Two-dimensional nanobody array, methods of making and using same

Abstract

The application provides a two-dimensional nanobody array, a preparation method and application thereof, and belongs to the field of biopharmaceuticals. The G-R two-dimensional antibody provides a new strategy for the treatment of lymphoma, further breaks through the technical difficulty of internal cross-linking of the second antibody, and creates a new method for antibody engineering, provides a new strategy for overcoming the limitations of rituximab in the treatment of non-Hodgkin lymphoma, and is expected to improve the prognosis of CD20-positive lymphoma patients, has important medical value, and has a wide clinical transformation and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a two-dimensional nanoantibody array, its preparation method, and its application. Background Technology

[0002] Lymphoma is a malignant tumor originating in the lymph nodes and / or extranodal lymphatic system. Based on pathological type, it can be divided into Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), with non-Hodgkin's lymphoma accounting for approximately 90% of all malignant lymphomas. According to the latest projections from the American Cancer Society, the United States is expected to see 80,350 new cases of NHL and 19,390 deaths by 2025, ranking among the top causes of cancer incidence and mortality. The disease burden is significantly higher in men than in women, posing a major public health challenge. Unlike solid tumors, which are primarily treated with surgical radical resection, NHL, due to its unique diffuse infiltration of the lymphatic system, is mainly treated with medical therapies such as radiotherapy and chemotherapy.

[0003] CD20 is a B-cell differentiation antigen that is highly expressed on the surface of the vast majority of B-NHL cells, but not expressed in hematopoietic stem cells, plasma cells, and normal tissues, making it an ideal target for the treatment of NHL. Rituximab is a human-mouse chimeric monoclonal antibody targeting CD20 and was the first anti-CD20 monoclonal antibody drug approved by the U.S. Food and Drug Administration (FDA). By specifically targeting the CD20 antigen on the surface of B cells, it has ushered in a new era of targeted therapy.

[0004] Since its launch in 1997, rituximab (RTX) has significantly improved the prognosis of patients with B-cell non-Hodgkin's disease (B-NHL), increasing the 5-year survival rate to 58%. RTX primarily kills tumor cells through complement-mediated cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). However, with the advancement of clinical application, the drug's therapeutic limitations have gradually become apparent, possibly due to the exhaustion of the immune effector system, the inhibition of key effector cell activity such as natural killer cells, and the depletion of the complement protein system, leading to a significant weakening of CDC and ADCC effects. To overcome the current therapeutic bottleneck, further development of novel anti-CD20 monoclonal antibodies or antibody derivatives that can induce more efficient programmed cell death (PCD) is needed.

[0005] Studies have shown that after CD20 antibodies bind to tumor cells, cross-linking the antigen-antibody complex with a secondary antibody can induce apoptosis of target cells by activating the Caspase pathway. However, this cross-linking mechanism is difficult to achieve in vivo. When anti-human IgG secondary antibodies enter the body, they undergo extensive cross-reactions with free CD20 antibodies in the bloodstream and even naturally occurring CD20 antigens on the surface of B cells. This non-specific binding not only weakens the precision of targeted cross-linking but also may trigger the risk of systemic immune activation. Therefore, constructing a safe and controllable in vitro cross-linking system has become a key strategy for overcoming therapeutic bottlenecks. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention aims to provide a two-dimensional nanobody array with a larger contact interface, higher antigen binding site density and higher cross-linking efficiency. By increasing the spatial cross-linking dimension, the cross-linking degree of the CD20 antigen-antibody complex is improved, thereby enhancing its apoptosis-inducing ability.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows: A two-dimensional nanobody array in which rituximab is coupled to carboxylated graphene oxide via amide bonds, wherein the loading of rituximab on the surface of carboxylated graphene oxide is 3-4 mg / mg of carboxylated graphene oxide.

[0008] Preferably, in the two-dimensional nanoantibody array, the carboxyl group density of the carboxylated graphene oxide is 0.1-5.0 mmol / g.

[0009] Preferably, a method for preparing a two-dimensional nanobody array includes the following steps: 1) Preparation of carboxylated graphene oxide, i.e. GO-COOH; includes mixing concentrated nitric acid and concentrated sulfuric acid, then adding graphene oxide dispersion, filtering after the reaction is complete, and freeze-drying to obtain GO-COOH; 2) GO-COOH is conjugated with rituximab to obtain a two-dimensional nanobody array, namely GR. GO-COOH is activated with EDC and NHS activation solution, rituximab is added, and the reaction yields the two-dimensional nanobody array.

[0010] The preparation of the carboxyl graphene oxide specifically includes: measuring equal volumes of concentrated nitric acid and concentrated sulfuric acid, mixing them evenly to obtain a mixed acid solution; taking a sample of the graphene oxide dispersion, and adding it dropwise to the above mixed acid solution while stirring; The specific steps for obtaining a two-dimensional nanobody array by conjugating GO-COOH with rituximab include: taking the GO-COOH stock solution, then adding EDC and NHS activation solution, adding rituximab solution, and shaking the reaction.

[0011] The ratio of graphene oxide to mixed acid is 0.2~0.3 mg / ml; the concentration of the graphene oxide dispersion is 0.5-0.7 mg / mL.

[0012] The mass ratio of GO-COOH to rituximab is 1:4~6.

[0013] Preferably, the two-dimensional nanobody array is used in the preparation of drugs for treating lymphoma.

[0014] Preferably, the lymphoma is non-Hodgkin lymphoma.

[0015] Preferably, the use of a pharmaceutical composition in the preparation of an anti-lymphoma drug, wherein the pharmaceutical composition comprises the two-dimensional nanobody array.

[0016] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0017] The beneficial effects of the technical solution of this invention are as follows: A two-dimensional nanobody array (GR) based on graphene oxide was successfully constructed, which has a typical uniform sheet-like structure and exhibits significant tumor-suppressive effects and good biocompatibility in in vitro and in vivo studies. It achieves an improvement in the spatial cross-linking dimension, upgrading the traditional "point" binding mode of monoclonal antibodies to a two-dimensional "surface" cross-linking, increasing the cross-linking degree of CD20, and successfully upgrading the CD20-targeted therapeutic structure, providing a new approach to improving the in vitro anti-tumor effect of CD20 antibodies. Secondly, the constructed two-dimensional nanobody array has significant anti-tumor efficacy and good biocompatibility, showing broad clinical application prospects and translational potential. Finally, this two-dimensional nanobody array provides a new strategy for the treatment of lymphoma, further overcoming the technical challenge of intra-antibody cross-linking, and providing a new potential strategy for improving the treatment and prognosis of CD20-positive lymphoma patients. Attached Figure Description

