A mannose-modified graphene oxide-based nanocarrier system, and a preparation method and application thereof

Abstract

The application discloses a kind of mannose modified graphene oxide-based nano drug delivery systems and its preparation method and application, belong to the technical field of biomedical materials and pharmaceutical preparations.The application solves the current free drug in vivo exposure shortage, lack of myeloid cell directional regulation, drug release uncontrollable and drug system biological compatibility problem.The application synthesizes mannose grafted ethylenediamine EDM, then with GO, EDM, EDC and NHS reaction, promote GO surface carboxyl and EDM amino form amide bond, obtain carrier, with DTX and Vadimezan loaded on carrier to obtain drug delivery system.The application uses GO as matrix, realizes receptor-mediated targeting nano-carrier system by surface functionalization of mannose, and it is used to deliver hydrophobic chemotherapeutic drugs and immune stimulants, which can be used for tumor treatment, including inhibiting primary tumor growth and metastasis-related lesions, and the effect is more obvious in combination with near-infrared light thermal response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and pharmaceutical formulation technology, and in particular relates to a mannose-modified graphene oxide-based nanoparticle drug delivery system, its preparation method and application. Background Technology

[0002] The characteristics and regulation of the tumor immune microenvironment are core factors influencing the efficacy of current cancer treatments. The tumor microenvironment of solid tumors typically includes tumor cells, blood vessels and stromal components, as well as various immune cells, among which tumor-associated macrophages (MAMs) are often the dominant infiltrating population. Numerous studies have shown that MAMs exhibit significant phenotypic heterogeneity and often tend to differentiate into an M2-like phenotype with immunosuppressive and pro-tumorigenic properties. This phenotypic polarization promotes tumor cell proliferation, invasion, and metastasis, and weakens the immunotherapy response. Simultaneously, the function of dendritic cells in the tumor microenvironment is often suppressed, manifesting as insufficient antigen uptake, processing, and presentation efficiency, thereby limiting the initiation of effective anti-tumor immunity. Docetaxel (DTX) is a commonly used anti-tumor drug in clinical practice, but its strong hydrophobicity and poor water solubility lead to non-specific distribution and dose-related toxicity after in vivo administration, and its effective drug concentration exposure in tumor tissues is limited. To improve the pharmacokinetics and safety of DTX, studies have proposed nano-formulations and drug delivery combinations of docetaxel (e.g., nanoparticle formulations). However, these approaches mainly focus on improving dissolution / pharmacokinetics and are usually difficult to simultaneously achieve targeted regulation of tumor immunosuppressive myeloid cells. STING pathway activation can induce immune-related signals such as type I interferon, thereby enhancing anti-tumor immune responses. Small molecule STING pathway agonists, represented by Vadimezan, have been used in previous studies for immune-related therapies or delivery system design, but their in vivo application still faces problems such as non-specific distribution and difficulty in maintaining local effective concentrations; without suitable delivery carriers, it is often difficult to achieve controlled, continuous or triggerable exposure at the tumor site. To address the aforementioned challenges, graphene oxide carriers and mannose receptor targeting strategies have demonstrated unique potential, but their individual and combined applications still have significant limitations. Graphene oxide (GO), due to its large specific surface area and various oxygen-containing functional groups, can interact with drugs through π–π interactions and hydrogen bonds, and can be used to construct photothermal conversion-related systems, thus finding applications in drug delivery and tumor therapy research. Existing research has disclosed the use of (nano)graphene oxide as an anti-tumor drug carrier and GO-based immunoadjuvant and / or vaccine delivery. However, existing GO as a carrier still has the following problems: (1) When GO is not modified or is not modified enough, it will exhibit safety hazards such as cytotoxicity and poor blood compatibility, which seriously restricts its applicability for intravenous administration; (2) GO drug loading mostly relies on non-covalent interaction, and when facing complex body fluid environment, the colloidal stability of the carrier and the drug binding stability may be insufficient, resulting in premature release or uncontrollable distribution of the drug before reaching the target; (3) At present, many GO-based regimens focus on a single treatment mechanism (such as chemotherapy or photothermal therapy alone), which has limited ability to remodel immunosuppressive myeloid cells. Mannose receptor (MR, CD206) is a type C lectin receptor, primarily expressed in specific subsets of macrophages and immature dendritic cells, and is involved in ligand recognition and endocytosis. Receptor-mediated uptake strategies based on mannose and / or mannose ligands have been used to construct targeted nanodelivery systems for dendritic cells or tumor-associated macrophages; studies have also reported the introduction of mannose modification into GO-type platforms for exploring immune-related therapies. However, current technologies for using mannose receptors still have limitations: on the one hand, mannose-targeting systems often focus on "delivering antigens or adjuvants or single immunomodulators," lacking sufficient coverage for the synergistic delivery and release design of "chemotherapeutic drugs + immune agonists"; on the other hand, a comprehensive technical solution that simultaneously addresses the blood compatibility, in vivo colloidal stability, tumor microenvironment-induced release response (such as acidity and exogenous energy triggering), and targeted action on myeloid cells required for intravenous administration has not yet been developed. In summary, current cancer treatments using hydrophobic chemotherapy drugs such as docetaxel suffer from poor water solubility, uneven distribution in vivo, and insufficient effective tumor exposure. While STING pathway agonists can enhance anti-tumor immunity, their free administration often results in difficulty maintaining effective concentrations at the target site and uncontrollable distribution in vivo. Meanwhile, CD206 in the tumor immune microenvironment... + Tumor-associated macrophages and tumor-infiltrating dendritic cells, among other myeloid cells, often exhibit an immunosuppressive phenotype, resulting in limited immune responses and an increased risk of tumor progression and metastasis.

[0003] Therefore, there is an urgent need to develop a nanodelivery system for tumor treatment that possesses the following capabilities: (1) stable loading and in vivo delivery of hydrophobic chemotherapeutic drugs and immune agonists; (2) good biocompatibility and blood compatibility for intravenous administration; (3) more controllable drug release under tumor-associated microenvironment conditions and synergistic effects with exogenous near-infrared stimulation; (4) improved efficacy against key myeloid cells in the tumor immune microenvironment to improve immunosuppression and enhance overall therapeutic effects; and (5) higher uptake and enrichment tendency for CD206-associated myeloid cells, thereby remodeling the tumor immune microenvironment and inhibiting primary and metastatic lesions. Summary of the Invention

[0004] To overcome the shortcomings of current free drug in vivo exposure, lack of myeloid cell-directed regulation, uncontrollable drug release, and insufficient biocompatibility of drug systems, this invention provides a mannose-modified graphene oxide-based nanocarrier system, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: One objective of this invention is to provide a method for preparing a mannose-modified graphene oxide-based nanocarrier system (GO-EDM-DTX-Vad), which includes the following steps: Step 1: Synthesis of mannose-grafted ethylenediamine (EDM) Ethylenediamine was dissolved in a solvent, followed by the addition of D-mannose and iodine. After ultrasonic treatment, the mixture was reacted under stirring to obtain mannose-grafted ethylenediamine (EDM). Step 2: Preparation of GO-EDM nanocarriers A graphene oxide dispersion was mixed with a mannose-grafted ethylenediamine dispersion to obtain a mixed dispersion. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added, and the mixture was reacted under stirring to promote the formation of amide bonds between the carboxyl groups on the GO surface and the amino groups of EDM. After the reaction, the system was washed, dialyzed, and lyophilized to obtain GO-EDM carrier powder. Step 3: Preparation of mannose-modified graphene oxide-based drug carrier nanoparticles Docetaxel (DTX) and Vadimezan (Vad) were dissolved in dimethyl sulfoxide to obtain a drug stock solution. The drug stock solution was added to a GO-EDM aqueous solution and reacted under light-protected and stirred conditions to fully load the drug onto the nanocarrier. After the reaction was completed, the system was dialyzed and lyophilized to obtain a mannose-modified graphene oxide-based nanocarrier system, namely the GO-EDM-DTX-Vad nanocomposite.

