Hf-based nanocomposite, method for preparing the same and use thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to an Hf-based nanocomposite material, its preparation method, and its applications. The Hf-based nanocomposite material is prepared by modifying Hf-based nanoparticles with fluorescent molecules bearing carboxyl groups, followed by the attachment of cell-penetrating peptides and carboxylic acid-functionalized polyethylene glycol. The Hf-based nanoparticles are formed by coordinating hafnium tetrachloride with adenosine diphosphate (ATP) and loading an A2AR antagonist via a reverse microemulsion method. This Hf-based nanocomposite material possesses biodegradability, radiosensitization, and immunomodulatory effects. When combined with X-ray irradiation, this Hf-based nanocomposite material significantly increases ROS content in tumor cells, significantly increases the accumulation of DNA damage within tumor cells, and increases extracellular ATP content, resulting in a significantly enhanced anti-tumor effect and broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an Hf-based nanocomposite material, its preparation method, and its application. Background Technology

[0002] Radiation therapy (RT) is a local treatment method that uses radiation to treat tumors. It has become one of the most commonly used and effective treatments for malignant tumors in clinical practice. Approximately 60%-70% of cancer patients require radiation therapy during their cancer treatment, and about 30%-40% of tumors can be cured with radiotherapy. The role and importance of radiation therapy in cancer treatment is increasingly prominent, and it has become one of the main methods for treating malignant tumors. X-ray therapy is a commonly used clinical cancer treatment method. The core mechanism by which X-rays exert their anti-tumor effect lies in inducing immunogenic death (ICD) in tumor cells. This death pattern prompts tumor cells to release tumor antigens and damage-associated molecular patterns (DAMPs). The released damage-associated molecule adenosine triphosphate (ATP) can be recognized by the P2Y2 receptor on the surface of monocytes, simultaneously activating the P2X7 receptor on dendritic (DC) cells, triggering the activation of the NALP3 inflammasome and the secretion of the pro-inflammatory cytokine IL-1β. This provides a key signal for the initiation of cytotoxic T cells, thereby constructing an effective anti-tumor immune response. However, this immune activation process faces a severe challenge: extracellular ATP is extremely unstable and can be rapidly degraded into immunosuppressive adenosine (ADO) within seconds by CD39 and CD73 enzymes on the cell membrane surface. ADO binds to the G protein-coupled receptor (A2AR) on the surface of immune cells, activating the downstream cAMP pathway. This significantly inhibits NF-κB pathway activity by increasing cAMP levels, activating protein kinase A and cAMP-reactive element-binding proteins, inducing the expression of Foxp3 and repressive cytokines, and ultimately suppressing the immune activity of effector T cells. After X-ray therapy is initiated, the inherent contradiction between ATP-mediated immune activation and ADO-mediated immunosuppression severely weakens the anti-tumor effect of radiotherapy, becoming a core obstacle restricting its therapeutic efficacy. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides an Hf-based nanocomposite material, its preparation method, and its application. The aim is to construct an Hf-based nanocomposite material that combines immunomodulatory and radiosensitizing effects.

[0004] To achieve the above objectives, the present invention mainly provides the following technical solutions: The first aspect of the present invention provides an Hf-based nanocomposite material, which is prepared by modifying Hf-based nanoparticles with fluorescent molecules bearing carboxyl groups and then linking them with cell-penetrating peptides and carboxylic acid-functionalized polyethylene glycol. The Hf-based nanoparticles are obtained by reverse microemulsion method using hafnium tetrachloride, adenosine diphosphate, and A2AR antagonist.

[0005] The Hf-based nanocomposite material described above has an average particle size of 40–150 nm for the Hf-based nanoparticles.

