A bimetallic nanoparticle-oncolytic virus intravenous delivery system and a preparation method and application thereof

Abstract

The application belongs to the technical field of drug delivery carrier, and particularly relates to a dual-metal nanodot-oncolytic virus intravenous delivery system and a preparation method and application thereof in tumor immunotherapy. The oncolytic adenovirus is used as raw material, and the dual-metal nanodot-adenovirus chimera is constructed by using the covalent force of 4-maleimide butyric acid-N-succinimidyl ester. The preparation performance, cytotoxicity, action mechanism, in-vivo anti-tumor effect, anti-tumor immunity, anti-tumor recurrence and anti-tumor metastasis effect of the chimera are evaluated. The preparation process is simple, the prepared dual-metal nanodot-adenovirus chimera has small and uniform particle size, is conducive to enrichment in tumor tissues through the enhanced permeability and retention (EPR) effect, has high drug loading capacity, good safety and can generate high-intensity MRI signals at the tumor site. The designed dual-metal nanodot-adenovirus chimera realizes the integration of tumor diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery carrier technology, specifically relating to a bimetallic nanodot-oncolytic virus intravenous delivery system, its preparation method, and its application in tumor immunotherapy. Background Technology

[0002] For many years, the incidence of cancer has been rising, becoming one of the leading causes of death worldwide. Traditionally, clinical treatment primarily involves surgical resection, radiotherapy, and chemotherapy. In recent years, with advancements in medicine, various new treatment methods have emerged, such as photothermal, photodynamic, and oncolytic virus therapy. Oncolytic virus therapy, as a promising new cancer treatment method, possesses unique advantages in its ability to selectively infect and kill tumor cells, specifically replicating and stimulating the body to produce an anti-tumor immune response. As a new force in the field of immunotherapy, oncolytic virus therapy has made significant progress after decades of development. However, the clinical application of oncolytic viruses still faces certain limitations, such as the predominantly intratumoral injection method of administration, the limited efficacy of using oncolytic viruses alone, and the lack of a visualized evaluation system for oncolytic virus therapy.

[0003] Therefore, how to enhance the efficacy of oncolytic viruses and establish an evaluation system have become major scientific issues that urgently need to be addressed in the field of oncolytic virus therapy. Summary of the Invention

[0004] To address the aforementioned deficiencies in existing technologies, this invention designs and constructs a novel intravenous delivery system based on bimetallic nanodots and oncolytic viruses. Bimetallic nanodots are formed by albumin mimicking the chelation of metal ions in organisms in nature. Under mild conditions, albumin can trigger the formation of metal ion complexes, which reside within the cavities of the albumin backbone, ultimately forming nanodots. These bimetallic nanodots not only do not alter the original properties of albumin, but also exert corresponding functions. Based on this, this invention constructs iron and gadolinium bimetallic nanodots, covalently linking them to oncolytic adenoviruses to achieve intravenous injection of the oncolytic virus, enhancing the tumor-killing ability of the oncolytic virus and enabling MRI.

[0005] Bimetallic nanodots are covalently linked to oncolytic adenoviruses, encapsulating the oncolytic adenoviruses with bimetallic nanodots to shield the antigens on the oncolytic virus surface, preventing clearance by the body's innate immune system and achieving long-term circulation in vivo. Simultaneously, bimetallic nanodots can enhance the tumor-killing ability of oncolytic viruses and can also be used as contrast agents for MRI. Oncolytic viruses can activate autophagy in cancer cells. The initiation of autophagy provides support for the inner membrane structure required for viral replication, thereby increasing viral yield. Autophagy can also enhance ferroptosis, an iron-dependent cell death pathway. During excessive autophagy, nuclear receptor coactivator 4 (NCOA4) is overexpressed, promoting ferroptosis. Notably, ferroptosis can trigger and amplify autophagy by generating reactive oxygen species (ROS), leading to autophagic cell death.

[0006] This invention provides a new solution for enhancing the efficacy of oncolytic adenovirus therapy and will provide a scientific basis for expanding the design of novel, efficient, and low-toxicity microbial delivery systems, thus possessing significant research value.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] In the first aspect, the bimetallic nanodot-oncolytic virus intravenous delivery system (i.e., bimetallic nanodot-adenovirus chimera) of the present invention is mainly achieved by chemically cross-linking protein technology.

[0009] Alternatively, the oncolytic virus may be an oncolytic adenovirus; the bimetallic nanodots may be iron nanodots and gadolinium nanodots generated inside bovine serum albumin; and the metal ions may be selected from gadolinium nitrate and ferric chloride.

[0010] Alternatively, the bimetallic nanodots are covalently linked to the oncolytic virus via 4-maleimide-butyric acid-N-succinimide ester.