[0006] Further specifying, the solvent in step 1 is at least one of methanol, ethanol, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

[0007] Further specifying, in step 1, the volume ratio of ethylene glycol to solvent is 1:6.

[0008] Further specifying, in step 1, the molar ratio of ethylenediamine to D-mannose is (1-5):1; the molar ratio of iodine to D-mannose is (0.1-0.5):1.

[0009] Further specifying, the ultrasonic treatment time in step 1 is 10-15 min to ensure uniform dispersion of the solution and promote the opening of mannose chains.

[0010] Further specified, the reaction time in step 1 is 12-24 h, and the stirring speed is 600 rpm.

[0011] Further specifying, in step 2, the concentration of the graphene oxide dispersion is 1-3 mg / mL, and the concentration of the mannose-grafted ethylenediamine dispersion is 4-10 mg / mL.

[0012] Further specifying, in step 2, the concentration of the graphene oxide dispersion is 2 mg / mL, and the concentration of the mannose-grafted ethylenediamine dispersion is 10 mg / mL.

[0013] Further specifying, the mass ratio of graphene oxide to EDM in step 2 is 1:(2-5).

[0014] Further specifying, in step 2, the mass ratio of graphene oxide, mannose-grafted ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(2-5):(0.5-2):(0.5-2).

[0015] Further specifying, the reaction time in step 2 is 12-24 h, and the stirring speed is 400-500 rpm.

[0016] Further specifying, the reaction time in step 2 is 24 hours.

[0017] Further specifying, in step 3, the mass ratio of GO-EDM to the sum of Dorcetathione and Vad is 2:1, 3:2, 1:1, 2:3 or 1:2.

[0018] Further specifying, in step 3, the mass ratio of GO-EDM to the sum of DTX and Vad is 1:1.

[0019] Further specifying, the reaction time in step 3 is 48-72 h, and the stirring speed is 400 rpm.

[0020] Further specifying, the reaction time in step 3 is 72 h.

[0021] The second objective of this invention is to provide a mannose-modified graphene oxide-based nano-drug delivery system prepared by the above method.

[0022] A third objective of this invention is to provide an application of the above-mentioned drug delivery system in the preparation of pharmaceutical compositions that deliver antitumor active ingredients. The present invention has the following beneficial effects: (1) The mannose-modified graphene oxide-based nanocarrier system of the present invention (denoted as GO-EDM-DTX-Vad) is a mannose-modified ethylenediamine-modified graphene oxide nanocarrier (GO-EDM) and its nanocomposite loaded with docetaxel and the STING agonist Vadimezan; a nanocarrier system with graphene oxide as the matrix and mannose surface functionalization to achieve receptor-mediated targeting, and is used to deliver hydrophobic chemotherapeutic drugs and immune agonists. This nanocomposite can be applied to tumor treatment, including inhibiting the growth of primary lesions and metastatic lesions, and can be combined with near-infrared photothermal response for more obvious effects.

[0023] (2) To optimize the ratio of carrier to drug and obtain better drug loading performance, this invention investigated the mass ratio of GO-EDM to drugs (DTX and Vad) in the range of 1:2-2:1 (2:1, 3:2, 1:1, 2:3, 1:2). By comparing the encapsulation efficiency, drug loading rate, and hydrated particle size of each group, the preferred ratio was 1:1. Under this preferred ratio, the constructed nanocomposite exhibited high drug encapsulation performance and drug loading capacity, with an encapsulation efficiency of 88.47% and a drug loading rate of 35.35% for DTX; and an encapsulation efficiency of 85.37% and a drug loading rate of 11.37% for Vadimezan (see [link to original text]). Figure 3 ).

[0024] (3) The nanocarrier GO-EDM of this invention is composed of graphene oxide (GO) and mannose-grafted ethylenediamine (EDM). EDM is covalently grafted onto carboxyl-containing sites on the GO surface via an amidation reaction mediated by EDC and NHS, forming a stable GO-EDM. While maintaining the two-dimensional sheet framework of the GO matrix, the carrier introduces mannose ligands and nitrogen-containing functional groups to improve its water dispersibility and cellular compatibility, and to endow it with potential adaptability to mannose receptor-mediated endocytosis (see [link]). Figure 4-6 ).

[0025] (4) To address the issues of free drug crystallization and uneven dispersion after in vivo injection, the nanocomposite of this invention exhibits solid-state and dispersed-state stability characteristics: the DSC spectrum of the nanocomposite XRD shows that the crystal form peaks or melting peaks of free DTX and Vad in the composite are significantly weakened or disappeared, indicating that the drug is in an amorphous and highly dispersed state on the carrier surface, which is beneficial for stable loading and consistent formulation output (see...). Figure 7 ).

[0026] (5) Compared with the poor biocompatibility and blood compatibility of traditional GO carriers and the limitations of intravenous administration, the nanocomposite of the present invention has good biocompatibility and blood compatibility: 1) Good cell compatibility: In DC2.4 cells, the overall cell viability of GO-EDM remained at a high level under the conditions of 0-100 μg / mL and 24 h; in the time gradient experiment of 10 μg / mL and 6-72 h, the overall cell viability of both types was maintained within the acceptable range of ≥80%, which was significantly better than the dose-dependent cytotoxicity trend of unmodified GO (see Figure 8 2) Low risk of hemolysis: After erythrocytes were treated with GO-EDM (1-50 μg / mL, 1 h), the absorbance of the supernatant was close to that of the PBS negative control and lower than that of the deionized water positive control, indicating that it had little impact on the integrity of the erythrocyte membrane and a low risk of hemolysis (see...). Figure 9 ).

[0027] (6) Compared with traditional drug delivery systems, which suffer from uncontrollable drug release and insufficient effective exposure at the tumor site, the nanocomposite of this invention exhibits dual-response release characteristics under acidic and near-infrared light conditions. Specifically, 1) Enhanced release in acidic environments: Under pH 5.5 conditions for 72 h, the cumulative release of DTX and Vad reached 58.39% and 55.45%, respectively. 2) Further enhanced release under near-infrared light irradiation: Under 808 nm irradiation, pH 5.5, and 72 h conditions, the cumulative release was further increased to 67.22% for DTX and 64.14% for Vad (see [link to relevant documentation]). Figure 10 The acidic + near-infrared light dual-response release mechanism of the nanocomposite of this invention can enhance the release rate of DTX and Vad in the acidic tumor microenvironment and achieve "on-demand enhanced release" under exogenous near-infrared light stimulation, thereby improving the spatiotemporally controllable exposure of tumor sites and reducing the risk of excessive exposure to non-target sites.