[0006] In the Hf-based nanocomposite material described above, the A2AR antagonist is one of vipadenant, imaradenant, ciforadenant, and SCH442416; And / or, the fluorescent molecule with the carboxyl group is one of IR808, IR806, and Cy5; And / or, the cell-penetrating peptide is one of cell-penetrating peptide TAT, cell-penetrating peptide R8, and cell-penetrating peptide R9; And / or, in the carboxylic acid-functionalized polyethylene glycol, the weight-average molecular weight of the polyethylene glycol is 700 to 20,000.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned Hf-based nanocomposite material, comprising the following steps: S1. Hafnium tetrachloride, adenosine diphosphate and A2AR antagonist were mixed using the reverse microemulsion method to obtain a microemulsion system; The microemulsion system was heated, subjected to pulsed ultrasound, and then stirred with triethylamine solution. The mixture was then centrifuged at a differential speed to obtain Hf-based nanoparticles. S2. The Hf-based nanoparticles are mixed with fluorescent molecules containing carboxyl groups and subjected to a light-shielded reaction. After a first centrifugation treatment, Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups are obtained. S3. The Hf-based nanoparticles modified with fluorescent molecules bearing carboxyl groups are mixed with cell membrane-penetrating peptides and subjected to a first stirring reaction, followed by a second centrifugation treatment to obtain an intermediate nanocomposite material. S4. The intermediate nanocomposite material is mixed with carboxylic acid-functionalized polyethylene glycol and subjected to a second stirring reaction. After a third centrifugation treatment, Hf-based nanocomposite material is obtained.

[0008] In the preparation method of the Hf-based nanocomposite material described above, for every 1.2 mg of the Hf-based nanoparticles used, 0.005 to 0.02 mg of the fluorescent molecule with carboxyl groups is required; And / or, for every 1.2 mg of the Hf-based nanoparticles used, 0.05–0.5 mg of the cell-penetrating peptide is required; And / or, for every 1.2 mg of the Hf-based nanoparticles used, 0.009 to 0.2 mg of the carboxylic acid-functionalized polyethylene glycol is required.

[0009] In the preparation method of Hf-based nanocomposite materials as described above, in step S1, the heating temperature is 100-130 °C and the time is 10-20 min; And / or, in step S1, the power of the pulsed ultrasound is 30-120 W, the temperature is 20-25 ℃, the total ultrasound time is more than 1 min, and each pulse cycle includes 3-5 s of running and 1-3 s of stopping; And / or, in step S1, the differential centrifugation process is as follows: first, centrifuge at a centrifugal force of 4000–10000 xg for 8–15 min and collect the supernatant; then, centrifuge the collected supernatant at a centrifugal force of 7000–15000 xg for 8–15 min and collect the precipitate; finally, wash the collected precipitate 2–5 times at a centrifugal force of 7000–15000 xg, each time for 8–15 min. And / or, in step S2, the light-protected reaction is: a light-protected reaction at room temperature for 4 to 10 hours; And / or, in step S2, the first centrifugation treatment is: centrifugation at 12000-16000 xg for 8-15 min.

[0010] In the preparation method of Hf-based nanocomposite materials as described above, in step S3, the first stirring reaction is: stirring reaction at room temperature for 12-24 h; And / or, in step S3, the second centrifugation treatment is: centrifugation at 12000-16000 xg for 8-15 min; And / or, in step S4, the second stirring reaction is: a stirring reaction at room temperature for 12 to 24 hours; And / or, in step S4, the third centrifugation treatment is: centrifugation at 12000-16000 xg for 8-15 min.

[0011] The preparation method of Hf-based nanocomposites described above, in step S1, involves mixing hafnium tetrachloride, adenosine diphosphate, and an A2AR antagonist using a reverse microemulsion method to obtain a microemulsion system. Specific steps include: (1) Hafnium tetrachloride solution was prepared by dissolving hafnium tetrachloride in an aqueous sodium hydroxide solution; (2) Dissolve adenosine diphosphate in water to prepare an ADP solution; (3) Dissolve the A2AR antagonist in dimethyl sulfoxide to prepare an A2AR antagonist solution; (4) Mix cyclohexane, n-hexanol and Triton X-100 to prepare an oil phase dispersion; (5) The hafnium tetrachloride solution is mixed with the A2AR antagonist solution, and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion A; (6) The ADP solution is mixed with the A2AR antagonist solution and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion B; (7) Mix the microemulsion A and the microemulsion B to obtain a microemulsion system.

[0012] In the preparation method of Hf-based nanocomposite materials as described above, in step (1), the concentration of the hafnium tetrachloride solution is 0.09–0.1 mmol / μL; In step (2), the concentration of the ADP solution is 0.4–0.5 mmol / μL; In step (3), the concentration of the A2AR antagonist solution is 25–40 mg / ml; In step (4), the volume ratio of the cyclohexane, the n-hexanol, and the Triton X-100 is (32-36):(8-12):(8-12). In step (5), the ratio of the amount of hafnium tetrachloride solution, the A2AR antagonist solution, and the oil phase dispersion is (150-300 μL): (1-5 μL): (1-1.5 mL). In step (6), the ratio of the ADP solution, the A2AR antagonist, and the oil phase dispersion is (150-300 μL): (1-5 μL): (1-3 mL).