[0011] The ratio of oncolytic adenovirus to total protein in bimetallic nanodots is 1:2 to 1:20, with a preferred ratio of 1:20 in one embodiment; the ratio of iron ions added during the biomimetic mineralization process to total protein is 50:1 to 1:1, with a preferred ratio of 46:1 in one embodiment; and the ratio of gadolinium ions added during the biomimetic mineralization process to total protein is 50:1 to 1:1, with a preferred ratio of 36:1 in one embodiment.

[0012] In a second aspect, the bimetallic nanodot-oncolytic adenovirus intravenous delivery system (i.e., bimetallic nanodot-adenovirus chimera) of the present invention is prepared by the following method:

[0013] The preparation method of the bimetallic nanodot-oncolytic virus intravenous delivery system of the present invention includes the following steps:

[0014] (1) Construction and validation of bimetallic nanodots:

[0015] ① Add gadolinium nitrate and ferric chloride solution to bovine serum albumin aqueous solution and mix under vigorous stirring;

[0016] ② The pH of the mixture was adjusted to 12 using a 2.0M sodium hydroxide solution, and then the mixture was stirred at 37°C for 12 hours to form a crude product containing gadolinium and iron bimetallic nanodots (Gd / Fe-ND);

[0017] ③ Centrifuge the crude product using a 100kDa ultrafiltration concentrator at 3000 rpm for 5 min to obtain the product;

[0018] ④ The product was dialyzed with distilled water for 24 hours to obtain the final product Gd / Fe-ND;

[0019] ⑤ The particle size, potential and morphology of the final product Gd / Fe-ND were verified by methods such as dynamic light scattering, transmission electron microscopy, elemental analysis and circular dichroism spectroscopy.

[0020] (2) Construction and validation of bimetallic nanodot-oncolytic virus:

[0021] ① Dissolve an appropriate amount of 4-maleimide butyric acid-N-succinimide ester in dimethyl sulfoxide (DMSO);

[0022] ② Dilute the solution prepared in step ① with an appropriate amount of phosphate buffer, add excess Gd / Fe-ND, and incubate for 30 minutes;

[0023] ③ Centrifuge the mixture to its original volume using a 30kDa ultrafiltration concentrator;

[0024] ④ Add the concentrated solution to the oncolytic adenovirus solution that has been diluted in an appropriate amount of phosphate buffer, and incubate again for 30 minutes;

[0025] ⑤ The particle size, potential, and morphology of the bimetallic nanodot-oncolytic virus were verified using dynamic light scattering and transmission electron microscopy.

[0026] In step (1)①, the specific operation is as follows: 2.0 mL of gadolinium nitrate solution with a concentration of 20.0 mM and 2.0 mL of ferric chloride solution with a concentration of 20.0 mM are added to 10.0 mL of bovine serum albumin aqueous solution with a concentration of 50.0 mg / mL and mixed under vigorous stirring.

[0027] As an optional method, in step (2)②, the specific operation is as follows: dilute the solution with an appropriate amount of phosphate buffer at pH 8, add excess Gd / Fe-ND, and incubate for 30 minutes.

[0028] As an optional approach, step (2)④ is specifically performed as follows: the concentrated solution is added to the oncolytic adenovirus solution that has been pre-diluted in an appropriate amount of phosphate buffer at pH 6.5, and incubated again for 30 minutes.

[0029] In a preferred embodiment, the preparation method of the bimetallic nanodot-oncolytic virus intravenous delivery system of the present invention includes the following steps:

[0030] (1) Construction and validation of bimetallic nanodots:

[0031] ① Add 2.0 mL of 20.0 mM gadolinium nitrate solution and 2.0 mL of 20.0 mM ferric chloride solution to 10.0 mL of 50.0 mg / mL bovine serum albumin aqueous solution and mix under vigorous stirring;

[0032] ② The pH of the mixture was adjusted to 12 using a 2.0M sodium hydroxide solution, and then the mixture was stirred at 37°C for 12 hours to form a crude product of gadolinium and iron bimetallic nanodots (Gd / Fe-ND);

[0033] ③ Centrifuge the mixture using a 100kDa ultrafiltration concentrator at 3000 rpm / min for 5 min to obtain the product;

[0034] ④ The product was dialyzed with distilled water for 24 hours to obtain the final product Gd / Fe-ND;

[0035] ⑤ The particle size, potential, and morphology of the final product Gd / Fe-ND were characterized by dynamic light scattering and transmission electron microscopy.

[0036] (2) Construction and validation of bimetallic nanodot-adenovirus chimeras:

[0037] ① Dissolve an appropriate amount of 4-maleimide butyric acid-N-succinimide ester in dimethyl sulfoxide (DMSO);

[0038] ② Dilute the solution with an appropriate amount of phosphate buffer (pH 8), add excess Gd / Fe-ND, and incubate for 30 minutes;

[0039] ③ Centrifuge the mixture to its original volume using a 30kDa ultrafiltration concentrator;

[0040] ④ Add the concentrated solution to the oncolytic adenovirus solution that has been pre-diluted in an appropriate amount of phosphate buffer (pH 6.5), and incubate again for 30 minutes;

[0041] ⑤ The particle size, potential, and morphology of the chimera were characterized by methods such as dynamic light scattering and transmission electron microscopy.