[0028] (7) The nanocomposite of the present invention has particle size, potential and dispersibility that meet the requirements for intravenous delivery, which can avoid uncontrollable drug behavior in vivo and reduce the risk of aggregation. 1) Narrow particle size distribution: The hydrated particle size of GO-EDM-DTX-Vad is 188.4 nm and the PDI is 0.136, indicating that the particle size distribution of the composite is concentrated and the dispersibility is good. 2) Moderate surface potential: The ζ potential is -23.6 mV, indicating that the composite system has a certain electrostatic stability, which is beneficial to maintaining colloidal dispersion and reducing the risk of non-specific aggregation.

[0029] (8) The nanocomposite of the present invention has an immune microenvironment remodeling effect, specifically a myeloid cell orientation effect and an immune synergistic effect. Evidence of receptor-mediated uptake is clear: in the DC2.4 cell (dendritic cell) uptake experiment, the fluorescence signal of the nanocomposite entering the cell increased over time; after pre-incubation with free mannose, uptake significantly decreased, proving the existence of the mannose receptor-related endocytosis pathway (see...). Figure 11 and Figure 12 ).

[0030] (9) This invention uses EDM to covalently mannose-modify GO, improving the dispersibility and biocompatibility of the drug delivery system while maintaining the photothermal and drug-carrying advantages of GO; it achieves stable loading of DTX and Vad on the same carrier, while also considering the drug loading rate; this invention utilizes nanocomposites, which have dual response release characteristics to the acidic environment of tumors and exogenous near-infrared light. In addition, the nanocomposites utilize the mannose receptor-related endocytosis properties to improve the efficiency of action on CD206-related myeloid cells, thereby achieving the remodeling of the immunosuppressive microenvironment of the M2 phenotype. This remodeling effect also produces a synergistic effect with chemotherapy and photothermal therapy (see...). Figure 13 ).

[0031] (10) To address the issues of rapid clearance of free drugs and short duration of effective drug concentration maintenance in tumor tissue, the nanocomposite of this invention exhibits a tumor enrichment effect, and the drug can maintain an effective therapeutic concentration in tumor tissue for a long time. Cy5.5 tracing results showed that after intravenous injection, the fluorescence signal of GO-EDM-DTX-Vad in the tumor area increased over time and peaked at 4-8 h, exhibiting the strongest signal. Furthermore, tumor signals could still be detected at 24-48 h (see [link to Cy5.5]). Figure 14 ).

[0032] (11) The GO-EDM-DTX-Vad of the present invention can be used for tumor treatment, especially suitable for scenarios that require simultaneous chemotherapy killing and immune activation, and immune microenvironment remodeling. It can be administered via intratumoral injection or intravenous injection (see Figures 15-17Near-infrared light can be applied to the tumor area after drug administration to generate a local temperature rise and promote drug release, achieving synergistic chemotherapy-photothermal-immunotherapy: After intratumoral administration, light irradiation can be performed within a short time; after intravenous administration, light irradiation can be performed within the time window during which the nanocomposite accumulates in the tumor area, inhibiting primary tumor growth and lung metastasis (see...). Figure 18 ).

[0033] (12) Compared with the instability and difficulty in reproducing defects of traditional drug delivery systems in photothermal environments, the nanocomposite of the present invention has stable photothermal properties, and after drug administration, combined with near-infrared laser irradiation, it can raise the temperature of tumor tissue to the effective therapeutic temperature range. 1) Under irradiation with 808 nm near-infrared light, the temperature rise curve shows a relationship between laser power density and material concentration, and has good stability under multiple heating / cooling cycles. 2) In vivo application: After intratumoral administration, irradiation with 808 nm near-infrared light can raise the local temperature of the tumor to 43°C ( Figure 16 After intravenous administration, irradiation within the tumor enrichment time window can raise the tumor temperature to 41-42°C. Figure 18 This temperature range meets the commonly used window for mild photothermal synergistic therapy, which is beneficial for synergistic effects with chemotherapy and immune activation to reduce the risk of overheating damage. Attached Figure Description

[0034] Figure 1 This is the reaction route for step 1 of Example 1, where ethylenediamine reacts with mannose under iodine as a catalyst to produce ethylenediamined mannose; Figure 2 This is the reaction route for step 2 of Example 1, where ethylenediamined mannose reacts with graphene oxide in EDC / NHS to generate GO-EDM. Figure 3 The images are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of graphene oxide in Example 1, (a) scanning electron microscope, (b) transmission electron microscope. Figure 4 The images are scanning electron microscope and transmission electron microscope images of GO-EDM in Example 1, (a) scanning electron microscope, (b) transmission electron microscope; Figure 5 The images are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of GO-EDM-DTX-Vad after drug loading in Example 1: (a) Scanning electron microscope, (b) Transmission electron microscope. Figure 6 The XRD patterns and DSC diagrams of graphene oxide, ethylenediamined mannose, GO-EDM support, DTX, Vad and GO-EDM-DTX-Vad in Example 1 are shown in (a) XRD pattern and (b) DSC diagram. Figure 7The concentration- and time-dependent effects of GO and GO-EDM vectors on mouse dendritic cell viability in Example 1 are shown in (a) concentration-dependent effects of GO, (b) concentration-dependent effects of GO-EDM, (c) time-dependent effects of GO, and (d) time-dependent effects of GO-EDM. Figure 8 The UV-Vis absorption spectra of erythrocyte culture supernatant in GO-EDM, PBS solution and deionized water treatment groups in Example 1 are shown. Figure 9 The drug release curves of GO-EDM-DTX-Vad prepared in Example 1 in different pH release media in vitro, (a) DTX, (b) Vad; Figure 10 The results of the quantitative analysis of the average fluorescence intensity of DC2.4 cells uptake of RhoB-GO-EDM-DTX-Vad by flow cytometry in Example 1 are shown. Figure 11 Laser confocal microscopy image of RhoB-labeled GO-EDM-DTX-Vad nanocomposite from Example 1 after co-incubation with DC2.4 cells (cell nuclei are blue, nanocomposite is red, scale bar is 50 μm). Figure 12 The results of the experiment on macrophage phenotype reprogramming induced by the GO-EDM-DTX-Vad nanocomposite in Example 1 are shown. Figure 13 The results show the differences in in vivo distribution and enrichment of the complexes prepared in Example 1 and Comparative Example 1. Figure 14 In the BALB / c mouse 4T1 subcutaneous tumor model established using the nanocomposite of Example 1, the size of the isolated tumor was adjusted after intratumoral injection of each group of mice; Figure 15 The temperature changes at the tumor site in the BALB / c mouse 4T1 subcutaneous tumor model established using the nanocomposite of Example 1 were observed after intratumoral injection of the tumor into mice irradiated with PBS+NIR and GO-EDM-DTX-Vad+NIR. Figure 16 In the BALB / c mouse 4T1 subcutaneous tumor model established using the nanocomposite of Example 1, the size of the isolated tumor was adjusted after intravenous injection of the drug into the tumor of each group of mice. Figure 17 The temperature changes at the tumor site in the BALB / c mouse 4T1 subcutaneous tumor model established using the nanocomposite of Example 1 were observed after intravenous injection of the mice into the PBS+NIR group and the GO-EDM-DTX-Vad+NIR group. Figure 18Images show metastatic nodules on the surface of isolated lung tissue and H&E staining of mouse lung tissue in an animal model of 4T1 subcutaneous tumor with lung metastasis established using the nanocomposite of Example 1. Detailed Implementation