[0013] A third aspect of the present invention provides the application of an Hf-based nanocomposite material in the preparation of a tumor therapeutic drug, wherein the Hf-based nanocomposite material is used in conjunction with X-ray irradiation; The Hf-based nanocomposite material is either the Hf-based nanocomposite material described above or the Hf-based nanocomposite material prepared by the preparation method described above.

[0014] In summary, the solution of the present invention has at least the following effects: The Hf-based nanocomposite material provided by this invention possesses degradability, radiosensitization, and immunomodulatory effects, and has potential application value in the field of tumor treatment. When combined with X-ray irradiation, this Hf-based nanocomposite material significantly increases the ROS content and DNA damage accumulation in tumor cells, resulting in better radiosensitization and effectively improving anti-tumor function. Under X-ray irradiation, the Hf-based nanocomposite material can induce tumor cells to produce a large amount of ATP, significantly increasing ATP content and enhancing ATP-mediated immune activation, thus significantly enhancing the anti-tumor effect. The Hf-based nanocomposite material degrades under X-ray irradiation, and the exposed A2AR antagonist can competitively bind to A2AR with ADO, weakening or blocking the interaction between ADO and A2AR, reducing or relieving ADO-mediated immunosuppression, thereby alleviating the tumor immunosuppressive microenvironment and further enhancing the anti-tumor effect. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 These are TEM images of the Hf-based nanoparticles obtained in Examples 1-3 of this invention, wherein... Figure 1 Image a is a TEM image of the Hf-based nanoparticles obtained in Example 1. Figure 1 b is a TEM image of the Hf-based nanoparticles obtained in Example 2. Figure 1 c is a TEM image of the Hf-based nanoparticles obtained in Example 3; Figure 2 The results of particle size testing of Hf-based nanoparticles obtained in Examples 1-3 of this invention are shown; the horizontal axis Size represents particle size (nm), and the vertical axis Number represents the number (%). Figure 3 The fluorescence imaging test results are those of the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1 of the present invention and the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2. Figure 4The ultraviolet absorption test results are for the Hf-based nanoparticles (Hf-ADP) obtained in Comparative Example 1, the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, the Hf-based nanocomposite material (V@Hf-ADP@IR-T) obtained in Comparative Example 3, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4. The horizontal axis Wavelength represents wavelength (nm), and the vertical axis Abs represents absorbance (au, dimensionless unit). Figure 5 The FTIR analysis results are shown for the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention; the horizontal axis, Wavenumber, represents the wavenumber (cm). -1 The vertical axis, Transmittance, represents the light transmittance (%). Figure 6 The particle size and zeta potential test results are for the Hf-based nanoparticles (Hf-ADP) obtained in Comparative Example 1, the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, the Hf-based nanocomposite material (V@Hf-ADP@IR-T) obtained in Comparative Example 3, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention. Figure 6 'a' represents the particle size test result. Figure 6 b represents the Zeta potential test result; Figure 7 The images shown are TEM images of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention after X-ray irradiation with different doses (2Gy, 4Gy, 6Gy, 8Gy). Figure 7 Image a is a TEM image of V@Hf-ADP@IR-TP after 2Gy X-ray irradiation. Figure 7 b is a TEM image of V@Hf-ADP@IR-TP after 4Gy X-ray irradiation. Figure 7 c is a TEM image of V@Hf-ADP@IR-TP after 6 Gy X-ray irradiation. Figure 7 d is a TEM image of V@Hf-ADP@IR-TP after 8Gy X-ray irradiation; Figure 8The figures show the particle size and zeta potential test results of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention after irradiation with different doses (2Gy, 4Gy, 6Gy, 8Gy) of X-rays. Figure 8 'a' represents the particle size test result. Figure 8 b represents the Zeta potential test result (the horizontal axis X-ray dose represents the X-ray irradiation dose); Figure 9 The results of cell survival tests for each group in this invention are shown; the horizontal axis represents concentration (μg / mL), and the vertical axis represents cell survival rate (%). Figure 10 The effects of each treatment group on the content of reactive oxygen species (ROS) in this invention; Figure 11 The effects of each treatment group in this invention on DNA damage; Figure 12 This invention illustrates the effect of each treatment group on the ATP production level. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0018] It should be noted that the descriptions involving "first," "second," "third," etc. in this invention are used to distinguish similar objects and are not used to describe a specific order or sequence, and therefore should not be construed as limiting the invention.

