe. 3+ .

[0011] The photothermal responsive carbon nanodot nanozyme has an amino acid-like structure at its edge, which can achieve intracellular transmembrane transport through the amino acid transporters LAT1 and ASCT2 on the cell surface.

[0012] The preparation method of the tumor-enhancing decomposable carbon dot composite system of the present invention includes the following steps:

[0013] (1) Preparation of iron-doped carbon nanoparticles: Citric acid, urea, disodium ethylenediaminetetraacetate and iron precursor were dissolved in formic acid solution, ultrasonically dispersed, and placed in a polytetrafluoroethylene bottle for reaction in a muffle furnace. Afterwards, ethanol was added to the reaction solution, centrifuged, the supernatant was discarded, and the precipitate was collected to obtain iron-doped carbon nanoparticles.

[0014] (2) Preparation of carbon dot-glucose oxidase complex system (FG): Iron-doped carbon dot nanozymes and glucose oxidase are mixed and stirred overnight. The carbon dot-glucose oxidase complex system is obtained by centrifugation and ultrafiltration to remove the uncomposite iron-doped carbon dot nanozymes.

[0015] (3) Preparation of hyaluronic acid-coated carbon dot composite system for enhancing tumor penetration: After co-incubation in hyaluronic acid solution, the FG complex can be obtained by ultrafiltration to enhance tumor penetration and depolymerizable red carbon dot composite system.

[0016] Preferably, in step (1), the muffle furnace temperature is set to 120-200℃, the reaction time is 1-6h, the volume ratio of the reaction stock solution to ethanol is 1:4-1:6, the centrifugation speed is 6000-10000rpm, the mass ratio of citric acid to urea is 1:1-1:3, the iron ion precursor is FeCl3, and the corresponding carbon dot nanozyme is Fe(III)-CDs.

[0017] Preferably, in step (2), the mass ratio of iron-doped carbon nanoparticles to glucose oxidase is 1:1 to 7:1, and the reaction time is 4 to 12 hours.

[0018] Preferably, in step (3), the mass ratio of hyaluronic acid to FG complex is 1:2 to 6:1, and the reaction time is 1 to 12 h.

[0019] The depolymerizable red carbon dot composite system for enhancing tumor penetration described in this invention can achieve synergistic tumor treatment through cascade catalysis / photothermal therapy / starvation therapy in the application of multimodal synergistic tumor therapy and enhanced tumor penetration of nanomedicines.

[0020] This invention first synthesizes iron-doped carbon dot nanozymes (Fe(III)-CDs) with amino acid-like edge structures. Then, the carbon dot nanozymes are combined with glucose oxidase using the self-assembly principle. Subsequently, hyaluronic acid is coated on the surface of the system to reduce the immunogenicity of the system, endow the system with hyaluronidase / photothermal responsive depolymerization, and enhance the system's targeting to tumor sites.

[0021] In this process, citric acid and urea undergo dehydration condensation polymerization to form a graphene-like structure. Formic acid is then added to the amino groups forming intramolecular hydrogen bonds, ultimately creating an amino acid-like edge structure with α-amino and carboxyl groups. Iron ions, acting as the active center of the nanozyme, combine with disodium ethylenediaminetetraacetate, citric acid, and urea through dehydration condensation to form a metal nanozyme active center with specific Fe-N4 coordination. The carbon dots Fe(III)-CDs exhibit an excitation wavelength of 560 nm, an emission wavelength of 610 nm, and a particle size of approximately 3.2 nm. They emit red fluorescence, thus achieving a tracking effect. The amino acid-like structure at the carbon dot edges facilitates intracellular transmembrane transport of the complex via the amino acid transporter ASCT2 / LAT1. 3+ The doping of ions endows this carbon dot with excellent peroxidase (POD), superoxide dismutase (SOD), and glutathione oxidase (GSH-OXD) activities, while also exhibiting excellent photothermal conversion capabilities. This carbon dot can generate a large amount of strongly oxidizing ·OH through a cascade reaction of SOD-like and POD-like enzymes. Simultaneously, the POD-like enzyme utilizes hydrogen peroxide to generate ·OH. To achieve the supply of hydrogen peroxide, glucose oxidase is combined with the carbon dot. Glucose oxidase consumes glucose and generates hydrogen peroxide, thus simultaneously producing more ·OH and inducing starvation therapy.