[0035] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification. Many specific details are set forth in the following description to provide a thorough understanding of the present invention; however, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0038] Example 1 Step 1: Synthesis of mannose-grafted ethylenediamine (EDM) D-mannose and ethylenediamine were reacted in a methanol system and condensed under iodine catalysis to obtain mannose-grafted ethylenediamine. The specific process was as follows: 12.0 mmol of ethylenediamine (0.8 mL) was dissolved in 6.0 mL of methanol, 2.77 mmol of D-mannose (500 mg) was added, and then 100 mg of iodine was added as a catalyst to obtain a mixed system. The system was placed in an ultrasonic cleaner and ultrasonically dispersed for 10-15 min to ensure uniform dispersion and promote mannose chain opening. After ultrasonication, the reaction was carried out at room temperature (25±2 ℃) with magnetic stirring at 600 rpm for 16 h. During the reaction, the system was kept away from light and properly ventilated to prevent solvent evaporation or side reactions. After the reaction, the solvent was removed by rotary evaporation or solvent precipitation to obtain EDM. The reaction in this step is shown below. Figure 1 Ethylenediamine and mannose undergo condensation or amination reactions under iodine as a catalyst to generate ethylenediamined mannose (EDM). This reaction introduces an amino-containing ethylenediamine group at the reducing end of mannose, giving the product both a glycosyl recognition structure and an amino active site that can be used for subsequent coupling, thus providing a key intermediate for subsequent covalent linkage with GO. Step 2: Preparation of GO-EDM nanocarriers The GO dispersion was reacted with EDM in the presence of EDC / NHS, causing the EDM to amidate the carboxyl groups on the GO surface to form GO-EDM. The specific reaction process is as follows: Add 20 mg of GO powder to 10 mL of DMF and sonicate for 30 min to obtain a GO dispersion with a concentration of 2 mg / mL; add 60 mg of EDM to 6 mL of DMF and sonicate for 20 min to obtain an EDM dispersion with a concentration of 10 mg / mL. The GO dispersion and EDM dispersion were mixed to obtain a mixed dispersion. 15 mg of EDC and 15 mg of NHS were added, and the mixture was magnetically stirred at 400 rpm for 18 h at room temperature (25±2 ℃). During the reaction, EDC / NHS first activated the carboxyl groups on the surface of GO to form a reactive intermediate. Subsequently, it underwent nucleophilic substitution with the primary amine on the EDM molecule to form an amide bond, thereby achieving covalent grafting of mannose ligands and generating mannose-functionalized graphene oxide carrier GO-EDM. After the reaction was completed, the system was washed repeatedly with PBS solution 5 times until the pH of the supernatant was adjusted to 7.0. Then, it was dialyzed in deionized water for 72 h using a 3.5 kDa molecular weight cutoff dialysis bag to remove unreacted raw materials and cross-linking reagents. The water was changed every 12-24 h during the dialysis. Finally, the dialyzed product was freeze-dried at -40 ℃ for 48 h using a vacuum freeze dryer to obtain dry GO-EDM carrier powder. The reaction diagram for this step is shown below. Figure 2 As shown. Through this method, ethylenediamined mannose and GO undergo efficient amidation coupling in an EDC / NHS activation system. The covalent grafting method can improve the stability of the modified layer and ensure the structural integrity of GO-EDM under physiological conditions, providing a stable carrier for subsequent drug loading. Step 3: Preparation of mannose-modified graphene oxide-based nanocarrier system (GO-EDM-DTX-Vad) Docetaxel and Vad (total 10 mg, with a mass ratio of DTX to Vad of 1:1) were dissolved in dimethyl sulfoxide to obtain a drug stock solution. The drug stock solution was then slowly added dropwise to a GO-EDM aqueous solution (containing 10 mg of GO-EDM). The mass ratio of GO-EDM to the sum of DTX and Vad was 1:1. The drug was fully loaded onto the nanocarrier by magnetic stirring at 400 rpm for 72 h under light-protected and room temperature conditions. After the reaction, the reaction system was placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa and dialyzed in deionized water for 72 h to remove unloaded free drug. The water was changed every 12 h during dialysis. After dialysis, the system was placed in a vacuum freeze dryer and freeze-dried at -40 ℃ for 72 h to obtain a dried mannose-modified graphene oxide-based nanocarrier system, namely the GO-EDM-DTX-Vad nanocomposite.

[0039] Example 2 The difference between this embodiment and embodiment 1 is that: in step 3, the amount of GO-EDM is kept constant, the mass ratio of GO-EDM to the sum of DTX and Vad is 2:1, and the remaining process operations and parameter settings are the same as in embodiment 1.

[0040] Example 3 The difference between this embodiment and embodiment 1 is that: in step 3, the amount of GO-EDM is kept constant, the mass ratio of GO-EDM to the sum of DTX and Vad is 3:2, and the remaining process operations and parameter settings are the same as in embodiment 1.

[0041] Example 4 The difference between this embodiment and embodiment 1 is that: in step 3, the amount of GO-EDM is kept constant, the mass ratio of GO-EDM to the sum of DTX and Vad is 2:3, and the remaining process operations and parameter settings are the same as in embodiment 1.

[0042] Example 5 The difference between this embodiment and embodiment 1 is that: in step 3, the amount of GO-EDM is kept constant, the mass ratio of GO-EDM to the sum of DTX and Vad is 1:2, and the remaining process operations and parameter settings are the same as in embodiment 1.

[0043] Comparative Example 1 The difference between this comparative example and Example 1 is that the GO was not modified, that is, steps 1 and 2 were not performed, only step 3 was performed. In step 3, GO-EDM is unmodified GO. The remaining process operations and parameter settings are the same as in Example 1. The resulting complex is denoted as GO-DTX-Vad.