[0042] In a third aspect, the present invention provides the application of the bimetallic nanodot-oncolytic virus intravenous delivery system described in the first aspect above in a drug delivery system.

[0043] In a fourth aspect, the present invention provides the application of the bimetallic nanodot-oncolytic virus intravenous delivery system described in the first aspect above in the preparation of antitumor drugs.

[0044] In a fifth aspect, the present invention provides the application of the bimetallic nanodot-oncolytic virus intravenous delivery system described in the first aspect above in the preparation of drug delivery systems for injection, oral administration or topical administration.

[0045] The present invention has the following beneficial effects:

[0046] (1) In this invention, a bimetallic nanodot-adenovirus chimera was constructed using oncolytic adenovirus and gadolinium iron bimetallic nanodots as raw materials and the covalent interaction of 4-maleiminobutyric acid-N-succinimide ester. The formulation performance, cytotoxicity, mechanism of action, in vivo antitumor effect, antitumor immunity and antitumor recurrence and metastasis effect of the chimera were evaluated.

[0047] The preparation process of this invention is simple, and the prepared bimetallic nanodot-adenovirus chimera has a small and uniform particle size, which is conducive to its enrichment in tumor tissue through the enhanced penetration and retention (EPR) effect; it has a high drug loading capacity, good safety, and can generate high-intensity MRI signals at the tumor site.

[0048] The bimetallic nanodot-adenovirus chimera designed in this invention realizes the integration of tumor diagnosis and treatment. Attached Figure Description

[0049] Figure 1 The DNTB results are for the bimetallic nanodots (Gd / Fe-ND) in Example 1 of this invention.

[0050] Figure 2 The results of circular dichroism spectroscopy for bimetallic nanodots (Gd / Fe-ND) in Example 1 of this invention are shown.

[0051] Figure 3 The elemental analysis results are for the bimetallic nanodots (Gd / Fe-ND) in Example 1 of this invention.

[0052] Figure 4 This is a particle size diagram of the bimetallic nanodot-adenovirus chimera in Example 2 of the present invention.

[0053] Figure 5 This is a potential diagram of the bimetallic nanodot-adenovirus chimera in Example 2 of the present invention.

[0054] Figure 6 This is a transmission electron microscope image of the bimetallic nanodot-adenovirus chimera in Example 2 of the present invention.

[0055] Figure 7 This is a confocal image of the bimetallic nanodot-adenovirus chimera in Example 2 of the present invention.

[0056] Figure 8 This is an MRI image of the bimetallic nanodot-adenovirus chimera in Example 2 of the present invention.

[0057] Figure 9 This is a confocal image showing how the composite microorganisms induced ROS production in cancer cells at the cellular level in Example 3 of this invention.

[0058] Figure 10 This is a confocal image of autophagosomes induced at the cellular level by the bimetallic nanodot-adenovirus chimera in Example 4 of the present invention.

[0059] Figure 11 This is a graph evaluating the ability of the bimetallic nanodot-adenovirus chimera to induce ferrous ion accumulation in cancer cells at the cellular level in Example 5 of this invention.

[0060] Figure 12 This is an in vitro evaluation diagram of how the bimetallic nanodot-adenovirus chimera promotes the replication of oncolytic adenovirus in Example 6 of the present invention.

[0061] Figure 13 This is an in vitro cytotoxicity evaluation diagram of the bimetallic nanodot-adenovirus chimera in Example 7 of the present invention.

[0062] Figure 14 This is an evaluation diagram of the in vivo tissue distribution of the bimetallic nanodot-adenovirus chimera after injection in Example 8 of the present invention.

[0063] Figure 15 This is an evaluation of the in vivo tissue distribution of oncolytic adenovirus after injection of the bimetallic nanodot-adenovirus chimera in Example 9 of the present invention.

[0064] Figure 16 This is an MRI image of the tumor site after injection of the bimetallic nanodot-adenovirus chimera in Example 10 of the present invention.

[0065] Figure 17 This is a WB evaluation diagram of ferroptosis, autophagy, and autophagy-dependent ferroptosis generated in the tumor of the bimetallic nanodot-adenovirus chimera in Example 11 of the present invention.

[0066] Figure 18 This is an evaluation diagram of the replication of bimetallic nanodot-adenovirus chimera intratumoral oncolytic adenovirus in Example 11 of the present invention.

[0067] Figure 19 This is an immunofluorescence evaluation image of the bimetallic nanodot-adenovirus chimeric tumor autophagy protein P62 in Example 11 of the present invention.

[0068] Figure 20 This is a diagram illustrating the in vivo antitumor effect of the bimetallic nanodot-adenovirus chimera in Example 12 of this invention.