[0018] Figure 1 This is the SDS-PAGE identification image of the GR nanocomposite in Example 1; Figure 2 Example 1: DLS detection of nanoparticle size and zeta potential of GO, GO-COOH, RTX and GR; The data include (A) hydrodynamic particle size comparison analysis; (B) hydrodynamic particle size distribution curves; and (C) zeta potential. All data are mean ± standard deviation (n=3). Figure 3 This is a topographic image of GR in Example 1; Among them, (A) TEM image; (B) AFM image; scale bar: A=500nm, B=600nm; Figure 4 The spectral characterization diagram of the material in Example 1 is shown below. Among them, (A) Fourier transform infrared spectrum; (B) Raman spectrum; Figure 5 Example 1: CLSM analysis of the binding of RTX and GR to CD20 antigen; Scale bar: 500 nm; Figure 6 This is a graph showing the results of flow cytometry in Example 1; Among them, (A) FCM analysis of the binding of RTX and GR to CD20 antigen; (B) Quantitative analysis of FCM results; Figure 7 Example 1: Secondary antibody labeling analysis of the dissociation capacity of free RTX and GR with CD20 antigen on the surface of (A) Jeko-1; (B) Mino; (C) Raji cells; All data are mean ± standard deviation (n=3). Figure 8 Biosafety evaluation of Example 1G-R; Figure 9 Live-dead staining of Jeko-1 and Mino cells after 48 h of treatment with GO, RTX, and GR, as shown in Example 2. Live cells were stained with calcein-AM (green), and dead cells were stained with PI (red); Scale bar: 100 µm; Figure 10 Example 2: (A) FCM analysis of apoptotic characteristics of Jeko-1 and Mino lymphoma cells after treatment with GO, RTX, and GR; (B) Quantitative analysis of FCM results for Jeko-1 cells and (C) Mino cells; Cells with Annexin V+ / PI± were classified as apoptotic cells. All data are mean ± standard deviation (n=3), **p<0.01; Figure 11 Example 2: Detection of mitochondrial membrane potential in JC-1 cells: (A) FCM results of changes in mitochondrial membrane potential in Jeko-1 and Mino cells after treatment with GO, RTX, and GR; (B) Quantitative analysis of FCM results in Jeko-1 cells and (C) Mino cells; (D) Fluorescence images of Jeko-1 and Mino cells after treatment with GO, RTX, and GR; Scale bar: 100 µm; Figure 12 This is a TMRE fluorescence staining image of Jeko-1 and Mino cells after 48 hours of treatment with GO, RTX, and GR, as shown in Example 2. Scale bar: 50µm. Figure 13Example 2: Antitumor mechanism of GR: (A) FCM results of ROS level changes in Jeko-1 cells and Mino cells; (B) Quantitative analysis of FCM results of Jeko-1 cells and Mino cells; Figure 14 Example 2: CLSM was used to analyze the release of mitochondrial cytochrome C in Jeko-1 and Mino cells after treatment in each group. Scale bar: 500 µm. Figure 15 Example 2: Antitumor mechanism of GR: Western blot was used to detect the expression levels of Caspase signaling pathway-related proteins and the intracellular protein expression levels of T-Akt, P-Akt, T-Erk, and P-Erk in Jeko-1 and Mino cells after treatment with GO, RTX, and GR. Figure 16 Example 2: Antitumor mechanism of GR: Flow cytometry was used to detect the inhibitory effect of the Caspase inhibitor Z-VAD-FMK on GR-induced apoptosis in (A) Jeko-1 cells and (B) Mino cells; Figure 17 Example 3 uses laser confocal scanning microscopy (CLSM) to visualize the biodistribution of GO, RTX, and GR complexes labeled with the Alexa Fluor-488 (green) fluorescent probe in mouse heart, liver, spleen, lung, kidney, and tumor tissue; DAPI (blue) is used to label cell nuclei. Scale bar: 50 µm. Figure 18 For Example 3, in vivo antitumor efficacy evaluation of GR two-dimensional nanobody array: (A) Solid tumor appearance comparison showed a significant reduction in tumor size in the treatment group; (B) Quantitative analysis of tumor growth curves; (C) Survival curves; (D) Weight change (n=3); Figure 19 H&E staining, Ki67 immunohistochemical staining and TUNEL staining of tumor tissue sections in Example 3, scale bar: 100 µm; Figure 20 For Example 3, the systemic biosafety analysis included: (A) Histological assessment of major organs after tail vein injection: H&E staining was used to perform histological morphology analysis of the heart, liver, spleen, lungs, and kidneys; scale bar: 100 µm. (B) Hematological safety testing: Whole blood samples were collected through the orbital venous plexus, and whole blood cell counts and serum biochemical indicators were performed, with a focus on assessing renal function-related parameters; all data are mean ± standard deviation (n=3). Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0020] Example 1: Preparation of two-dimensional nanobody arrays 1. Drugs and materials Rituximab (RTX, C2B8) (100 mg / 10 mL) was purchased from Roche Pharmaceuticals Ltd., and graphene oxide (GO, GO1231) was purchased from Angxing New Carbon Materials Changzhou Co., Ltd.

[0021] Reagent (kit) brand Phosphate-buffered saline (PBS) BasalMedia 70% HNO3 Sigma Aldrich 95% H2SO4 Codow Penicillin-Streptomycin (PS) ThermoFisher 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) Wokai Bio N-hydroxysuccinimide (NHS) Merck DMEM high-glucose medium Pronosel SDS-PAGE Protein Loading Buffer (5×) (Source: Beyotime) RPMI-1640 medium Pronosel PAGE Gel Rapid Preparation Kit (Yaxin) serum-free cell cryopreservation solution MCE Tris / glycine / SDS electrophoresis buffer (YAG) Tris(hydroxymethyl)aminomethane (Tris) Aladdin Glycine (electrophoresis grade) McLean Methanol Guoyao Reagent Color-stained prestained protein molecular weight standard new sema Coomassie Brilliant Blue Rapid Staining Solution (Biyuntian) Coomassie Brilliant Blue Staining Decolorizing Solution (Biyuntian) Fetal bovine serum (FBS) ThermoFisher Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 ThermoFisher EDTA.2K anticoagulant Regen Biotech Cell Counting Kit-8 (CCK-8) Bimake 2.1 Cell line culture and cryopreservation All cell lines used in the study were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Regarding culture conditions, the human B-cell lymphoma cell lines Mino, Raji, and Jeko-1 used RPMI-1640 basal medium; while the human renal epithelial cell line 293T and the rat cardiomyocyte cell line H9C2 used high-glucose DMED medium containing 4.5 g / L glucose. All culture systems were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution and cultured in a 37°C incubator containing 5% CO2 for standardized cell culture. For cell cryopreservation, serum-free cryopreservation solution was used; cryovials containing cell suspension were directly placed in a -80°C freezer, and after 24 hours, transferred to liquid nitrogen for long-term storage.

[0022] 2.2 Preparation of carboxylated graphene oxide Measure 10 mL of concentrated nitric acid (70%) and 10 mL of concentrated sulfuric acid (95%) using a graduated cylinder, and slowly pour them into a 50 mL round-bottom flask, gently shaking to mix the acids thoroughly. Using a pipette, take 10 mL of graphene oxide dispersion (0.5 mg / mL) and slowly add it dropwise to the mixed acid solution under continuous magnetic stirring. Transfer the reaction system to a 60°C constant-temperature water bath and stir at a medium speed (approximately 500 rpm) for 1 h. During the reaction, the solution color gradually changes from an initial brownish-black to a dark brown, indicating that the functional groups on the graphene oxide surface are beginning to transform. After the reaction system reaches the predetermined time point, quickly remove the flask from the constant-temperature device and allow it to cool naturally. Under ice-water bath conditions, slowly add 500–1000 mL of pre-cooled deionized water for dilution, maintaining stirring during dilution to prevent localized overheating. After dilution, the solution volume expands, and the color further lightens. The diluted solution was filtered using a vacuum filtration device with a 0.22 μm PTFE membrane. The filtration process was repeated three times to thoroughly remove residual acid and unreacted impurities. The filter cake was collected and transferred to a freeze dryer, where it was freeze-dried at -50°C and a vacuum of <10 Pa for 24 h. After drying, a fluffy carboxylated graphene oxide (GO-COOH) solid was obtained, with a carboxyl group density of 2 mmol / g. A suitable amount of GO-COOH solid was scraped off, and 10 mL of deionized water was added. The mixture was ultrasonically treated for 30 min using an ultrasonic cleaner (200 W, 40 kHz) until the supernatant was a uniform light brown color and free of visible solid particles. Finally, the supernatant dispersion was collected, transferred to sterile centrifuge tubes, sealed, and stored at 4°C for later use.