[0044] The encapsulation efficiency (EE), drug loading rate (LE), and nanoparticle size of GO-EDM carrier and DTX+Vad in Examples 1-5 were tested under different mass ratios. Drug encapsulation efficiency and drug loading rate were determined by high-performance liquid chromatography (HPLC, Agilent 1260 Infinity Analytical SFC System): 10 mg of graphene oxide-based nanoparticle drug delivery system powder was weighed and dissolved in 50 mL of methanol or acetonitrile, and the mixture was ultrasonically dispersed to release the drug. A TC-C18 reversed-phase column (4.6 × 250 mm, 5 μm) was used, with an acetonitrile / water ratio of 70:30 (v / v) as the mobile phase, a flow rate of 1.0 mL / min, and detection wavelengths of 227 nm (DTX) and 345 nm (Vad). The drug content in the sample was calculated based on the standard curve to obtain the encapsulation efficiency and drug loading rate; the hydrated particle size was determined using a Zetasizer Nano ZS90.

[0045] The test results are shown in Table 1. It can be seen that with increasing drug dosage, the drug loading rate (LE) of DTX increased from 24.56% to 39.60%, while the encapsulation efficiency (EE) decreased from 97.41% to 56.53%; the drug loading rate (LE) of Vad increased from 8.22% to 13.70%, while the encapsulation efficiency (EE) decreased from 97.85% to 58.66%. When the ratio of carrier to drug exceeds 1:1, the increase in LE significantly weakens, while the decrease in EE significantly accelerates, indicating that the available binding sites on the surface of the GO-EDM carrier gradually become saturated.

[0046] Table 1

[0047] Taking into account LE, EE, and nanoparticle size variations, a 1:1 ratio (Example 1) was ultimately selected as the preferred mass ratio for subsequent performance tests. Under these conditions, the LE of DTX was 35.35%, and the EE was 88.47%, while the LE of Vad was 11.37%, and the EE was 85.37%, which were used to evaluate the nanocomposite. At this point, the hydrated particle size of the final product was 188.4 nm, the PDI was 0.136, and the zeta potential was -23.6 mV. These physicochemical parameters are beneficial to the dispersion stability of GO-EDM-DTX-Vad and its in vivo behavior after intravenous administration.

[0048] The SEM and TEM images of GO used in Example 1 are as follows: Figure 3 As shown in (a) and (b). Figure 3This indicates that GO exhibits a typical two-dimensional lamellar structure. SEM images show that GO has a thin, lamellar morphology with obvious wrinkles or undulations, with curling and stacking at the edges, exhibiting an overall loose network structure formed by overlapping lamellar sheets. TEM images further confirm that GO is an ultrathin lamellar structure, with low intra-sheet contrast and high edge contrast, and slight wrinkles and overlapping areas are visible in some areas, indicating that GO has a large specific surface area and flexible lamellar characteristics.

[0049] The SEM and TEM images of the GO-EDM prepared in step 2 of Example 1 are shown below. Figure 4 As shown in (a) and (b). Figure 4 This indicates that GO retains its lamellar framework structure after EDM surface functionalization. In the SEM images, GO-EDM still exhibits a thin, lamellar morphology, but the surface roughness of the lamellars is relatively increased, and the stacking and / or overlapping structures between the lamellars are more pronounced, indicating that the interfacial properties of the material have changed after GO surface modification. In the TEM images, the GO-EDM lamellars are observed to be relatively thin and continuous, with enhanced local contrast and more obvious edge and / or adhesion features, indicating that the functionalized EDM molecules form a modification layer on the GO lamellar surface or are distributed in local areas of the GO lamellars, while not disrupting the two-dimensional lamellar morphology of GO.

[0050] The SEM and TEM images of GO-EDM-DTX-Vad prepared after drug loading in step 3 of Example 1 are shown below. Figure 5 As shown in (a) and (b). Figure 5 This indicates that GO-EDM forms a stable nanocomposite after loading DTX and Vad, and its sheet structure remains identifiable. SEM images show more pronounced particle and / or roughening features on the carrier surface, with tighter connections between sheet edges and layers, indicating effective binding between the drug molecules DTX and Vad and the GO-EDM carrier, altering the sheet interface morphology. TEM images show further increased contrast in the sheet regions, with localized dark areas or adhesion patches exhibiting high electron density, indicating drug distribution and enrichment on the carrier sheet surface and / or between layers after loading. Furthermore, the overall GO-EDM-DTX-Vad SEM image retains the predominantly two-dimensional sheet-like composite structure.

[0051] The XRD and DSC test results of GO-EDM-DTX-Vad prepared after drug loading in step 3 of Example 1 are as follows: Figure 6 As shown in (a) and (b), it can be seen that the crystal form peaks or melting peaks of free DTX and Vad in the nanocomposite are significantly weakened or disappear, indicating that the drug is in an amorphous and highly dispersed state on the surface of the GO-EDM carrier. This is beneficial for stable loading and consistent formulation output (corresponding to the problem of free drug being prone to crystallization and uneven dispersion).

[0052] Comparative Example 1 The difference between this comparative example and Example 1 is that the GO was not modified, that is, steps 1 and 2 were not performed, only step 3 was performed. In step 3, GO-EDM is unmodified GO. The remaining process operations and parameter settings are the same as in Example 1. The resulting complex is denoted as GO-DTX-Vad.

[0053] Performance testing (a) Cell compatibility and blood compatibility (1) Cell compatibility: DC2.4 (mouse dendritic cells, purchased from Wuhan Pronosei Biotechnology Co., Ltd.) were seeded at an appropriate density in 96-well culture plates to allow the cells to adhere fully and reach a stable state. In the first experiment, for the dose-dependent effect, the culture medium was replaced with complete culture medium containing different concentrations of GO or GO-EDM (0, 1, 5, 10, 25, 50, 100 μg / mL) and incubated for another 24 h to assess the effect. In the second experiment, to investigate the time-dependent effect, after the DC2.4 cells were stably adhered, the culture medium was replaced with medium containing 10 μg / mL GO or GO-EDM and incubated for 6, 12, 24, 36, 48, and 72 h, respectively. After treatment, CCK-8 reagent (CCK-8 kit purchased from Dongren Chemical Technology Co., Ltd.) was added according to the reagent instructions and incubated for 2 h. Then, the absorbance of each well was measured at 450 nm using a microplate reader. CCK-8 cell viability was calculated based on the absorbance (OD) value, using the formula (1). The relative cell viability was normalized to 100% with the untreated control group, thereby assessing the effects of GO or GO-EDM on the cytotoxicity and proliferation of DC2.4 cells. Equation (1) Cell compatibility results as follows Figure 7 As shown in the figure. The results showed that in DC2.4 cells, GO-EDM maintained a high level of cell viability overall under conditions of 0-100 μg / mL and 24h; in the 10 μg / mL, 6-72h time gradient experiment, the overall cell viability of both types was maintained within the acceptable range of ≥80%, which was significantly better than the dose-dependent cytotoxicity trend of unmodified GO.

[0054] (2) Hemolytic assay: Healthy female Balb / c mice aged 4-6 weeks and weighing 16-20 g were collected. Blood was collected and anticoagulated with heparin. After centrifugation at 1000 g for 15 min, red blood cells were collected. The red blood cells were resuspended in PBS to prepare a homogeneous red blood cell suspension. Then, an equal volume of the red blood cell suspension was mixed with PBS containing different concentrations of GO-EDM (1, 5, 10, 20, 50 μg / mL) and incubated at 37°C for 1 h. The experimental setup included a PBS group as a negative control to reflect the normal state of red blood cells and a deionized water group as a positive control to induce complete hemolysis. After incubation, the mixture was centrifuged, the supernatant was collected, and the absorbance was measured at an appropriate wavelength using a UV-Vis spectrophotometer to assess the hemolysis status of red blood cells.