[0019] It should be noted that in the specification and claims, "and / or" means at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0020] Those skilled in the art should understand that, in the following description of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0021] Those skilled in the art will understand that the numerical ranges in this invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail. The first aspect of the present invention provides an Hf-based nanocomposite material, which is prepared by modifying Hf-based nanoparticles with fluorescent molecules bearing carboxyl groups and then linking them with cell-penetrating peptides and carboxylic acid-functionalized polyethylene glycol. The Hf-based nanoparticles are obtained by reverse microemulsion method using hafnium tetrachloride, adenosine diphosphate, and A2AR antagonist.

[0023] In this invention, the cell-penetrating peptide is a cell-penetrating peptide with a carboxyl group.

[0024] The principle of preparing Hf-based nanoparticles according to this invention is explained: Hf in hafnium tetrachloride (HfCl4) 4+ Hf-ADP nanoparticles were synthesized by coordination with two hydroxyl groups on the β-phosphate group of the diphosphate group of adenosine diphosphate (ADP) and two hydroxyl groups on the carbon atoms at the 2' and 3' positions of the five-membered ribose ring in a microemulsion. The A2AR antagonist was dispersed in the microemulsion through hydrophilic-hydrophobic interactions and was embedded inside the Hf-ADP nanoparticles during the coordination synthesis process, thus obtaining V@Hf-ADP nanoparticles, i.e., Hf-based nanoparticles.

[0025] In this invention, the Hf-based nanoparticles are obtained from hafnium tetrachloride, adenosine diphosphate, and an A2AR antagonist via a reverse microemulsion method, specifically including the following steps: (1) Hafnium tetrachloride solution was prepared by dissolving hafnium tetrachloride in an aqueous sodium hydroxide solution; (2) Dissolve adenosine diphosphate in water to prepare an ADP solution; (3) Dissolve the A2AR antagonist in dimethyl sulfoxide to prepare an A2AR antagonist solution; (4) Mix cyclohexane, n-hexanol and Triton X-100 to prepare an oil phase dispersion; (5) The hafnium tetrachloride solution is mixed with the A2AR antagonist solution, and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion A; (6) The ADP solution is mixed with the A2AR antagonist solution and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion B; (7) Mix the microemulsion A and the microemulsion B to obtain a microemulsion system; (8) The microemulsion system is heated, pulsed sonicated, triethylamine solution is added and stirred, and differential centrifugation is performed to obtain Hf-based nanoparticles.

[0026] In some embodiments, the average particle size of the Hf-based nanoparticles is 40–150 nm.

[0027] In some embodiments, the A2AR antagonist is one of vipadenant, imaradenant, ciforadenant, and SCH442416, preferably vipadenant.

[0028] In this invention, A2AR antagonists can competitively bind to A2AR with ADO, thereby reducing or eliminating ADO-mediated immunosuppression, thus alleviating the tumor immunosuppressive microenvironment and enhancing the anti-tumor effect.

[0029] In some embodiments, the fluorescent molecule with a carboxyl group is one of IR808, IR806, and Cy5, preferably IR808.

[0030] In this invention, fluorescent molecules with carboxyl groups possess fluorescence imaging properties, enabling Hf-based nanocomposites to achieve imaging-guided precision treatment and improve anti-tumor effects.

[0031] In some embodiments, the cell-penetrating peptide is one of cell-penetrating peptide TAT, cell-penetrating peptide R8, and cell-penetrating peptide R9, preferably cell-penetrating peptide TAT.

[0032] In this invention, the Hf-based nanocomposite material contains the cell-penetrating peptide TAT, which effectively enhances the enrichment of the Hf-based nanocomposite material in tumor cells, resulting in a better radiosensitization effect.

[0033] In some embodiments, the weight-average molecular weight of the carboxylic acid-functionalized polyethylene glycol is 700 to 20,000, preferably 2,000.