[0022] Hyaluronic acid is coated on the surface of the FG complex, increasing particle size and reducing immunogenicity while enabling hyaluronidase / photothermal-responsive depolymerization at the tumor site. Following tumor-responsive depolymerization, the system releases smaller-sized FG complexes and exposes the amino acid-like edge structure of the complex. The complex then diffuses deeper into the tumor and binds to the ASCT2 / LAT1 transporters on the tumor cell surface to promote cellular uptake of the FG complex. After uptake, the nanoparticles are transported to lysosomes. GOx then consumes glucose and produces hydrogen peroxide. The POD-like activity of the carbon dots catalyzes the generation of highly oxidizing ·OH from the hydrogen peroxide, destroying the lysosomes and allowing the nanoparticles to escape. Under photothermal heating, the system depolymerizes and diffuses deeper into the tumor, achieving a synergistic tumor-killing effect through photothermal therapy, cascade catalytic therapy, and starvation therapy.

[0023] The beneficial effects of this invention are:

[0024] (1) The carbon dot nanozymes constructed in this invention possess POD-like, SOD-like, and GSH-OXD-like activities, which can enhance oxidative stress at the tumor site and greatly improve the anti-tumor therapeutic effect of nanozyme-mediated cascade catalysis. Furthermore, due to their red light emission characteristics, they can be used for fluorescent tracing of tumor sites. Simultaneously, the amino acid-like structure on the surface of the carbon dot nanozymes can achieve efficient cellular uptake via the amino acid transporter ASCT2 / LAT1 in tumor cells, effectively enhancing the transmembrane transport of GOx.

[0025] (2) This invention achieves responsive depolymerization at the tumor site by coating the system with hyaluronic acid, which enhances delivery deep into the tumor and generates ROS to enhance oxidative stress and stimulate apoptosis at the tumor site. In vivo pharmacodynamic studies revealed that under the multiple effects of cascade catalytic therapy mediated by nanozymes and natural enzymes, photothermal therapy, and starvation therapy, tumor growth was severely inhibited, achieving synergistic tumor treatment with multiple therapies. Attached Figure Description

[0026] Figure 1 The image shows a transmission electron microscope (TEM) image of the Fe(Ⅲ)-CDs prepared in Example 1.

[0027] Figure 2 The image shows the carbon NMR spectrum of the Fe(III)-CDs prepared in Example 1.

[0028] Figure 3 The image shows the X-ray absorption near-edge structure of Fe(III)-CDs prepared in Example 1.

[0029] Figure 4 The image shown is a transmission electron microscope (TEM) image of the FG prepared in Example 2.

[0030] Figure 5 The image shows the UV-Vis spectrum of FG@HA prepared in Example 3.

[0031] Figure 6 This is a transmission electron microscope image of FG@HA prepared in Example 3.

[0032] Figure 7 This is a transmission electron microscope image of the depolymerized FG@HA prepared in Example 3.

[0033] Figure 8 Fluorescence properties of Fe(Ⅲ)-CDs, FG, and FG@HA prepared in Examples 1, 2, and 3.

[0034] Figure 9 The ESR spectra of FG and FG@HA after depolymerization prepared in Examples 2 and 3 are shown.

[0035] Figure 10 Cytotoxicity of Fe(Ⅲ)-CDs, FG and FG@HA prepared in Examples 1, 2 and 3.

[0036] Figure 11 Cellular uptake effects of Fe(Ⅲ)-CDs, FG, and FG@HA prepared in Examples 1, 2, and 3.

[0037] Figure 12The diagram shows the intracellular ROS production effects of Fe(Ⅲ)-CDs, FG, and FG@HA prepared in Examples 1, 2, and 3.

[0038] Figure 13 The penetration depth of Fe(Ⅲ)-CDs, FG and FG@HA prepared in Examples 1, 2 and 3 to 4T1 tumor spheres.

[0039] Figure 14 (a) Mouse tumor growth curve, (b) Mouse body weight change curve, (c) Tumor photograph and (d) Tumor anatomical quality of Fe(Ⅲ)-CDs, FG and FG@HA prepared in Examples 1, 2 and 3. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0041] Example 1

[0042] Preparation and characterization of Fe(III)-CDs

[0043] (1) Preparation of Fe(Ⅲ)-CDs

[0044] Weigh 2.0 g citric acid, 2.0 g urea, 0.5 g disodium ethylenediaminetetraacetate (EDTA), and 0.35 g ferric chloride hexahydrate, dissolve them in 20 mL formic acid solution, and stir for 25 min. Then transfer the solution to a 50 mL polytetrafluoroethylene (PTFE)-stainless steel reactor, place the reactor in a muffle furnace, and react at 160 °C for 4 h, followed by cooling to room temperature. After the reaction is complete, mix the original reaction solution with 5 times its volume of anhydrous ethanol, centrifuge at 10,000 rpm for 5 min, and collect the precipitate. Repeat this operation three times to obtain a black solid, Fe(III)-CDs.