[0055] Cell compatibility results as follows Figure 8 As shown in the figure. The results showed that the absorbance of the supernatant after GO-EDM treatment (1-50 μg / mL, 1 h) was close to that of the PBS negative control, but significantly lower than that of the deionized water positive control, indicating that GO-EDM has little impact on the integrity of erythrocyte membranes, low risk of hemolysis, and high safety for use in vivo.

[0056] The test results above show that, compared with traditional GO carriers, which have poor biocompatibility and blood compatibility and are limited by intravenous administration, the nanocomposite of the present invention has good biocompatibility and blood compatibility and is suitable for intravenous administration.

[0057] (ii) In vitro release performance In vitro drug release assays were performed using dynamic dialysis: 10.0 mg of GO-EDM-DTX-Vad nanocomposite was accurately weighed and dispersed in 5.0 mL of release medium (PBS containing 0.1% Tween-80), and sonicated for 5 min to ensure uniform dispersion. The entire dispersion was transferred to a pretreated dialysis bag (molecular weight cutoff 8-14 kDa) and placed in a transparent glass beaker containing 50 mL of the same release medium. The release medium was adjusted to different pH values ​​(8.4, 7.4, 5.5) to simulate the pH conditions of normal tissue, tumor microenvironment, and intracellular lysosomes. The entire system was placed in a constant-temperature shaking incubator and continuously shaken at 37 ℃ and 100 rpm.

[0058] At preset time points (0.5, 1, 2, 4, 8, 12, 24, and 48 h), 0.5 mL of sample was precisely transferred from the external liquid in the beaker, and an equal volume of fresh release medium at the same temperature and pH was immediately added to maintain a constant total volume and avoid diffusion slowdown caused by concentration gradients. The samples were filtered through a 0.22 μm microporous membrane, and the DTX content was determined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated.

[0059] To investigate the effect of near-infrared photothermal effect on drug release behavior, in some experimental groups (pH 7.4 and 5.5), the area containing the dialysis bag was irradiated with an 808 nm near-infrared laser (power density 1.0 W / cm², irradiation time 5 min) before each sampling. Samples were taken and analyzed immediately after irradiation. This procedure simulates the local thermal effect triggered by laser in in vivo therapy, thereby evaluating the drug release characteristics under the synergistic effect of photothermal action. All experiments were performed in triplicate, and results are expressed as mean ± standard deviation.

[0060] Figure 9 This document describes the in vitro drug release behavior of GO-EDM-DTX-Vad under different pH conditions and in conjunction with infrared light treatment, as described in Example 1. The release curves were used to compare the release differences of the GO-EDM-DTX-Vad nanocomposite in neutral and acidic environments, thereby simulating drug release characteristics in acidic compartments such as blood or normal tissue, tumor microenvironment, or intracellular bodies and lysosomes. The results show that GO-EDM-DTX-Vad exhibits stimulus-responsive release characteristics, displaying distinguishable release kinetics (different pH partitions simulate the acidity / alkalinity of different sites in vivo) under different pH conditions and near-infrared irradiation. This demonstrates that the nanocomposite exhibits an environmentally responsive release trend, which is beneficial for improving drug availability in tumor-associated acidic microenvironments and reducing exposure to non-target sites.

[0061] (III) Uptake performance of nanocomposites by dendritic cells To evaluate the uptake efficiency of the GO-EDM-DTX-VAD nanocomposite of Example 1 by DC2.4 dendritic cells, the nanocomposite was fluorescently labeled with Rhodamine B (RhoB). Specifically, DC2.4 cells were charged at a concentration of 1 × 10⁻⁶ cells / cells. 6 Nanocomposite particles were seeded per well in 24-well plates or 35 mm diameter confocal culture dishes and cultured at 37 °C and 5% CO2 until stable adhesion. After adhesion, the medium was replaced with complete medium containing 10 μg / mL RhoB-GO-EDM-DTX-Vad and incubated for 3, 6, and 12 h to observe the time-dependent uptake of the nanocomposite.

[0062] To investigate mannose receptor-mediated competitive uptake, 1 h before the addition of the nanocomposite, complete culture medium containing 1 mg / mL of mannose was added to some wells and incubated continuously to competitively block receptor-carrier binding. For the photothermal effect experiment, DC2.4 cells were incubated at 1×10⁻⁶ cells / well. 6DC2.4 dendritic cells were seeded per well in 24-well plates or 35 mm diameter confocal culture dishes and cultured at 37°C and 5% CO2 until stable adhesion. After adhesion, the culture medium was replaced with complete medium containing GO-EDM-DTX-Vad (10 μg / mL) from Example 1, and incubated for 3 h. After incubation, the cells were exposed to an 808 nm near-infrared laser at a power of 1 W / cm² for 5 min to evaluate the effect of photothermal stimulation on cell membrane permeability and nanocomposite uptake efficiency.

[0063] After incubation, cells were collected and fluorescence intensity was detected in the PE channel using a DxFLEX flow cytometer (Beckman Coulter, USA). The uptake level of the nanocomposite was quantified by mean fluorescence intensity (MFI). In confocal imaging experiments, DC2.4 dendritic cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, and stained with DAPI for 10 min. Fluorescence images were acquired using a laser confocal microscope (CLSM, Nikon A1, Nikon Corporation, Japan).

[0064] Flow cytometry ( Figure 11 ) and confocal imaging results ( Figure 12 The results showed that in DC2.4 cells, the intracellular fluorescence signal of RhoB-labeled GO-EDM-DTX-VAD continuously increased with prolonged incubation time, exhibiting typical time-dependent uptake characteristics. In the mannose competitive blocking group, intracellular fluorescence decreased significantly, indicating that the nanocomposite mainly entered the cell via mannose receptor-mediated active endocytosis. Furthermore, after irradiation with 808 nm near-infrared light, the intracellular fluorescence signal was further significantly enhanced, suggesting that the photothermal effect can improve cell membrane permeability and endocytosis rate, thereby synergistically enhancing intracellular delivery efficiency.

[0065] Figure 10 The mean fluorescence intensity of DC2.4 cells uptake of RhoB-GO-EDM-DTX-Vad was quantitatively analyzed by flow cytometry. Figure 11 Laser confocal microscopy images of DC2.4 cells after co-incubation of RhoB-labeled GO-EDM-DTX-Vad nanocomposite materials. In the images, Merge represents the superposition of fluorescence images from the DAPI blue and RohB red channels; NPs represents cells treated only with the nanocomposite material from Example 1; NPs+Mannose represents the group treated with both the nanocomposite material and mannose; and NPs+NIR represents the group treated with a combination of the nanocomposite material and near-infrared light. Figure 10 and Figure 11As can be seen, the intracellular fluorescence signal of RhoB-labeled GO-EDM-DTX-Vad in DC2.4 dendritic cells continuously increased with prolonged incubation time, exhibiting typical time-dependent uptake characteristics. In the mannose competitive blocking group, intracellular fluorescence decreased significantly, indicating that the nanocomposite mainly entered the cell via mannose receptor-mediated active endocytosis. Furthermore, after irradiation with 808 nm near-infrared light, the intracellular fluorescence signal was further significantly enhanced, indicating that the photothermal effect can improve cell membrane permeability and endocytosis rate, thereby synergistically enhancing intracellular delivery efficiency.