[0034] A second aspect of the present invention provides a method for preparing the above-mentioned Hf-based nanocomposite material, comprising the following steps: S1. Hafnium tetrachloride, adenosine diphosphate and A2AR antagonist were mixed using the reverse microemulsion method to obtain a microemulsion system; The microemulsion system was heated, subjected to pulsed ultrasound, and then stirred with triethylamine solution. The mixture was then centrifuged at a differential speed to obtain Hf-based nanoparticles. S2. The Hf-based nanoparticles are mixed with fluorescent molecules containing carboxyl groups and subjected to a light-shielded reaction. After a first centrifugation treatment, Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups are obtained. S3. The Hf-based nanoparticles modified with fluorescent molecules bearing carboxyl groups are mixed with cell membrane-penetrating peptides and subjected to a first stirring reaction, followed by a second centrifugation treatment to obtain an intermediate nanocomposite material. S4. The intermediate nanocomposite material is mixed with carboxylic acid-functionalized polyethylene glycol and subjected to a second stirring reaction. After a third centrifugation treatment, Hf-based nanocomposite material is obtained.

[0035] In step S1, heating is used to increase the temperature of the microemulsion system, thereby increasing the average molecular kinetic energy of the reacting components and promoting the formation of Hf-based nanoparticles; pulsed ultrasound is used to break down the aqueous phase in the large-particle microemulsion system, allowing Hf to... 4+ The chamber volume of the reaction with ADP decreases, thereby reducing the size of the generated Hf-based nanoparticles. The role of adding triethylamine solution and stirring is that triethylamine increases the interfacial charge and refines the droplet size through neutralization reaction, transforming the system from a kinetically stable coarse emulsion into a thermodynamically stable, nanoscale transparent microemulsion.

[0036] This invention employs a reverse microemulsion method, which utilizes the spatial confinement effect of water-in-oil droplets to control nucleation and growth, prevent aggregation, and thus synthesize Hf-based nanoparticles with uniform size and good dispersibility.

[0037] In step S2, the light-shielding reaction is to prevent the "backbone" of the fluorescent molecules with carboxyl groups from being destroyed by light and heat, thereby ensuring the stability of their fluorescence performance; the first centrifugation treatment is to remove unreacted fluorescent molecules with carboxyl groups.

[0038] In step S3, the first stirring reaction serves to ensure that the nucleation and growth processes are consistent through forced convection, while preventing particle aggregation and sedimentation, thereby obtaining a highly dispersed intermediate nanocomposite material; the second centrifugation process is to remove unreacted cell-penetrating peptides.

[0039] In step S4, the second stirring reaction serves to ensure that the nucleation and growth processes are consistent through forced convection, while preventing particle agglomeration and sedimentation, thereby obtaining Hf-based nanocomposites with uniform size, regular morphology, and high dispersion; the third centrifugation treatment is to remove unreacted carboxylic acid-functionalized polyethylene glycol.

[0040] In this invention, the fluorescent molecule with a carboxyl group is obtained by activating the carboxyl group of the fluorescent molecule. The cell-penetrating peptide is obtained by activating the carboxyl group of the cell-penetrating peptide. The carboxylic acid-functionalized polyethylene glycol is obtained by activating the carboxyl group of the carboxylic acid-functionalized polyethylene glycol. The activation of the carboxyl group is performed using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0041] In some embodiments, for every 1.2 mg of the Hf-based nanoparticles used, 0.005 to 0.02 mg of the carboxyl-containing fluorescent molecule is required.

[0042] In some embodiments, for every 1.2 mg of the Hf-based nanoparticles used, 0.05–0.5 mg of the cell-penetrating peptide is required.

[0043] In some embodiments, for every 1.2 mg of the Hf-based nanoparticles used, 0.009 to 0.2 mg of the carboxylic acid-functionalized polyethylene glycol is required.

[0044] In some embodiments, in step S1, the heating temperature is 100–130 °C and the heating time is 10–20 min.

[0045] In some embodiments, in step S1, the power of the pulsed ultrasound is 30-120 W, the temperature is 20-25°C, the total ultrasound time is more than 1 min, and each pulse cycle includes 3-5 s of operation and 1-3 s of stop.

[0046] In some embodiments, in step S1, the differential centrifugation process is as follows: first, centrifuge at a centrifugal force of 4000–10000 xg for 8–15 min and collect the supernatant; then, centrifuge the collected supernatant at a centrifugal force of 7000–15000 xg for 8–15 min and collect the precipitate; finally, wash the collected precipitate 2–5 times at a centrifugal force of 7000–15000 xg, each time for 8–15 min.

[0047] In some embodiments, in step S2, the light-protected reaction is carried out at room temperature for 4 to 10 hours.

[0048] In some embodiments, in step S2, the first centrifugation process is: centrifugation at 12000-16000 xg for 8-15 min.