[0023] 2.3 Preparation of rituximab-functionalized graphene oxide Add 50 μL of GO-COOH stock solution (1 mg / mL) to a 1.5 mL centrifuge tube, and dilute with 950 μL of ddH2O to a final concentration of 50 μg / mL. Then, add 500 μL of freshly prepared EDC / NHS activation solution (containing 77.5 mg EDC and 11.6 mg NHS dissolved in 1 mL ddH2O) and incubate at room temperature for 20 min. After activation, centrifuge the tube at 4°C and 10,000 rpm for 5 min. Carefully remove the supernatant to remove unreacted EDC / NHS, retaining the bottom precipitate. Add 1 mL of pre-chilled PBS buffer and gently resuspend the precipitate by pipetting, avoiding air bubbles. Add 25 μL of rituximab solution (10 mg / mL) to the resuspended system to a final antibody concentration of 0.25 mg / mL. The reaction system was kept in a metal thermostatic bath and reacted at 25°C and 600 rpm for 3 h with shaking. The solution status needed to be observed regularly during the reaction to ensure no precipitation occurred. After the reaction was complete, the centrifuge tubes were transferred to a low-temperature centrifuge and centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was carefully removed to remove unbound antibodies, retaining the bottom functionalized product. The antibody concentration was 28.43 μg / mL. The precipitate was centrifuged and weighed; the antibody loading rate was 71.08%, and the rituximab loading on the carboxylated graphene oxide surface was 3.55 mg / mg carboxylated graphene oxide. The precipitate was washed three times with pre-cooled PBS buffer, centrifuged after each wash. Finally, 200 μL of PBS buffer was added to the precipitate and gently pipetted until completely dispersed to obtain rituximab-functionalized graphene oxide, which will be described in the following description as a two-dimensional nanobody array GR.

[0024] 2.4 SDS-PAGE Identification of Two-Dimensional Nanobody Arrays Following the instructions of the rapid PAGE gel preparation kit, prepare a 10% SDS-PAGE gel. Take 20 μL each of RTX and GR samples, mix them thoroughly with an equal volume of 2×SDS loading buffer, and then load the samples. Set the stacking gel to a constant voltage of 80 V. After the bromophenol blue indicator band enters the separating gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue migrates to the bottom of the gel. After electrophoresis, carefully remove the gel and place it in an appropriate amount of Coomassie Brilliant Blue staining solution. Stain on a horizontal shaker at room temperature for 30 min. Recover the staining solution, rinse the gel surface with ultrapure water, add Coomassie Brilliant Blue destaining solution, and continue shaking on a shaker. Change the destaining solution at 30-minute intervals, adding pre-cooled destaining working solution each time. Perform 3 to 4 solution change cycles until the gel background is transparent and the protein bands are clear, then stop destaining. Finally, scan using a gel imaging system.

[0025] 2.5 Characterization of Two-Dimensional Nanobody Arrays 2.5.1 Dynamic Light Scattering (DLS) for detecting hydrodynamic particle size and zeta potential Appropriate amounts of GO, GO-COOH, RTX, and GR solutions were diluted in 1 mL of PBS buffer. The hydrodynamic particle size and distribution, as well as the zeta potential, were measured using DLS to characterize the surface charge properties and dispersion stability of the nanoparticles.

[0026] 2.5.2 Morphological observation by transmission electron microscope (TEM) Take 20 μL of the sample to be tested, dilute it with 80 μL of PBS buffer, vortex mix it, and then place it in an ultrasonic processor for dispersion treatment, sonicating for 5 min. Fix the sealing film on the surface of the glass slide, use tweezers to pick up the copper mesh and lay it flat in the center of the membrane, accurately measure 10 μL of the pretreated sample using a pipette system, and vertically drop it into the copper mesh carbon film area. The entire adsorption process is timed for 10 min. After the sample settles naturally, use filter paper to gently touch the edge of the copper mesh at a 45° angle to remove residual liquid. Transfer the treated copper mesh to the sample holder and place it in a vacuum desiccator to stand for 1 h. After the copper mesh is completely dry, use a field emission electron microscope for imaging analysis.

[0027] 2.5.3 Morphological observation using atomic force microscopy (AFM) The sample to be tested was appropriately diluted and ultrasonically dispersed at 100 W for 3 min. 5 μL of the ultrasonically dispersed solution was dropped onto a clean mica sheet and dried under ambient pressure before testing.

[0028] 2.6 Spectroscopic Detection of Two-Dimensional Nanobody Arrays 2.6.1 Fourier Transform Infrared Spectroscopy (FTIR) Measurement (1) Preparation of reference sample: Take 100-200 mg of dry potassium bromide in a clean agate mortar and grind it in one direction until there is no obvious particle feel and the crystals are uniformly snowflake-like. Spread the ground potassium bromide powder evenly in a circular mold and then place it on the operating table of a vacuum tablet press. After the vacuum pump is started and the pressure stabilizes, slowly increase the pressure to 520 kgf / cm³. 2 (Approximately 51 MPa), maintain constant pressure for 120 s. Close the pressurization valve, slowly loosen the exhaust valve until the vacuum level returns to zero, remove the transparent KBr pellet as a blank control, seal and store it in a desiccator for later use.

[0029] (2) Sample preparation: 50 μL of GO, GO-COOH, RTX, and GR solutions were respectively pipetted into four agate mortars containing 150 mg of dried potassium bromide powder. The solutions were lightly ground to initially mix with KBr, and then dried under an infrared lamp at 45°C until no liquid flowed on the surface. The mixture was then ground further until it reached a powder state, and transferred to a mold to prepare sample pieces according to the pressing conditions described in (1). Finally, each sample was scanned by infrared spectroscopy according to the instrument operation.

[0030] 2.6.2 Raman Spectroscopy Detection GO, GO-COOH, and GR samples were diluted and sonicated. 5 μL of the sample solution was dropped onto a silicon wafer substrate and rapidly dried in a vacuum oven. The prepared sample slide was then fixed onto the motorized stage of a micro Raman spectrometer, and the uniformity of sample distribution was confirmed using a white light microscopy imaging system. A confocal optical path system was used to achieve spatial focusing of the laser beam, enabling linear scanning of the sample surface. Spectral acquisition was performed using software, ultimately generating Raman shift-intensity curves. The D peak (~1350 cm⁻¹) was analyzed in detail. -1 ) and G peak (~1580 cm) -1 Structural parameters such as strength ratio.

[0031] 2.7 Detection of the binding ability of two-dimensional nanobody arrays to CD20 antigen on the surface of lymphoma cells Raji, Mino, and Jeko-1 lymphoma cells in the logarithmic growth phase were collected separately (1×10⁻⁶). 5 Cells were washed with pre-chilled PBS containing 5% BSA, and incubated with 10 μg / mL RTX and GR in PBS containing 5% BSA for 1 h. After washing and centrifugation at 1200 rpm for 5 min, the cells were resuspended in PBS buffer, and Alexa Fluor-488goat anti-human IgG (H+L) dye diluted 1:500 was added and incubated on ice in the dark for 15 min. After staining, the cells were washed twice with pre-chilled PBS containing 5% BSA. A portion of the cells were analyzed by flow cytometry to detect the fluorescence intensity on the cell surface, while the other portion of the cell suspension was dropped onto a laser confocal microscope dish coated with poly-L-lysine and allowed to stand for 15 min. After the cells had fully adhered to the bottom of the culture dish, the culture medium was discarded, and the cells were slowly rinsed with a small amount of PBS. Finally, the distribution of green fluorescence on the cell surface was observed using a laser confocal microscope.

[0032] 2.8 Detection of the dissociation ability of two-dimensional nanobody arrays and CD20 antigen on the surface of lymphoma cells Lymphoma cells Raji, Mino, and Jeko-1 (1×10⁻⁶) in the logarithmic growth phase were collected respectively. 5 Cells were washed with pre-cooled PBS and incubated with 10 μg / mL RTX and GR for 1 h, respectively. After washing twice with PBS, the three types of cells with antibodies on their surface were resuspended in RPMI 1640 medium containing 10% heat-inactivated FBS and cultured under normal conditions. Cells were harvested at different time points (0 h, 4 h, 8 h, 16 h, 24 h, and 36 h), labeled with 1:500 Alexa Fluor-488 goat anti-human IgG (H+L) fluorescent secondary antibody, incubated at 4°C in the dark for 15 min, washed with PBS buffer by centrifugation, and resuspended in 300 μL PBS for analysis. Finally, quantitative detection was performed by flow cytometry to obtain the mean fluorescence intensity (MFI) of the cell surface, and the proportion of residual antibodies on the cell surface was calculated according to the following formula: % Initial Binding = MFI 样品 / MFI 0h ×100% 2.9 Effect of Two-Dimensional Nanobody Arrays on Erythrocyte Hemolysis (1) Blood anticoagulation treatment: Take a 1.5 mL sterile EP tube, prefill it with anticoagulation solution at a blood:anticoagulant volume ratio of 9:1, mix it by inverting and then store it in an ice bath at 4℃.