[0066] (iv) The regulatory role of nanocomposites in macrophage phenotypic reprogramming Macrophage phenotype reprogramming: RAW264.7 cells (mouse macrophages, purchased from Wuhan Pronosei Life Sciences Co., Ltd.) were programmed at a rate of 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 10 cells / well in 12-well plates and cultured at 37 °C and 5% CO2 until stable adhesion. 20 ng / mL interleukin-4 (IL-4) and 20 ng / mL interleukin-13 (IL-13) were added to the culture medium to stimulate cells for 24 h to induce M2 polarization. The supernatant was then discarded and replaced with complete culture medium containing different nanocomposites such as PBS, GO-EDM, GO-EDM-DTX, GO-EDM-Vad, or GO-EDM-DTX-Vad+NIR, and incubated for another 24 h.

[0067] After incubation, cells from each group were collected and washed with PBS to adjust the cell concentration to 1×10⁻⁶. 7 Cells were analyzed by flow cytometry after reaching a concentration of cells / mL. For M1 phenotypic analysis, surface staining was performed directly using anti-mouse PE-CD86 antibody (clone A17199A, BioLegend). For M2 phenotypic analysis, cells were first fixed and permeabilized using Cyto-Fast™ Fix / PermBuffer Set (BioLegend), followed by staining with anti-mouse PE-Cy7-CD206 antibody (clone C068C2, BioLegend). After staining, the positive expression ratios of CD86 and CD206 in each group of cells were detected by flow cytometry.

[0068] The experimental results of macrophage phenotypic reprogramming induced by the GO-EDM-DTX-Vad nanocomposite in Example 1 are as follows: Figure 12 As shown in the figure. The results showed that, compared with the control group (PBS only), the fluorescence signal of CD206 gradually decreased in each treatment group, while the signal of M1-related markers significantly increased. Flow cytometry quantitative analysis further indicated that the nanocomposite treatment could significantly reduce CD206. +Cell ratio, while increasing CD86 + The cell ratio indicates that it has the effect of inhibiting M2 polarization and promoting the transformation of macrophages to the M1 phenotype.

[0069] (v) In vivo tumor targeting performance Figure 13 The results show the differences in in vivo distribution and enrichment between GO-EDM-DTX-Vad of Example 1 and GO-DTX-Vad of Comparative Example 1.

[0070] 4T1 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., 1×10⁴ cells) were used. 6 (Each mouse was subcutaneously injected with one tumor sample per mouse, suspended in 100 μL PBS) into the right subcutaneous region. Tumor volume increased to approximately 150-200 mm. 3 Tumor-bearing mice were randomly divided into two groups (n=3), and injected intravenously with either 100 μL of Cy5.5-GO or Cy5.5-GO-EDM-DTX-Vad solution, with drug concentration and dosage calculated based on a DTX 20 mg / kg equivalent. Under isoflurane inhalation anesthesia, fluorescence imaging was acquired at 0, 0.5, 2, 4, 8, 16, 24, and 48 h after administration using a small animal in vivo imaging system (IVIS Spectrum, PerkinElmer, USA). Forty-eight h after intravenous injection, the mice were sacrificed under anesthesia, and the heart, liver, spleen, lung, kidney, and tumor tissue were isolated for ex vivo fluorescence imaging.

[0071] from Figure 13 As can be seen, compared with GO without mannose modification, GO-EDM-DTX-Vad exhibited a more significant enrichment signal and a higher contrast between the tumor and the background in 4T1 subcutaneous tumors, indicating that GO-EDM-DTX-Vad of Example 1 of this invention has enhanced tumor targeting. This enhanced targeting is mainly due to the recognition and uptake of myeloid-related receptors mediated by mannose ligands on the surface of the nanocomposite, as well as the passive enrichment effect of the nanocomposite in the tumor microenvironment, thereby improving local drug exposure in the tumor and enhancing the therapeutic effect.

[0072] (vi) Intratumoral injection in mice to treat subcutaneous tumors, producing a mild photothermal effect under 808nm laser irradiation. All animal experiments of this invention were conducted at the Laboratory Animal Department of Capital Medical University, and all protocols were approved by the Animal Experimentation and Laboratory Animal Welfare Committee of Capital Medical University. A BALB / c mouse 4T1 subcutaneous tumor model was established using female BALB / c mice (4-6 weeks old, 16-20g), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in a standard SPF-grade animal laboratory.

[0073] After one week of acclimatization, 4T1 cells (1×10⁻⁶) were introduced. 6 (Each mouse was subcutaneously injected with one tumor sample per mouse, suspended in 100 μL PBS) into the right subcutaneous region. Tumor volume increased to approximately 150-200 mm. 3 At that time, the tumor-bearing mice were randomly divided into 6 groups (n=5): G1 group: PBS group, G2 group: vector group (GO-EDM group), G3 group: GO-EDM-DTX group, G4 group: GO-EDM-Vad group, G5 group: GO-EDM-DTX-Vad group and G6 group: GO-EDM-DTX-Vad+NIR group.

[0074] Day 0 was designated as the day of grouping for each group. Administration began with intratumoral injection of the corresponding formulation every 3 days, for a total of 3 administrations. The administration volume was 100 μL, calculated based on a docetaxel 20 mg / kg equivalent. Mouse body weight was measured every two days from tumor formation, and the long diameter (a) and short diameter (b) of the tumor were recorded. The value was calculated using the formula V = a × b. 2 / 2 Calculate tumor volume. In the GO-EDM-DTX-Vad+ NIR group, 808 nm near-infrared laser (1 W / cm²) was used 30 min after each administration. 2 The tumor was irradiated locally for 10 minutes. Eight days after the last treatment, the mice were euthanized, the primary tumor was dissected and weighed to assess the efficacy, and major organs were collected for subsequent analysis.

[0075] The size of the isolated tumors after intratumoral administration to mice in each group was as follows: Figure 14 As shown, the tumors in the PBS group and the GO-EDM group grew rapidly with no significant difference. The GO-EDM-DTX or GO-EDM-Vad groups could delay tumor progression to some extent, but the inhibitory effect was limited. Compared with the control group (PBS group), the GO-EDM-DTX-Vad group showed a significant tumor growth inhibition effect. When combined with NIR, its tumor-suppressing effect was further enhanced, which confirmed that the nanocomposite can promote drug release in vivo through the local temperature rise effect triggered by near-infrared light, achieving synergistic chemotherapy-photothermal-immunotherapy, and has good biosafety.