[0049] In some embodiments, in step S3, the first stirring reaction is: stirring reaction at room temperature for 12 to 24 hours.

[0050] In some embodiments, in step S3, the second centrifugation process is: centrifugation at 12000–16000 xg for 8–15 min.

[0051] In some embodiments, in step S4, the second stirring reaction is: stirring at room temperature for 12 to 24 hours.

[0052] In some embodiments, in step S4, the third centrifugation treatment is: centrifugation at 12000–16000 xg for 8–15 min.

[0053] In some embodiments, in step S1, the mixing of hafnium tetrachloride, adenosine diphosphate, and the A2AR antagonist using a reverse microemulsion method to obtain a microemulsion system specifically includes the following steps: (1) Hafnium tetrachloride solution was prepared by dissolving hafnium tetrachloride in an aqueous sodium hydroxide solution; (2) Dissolve adenosine diphosphate in water to prepare an ADP solution; (3) Dissolve the A2AR antagonist in dimethyl sulfoxide to prepare an A2AR antagonist solution; (4) Mix cyclohexane, n-hexanol and Triton X-100 to prepare an oil phase dispersion; (5) The hafnium tetrachloride solution is mixed with the A2AR antagonist solution, and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion A; (6) The ADP solution is mixed with the A2AR antagonist solution and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion B; (7) Mix the microemulsion A and the microemulsion B to obtain a microemulsion system.

[0054] The principle of preparing Hf-based nanocomposites according to this invention is explained as follows: Hf-based nanocomposites are synthesized via a reverse microemulsion method. Triton X-100 is used as the surfactant, n-hexanol as the co-surfactant, and cyclohexane as the oil phase to prepare an oil-phase dispersion. Water is used as the aqueous phase, and the mixture forms a water-in-oil microemulsion. Hf 4+Both ADP and Hf are water-soluble and coordinate in the aqueous phase of a microemulsion system. Under ultrasound, the volume of the microemulsion decreases, and upon heating, stable Hf-ADP nanoparticles are generated. Therefore, the dispersion uniformity and volume of the aqueous phase in the microemulsion can be controlled by adjusting the ultrasonic power of the pulsed ultrasound, thereby controlling the size of the generated Hf-based nanocomposite material. The A2AR antagonist is dispersed in the microemulsion through hydrophilic-hydrophobic interactions and is embedded within the Hf-ADP nanoparticles during their coordination synthesis, resulting in Hf-based nanoparticles. Carboxyl-containing fluorescent molecules, cell-penetrating peptides, and carboxylic acid-functionalized polyethylene glycol undergo an amide reaction with the amino groups on the exposed ADP on the material surface, ultimately yielding the Hf-based nanocomposite material.

[0055] In some embodiments, in step (1), the concentration of the hafnium tetrachloride solution is 0.09 to 0.1 mmol / μL.

[0056] In some embodiments, in step (2), the concentration of the ADP solution is 0.4 to 0.5 mmol / μL.

[0057] In some embodiments, in step (3), the concentration of the A2AR antagonist solution is 25–40 mg / ml.

[0058] In some embodiments, in step (4), the volume ratio of the cyclohexane, the n-hexanol and the Triton X-100 is (32-36):(8-12):(8-12).

[0059] In some embodiments, in step (5), the ratio of the amount of hafnium tetrachloride solution, the A2AR antagonist solution and the oil phase dispersion is (150-300 μL): (1-5 μL): (1-1.5 mL).

[0060] In some embodiments, in step (6), the ratio of the ADP solution, the A2AR antagonist, and the oil phase dispersion is (150-300 μL): (1-5 μL): (1-3 mL).

[0061] A third aspect of the present invention provides the application of an Hf-based nanocomposite material in the preparation of a tumor therapeutic drug, wherein the Hf-based nanocomposite material is used in conjunction with X-ray irradiation; wherein the Hf-based nanocomposite material is the Hf-based nanocomposite material described above or an Hf-based nanocomposite material prepared by the above preparation method.

[0062] In some implementations, the tumor is breast cancer.

[0063] The present invention will be further described below through specific embodiments.

[0064] In the following examples, adenosine diphosphate (ADP) was purchased from Anaiji (CAS No. 58-64-0); vipadenant was purchased from Anaiji (CAS No. 442908-10-3); IR808 was purchased from Anaiji (CAS No. 172971-76-5); cell-penetrating peptide TAT was purchased from Aladdin (CAS No. 191936-91-1); and mPEG2000-COOH (i.e., carboxylic acid-functionalized polyethylene glycol (PEG with a weight average molecular weight of 2000)) was purchased from Vanshuo Biotechnology (CAS No. 67665-18-3).