[0045] Weigh 2.0 g citric acid, 4.0 g urea, 0.5 g disodium ethylenediaminetetraacetate, and 0.35 g ferric chloride hexahydrate, dissolve them in 20 mL formic acid solution, stir for 25 min, then transfer the solution to a 50 mL polytetrafluoroethylene-stainless steel reactor. Place the reactor in a muffle furnace and react at 160 °C for 4 h, then cool to room temperature. After the reaction, mix the reaction mixture with 5 times its volume of anhydrous ethanol, centrifuge at 10,000 rpm for 5 min, and collect the precipitate. Repeat this operation three times to obtain a black solid, Fe(III)-CDs, which was used in Example 2 to prepare the FG complex.

[0046] Weigh 2.0 g citric acid, 6.0 g urea, 0.5 g disodium ethylenediaminetetraacetate (EDTA), and 0.35 g ferric chloride hexahydrate, dissolve them in 20 mL formic acid solution, and stir for 25 min. Then transfer the solution to a 50 mL polytetrafluoroethylene (PTFE)-stainless steel reactor, place the reactor in a muffle furnace, and react at 160 °C for 4 h, followed by cooling to room temperature. After the reaction is complete, mix the original reaction solution with 5 times its volume of anhydrous ethanol, centrifuge at 10,000 rpm for 5 min, and collect the precipitate. Repeat this operation three times to obtain a black solid, Fe(III)-CDs.

[0047] (2) Characterization of Fe(Ⅲ)-CDs

[0048] The particle size of the carbon dots was measured using transmission electron microscopy, and the results are shown in the attached figure. Figure 1 As shown, the carbon dots have a particle size of approximately 3.2 nm and a uniform particle size distribution. High-resolution transmission electron microscopy reveals that these carbon dots exhibit distinct lattice fringes. The lattice spacing is similar to that of graphite sp. 2 The uniform interplanar spacing of the hybrid carbon indicates a graphite-like structure. This demonstrates the successful preparation of Fe(III)-CDs.

[0049] The carbon nanozyme was then characterized by carbon NMR spectroscopy, and relevant structures were labeled in the spectra. The results are shown in the attached figure. Figure 2 As shown.

[0050] X-ray absorption near-edge structure analysis of the iron active center of this carbon dot nanozyme was performed, and the results are shown in the attached figure. Figure 3 As shown.

[0051] Example 2

[0052] Preparation of carbon dot-glucose oxidase complex (FG)

[0053] 10 mg of the carbon dot nanozyme Fe(III)-CDs prepared in Example 1 was dispersed in 25 mL of pH 7.4 PBS. 5 mL of 1 mg / mL glucose oxidase solution was added dropwise. After stirring at room temperature for 4 h, the reaction solution was transferred to a 50 KD ultrafiltration centrifuge tube. After centrifugation at 5000 rpm for 5 min, the nanoparticles in the inner tube and the solution in the outer tube were collected. This process was repeated three times. Finally, the solid material in the inner tube was removed, which was the carbon dot-glucose oxidase composite system.

[0054] 20 mg of the carbon dot nanozyme Fe(III)-CDs prepared in Example 1 was dispersed in 25 mL of pH 7.4 PBS. 5 mL of 1 mg / mL glucose oxidase solution was added dropwise. After stirring at room temperature for 4 h, the reaction solution was transferred to a 50 KD ultrafiltration centrifuge tube. After centrifugation at 5000 rpm for 5 min, the nanoparticles in the inner tube and the solution in the outer tube were collected. This process was repeated three times. Finally, the solid material in the inner tube was removed, which was the carbon dot-glucose oxidase composite system, and it was used in Example 3 to prepare FG@HA.