[0033] (2) Anesthesia and blood collection in mice: Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.2 mL / 10 g). After the pain reflex disappeared, the skin around the eyes was disinfected. The mouse head was fixed with the left hand, and the eyeballs were quickly removed with sterile curved ophthalmic forceps in the right hand. At the same time, the heart area was moderately compressed with the left thumb to make the blood flow into the pre-placed centrifuge tube along the direction of gravity (blood collection volume ≥200 μL).

[0034] (3) Blood pretreatment: Centrifuge at 4℃ and 2000 rpm for 10 min to separate plasma. After discarding the supernatant, add pre-cooled PBS buffer (pH 7.4), vortex to resuspend, and wash 3-5 times until the supernatant is clear. Finally, use PBS to bring the red blood cell suspension to a final volume of 10 mL.

[0035] (4) Concentration gradient treatment: Take 200 μL of erythrocyte suspension and mix it with GO and GR complexes at final concentrations of 25, 50, 75, 100, and 200 μg / mL respectively (total volume 500 μL). Also set up a PBS negative control group (500 μL) and a deionized water positive control group (500 μL). All groups were incubated at 37℃ with shaking for about 4 h.

[0036] (5) Hemolysis rate detection: After incubation, centrifuge at 10,000×g for 5 min, and use a pipette to aspirate 100 μL of the supernatant liquid, transferring it to a clear 96-well microplate. Each group has 3 replicates. Use a full-wavelength multi-mode microplate reader, set the detection wavelength to 540 nm, to measure the absorbance (OD value), and calculate the hemolysis rate according to the following formula: Hemolysis rate = OD （实验组-阴性对照） / OD （阳性对照-阴性对照） ×100% 2.10 Cytotoxicity of Two-Dimensional Nanobody Arrays (1) Cell seeding and culture: Human kidney epithelial cell line 293T cells and rat cardiomyocyte H9C2 cells in logarithmic growth phase were prepared into cell suspensions, counted, and then diluted to 3×10⁻⁶ cells. 3 Cells / μL. Using a pipette, seed 90 μL / well into a 96-well culture plate, with 4-6 parallel wells per treatment group. Add 200 μL of sterile PBS solution to the outer wells of the culture plate to form a humidity barrier and prevent evaporation of the culture system.

[0037] (2) Drug treatment and cell adhesion: After the cells adhered and grew in a 37℃, 5% CO2 incubator for 24 h, when the cell confluence reached 80%-85% under a microscope, the old culture medium was carefully removed, and 10 μL of GO and GR complexes of different concentration gradients were added to each well, and the cells were cultured for another 48 h.

[0038] (3) CCK-8 reaction: After drug treatment, discard the culture medium in each well, add 100 μL of premixed detection working solution (containing 90 μL of serum-free DMEM and 10 μL of CCK-8 reagent) to each well, shake gently to mix, and incubate in a 37℃ light-protected incubator for about 1 hour. The incubation endpoint is defined as the solution turning a uniform orange-brown color and the OD value at 450 nm being in the range of 0.8 to 1.5 (OD of the negative control group is 1.0).

[0039] (4) OD value detection and data analysis: The OD value at 450 nm in each well was measured using a full-wavelength microplate reader. Each well was measured three times, and the average value was taken. Cell viability was calculated using the following formula: Cell viability = OD （实验组-空白） / OD （对照组-空白） ×100% In the formula: the experimental group is the drug-treated group; the control group is the cell group without drug treatment; and the blank group is the group containing only PBS and no cells.

[0040] 3. Experimental Results 3.1 Successful Construction of Two-Dimensional Nanobody Arrays To verify the covalent link between RTX and GO-COOH, we first used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to characterize the molecular weight distribution of free RTX and GR two-dimensional nanobody arrays. Figure 1 As shown, free RTX exhibits clearly distinguishable protein bands in the gel, with a molecular weight of approximately 140 kDa (corresponding to lanes 1 and 2). However, the antibody-modified GR, due to its significantly increased molecular weight (multiple RTX molecules covalently coupled to the graphene oxide surface), failed to achieve effective separation, with the sample mainly remaining at the bottom of the gel wells (corresponding to lanes 3 and 4). This result suggests that the connection between RTX and GO-COOH may be achieved through a multi-molecular cross-linking mechanism, and their molecular weight distribution characteristics provide direct experimental evidence for successful coupling.

[0041] 3.2 Two-dimensional nanobody arrays exhibit uniform particle size and good dispersibility. After confirming the successful construction of the GR two-dimensional nanobody array, we used DLS technology to detect its hydrodynamic particle size and zeta potential. Figure 2 A shows that the particle size of GO is mainly around 621 nm. After carboxylation modification, the particle size is reduced, enhancing the dispersion stability. Furthermore, after ultrasonic-assisted centrifugation purification, the particle size of GR is further reduced to around 357 nm, indicating that unbound free RTX and small-sized GO-COOH fragments are effectively removed, and the particle size dispersibility is good (e.g., ...). Figure 2 B). The zeta potential of the GR nanocomposite is as follows: Figure 2 As shown in Figure C, a significant polarity reversal is observed compared to GO and GO-COOH, with a Zeta potential of +5.54 mV. The charge sign retains the RTX characteristic, further indicating that multiple RTX molecules may be covalently bonded to the surface of a single graphene oxide support. All these results demonstrate that the covalent coupling of RTX with GO-COOH significantly alters the interfacial charge distribution and particle size characteristics of the composite material, and the GR two-dimensional nanobody array exhibits excellent performance.

[0042] 3.3 The two-dimensional nanobody array exhibits a uniform sheet-like structure. TEM and AFM characterization results jointly confirmed the morphological characteristics of the two-dimensional nanobody array GR. TEM images showed that GR exhibited a typical sheet-like structure. Figure 3 AFM results further revealed that its thickness was approximately 2.5 nm. Figure 3 (B) The thickness is greater than the theoretical thickness of carboxylated graphene oxide GO-COOH, indicating that RTX molecules were successfully loaded onto the graphene oxide surface. Furthermore, the lateral dimensions measured by AFM and TEM show spatial scale consistency with the DLS results, further demonstrating the structural homogeneity of the GR nanocomposite in solution.

[0043] 3.4 The two-dimensional nanobody array exhibits good spectral characteristics. Fourier transform infrared spectroscopy was used to characterize the chemical bond vibration modes of four samples: GO, GO-COOH, RTX, and GR. Figure 4 Results A show that the GR nanocomposite simultaneously possesses the characteristic absorption peaks of both GO and RTX, exhibiting significant peak shifts and intensity changes. These results indicate that RTX and GO-COOH are covalently coupled via amide bonds, and their interaction significantly alters the functional group state of the material surface. Next, we characterized the defect levels of GO, GO-COOH, and GR based on the Raman spectral characteristic peak intensity ratio (ID / IG), as shown in the results. Figure 4 As shown in Figure B, compared to GO, the ID / IG value of GO-COOH significantly increased to 1.18, indicating that carboxylation modification further exacerbated the defect density of the material. The GR composite, on the other hand, had the lowest ID / IG value at 1.03, indicating the lowest degree of defect, further demonstrating that the covalent coupling of RTX repaired the surface defects of graphene and reduced disorder.

[0044] 3.5 The binding affinity of the two-dimensional nanobody array to the CD20 antigen was not affected by the construction process. In this study, we used Alexa Fluor-488 to fluorescently label free RTX and GR (denoted as RTX-488 and GR-488, respectively), and co-incubated them with CD20-positive lymphoma cells Raji, Mino, and Jeko-1. We then analyzed their targeting and binding characteristics using laser confocal microscopy and flow cytometry. The results of the laser confocal microscopy images show (…). Figure 5 The cell membrane surfaces of both the RTX and GR groups showed green fluorescence, indicating that the binding ability of the GR two-dimensional nanobody array to the CD20 antigen on the cell surface was not significantly affected by various chemical modifications during the construction process, and it could still target and bind to the CD20 antigen well.