[0069] Figure 21 This is an in vivo positivity evaluation diagram of the bimetallic nanodot-adenovirus chimera in Example 12 of the present invention.

[0070] Figure 22 This is an in vivo immunological study of the bimetallic nanodot-adenovirus chimera in Example 13 of the present invention (G1: PBS, G2: Ad, G3: 3-MA+Ad@Gd / Fe-ND, G4: Gd / Fe-ND, G5: Ad@BSA, G6: Ad@Gd / Fe-ND);

[0071] Figure 23 This is a tumor growth curve of the bimetallic nanodot-adenovirus chimera in vivo against recurrent tumors in Example 14 of the present invention (G1: PBS, G2: Ad, G3: 3-MA+Ad@Gd / Fe-ND, G4: Gd / Fe-ND, G5: Ad@BSA, G6: Ad@Gd / Fe-ND);

[0072] Figure 24 The images shown are pre- and post-operative images of the bimetallic nanodot-adenovirus chimera in vivo for anti-recurrent tumors in Example 14 of this invention (G1: PBS, G2: Ad, G3: 3-MA+Ad@Gd / Fe-ND, G4: Gd / Fe-ND, G5: Ad@BSA, G6: Ad@Gd / Fe-ND).

[0073] Figure 25 This is a tumor growth curve of the bimetallic nanodot-adenovirus chimera in vivo against metastatic tumors in Example 14 of the present invention (G1: PBS, G2: Ad, G3: 3-MA+Ad@Gd / Fe-ND, G4: Gd / Fe-ND, G5: Ad@BSA, G6: Ad@Gd / Fe-ND);

[0074] Figure 26 This is an in vivo tumor imaging image of the bimetallic nanodot-adenovirus chimera in Example 14 of the present invention, which shows its anti-metastatic effect on tumors (G1: PBS, G2: Ad, G3: 3-MA+Ad@Gd / Fe-ND, G4: Gd / Fe-ND, G5: Ad@BSA, G6: Ad@Gd / Fe-ND). Detailed Implementation

[0075] The following specific examples further illustrate the above-mentioned content of the present invention in detail, but do not imply that the embodiments limit the present invention.

[0076] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0077] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0078] Example 1: Preparation and Characterization of Bimetallic Nanodots

[0079] Bimetallic nanodots (Gd / Fe-ND) were prepared by adding 2.0 mL of 20.0 mM gadolinium nitrate solution and 2.0 mL of 20.0 mM ferric chloride solution to 10.0 mL of 50.0 mg / mL bovine serum albumin aqueous solution and mixing under vigorous stirring. The pH of the mixture was adjusted to 12 using 2.0 M sodium hydroxide solution. The mixture was then stirred at 37 °C for 12 hours to form Gd / Fe-ND. Subsequently, the mixture was centrifuged using a 100 kDa ultrafiltration concentrator, and the product was dialyzed through distilled water for 24 hours to finally obtain Gd / Fe-ND. The structure and composition of the bimetallic nanodots were verified by DTNB, circular dichroism spectroscopy, and elemental analysis. The results are shown below. Figure 1 , Figure 2 and Figure 3 As shown.

[0080] Example 2: Preparation and characterization of bimetallic nanodot-adenovirus chimeras

[0081] A bimetallic nanodot-oncolytic virus chimera (Ad@Gd / Fe-ND) was prepared by linking Gd / Fe-ND to Ad using 4-maleimide-butyric acid-N-succinimidyl ester. Maleimide and thiol groups form stable thioether bonds under mild conditions (pH 6.5-7.5), while succinimidyl reacts with primary amines under slightly alkaline conditions (pH 7.2-9) to generate stable amide bonds. By adjusting the solution pH, Gd / Fe-ND or Ad can be selectively linked; succinimidyl is linked to Gd / Fe-ND, and maleimide is linked to Ad.

[0082] First, an appropriate amount of 4-maleimide butyric acid-N-succinimide ester was dissolved in dimethyl sulfoxide (DMSO), diluted with an appropriate amount of PBS (pH 8), and excess Gd / Fe-ND was added. The mixture was incubated for 30 minutes. Subsequently, the mixture was centrifuged to its original volume using an ultrafiltration concentrator. The concentrated solution was added to Ad solution pre-diluted in an appropriate amount of PBS (pH 6.5), and incubated again for 30 minutes. Finally, the mixture was concentrated to the desired volume. The successful preparation of the bimetallic nanodot-adenovirus chimera was confirmed by particle size, potential, electron microscopy, and confocal microscopy. The results are as follows: Figure 4 , Figure 5 , Figure 6 , Figure 7 As shown. Furthermore, MRI experiments demonstrated the imaging capabilities of the bimetallic nanodot-adenovirus chimera, as shown... Figure 8 As shown.