[0055] 30 mg of the carbon dot nanozyme Fe(III)-CDs prepared in Example 1 was dispersed in 25 mL of pH 7.4 PBS. 5 mL of 1 mg / mL glucose oxidase solution was added dropwise. After stirring at room temperature for 4 h, the reaction solution was transferred to a 50 KD ultrafiltration centrifuge tube. After centrifugation at 5000 rpm for 5 min, the nanoparticles in the inner tube and the solution in the outer tube were collected. This process was repeated three times. Finally, the solid material in the inner tube was removed, which was the carbon dot-glucose oxidase composite system.

[0056] The particle size of this carbon dot-glucose oxidase complex system was measured using transmission electron microscopy, and the results are shown in the attached figure. Figure 4 As shown, the particle size of the composite is approximately 30.8 nm.

[0057] Example 3

[0058] Preparation of FG@HA

[0059] 10 mg of the FG complex prepared in Example 2 was dispersed in 2 mL of pH 7.4 PBS and added dropwise to 5 mL of 1 mg / mL hyaluronic acid (90K) solution. After stirring at room temperature for 6 h, the reaction solution was transferred to a 150 KD ultrafiltration centrifuge tube and centrifuged at 5000 rpm for 5 min. The nanoparticles in the inner tube and the solution in the outer tube were collected, and this process was repeated three times. Finally, the solid material in the inner tube was removed, which was FG@HA.

[0060] 10 mg of the FG complex prepared in Example 2 was dispersed in 2 mL of pH 7.4 PBS and added dropwise to 5 mL of 2 mg / mL hyaluronic acid (90 K) solution. After stirring at room temperature for 6 h, the reaction solution was transferred to a 150 KD ultrafiltration centrifuge tube and centrifuged at 5000 rpm for 5 min. The nanoparticles in the inner tube and the solution in the outer tube were collected, and this process was repeated three times. Finally, the solid material in the inner tube was removed, which was FG@HA.

[0061] 10 mg of the FG complex prepared in Example 2 was dispersed in 2 mL of pH 7.4 PBS and added dropwise to 5 mL of 3 mg / mL hyaluronic acid (90K) solution. After stirring at room temperature for 6 h, the reaction solution was transferred to a 150 KD ultrafiltration centrifuge tube and centrifuged at 5000 rpm for 5 min. The nanoparticles in the inner tube and the solution in the outer tube were collected, and this process was repeated three times. Finally, the solid material in the inner tube was removed, which was FG@HA.

[0062] Fe(III)-CDs, FG, and FG@HA were characterized by UV-Vis spectroscopy, and the results are shown in the appendix. Figure 5 As shown.

[0063] The morphology and particle size of the FG@HA complex were measured using transmission electron microscopy, and the results are shown in the attached figure. Figure 6 As shown, the particle size of the composite is approximately 72.6 nm.

[0064] FG@HA was dispersed in pH 7.4 PBS and irradiated with a NIR laser for 5 min and treated with hyaluronidase for 30 min, respectively. The depolymerization of the system was analyzed by transmission electron microscopy, and the results are shown in the attached figure. Figure 7 As shown, FG@HA can be depolymerized in a slightly acidic environment and under NIR irradiation.

[0065] Example 4

[0066] The fluorescence properties of the carbon dots and FG@HA (FG:HA = 1:1, m / m) were characterized using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in the attached figure. Figure 8 As shown, the emission peaks of carbon dots Fe(III)-CDs, FG complex and FG@HA are all around 630 nm, emitting red fluorescence, and FG and FG@HA have stronger fluorescence intensity.

[0067] Example 5

[0068] FG and HAase+NIR-treated FG@HA were placed in a 5 mM glucose solution, and the types of ROS generated in the system were investigated by electron spin resonance. The results are attached. Figure 9 As shown, the experimental group exhibited the classic 1:2:2:1 hydroxyl radical peak pattern. This result indicates that FG can catalyze the generation of hydroxyl radicals from glucose through a cascade, and that FG@HA can generate hydroxyl radicals under co-treatment with HAase+NIR.