[0045] Flow cytometry results showed ( Figure 6The average fluorescence intensity of the GR group was significantly higher than that of the free RTX group. This is because multiple RTX molecules are simultaneously bound to the GR nanoarray and bind to the CD20 antigen, resulting in a higher fluorescence level. These results confirm that the construction process of the two-dimensional nanobody array did not significantly affect the specific targeting and binding activity of the antibody and antigen.

[0046] 3.6 The dissociation rate of the two-dimensional nanobody array from the CD20 antigen was significantly reduced. The binding stability of GR to CD20 antigen on the surface of Raji, Mino, and Jeko-1 positive lymphoma cells was evaluated by dissociation experiments. We used logarithmic growth phase cells (density 1×10⁻⁶) as an example. 5 Cells (number / mL) were co-incubated with RTX and GR at a final concentration of 10 μg / mL for 1 h, washed with pre-chilled PBS buffer, and resuspended in RPMI 1640 complete medium containing 10% fetal bovine serum. Cells were collected at different time points (0, 6, 12, 24, 36 h), and the fluorescence intensity on the cell surface was detected by indirect immunofluorescence labeling (secondary antibody labeling). Flow cytometry analysis results are shown below. Figure 7 As shown, in the three cell lines, the mean fluorescence intensity (MFI) of the GR group was above 60% at 36 h, while the fluorescence retention of the free RTX group was less than 10%. Dynamic monitoring showed that the decay rate of GR-488 fluorescence on the cell surface was significantly lower than that of free RTX-488. These results indicate that the GR complex can significantly reduce the dissociation rate of the antibody on the cell surface through covalent bonding, and its binding stability is significantly improved compared with the free antibody, indicating that this carrier system has a significant advantage in maintaining the biological activity of the targeting ligand.

[0047] 3.7 The two-dimensional nanobody array exhibits good biocompatibility and safety. In the experiment, we set up different concentrations of GO and GR, and incubated them with red blood cells at room temperature. PBS and deionized water were used as negative and positive controls, respectively, for comparative analysis. Figure 8 Results A show that both GO and the modified GR exhibited extremely low hemolysis rates (<5%) at various concentrations. This strongly demonstrates that our constructed two-dimensional nanobody array possesses good blood compatibility and can effectively avoid damage to erythrocytes. To further verify biosafety and ensure that it only has a killing effect on tumor cells and no significant toxicity to normal cells, we treated normal human renal epithelial cells 293T and rat cardiomyocytes H9C2 with GO and GR, and detected cell viability using the CCK 8 assay. The results are as follows: Figure 8As shown in Figures BC, within the selected concentration range, cell viability remained above 90%, indicating that GO and GR possess excellent safety at the cellular level. These experimental results fully demonstrate the significant advantages of GO and GR in terms of biocompatibility and safety, providing strong support for their in vitro and in vivo experiments.

[0048] summary A two-dimensional nanobody array (GR) was constructed and systematically characterized. The results showed that this two-dimensional nanobody array had a large molecular weight (…). Figure 1 The hydrodynamic particle size is approximately 357 nm and uniformly distributed. The surface zeta potential retains the characteristics of RTX, confirming that the covalent modification process did not destroy the antibody functional groups. Figure 2 Both TEM and AFM results show that GR has a uniform sheet-like structure, and its lateral dimensions are consistent with those of DLS. Figure 3 Further spectroscopic analysis revealed that the RTX heavy chain amino groups and the carboxyl groups of GO-COOH are covalently bonded via stable amide bonds, and this coupling process significantly repairs the surface lattice defects of the graphene material. Figure 4 The above multi-dimensional characterization results collectively verify the successful construction of the GR two-dimensional nanobody array and its structural integrity and superior performance.

[0049] Further biological function identification and in vitro safety testing yielded the following conclusions: Compared with free antibodies, the GR two-dimensional nanobody array completely retained its binding ability to the target cell CD20 antigen during the construction process. Figure 5 and Figure 6 However, its ability to dissociate CD20 antigen is significantly reduced. Figure 7 This characteristic stems from the fact that antibody molecules loaded on the GR surface can simultaneously bind to multiple neighboring CD20 antigens, forming a multi-site anchoring effect. The covalent modification strategy enhances the binding stability of the antibody on the graphene oxide surface. When some antigen-antibody dissociates, other antibody molecules in the GR remain tightly bound to the antigen, and the residual antibody can maintain the overall anchoring state of the complex on the tumor cell membrane. Furthermore, these dissociated antibody molecules still have a significant chance to bind to neighboring antigen molecules, forming a dynamic "binding-dissociation-rebinding" process. This multivalent synergistic effect and dynamic equilibrium mechanism together constitute the molecular basis of its low dissociation characteristics and are also an important microscopic biological basis for its sustained antitumor effect. In addition, after co-incubation with erythrocytes, the hemolysis rate is less than 5%, and it did not show significant cytotoxicity to normal human epithelial cells 293T and rat cardiomyocytes H9C2. Figure 8 The above studies confirm that the nanocomposite exhibits excellent in vitro biocompatibility while maintaining its bioactivity and low dissociation rate, providing key experimental evidence for subsequent in vivo and in vitro efficacy evaluation.

[0050] Example 2: Study on the in vitro antitumor effect and mechanism of two-dimensional nanobody arrays 1 Experimental Methods 1.1 Cell line culture and cryopreservation All cell lines used were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. For culture conditions, the human B lymphoma cell lines Mino and Jeko-1 were cultured in RPMI-1640 basal medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and placed in a 37°C incubator containing 5% CO2 for standardized cell culture. For cell cryopreservation, serum-free cryopreservation solution was used; cryovials containing cell suspension were directly placed in a -80°C freezer, and after 24 hours, transferred to liquid nitrogen for long-term storage.

[0051] 1.2 In vitro antitumor efficacy detection 1.2.1 Live / Dead Cell Detection Mino and Jeko-1 lymphoma cells in the logarithmic growth phase were selected, and single-cell suspensions were prepared. Viable cell counts were performed using a cell counter at a concentration of 3 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 1 / well in 12-well cell culture plates, and each well was replenished with complete culture medium to a total volume of 1.5 mL. Different groups of drugs (GO, RTX, GR) at the same concentration (10 μg / mL) were added to treat the cells. After 48 h, the cells were collected and washed twice with pre-chilled PBS. Following the instructions of the Calcein / PI cell viability and cytotoxicity assay kit, staining working solution was prepared. 500 μL of staining working solution (containing 0.2 μL Calcein AM and 0.2 μL LPI staining solution) was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were centrifuged at 1200 rpm at 4°C for 5 min, resuspended in PBS, and observed and photographed under a fluorescence microscope.

[0052] 1.2.2 Apoptosis Detection Mino and Jeko-1 lymphoma cells in the logarithmic growth phase were selected, and single-cell suspensions were prepared. Viable cell counts were performed using a cell counter at a concentration of 3 × 10⁻⁶ cells / cells. 5Cells were seeded at a density of 1 / well in 12-well cell culture plates, with each well replenished with complete culture medium to a total volume of 1.5 mL. Cells were treated with different drugs (GO, RTX, GR) at the same concentration (10 μg / mL). After 48 h, cells were collected and washed twice with pre-chilled PBS at 1200 rpm. The positive control group was heated in a 55°C water bath for 3-5 min before the final PBS wash. Cells were resuspended in 100 μL of 1× Binding Buffer. In both the experimental and positive control groups, an additional 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution were added to each tube. Three control groups were also established: unstained, PI-stained, and Annexin V-FITC-stained. After staining, all groups were gently vortexed and incubated at room temperature in the dark for 15 min. After incubation, 200 μL of pre-chilled binding buffer was added to each tube, and the entire process was strictly performed in the dark. Flow cytometry analysis of the samples was performed within 1 h of preparation.