[0083] Example 3: Evaluation of the ability of bimetallic nanodot-adenovirus chimera to induce ROS generation at the cellular level

[0084] Non-small cell lung cancer cells (TC-1) were cultured in 12-well cell culture plates. After the cells reached 80% confluence, the culture medium was discarded, and the cells were washed twice with PBS. An appropriate volume of blank working medium containing 10 μM DCFH-DA fluorescent dye was added, and the cells were incubated at 37°C in the dark for 1 hour. The DCFH-DA working dye solution was discarded, and the cells were washed twice with PBS. Cell dispersions containing PBS, Ad, and Ad@Gd / Fe-ND were added according to the group, and the cells were incubated at 37°C in the dark for 3 hours. The drug solution was discarded, and the cells were washed twice with PBS. After digestion, the cells were resuspended in an appropriate amount of cell staining buffer. Fluorescence intensity was assessed by confocal microscopy to evaluate the ability of the complex organism to induce ROS production in cells. Figure 9 As shown, both confocal and flow cytometry results indicate that the bimetallic nanodot-adenovirus chimera can improve ROS levels at the in vitro cellular level.

[0085] Example 4: Evaluation of the ability of bimetallic nanodot-adenovirus chimera to induce autophagy in cancer cells at the cellular level

[0086] TC-1 cells (1×10⁻⁶) were seeded in 12-well plates. 5 Cells were cultured in a cell culture incubator for 24 hours to allow them to adhere and grow. Then, the culture medium was aspirated using a pipette, and PBS and Ad (10 mg / well) were added. 7 pfu) and Ads@Gd / Fe-ND (10 7Cells were cultured in serum-free medium (PFU) and incubated in a cell culture incubator for 24 or 48 hours. After incubation, the medium was aspirated, and 1 mL of MDC staining solution was added to each well. Cells were incubated at 37°C in the dark for 30 min. The MDC staining solution was aspirated, and the cells were washed three times with assay buffer, 1 mL of assay buffer each time. The assay buffer was aspirated, and then 1 mL of assay buffer was added. EB staining solution was then added for staining. After incubation for 10 min, the cells were washed three times with serum-free cell culture medium to thoroughly remove any EB staining solution that had not entered the cells. Cells were then photographed under a CLSM microscope to examine the production of autophagosomes within the cells. The autophagosome level in the Ad@Gd / Fe-ND group was significantly higher than that in the PBS group and the Ad group, indicating that Ad@Gd / Fe-ND has a higher level of autophagy. The results are as follows: Figure 10 As shown.

[0087] Example 5: Evaluation of the ability of bimetallic nanodot-adenovirus chimera to induce ferrous ion accumulation in cancer cells at the cellular level

[0088] TC-1 cells (1×10⁻⁶) will be seeded in 12-well plates. 5 Cells were cultured in a cell culture incubator for 24 hours to allow them to adhere and grow. Subsequently, the culture medium was aspirated using a pipette, and PBS, Gd / Fe-ND, and Ad@Gd / Fe-ND (10⁻⁶ cells / well) were added. 7 Cells were cultured in serum-free medium (PFU) and incubated for 24 or 48 hours. Afterward, the medium was removed, and 1 μmol / L FerroOrange working solution was added to each well for staining. The cells were incubated for 30 min, followed by 10 min of DAPI working solution. The cells were then washed three times with serum-free cell culture medium to thoroughly remove any uninfiltrated FerroOrange and DAPI. The cells were then imaged under a CLSM microscope to examine the Fe... 2+ At the intracellular level, compared with the PBS group and the Ad group, the Ad@Gd / Fe-ND group had a higher level of intracellular ferrous ions, indicating a higher level of ferroptosis in the Ad@Gd / Fe-ND group. Results are as follows... Figure 11 As shown.

[0089] Example 6: In vitro evaluation of the effect of bimetallic nanodot-adenovirus chimera on promoting Ad replication

[0090] TC-1 cells were seeded into cell culture plates. When the cells reached 80% coverage at the bottom, the culture medium was aspirated using a 2 mL syringe, and the cells were washed twice with PBS. Then, different concentrations of Ad@Gd / Fe-ND (5 pfu / cell, 10 pfu / cell, 50 pfu / cell, and 100 pfu / cell) were added to each group, and the plates were incubated at 37°C for different times. After incubation, the solution was aspirated, and the cells were washed twice with PBS. An equal volume of fresh culture medium was added, and the plates were subjected to three freeze-thaw cycles at -80°C. The freeze-thawed solutions were then collected and centrifuged at 840 × g for 30 minutes at 4°C. The supernatant was collected, and 1% Triton was added. Pre-denaturation was then performed at 98°C, followed by centrifugation at 3600 × g for 10 minutes to remove denatured proteins. Finally, the supernatant was collected, and RT-qPCR was performed for quantitative detection. The 50 pfu / cell concentration of Ad@Gd / Fe-ND showed a good ability to enhance viral replication. The Ad@Gd / Fe-ND at 100 pfu / cell concentration showed poor replication enhancement, possibly because excessively high Ad concentrations cause rapid cell death before the virus can replicate extensively, resulting in a low quantitative level. The 5 pfu / cell and 10 pfu / cell concentrations, due to their lower concentrations, also showed poor replication enhancement within the specified time. Results are as follows... Figure 12 As shown.