[0069] Example 6

[0070] In vitro cytotoxicity of the composite system against 4T1 tumor cells

[0071] 4T1 tumor cells in logarithmic growth phase were collected at a concentration of 5 × 10⁻⁶. 3Cells were seeded at a density of 1 cell / well in 96-well plates, with 5 replicates per group, and cultured in a 37°C, 5% CO2 incubator. After cell attachment, the cells were incubated for 24 h in 1640 medium containing Fe(III)-CDs, GOx, FG, and FG@HA. For the NIR laser irradiation group, after 12 h of incubation, the cells were treated with 1.0 W / cm² medium. 2 Cells were irradiated with laser power for 3 min. GOx concentrations were 0.01, 0.05, 0.1, 0.5, and 1.0 μg / mL (concentrations in other groups were converted from the GOx group concentrations). After incubation, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C in a 5% CO2 cell culture incubator for 4 h. The solution in the 96-well plate was discarded, and 200 μL of dimethyl sulfoxide solution was added to each well. The plate was shaken for 10 min, and the absorbance at 570 nm was measured using a microplate reader to calculate cell viability. Results are attached. Figure 10 As shown, Fe(III)-CDs alone did not produce significant cytotoxicity to 4T1 cells. The FG and FG@HA groups significantly reduced the survival rate of 4T1 cells. In addition, irradiation with NIR laser further reduced the survival rate of 4T1 cells.

[0072] Example 7

[0073] Uptake of the complex system in 4T1 cells

[0074] 4T1 cells in logarithmic growth phase were divided into 10 groups. 5 Cells were seeded per well in 24-well plates with spreader slides and cultured for 12 h. A suspension of Fe(III)-CDs, GOx-FITC, FG, and FG@HA prepared in FBS-free 1640 medium was added to bring the final GOx concentration to 0.05 μg / mL. Cells were co-cultured for 2 h. Cells were washed three times with PBS, and 400 μL of 4% paraformaldehyde was added. Cells were incubated at room temperature for 15 min, washed three times with PBS, and then 100 μL of Hoechst (10 μg / mL) was added. Cells were incubated at 37°C for 10 min, washed three times with PBS, and the spreader slides were inverted onto slides with mounting medium for observation under a CLSM microscope. Results are attached. Figure 11 As shown, red represents the red fluorescence of carbon dots, and green represents the fluorescence of FITC-labeled glucose oxidase. It can be seen that carbon dots, after forming an FG complex with GOx, can increase cellular uptake of GOx, and NIR laser irradiation further increases cellular uptake of the system.

[0075] Example 8

[0076] Intracellular reactive oxygen species production in the complex system

[0077] 4T1 tumor cells in logarithmic growth phase were seeded into 24-well plates with cell spreaders and incubated for 24 h. The culture medium was then replaced with 1640 medium containing Fe(III)-CDs, GOx, FG, and FG@HA and incubated for another 4 h. The NIR laser irradiation group was treated with 1.0 W / cm² laser after incubation. 2 Cells were irradiated with a laser at a specific power for 3 min. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. 500 μL of 10 μM DCFH-DA staining solution was added, and the cells were incubated for 30 min to stain for intracellular reactive oxygen species. After incubation, the cells were washed three times with PBS, and 500 μL of 10 μg / mL Hoechst staining solution was added, and the cells were incubated for 15 min to stain the nuclei. After staining, the cells were washed three times with PBS, mounted with anti-fluorescence quenching mounting medium, and then subjected to fluorescence imaging under a confocal laser microscope. The results are attached. Figure 12 As shown, the green fluorescence of the FG complex-treated group was significantly higher than that of other nanozyme groups. Furthermore, NIR laser irradiation further enhanced the production of intracellular reactive oxygen species, indicating that FG@HA has excellent intracellular reactive oxygen species production capabilities.

[0078] Example 9

[0079] The penetration depth of the composite system into 4T1 tumor spheres

[0080] Weigh 100 mg of agarose and dissolve it in an Erlenmeyer flask containing 10 mL of distilled water. Seal the flask with a breathable membrane and autoclave at 127°C for 15 min. After sterilization, add 100 μL of the hot agarose solution to each well of a 96-well plate. Once the agarose has solidified, inoculate 4 T1 cells at a rate of 2 × 10⁻⁶ cells / well. 3 Cells were seeded at a density of 4T1 cells / well in the above-mentioned 96-well plates and cultured in an incubator. Fresh culture medium was changed every 3 days, and the cells aggregated into spherical growths. When the diameter of the tumor spheres reached approximately 300 μm, a suspension of Fe(III)-CDs, GOx-FITC, FG, and FG@HA prepared in FBS-free 1640 medium was added to bring the final concentration of GOx to 0.05 μg / mL. The cells were co-cultured for 4 h, and then the uptake of the nanosystem by the 4T1 tumor spheres and the penetration of the nanosystem into the 4T1 tumor spheres were observed using CLSM. The results are attached. Figure 13 As shown, the FG complex group exhibited significantly greater penetration depth into tumor spheres than the GOx group. Furthermore, NIR irradiation significantly enhanced the penetration depth of FG@HA into tumor spheres. These results indicate that the formation of the FG complex significantly enhanced the uptake of nanoparticles by tumor spheres, and NIR irradiation also promoted the penetration of the nanosystem.