[0053] 2 Experimental Results 2.1 Two-dimensional nanobody arrays exhibit significant in vitro antitumor effects. 2.1.1 Two-dimensional nanobody arrays exhibit significant lymphoma cell killing effects. To investigate the antitumor effect of GR in vitro, a Calcein / PI cell viability and cytotoxicity assay kit was used, and cell viability was observed using a fluorescence microscope. Calcein Acetoxymethyl Ester (Calcein AM) can penetrate the cell membrane into living cells, where it is hydrolyzed by esterases to produce the green fluorescent product Calcein. In contrast, the red fluorescent dye Propidium Iodide (PI) can only penetrate damaged cell membranes into dead cells, where it binds to nucleic acids and produces red fluorescence. Therefore, based on the metabolic differences between living and dead cells for the two dyes, the combined use of Calcein AM and Propidium iodide allows for the simultaneous identification of living (green) and dead (red) cells. Figure 9 As shown, in Jeko-1 and Mino cells, GO and RTX treatment for 48 h only showed a small amount of red fluorescence, indicating that these two drugs had relatively weak killing effects on tumor cells. In contrast, the GR-treated group showed a decrease in cell number and a significant increase in the number of dead red cells. This demonstrates that GR has a significant tumor cell killing effect.

[0054] 2.1.2 Two-dimensional nanobody arrays can significantly induce apoptosis in lymphoma cells. Apoptosis, an evolutionarily conserved programmed cell death mechanism, is significantly associated with the development and progression of malignant tumors due to imbalances in its regulatory network. In the early stages of apoptosis, a conformational change occurs in the cell membrane phospholipid bilayer, with phosphatidylserine (PS), originally located on the inner side of the cell membrane, everting to expose the cell surface. Based on this pathological feature, Annexin V protein, leveraging its Ca2+... 2+ The phospholipid-dependent binding property allows for the specific recognition and binding of everted PS molecules, forming a stable protein-lipid complex. Complementing this detection method is propidium iodide (PI) dye: this fluorescent probe, due to its large molecular weight and positive charge, cannot penetrate the intact cell membrane structure. It can only pass through the damaged plasma membrane barrier in late-stage apoptosis or necrotic cells, binding to DNA molecules in the cell nucleus and emitting a characteristic red fluorescent signal. Therefore, based on the difference in cell membrane integrity recognition between these two methods, the staging of apoptotic cells can be achieved, classifying cells with Annexin V+ / PI± as apoptotic cells. Figure 10 The streaming results of A and Figure 10 Quantitative analysis by BC showed that GO treatment had no effect on Jeko-1 and Mino cells. Free RTX treatment induced low levels of apoptosis in Jeko-1 and Mino cells, respectively, at 7.69% and 9.87%, while GR significantly enhanced tumor cell apoptosis, with apoptosis rates of 32.02% and 35.36% in Jeko-1 and Mino cells, respectively.

[0055] 2.2 Two-dimensional nanobody arrays can activate the caspase-dependent endogenous apoptosis pathway in target cells. 2.2.1 Two-dimensional nanobody arrays can significantly induce mitochondrial depolarization in target cells. Mitochondrial depolarization, a key early event in the initiation of the apoptosis process, triggers the outer mitochondrial membrane permeability transition (MOMP), leading to the release of pro-apoptotic factors such as cytochrome C into the cytoplasm, thereby activating downstream signaling pathways. The JC-1 fluorescent probe can detect the mitochondrial membrane potential (∆Ψm). At high ∆Ψm, JC-1 forms red fluorescent polymers (J-aggregates) in the mitochondrial matrix; at low ∆Ψm, JC-1 disperses as green fluorescent monomers. Therefore, the relative ratio of red and green fluorescence can quantify the degree of mitochondrial depolarization. This color change not only reflects a decrease in mitochondrial membrane potential but also serves as a specific indicator for early apoptosis. Flow cytometry results are shown below. Figure 11As shown in AC, the mitochondrial membrane potential of both Jeko-1 and Mino cells was normal in the negative control group. Treatment with free GO and RTX also maintained a certain degree of stability in the mitochondrial membrane potential. However, after GR treatment, the mitochondrial membrane potential of both Jeko-1 and Mino cells significantly decreased, and cell clumping occurred. Figure 11 Consistent with the fluorescence microscopy results of D, treatment with the GR complex resulted in a decrease in cell number, a reduction in red fluorescence, and an enhancement in green fluorescence.

[0056] Similarly, the cationic probe tetramethylrhodamine ethyl ester (TMRE) can also be used to detect mitochondrial membrane potential. In normal mitochondria, TMRE accumulates in the matrix and produces red fluorescence; during apoptosis, the mitochondrial permeability transition pore opens, ΔΨm decreases, and TMRE is released from the mitochondria into the cytoplasm, resulting in a significant decrease in fluorescence intensity. The intensity of the signal is observed by fluorescence microscopy, thereby determining the changes in mitochondrial membrane potential. Figure 12 The results showed that the red fluorescence intensity of both types of lymphoma cells was significantly reduced after treatment with GR two-dimensional nanobody arrays, further indicating that mitochondria depolarization occurred.

[0057] 2.2.2 Two-dimensional nanobody arrays can increase the level of reactive oxygen species (ROS) in target cells. Changes in ROS in Jeko-1 and Mino cells were detected using a reactive oxygen species (ROS) assay kit. The results are as follows: Figure 13 As shown, ROS levels in both cell types increased after treatment with the GR complex, while they remained unchanged in other groups.

[0058] 2.2.3 Two-dimensional nanobody arrays can induce the release of mitochondrial cytochrome C. In normal cells, cytochrome C is mainly located in mitochondria, participating in electron transport as an important component of the mitochondrial respiratory chain. When apoptosis is initiated, the mitochondrial membrane undergoes structural changes, primarily manifested as the abnormal opening of the membrane permeability transition pore. This process leads to the transmembrane translocation of cytochrome C, which was originally isolated within the mitochondria, releasing it into the cytoplasm. Therefore, labeling cytochrome C can be used to assess the functional state of mitochondria and further elucidate the mechanism of apoptosis. The results of fluorescence confocal microscopy are shown below. Figure 14 As shown, in the negative control group, cytochrome C appeared as dots, and treatment with free GO and RTX did not have a significant effect on its release. However, after GR treatment, fluorescence in Jeko-1 and Mino cells increased significantly and appeared diffuse, indicating that cytochrome C was released from the mitochondria into the cytoplasm.

[0059] 2.2.4 Two-dimensional nanobody arrays can significantly induce the activation of intracellular caspase in target cells and inhibit tumor cell proliferation. To verify the activation of the caspase pathway in tumor cells, we performed direct detection using Western blotting. See [link to article / reference]. Figure 15 The results showed that GR complex treatment significantly altered the expression levels of apoptosis-related proteins, such as decreasing the expression level of the anti-apoptotic protein Bcl-2 and increasing the expression level of the pro-apoptotic protein Bax, promoting the activation of Cleaved-caspase 9, and consequently increasing the expression level of Cleaved-caspase 3, the final apoptosis executor, in both cell lines. To further explore the mechanism, the expression levels of P-Akt and P-Erk, core proteins of the PI3K-Akt and Raf-MEK1 / 2 signaling pathways, were also evaluated. In both cell lines, GR treatment did not significantly change the expression levels of T-Akt and T-Erk proteins, while the expression levels of P-Akt and P-Erk were significantly reduced. These results indicate that GR can activate the endogenous apoptosis pathway, inducing apoptosis in tumor cells and inhibiting tumor cell proliferation.

[0060] In subsequent apoptosis inhibition experiments, Z-VAD-FMK, a caspase inhibitor with cell membrane penetration capability, was used. This inhibitor can effectively block the apoptosis process by specifically binding to activated caspase enzymes. The experimental results are as follows: Figure 16 As shown, for Jeko-1 and Mino cells, the GR two-dimensional nanobody array exhibited significant pro-apoptotic activity at the same concentration. Z-VAD-FMK at concentrations of 20-80 μM failed to significantly inhibit free GO and RTX-induced apoptosis, but it significantly inhibited GR-induced apoptosis in a dose-dependent manner. These results not only validate the apoptosis-inducing efficacy of the GR complex, but also provide important experimental evidence that GR functions through the mitochondrial-mediated endogenous apoptosis pathway via a caspase-dependent inhibitory effect.