[0091] Example 7: In vitro cytotoxicity evaluation of bimetallic nanodot-adenovirus chimera

[0092] After digesting and centrifuging healthy cells, discard the supernatant and resuspend the cells in 1 mL of culture medium. Add 10 μL of the cell suspension to a cell counting chamber and count the cells under a microscope. Dilute the cell suspension to 1 × 10⁻⁶. 4Cells were seeded at a density of 100-150 μL / mL into 96-well plates. After complete cell attachment, the old culture medium was discarded, and 150 μL of solutions prepared with serum-free culture medium were added to each well: PBS, BSA, Ad, Gd / Fe-ND, Ad@Gd / Fe-ND, 3-MA (3-methyladenine) + BSA, 3-MA + Ad, 3-MA + Gd / Fe-ND, and 3-MA + Ad@Gd / Fe-ND. Three parallel wells were set up for each group, with the zero-adjustment well containing no cells and an equal volume of cell culture medium. After 24 and 48 hours of culture, 50 μL of LTT solution was added to each well under dark conditions, and the plates were cultured for another 4 hours. The culture medium was then discarded, and 150 μL of DMSO solution was added. The plates were then shaken for 10 minutes. The absorbance of each well was measured at 490 nm using a microplate reader, and the corresponding cell survival percentage was calculated. A comparison between the BSA group and the 3-MA+BSA group showed that a certain amount of 3-MA did not exhibit cytotoxicity. Therefore, 3-MA can be used as an autophagy inhibitor in subsequent drug administration groups. Compared to the Ad group, the addition of 3-MA reduced the cytotoxicity of Ad by inhibiting autophagy, indicating that autophagy plays a crucial role in the killing of tumor cells by oncolytic viruses. Compared to the Gd / Fe-ND group and the Ad@Gd / Fe-ND group, the Ad@Gd / Fe-ND group showed more significant cytotoxicity. Results are as follows... Figure 13 As shown.

[0093] Example 8: Evaluation of in vivo tissue distribution of bimetallic nanodot-adenovirus chimera after injection

[0094] When the tumor grows to approximately 200-300 mm 3 C57BL / 6 mice bearing lung cancer (LUC-TC-1) were randomly divided into two groups of three. D-luciferin potassium solution, the most commonly used substrate for firefly luciferase, was injected. In the presence of ATP and other substances, D-luciferin potassium emitted fluorescence at a wavelength of approximately 560 nm through an enzymatic reaction. In vivo imaging of TC-1 tumors bearing LUC was performed using D-luciferin potassium. Subsequently, Ad@Gd / Fe-ND and Ad, pre-stained with CY7-NHS, were injected via the tail vein, with an equivalent concentration of 10... 7PFU. In vivo imaging was performed at 6, 12, and 24 hours post-administration. After the final imaging, mice were euthanized, and the heart, liver, spleen, lungs, kidneys, and tumors were dissected. The surfaces of each tissue were thoroughly rinsed for imaging of the ex vivo tissues. Within 24 hours of intravenous administration, Ad@Gd / Fe-ND showed a stronger CY7 signal overlap with tumor fluorescence compared to Ad, indicating that Ad@Gd / Fe-ND has stronger tumor targeting and accumulation capabilities. Specifically, the highest accumulation of Ad@Gd / Fe-ND in tumors was observed within 6 hours of administration, while the fluorescence intensity at the tumor site gradually decreased at 12 and 24 hours. This indicates that Ad@Gd / Fe-ND can effectively target tumor sites. Next, we investigated drug accumulation in ex vivo organs. In contrast, at 24 hours post-administration, the Ad@Gd / Fe-ND group still showed higher tumor accumulation, while the Ad group showed higher accumulation in the liver. Results are as follows... Figure 14 As shown.

[0095] Example 9: Evaluation of in vivo tissue distribution of Ad after injection of bimetallic nanodot-adenovirus chimera

[0096] When the tumor grows to approximately 200-300 mm 3 Mice were randomly divided into two groups of three. Ad@Gd / Fe-ND and Ad were injected via tail vein, with Ad having an equivalent concentration of 10... 7 PFU was present. Six hours after administration, mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, and tumors were rapidly dissected. The surfaces of each tissue were rinsed thoroughly with physiological saline, blotted dry with filter paper, and approximately 200 mg of each tissue sample was accurately weighed, placed in 10 mL EP tubes, and minced. DNA was extracted using a tissue DNA extraction kit, and Ad was quantified using RT-qPCR. In liver tissue, the Ad count in the Ad@Gd / Fe-ND group was approximately four times that of the Ad@Gd / Fe-ND group. In tumor tissue, the Ad count in the Ad@Gd / Fe-ND group was approximately five times that of the Ad@Gd / Fe-ND group. Results are as follows... Figure 15 As shown.