[0081] Example 10

[0082] Composite system for mouse tumor treatment

[0083] 4T1 tumor cells in the logarithmic growth phase were collected and processed at a concentration of 1×10⁻⁶. 6 Inoculate subcutaneously into mice at a density of 1 mouse / mouse, and wait until the tumor volume in the mice reaches approximately 100 mm. 3 Mice were randomly divided into saline, Fe(III)-CDs, FG, FG@HA, and FG@HA+NIR groups. The nanozymes in each group were diluted and intravenously injected into mice at a concentration of 100 μL (20 mg / kg) per mouse. The administration was repeated every two days for a total of three treatments. Changes in mouse body weight and tumor volume were recorded every other day during the treatment period. Results are attached. Figure 14 As shown, compared to the saline group, each composite treatment group exhibited a certain degree of inhibitory effect on tumor growth in mice. Furthermore, after NIR laser irradiation, the tumor-inhibiting effect of FG@HA was further enhanced, and the tumor volume showed a significant decreasing trend.

[0084] The above description is only the best specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A decomposable carbon dot composite system for enhancing tumor penetration, characterized in that, The composite system consists of photothermal responsive carbon nanoparticles, glucose oxidase, and hyaluronic acid.

2. The depolymerizable carbon dot composite system for enhancing tumor penetration according to claim 1, characterized in that, The composite system mainly consists of photothermal responsive carbon nanoparticles and glucose oxidase, with hyaluronic acid coating the outer layer of the composite.

3. The depolymerizable carbon dot composite system for enhancing tumor penetration according to claim 1, characterized in that, The photothermal responsive carbon nanoparticle nanozyme is synthesized by carbonization polymerization of citric acid, urea, disodium ethylenediaminetetraacetate, and iron ion precursor under high temperature and high pressure, wherein the iron ion is Fe. 3+ .

4. The depolymerizable carbon dot composite system for enhancing tumor penetration according to claim 1, characterized in that, The photothermal responsive carbon nanodot nanozyme has an amino acid-like structure at its edge, which enables transmembrane transport within the cell through the amino acid transporters LAT1 and ASCT2 on the cell surface.

5. A method for preparing the depolymerizable carbon dot composite system for enhancing tumor penetration according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Dissolve citric acid, urea, disodium ethylenediaminetetraacetate and iron ion precursor in formic acid solution, disperse evenly by ultrasonication, place in polytetrafluoroethylene bottle, and react in muffle furnace. (2) After adding ethanol to the reaction stock solution in step (1), centrifuge, discard the supernatant and collect the precipitate to obtain iron ion-doped carbon dot nanozymes. (3) Mix the iron ion-doped carbon dot nanozyme and glucose oxidase in step (2), stir, and remove the unreacted iron ion-doped carbon dot nanozyme by centrifugation and ultrafiltration to obtain the carbon dot-glucose oxidase composite system. (4) Add the carbon dot-glucose oxidase complex system from step (3) to the hyaluronic acid solution, mix evenly, and then ultrafilter to obtain the carbon dot complex system coated with hyaluronic acid.

6. The preparation method according to claim 5, characterized in that, In step (1), the temperature of the muffle furnace is set to 120-200 degrees Celsius, and the reaction time is 1-6 hours; the mass ratio of citric acid to urea is 1:1-1:

3.

7. The preparation method according to claim 5, characterized in that, In step (2), the volume ratio of the reaction stock solution to ethanol is 1:4 to 1:6, and the centrifugation speed is 6000 to 10000 rpm.

8. The preparation method according to claim 5, characterized in that, In step (3), the mass ratio of iron ion-doped carbon nanoparticles to glucose oxidase is 1:1 to 7:1, and the reaction time is 4 to 12 hours.

9. The preparation method according to claim 5, characterized in that, In step (4), the mass ratio of hyaluronic acid to carbon dot-glucose oxidase complex is 1:2 to 6:1, and the reaction time is 1 to 12 hours.

10. The application of the tumor-penetrating depolymerizable carbon dot composite system according to any one of claims 1-4 in achieving synergistic tumor therapy of cascade catalysis / photothermal therapy / starvation therapy.

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