[0061] summary The antitumor activity of GR nanocomposites was investigated, and the underlying mechanism by which they induce tumor cell apoptosis was explored. This was determined by live / dead cell viability assays. Figure 9 ) and Annexin V FITC / PI dual staining flow cytometry analysis ( Figure 10 The results showed that, compared with free RTX, the GR two-dimensional nanobody array exhibited superior efficacy in inhibiting malignant tumors. Specifically, it significantly reduced the survival rate of tumor cells and significantly increased the apoptosis rate. Based on the above experimental results, this study explored the mechanism of action of the GR two-dimensional nanobody array in depth. Figures 11 to 16Studies have found that its potent inhibitory effect on lymphoma mainly involves the following two aspects: (1) Intracellular crosslink-induced endogenous apoptosis pathway: A single GR nanocomplex can be loaded with multiple RTX molecules. When it binds to the CD20 antigen on the surface of lymphoma cells, it can form a multivalent antigen-antibody complex, which in turn triggers intracellular crosslink. This intracellular crosslink can cause depolarization of the mitochondrial membrane potential of tumor cells and release of cytochrome C, thereby activating the Caspase-9 / 3 cascade reaction and ultimately inducing tumor cell death through the mitochondrial apoptosis pathway. (2) Target retention effect: Previous results have shown that GR nanocomposites can form a stable structure on the surface of tumor cells through specific binding mediated by surface antibodies. This persistent binding on the surface of tumor cells not only enhances the local concentration of drugs in the tumor microenvironment, but also blocks the continuous activation of survival signaling pathways (such as PI3K / AKT) on the surface of tumor cells through steric hindrance, forming a "double-hit" effect and causing a more persistent anti-tumor effect. This may also be one of the reasons why GR nanocomposites can induce apoptosis of target cells more efficiently.

[0062] Example 3: In vivo antitumor activity and safety of two-dimensional nanobody arrays 1 Experimental Methods 1.1 Cell line culture and cryopreservation All cell lines used in the research were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Regarding culture conditions, the human B-lymphoma cell line Jeko-1 was cultured in RPMI-1640 basal medium, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and placed in a 37°C incubator containing 5% CO2 for standardized cell culture. For cell cryopreservation, serum-free cryopreservation solution was used; cryovials containing cell suspension were directly placed in a -80°C freezer, and after 24 hours, transferred to liquid nitrogen for long-term storage.

[0063] 1.2 Establishment of a tumor-bearing mouse model (1) Collect Jeko-1 cells in the logarithmic growth phase, centrifuge at 1200 rpm for 5 min in PBS buffer pre-chilled at 4°C, and wash 3 times. After cell counting, resuspend the cell pellet in pre-chilled PBS, and maintain cell viability throughout the operation in an ice bath at 4°C.

[0064] (2) Using a 1 mL sterile syringe, 200 μL of cell suspension (concentration 3×10⁻⁶) was subcutaneously injected into the right dorsal and ventral region of nude mice. 7 Cells / mouse, constructing a human tumor xenograft model (Cell line derived xenograft, CDX).

[0065] (3) Tumor growth was continuously monitored, and a CDX model was successfully constructed after approximately 3 weeks. The tumor volume was calculated as follows: Tumor volume V = 1 / 2 × L1 × L2 2 1.3 In vivo targeting of two-dimensional nanobody arrays.

[0066] To evaluate the tumor-targeting accumulation ability and in vivo distribution of GR nanocomposites, the following research model was established: a tumor with a volume of approximately 200 mm² was selected. 3 Xenograft model mice were randomly divided into groups and administered 200 μL of different formulations (PBS, GO, RTX, and GR) via tail vein. Twelve h after administration, the mice were euthanized, and major organs (heart, liver, spleen, lung, and kidney) and tumor tissues were collected. The collected tissues were pre-cooled to 4°C, washed with PBS, fixed in 4% paraformaldehyde for 4 h, and then thoroughly dehydrated using a gradient ethanol solution. The dehydrated tissues were immersed twice in xylene for 1 h each time. Finally, the cleared tissues were infiltrated and embedded in paraffin, and paraffin sections approximately 10 µm thick were prepared for preservation. The immunofluorescence staining procedure for the paraffin sections of each tissue and organ is as follows: (1) Dewaxing and hydration: Place the paraffin slices in a white box and immerse them in the dewaxing solution twice, 30 min each time; then rehydrate them with ethanol solutions of varying concentrations (anhydrous ethanol, 95%, 80%, 75%), 5 min each time.

[0067] (2) Washing: Wash three times with PBS solution on a shaker for 5 min each time.

[0068] (3) Antigen retrieval: Place the slide in preheated 1× citrate buffer (pH 6.0), microwave on high and bring to a boil, then maintain low power for 20 min.

[0069] (4) Cooling and washing: Remove the white box from the microwave oven and allow it to cool naturally to room temperature. Place it on a shaker and wash it three times with PBS solution for 5 minutes each time.

[0070] (5) Blocking: 3% BSA at room temperature for 1 h (the tissue area was delineated using an immunohistochemical pen).

[0071] (6) Secondary antibody incubation: Remove the blocking solution, add 20 µL of diluted fluorescent secondary antibody Alexa Fluor-488 goat anti-human IgG (H+L) to the tissue, incubate at room temperature in the dark for 1 h, and after incubation, place on a shaker and wash three times with PBS solution for 5 min each time.

[0072] (7) DAPI staining: After the secondary antibody incubation, stain with DAPI in the dark for 10 min, and wash with PBS 3 times on a shaker for 5 min each time.

[0073] (8) Mounting: Add 10 µL of anti-fluorescence quenching agent to the tissue, cover it with a coverslip and mount it. It can be used as nail polish to fix the tissue by applying it to the four corners of the coverslip.

[0074] (9) Photographs: Observe the slices under a confocal fluorescence microscope and collect images.

[0075] 1.4 In vivo antitumor effect of two-dimensional nanobody arrays When the xenograft tumor volume reached 30 mm³, a randomized grouping design was used to divide the tumor-bearing mouse model into 4 groups (n=3). The mice were administered 200 μL of PBS, GO, RTX, and GR via tail vein infusion, respectively. The antibody dose was 20 mg / kg / dose, with weekly single-dose intervention, for a total of 3 drug exposures. Systematic monitoring continued for 60 days, until the tumor volume reached 1500 mm³. 3 The experimental mice were euthanized. During the study, body weight and survival status were dynamically monitored every two days, and tumor volume was recorded every three days using calipers. At the end of the experimental period, tumor tissues from each group of mice were collected for histological analysis: H&E staining, Ki67 immunohistochemical staining, and TUNEL cell apoptosis staining, further revealing the overall morphological characteristics of the tumor tissue, the proliferation of tumor cells, and the inhibitory effect of drugs on cell proliferation.

[0076] 1.5 In vivo biosafety assessment of two-dimensional nanobody arrays (1) Anesthesia and blood sample preparation: Mice were anesthetized by intraperitoneal injection of tribromoethanol at a dose of 0.2 mL / 10 g. After anesthesia, blood was collected from the mice by orbital sampling and placed in 1.5 mL EP tubes containing anticoagulant (anticoagulant:blood = 1:9) and without anticoagulant.

[0077] (2) Blood routine test: After the blood sample containing anticoagulant is thoroughly mixed, it is immediately placed on ice to maintain the stability of the sample and wait for subsequent blood routine index testing and analysis.

[0078] (3) Blood biochemistry analysis: Blood samples without anticoagulants were left to stand at room temperature for 2 h, then transferred to a 4 ℃ environment and centrifuged at 3000 xg for 20 min. After centrifugation, the upper light yellow serum was collected, aliquoted and stored for subsequent mouse blood biochemistry analysis.