[0097] Example 10: MRI of tumor sites after injection of bimetallic nanodot-adenovirus chimera

[0098] When the tumor grows to approximately 200-300 mm 3 At that time, Ad@Gd / Fe-ND and PBS were injected via tail vein, with an effective concentration of Ad of 10... 7PFU. Mice were anesthetized with isoflurane at 0h, 4h, and 12h after administration, followed by MRI. At 0h, there was no significant difference in tumor imaging between the two groups. Compared to the PBS group, the Ad@Gd / Fe-ND group showed a brighter trend in tumor MRI 4h after administration. At 6h after injection, the tumor tissue in the Ad@Gd / Fe-ND group showed a more distinct boundary with other tissues, allowing for a more direct assessment of tumor size. Figure 16 As shown.

[0099] Example 11: Evaluation of the mechanism of action of bimetallic nanodot-adenovirus chimera in tumors

[0100] A TC-1 ectopic tumor-bearing mouse model was constructed and divided into the following groups: PBS group, Ad group, 3-MA+Ad@Gd / Fe-ND group, Gd / Fe-ND group, Ad@BSA group, and Ad@Gd / Fe-ND group. After 72 hours, the mice were dissected and tumor tissue was collected. The obtained tumor tissue was subjected to in vivo Western blot experiments for LC3 autophagy protein, GPX4 ferroptosis protein, and autophagy-dependent ferroptosis NCOOA4 protein; immunofluorescence sectioning of P62 autophagy protein; and H&E sectioning of tumor tissue. Simultaneously, an appropriate amount of tumor tissue was placed in an EP tube, minced, and homogenized. 150 μL of each homogenate was used to separate and purify DNA according to the DNA separation and extraction kit steps, and then detected using a real-time PCR instrument. Figure 17 , Figure 18 and Figure 19 As shown, the results indicate that the bimetallic nanodot-adenovirus chimera, after tail injection, can promote ROS production at the tumor site, induce tumor cell autophagy, enhance ferroptosis due to excessive autophagy, and enhance Ad replication at the tumor site.

[0101] Example 12: In vivo antitumor efficacy and safety evaluation of bimetallic nanodot-adenovirus chimera

[0102] A TC-1 ectopic tumor-bearing mouse model was constructed, and the tumor volume was increased to approximately 250 mm. 3 Tumor-bearing mice were randomly divided into six groups: PBS group, Ad group, 3-MA+Ad@Gd / Fe-ND group, Gd / Fe-ND group, Ad@BSA group, and Ad@Gd / Fe-ND group. PBS, Ad, 3-MA (intraperitoneal injection)+Ad@Gd / Fe-ND, Gd / Fe-ND, Ad@BSA, and Ad@Gd / Fe-ND were administered via tail vein injection, respectively. The effective concentration of Ad was 10... 7 PFU was administered via tail vein injection, once every two days, for a total of four administrations (on days 5, 7, 9, and 11 after tumor inoculation). Tumor volume and body weight were measured and recorded daily from the first day after administration to monitor tumor growth. Figure 20 , Figure 21 As shown, the results indicate that, compared with oncolytic viruses and bimetallic nanodots alone, the bimetallic nanodot-adenovirus chimera exhibits better antitumor efficacy after tail injection, and no significant safety issues were found.

[0103] Example 13: In vivo immunological evaluation of bimetallic nanodot-adenovirus chimera

[0104] Blood, tumor tissue, and spleen were collected from mice euthanized after treatment in Example 12. Single-cell suspensions were prepared using collagenase IV, hyaluronidase, and deoxyribonuclease I. 100 μL, 1×10 7 One sample consists of 1 cell / mL, and is co-incubated with the relevant antibody. The antibody content is then determined by flow cytometry. Figure 22 As shown, the results indicate that, compared with oncolytic viruses and bimetallic nanodots alone, the bimetallic nanodot-adenovirus chimera has a better ability to remodel the inhibitory immune microenvironment at the tumor site after tail injection.

[0105] Example 14: In vivo pharmacodynamic evaluation of bimetallic nanodot-adenovirus chimera in a model of antitumor recurrence and metastasis