[0076] To evaluate the photothermal properties of GO-EDM-DTX-Vad in vivo, a BALB / c mouse 4T1 subcutaneous tumor-bearing model was first established, using the same modeling method as (1). The tumor volume was allowed to grow to 150-200 mm. 3Mice were then randomly divided into two groups (n=3): a PBS group and a GO-EDM-DTX-Vad+NIR group, receiving intratumoral injections of 100 μL PBS or GO-EDM-DTX-Vad (calculated as a docetaxel 20 mg / kg equivalent), respectively. Thirty min after administration, the tumor site was irradiated with an 808 nm near-infrared laser for 10 min. The temperature changes at the tumor site after laser irradiation in both groups are shown below. Figure 15 As shown in the figure, thermal imaging revealed limited local temperature rise with PBS+NIR; while GO-EDM-DTX-Vad, under the same irradiation conditions, could rapidly raise the tumor temperature to a mild photothermal therapy window of approximately 40-45°C (approximately 43°C) and maintain it for a certain period, suggesting that this window balances therapeutic sensitization (enhancing the therapeutic effect of chemotherapy drugs) with a low risk of thermal damage to surrounding tissues. Only mild and transient skin reactions were observed, with no obvious burns or ulcers. Real-time monitoring showed that the local tumor temperature in this group could rapidly rise and be maintained within the mild photothermal therapy window of 40-45°C, and the skin in the irradiated area showed only mild and reversible reactions.

[0077] (vii) Tail vein administration inhibits the primary tumor and reduces the burden of lung metastases. An animal model of 4T1 subcutaneous tumor with lung metastasis was established. Following the method described above, a 4T1 subcutaneous tumor-bearing model was first established, with the primary tumor volume approximately 50 mm². 3 At that time, 1.0×10 6 4T1 cells were slowly injected via the tail vein in 100 μL of PBS suspension to induce lung metastasis. After modeling, mice were randomly divided into five groups (n=5): G1: control group, G2: GO-EDM vector group, G3: free DTX+Vad group, G4: GO-EDM-DTX-Vad group, and G5: GO-EDM-DTX-Vad+NIR group.

[0078] Starting on day 4 after tail vein injection of tumor cells, docetaxel was administered via tail vein at a dose of 20 mg / kg equivalent, once every 3 days for a total of 3 times, with each administration volume being 100 μL. In the GO-EDM-DTX-Vad+NIR group, the primary tumor area was irradiated with 808 nm near-infrared laser (1 W / cm²) 8 hours after each administration. 2 (5 min). Mice were sacrificed on day 8 after the last administration. After treatment, mice were sacrificed and tumors were removed. They were arranged and displayed according to the treatment groups. Lungs were harvested for observation and the number of visible metastatic nodules was recorded. H&E staining was performed to assess pathological changes.

[0079] Figure 16 The size of isolated tumors was determined after intravenous administration of the drug to mice in each group. Figure 16It can be seen that, compared with the PBS and GO-EDM groups, the GO-EDM-DTX-Vad group significantly inhibited primary tumor growth and reduced the endpoint tumor weight. Compared with the free DTX+Vad group, nanodelivery improved effective intratumoral exposure, resulting in stronger efficacy. GO-EDM-DTX-Vad combined with NIR irradiation showed a stronger inhibitory effect. Figure 17 As shown, infrared thermal imaging shows that the tumor site can be heated to about 41-42°C after tail vein injection of the nanocomposite of Example 1, further demonstrating that the GO-EDM-DTX-Vad nanocomposite of the present invention has the ability to enrich tumors and perform photothermal conversion in vivo, and the combined effect of NIR irradiation on inhibiting primary tumors is the most significant.

[0080] Figure 18 This image shows metastatic nodules on the surface of isolated mouse lungs. Lungs were removed from mice, and nodules representing metastatic tumors were observed on their surface. Based on these images, H&E staining of lung tissue from each group of mice was used to assess the lung metastasis burden. Figure 18 It can be seen that: in terms of lung metastasis, the PBS group and GO-EDM group had more lung surface nodules and a heavier pathological burden. The free DTX+Vad group only slightly reduced the number and volume of nodules. The GO-EDM-DTX-Vad group could significantly reduce metastatic lesions. After combining with NIR, only a few small and scattered nodules remained. H&E showed that the alveolar structure of the GO-EDM-DTX-Vad+NIR group was more intact.

[0081] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a mannose-modified graphene oxide-based nanocarrier system, characterized in that, The method includes the following steps: Step 1: Synthesis of mannose-grafted ethylenediamine Ethylenediamine was dissolved in a solvent, then D-mannose and iodine were added, and the mixture was ultrasonically treated and reacted under stirring to obtain mannose-grafted ethylenediamine. Step 2: Preparation of GO-EDM nanocarriers The graphene oxide dispersion was mixed with the mannose-grafted ethylenediamine dispersion to obtain a mixed dispersion. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added and reacted under stirring. After the reaction, the system was washed, dialyzed and lyophilized to obtain GO-EDM carrier powder. Step 3: Preparation of mannose-modified graphene oxide-based drug carrier nanoparticles Docetaxel and Vadimezan were dissolved in dimethyl sulfoxide to obtain a drug stock solution. The drug stock solution was added to a GO-EDM aqueous solution and reacted under light-protected and stirred conditions. After the reaction was completed, the system was dialyzed and lyophilized to obtain a mannose-modified graphene oxide-based nano-drug delivery system.

2. The preparation method according to claim 1, characterized in that, In step 1, the volume ratio of ethylene glycol to solvent is 1:6, the molar ratio of ethylenediamine to D-mannose is (1-5):1, and the molar ratio of iodine to D-mannose is (0.1-0.5):

1.

3. The preparation method according to claim 1, characterized in that, In step 1, the ultrasonic treatment time is 10-15 min, the reaction time is 12-24 h, and the stirring speed is 600 rpm.

4. The preparation method according to claim 1, characterized in that, In step 2, the concentration of the graphene oxide dispersion is 1-3 mg / mL, and the concentration of the mannose-grafted ethylenediamine dispersion is 4-10 mg / mL.

5. The preparation method according to claim 1, characterized in that, In step 2, the mass ratio of graphene oxide, mannose-grafted ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(2-5):(0.5-2):(0.5-2).

6. The preparation method according to claim 1, characterized in that, In step 2, the reaction time is 12-24 h and the stirring speed is 400-500 rpm.

7. The preparation method according to claim 1, characterized in that, In step 3, the mass ratio of GO-EDM to the sum of Dorcetathione and Vad is 2:1, 3:2, 1:1, 2:3 or 1:

2.

8. The preparation method according to claim 1, characterized in that, In step 3, the reaction time is 48-72 h and the stirring speed is 400 rpm.

9. A mannose-modified graphene oxide-based nanocarrier system prepared by the preparation method according to any one of claims 1-8.

10. The use of the mannose-modified graphene oxide-based nanocarrier system of claim 9 in the preparation of pharmaceutical compositions for delivering antitumor active ingredients.

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