[0079] (4) Collection and staining of tissues and organs: Immediately after blood collection, cervical cervical dislocation euthanasia was performed, and core organs such as the heart, liver, spleen, lungs, and kidneys were quickly collected. After rinsing with physiological saline to remove residual blood, the tissues were immersed in 4% paraformaldehyde solution according to experimental groups. After fixing at room temperature for 48 h, the tissues were dehydrated, cleared, and then routinely embedded in paraffin and stained with H&E for subsequent observation and analysis.

[0080] 2 Experimental Results 2.1 Two-dimensional nanobody arrays exhibit good in vivo tumor targeting properties. Previous in vitro experiments confirmed that the GR two-dimensional nanobody array has high affinity for the surface antigen of CD20-positive B lymphoma cells. To verify its in vivo targeted delivery efficiency, we conducted further validation. Laser confocal microscopy results are shown below. Figure 17 As shown, in the control group and the free RTX treatment group, only blue fluorescence signals were observed in the cell nuclei of mice in various organs and tumor tissues. However, the aggregation degree of the GR complex treatment group in tumor tissues was significantly higher than that in the free RTX treatment group, specifically manifested as a significant increase in the content of green fluorescently labeled monoclonal antibodies in tumor tissues. These results indicate that the GR nanocomposite can maintain and exert its excellent targeting ability in vivo.

[0081] 2.2 Two-dimensional nanobody arrays exhibit significant in vivo antitumor efficacy. Based on the highly efficient tumor-suppressive activity demonstrated by GR two-dimensional nanobody arrays in in vitro experiments, this study further validated the in vivo efficacy using a CDX model. The study found that after GR treatment, the tumor growth curve in mice showed a significant inhibitory trend, and the tumor volume was significantly reduced compared to the PBS control group, the free GO group, and the RTX group. Figure 18 AB). Survival analysis showed that the median survival was significantly prolonged in the GR treatment group ( Figure 18 C). Meanwhile, the weight fluctuations of animals in each treatment group were small during the experimental period, and no obvious toxic side effects were observed, confirming that the two-dimensional nanobody array has good in vivo safety. Figure 18 D).

[0082] 2.3 Two-dimensional nanobody arrays significantly inhibit tumor cell proliferation and promote tumor cell apoptosis. To systematically investigate the in vivo therapeutic efficacy of GR two-dimensional nanobody arrays against lymphoma, this study performed histopathological analysis on tumor specimens from each treatment group. Figure 19Hematoxylin and eosin (H&E) staining results showed that a large number of surviving tumor cells with intact morphology and dense arrangement were still visible in the tumor tissue of the RTX monotherapy group. In contrast, the GR treatment group exhibited extensive tumor necrosis, characterized by large areas of coagulative necrosis, severe cell structure destruction, significant nuclear fragmentation and karyolysis, with only a small number of nuclear fragments remaining. This demonstrates the high therapeutic potential of this two-dimensional nanobody array. Ki67 immunohistochemical staining analysis showed that the tumor cell proliferation index (Ki67 positivity rate) in the GR treatment group was significantly lower than in other groups, indicating a significant inhibitory effect on tumor proliferation. Furthermore, TUNEL fluorescence staining analysis showed that GR induced significant apoptosis in tumor cells, with the number and degree of apoptotic cells far exceeding those in other groups.

[0083] In summary, the research results validate the remarkable efficacy of GR nanocomposites in anti-tumor therapy from multiple levels and perspectives. They can not only significantly inhibit tumor cell growth and proliferation, but also effectively induce apoptosis, providing a solid experimental basis for exploring their clinical applications.

[0084] 2.4 Two-dimensional nanobody arrays exhibit good in vivo safety. Biosafety is always a core consideration when evaluating the clinical translational potential of nanomedicines. Through a systematic safety evaluation system, the in vivo safety of the GR complex was verified from both histopathological and hematological dimensions: First, H&E staining technology was used to perform histomorphological analysis of the major organs (heart, liver, spleen, lungs, and kidneys) of mice. Figure 20 Results showed that the organs in each treatment group were structurally intact, with no inflammatory cell infiltration or histopathological changes, exhibiting high consistency with the control group, preliminarily confirming that GR did not cause damage to vital organs. To further determine its biocompatibility, we performed complete blood counts and serum biochemical tests by puncturing the orbital venous plexus. Figure 20 The B test results showed that all indicators were within the normal range.

[0085] Based on the above analysis, the multi-level safety evaluation from histopathology to hematophysiology jointly verified that the GR two-dimensional nanobody array has excellent biocompatibility characteristics. It not only ensures low toxicity risk to healthy tissues during the treatment process, but also maintains the homeostasis of important physiological systems in the body, providing key safety assurance for its subsequent clinical translation and application.

[0086] summary Using a subcutaneous CDX tumor model constructed with the Jeko-1 cell line, the targeting properties, antitumor efficacy, and biosafety of GR two-dimensional nanobody arrays in tumor-bearing mice were systematically evaluated. The results showed that, compared to free RTX, the GR two-dimensional nanobody array exhibited significantly enhanced tumor tissue enrichment capacity. Figure 17 In the treatment of tumor-bearing mice, the GR two-dimensional nanobody array exhibited the strongest anti-tumor effect, significantly inhibiting tumor growth and prolonging mouse survival time (Figure 18). Pathological analysis of tumor tissue further demonstrated its ability to significantly induce tumor cell apoptosis and inhibit tumor cell growth and proliferation. Figure 19 Meanwhile, H&E staining showed no histological abnormalities in the five major organs: heart, liver, spleen, lungs, and kidneys. Hematological tests confirmed that blood routine parameters and liver and kidney function indicators were all within the normal physiological range. Figure 20 The above multi-dimensional data indicate that the GR two-dimensional nanobody array achieves efficient tumor inhibition while maintaining good tissue compatibility and system safety, providing key experimental evidence for its translation from basic research to clinical application.

[0087] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A two-dimensional nanoantibody array, characterized in that, Rituximab is coupled to carboxylated graphene oxide via an amide bond, wherein the loading of rituximab on the surface of carboxylated graphene oxide is 3-4 mg / mg of carboxylated graphene oxide.

2. The two-dimensional nanobody array as described in claim 1, characterized in that, The carboxyl group density of the carboxylated graphene oxide is 0.1-5.0 mmol / g.

3. A method for preparing a two-dimensional nanobody array, characterized in that, Includes the following steps: 1) Preparation of carboxylated graphene oxide, i.e. GO-COOH; includes mixing concentrated nitric acid and concentrated sulfuric acid, then adding graphene oxide dispersion, filtering after the reaction is complete, and freeze-drying to obtain GO-COOH; 2) GO-COOH is conjugated with rituximab to obtain a two-dimensional nanobody array, namely GR. GO-COOH is activated with EDC and NHS activation solution, rituximab is added, and the reaction yields the two-dimensional nanobody array.

4. The method according to claim 3, characterized in that, The preparation of carboxylated graphene oxide specifically includes: measuring equal volumes of concentrated nitric acid and concentrated sulfuric acid, mixing them evenly to obtain a mixed acid solution; taking a sample of graphene oxide dispersion, and adding it dropwise to the above mixed acid solution while stirring; The specific steps for obtaining a two-dimensional nanobody array by conjugating GO-COOH with rituximab include: taking the GO-COOH stock solution, then adding EDC and NHS activation solution, adding rituximab solution, and shaking the reaction.

5. The method according to claim 4, characterized in that, The ratio of graphene oxide to mixed acid is 0.2~0.3 mg / ml; the concentration of the graphene oxide dispersion is 0.5-0.7 mg / mL.

6. The method according to claim 4, characterized in that, The mass ratio of GO-COOH to rituximab is 1:4~6.

7. Application of two-dimensional nanobody arrays in the preparation of drugs for treating lymphoma.

8. The application according to claim 7, characterized in that, The lymphoma is non-Hodgkin's lymphoma.

9. The use of a pharmaceutical composition in the preparation of an anti-lymphoma drug, characterized in that, The pharmaceutical composition described herein comprises the two-dimensional nanobody array of claim 1.

10. The application according to claim 9, characterized in that, The pharmaceutical composition described therein also contains a pharmaceutically acceptable carrier or excipient.

Images