[0106] A mouse model of tumor recurrence and metastasis was established by subcutaneous inoculation of TC-1 cells in the left and right abdomens to evaluate the antitumor immunogenicity of Ad@Gd / Fe-ND. First, the ability of Ad@Gd / Fe-ND to prevent tumor recurrence was analyzed by plotting tumor curves based on the volume of recurrent tumors. The results are as follows: Figure 23 As shown in the figure, the tumor volume continued to increase after surgery in the PBS group, Ad group, 3-MA+Ad@Gd / Fe-ND group, and Gd / Fe-ND group, indicating that PBS, Ad, 3-MA+Ad@Gd / Fe-ND, and Gd / Fe-ND could not inhibit tumor growth and had little effect on recurrent tumors. Ad@BSA and Ad@Gd / Fe-ND could inhibit tumor growth, with Ad@Gd / Fe-ND showing the most significant effect, controlling the tumor volume to 500 mm. 3 Within. Pre- and post-operative photos as follows. Figure 24 As shown. Secondly, metastatic tumors were analyzed. Metastatic tumors were inoculated on day 16, and measurements began on day 20. The tumor curve is shown in the figure. Figure 25 As shown, the 3-MA+Ad@Gd / Fe-ND group, Gd / Fe-ND group, Ad@BSA group, and Ad@Gd / Fe-ND group all exhibited tumor-suppressive effects, with the Ad@Gd / Fe-ND group showing the strongest tumor-suppressive effect. In vivo imaging results are shown below. Figure 26 As shown.

[0107] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A bimetallic nanodot-oncolytic virus intravenous delivery system, characterized in that: The system uses bimetallic nanodots as a carrier to encapsulate oncolytic adenovirus; the ratio of oncolytic adenovirus to total protein in bimetallic nanodots is 1:2 to 1:20; the ratio of iron ions to albumin added during the formation of bimetallic nanodots is 50:1 to 1:1, and the ratio of gadolinium ions to total protein is 50:1 to 1:1; the oncolytic virus is oncolytic adenovirus; the bimetallic nanodots are nanodots formed by bovine serum albumin and metal ions binding within them; the metal ions are selected from gadolinium nitrate and ferric chloride; the bimetallic nanodots and the oncolytic virus are covalently linked via 4-maleimide butyrate-N-succinimide ester.

2. The preparation method of the bimetallic nanodot-oncolytic adenovirus intravenous delivery system according to claim 1, characterized in that: Includes the following steps: (1) Construction and verification of bimetallic nanodots: ① Add gadolinium nitrate and ferric chloride solution to bovine serum albumin aqueous solution and mix under vigorous stirring; ② The pH of the mixture was adjusted to 12 using a 2.0 M sodium hydroxide solution, and then the mixture was stirred at 37°C for 12 hours to form a crude product containing gadolinium and iron bimetallic nanodots (Gd / Fe-ND); ③ Centrifuge the crude product using a 100 kDa ultrafiltration concentrator at 3000 rpm for 5 min to obtain the product; ④ The product was dialyzed with distilled water for 24 hours to obtain the final product Gd / Fe-ND; ⑤ The particle size, potential and morphology of the final product Gd / Fe-ND were verified by dynamic light scattering, transmission electron microscopy, elemental analysis and circular dichroism spectroscopy. (2) Construction and validation of bimetallic nanodot-oncolytic virus chimera: ① Dissolve an appropriate amount of 4-maleimide butyric acid-N-succinimide ester in dimethyl sulfoxide (DMSO); ② Dilute the solution prepared in step ① with an appropriate amount of phosphate buffer, add excess Gd / Fe-ND, and incubate for 30 minutes; ③ Centrifuge the mixture to its original volume using a 30 kDa ultrafiltration concentrator; ④ Add the concentrated solution to the oncolytic adenovirus solution that has been diluted in an appropriate amount of phosphate buffer, and incubate again for 30 minutes; ⑤ Dynamic light scattering and transmission electron microscopy were used to verify the particle size, potential and morphology of the chimera.

3. The preparation method of the bimetallic nanodot-oncolytic virus intravenous delivery system according to claim 2, characterized in that: In step (1)①, the specific operation is as follows: 2.0 mL of gadolinium nitrate with a concentration of 20.0 mM and 2.0 mL of ferric chloride solution with a concentration of 20.0 mM are added to 10.0 mL of bovine serum albumin aqueous solution with a concentration of 50.0 mg / mL, and mixed under vigorous stirring.

4. The preparation method of the bimetallic nanodot-oncolytic virus intravenous delivery system according to claim 2, characterized in that: In step (2) ②, the specific operation is as follows: dilute the solution with an appropriate amount of phosphate buffer at pH 8, add excess Gd / Fe-ND, and incubate for 30 minutes.

5. The method for preparing the bimetallic nanodot-oncolytic virus intravenous delivery system according to claim 2, characterized in that: In step (2) ④, the specific operation is as follows: add the concentrated solution to the oncolytic adenovirus solution that has been diluted in an appropriate amount of phosphate buffer at pH 6.5, and incubate again for 30 minutes.

6. The application of the bimetallic nanodot-oncolytic virus intravenous delivery system according to claim 1 in the preparation of drug delivery systems.

7. The application of the bimetallic nanodot-oncolytic virus intravenous delivery system according to claim 1 in the preparation of antitumor drugs.

8. The application of the bimetallic nanodot-oncolytic virus intravenous delivery system according to claim 1 in the preparation of drug delivery systems for injection, oral administration or local administration.

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