[0065] Example 1 This embodiment provides a method for preparing Hf-based nanoparticles, including the following steps: (1) Dissolve 18.7 mmol hafnium tetrachloride in 200 μL of 0.5 M sodium hydroxide aqueous solution to prepare a hafnium tetrachloride solution with a concentration of 0.0935 mmol / μL.

[0066] (2) Dissolve 93.5 mmol of adenosine diphosphate (ADP) in 200 μL of ultrapure water to prepare an ADP solution with a concentration of 0.4675 mmol / μL.

[0067] (3) Vipadenant (A2AR antagonist) was dissolved in dimethyl sulfoxide (DMSO) to prepare an A2AR antagonist solution with a concentration of 30 mg / ml.

[0068] (4) Prepare an oil phase dispersion by mixing cyclohexane, n-hexanol and Triton X-100 in a volume ratio of 34:9:9.

[0069] (5) Mix 200 μL of hafnium tetrachloride solution with 1 μL of A2AR antagonist solution, then mix with 1 mL of oil phase dispersion, stir for 10 min to obtain water-in-oil microemulsion A.

[0070] (6) Mix 200 μL of ADP solution with 1 μL of A2AR antagonist solution, then mix with 1 mL of oil phase dispersion, stir for 10 min to obtain water-in-oil microemulsion B.

[0071] (7) Mix microemulsion A and microemulsion B to obtain a microemulsion system; (8) Stir the microemulsion system for 10 min, heat it at 120 °C for 15 min, perform pulsed ultrasound after heating, add 20 µL of triethylamine after pulsed ultrasound and stir for 20 min, and perform differential centrifugation with anhydrous ethanol to obtain Hf-based nanoparticles with an average particle size of 40 nm (denoted as V@Hf-ADP). The pulsed ultrasound has a power of 120 W, a temperature of 25 ℃, and a duration of 1 min, with each pulse cycle consisting of 4 s of running and 2 s of stopping. The differential centrifugation process is as follows: First, centrifuge at 10000 xg for 10 min and collect the supernatant; then, centrifuge the collected supernatant at 15000 xg for 10 min and collect the precipitate; finally, wash the collected precipitate three times at 15000 xg for 10 min each time.

[0072] Example 2 This embodiment provides a method for preparing Hf-based nanoparticles, including the following steps: Steps (1) to (7) are the same as steps (1) to (7) in Example 1; (8) Stir the microemulsion system for 10 min, heat it at 120 °C for 15 min, perform pulsed ultrasound after heating, add 20 µL of triethylamine after pulsed ultrasound and stir for 20 min, and perform differential centrifugation with anhydrous ethanol to obtain Hf-based nanoparticles with an average particle size of 100 nm (denoted as V@Hf-ADP). The pulsed ultrasound has a power of 60 W, a temperature of 25 ℃, and a duration of 1 min, with each pulse cycle consisting of 4 s of running and 2 s of stopping. The differential centrifugation process is as follows: First, centrifuge at 7000 xg for 10 min and collect the supernatant; then, centrifuge the collected supernatant at 10000 xg for 10 min and collect the precipitate; finally, wash the collected precipitate three times at 10000 xg for 10 min each time.

[0073] Example 3 This embodiment provides a method for preparing Hf-based nanoparticles, including the following steps: This embodiment provides a method for preparing Hf-based nanoparticles, including the following steps: Steps (1) to (7) are the same as steps (1) to (7) in Example 1; (8) Stir the microemulsion system for 10 min, heat it at 120 °C for 15 min, perform pulsed sonication after heating, add 20 µL of triethylamine after pulsed sonication and stir for 20 min, and perform differential centrifugation with anhydrous ethanol to obtain Hf-based nanoparticles with an average particle size of 150 nm (denoted as V@Hf-ADP). The pulsed ultrasound has a power of 30 W, a temperature of 25 ℃, and a duration of 1 min, with each pulse cycle consisting of 4 s of running and 2 s of stopping. The differential centrifugation process is as follows: First, centrifuge at 4000 xg for 10 min and collect the supernatant; then, centrifuge the collected supernatant at 7000 xg for 10 min and collect the precipitate; finally, wash the collected precipitate three times at 7000 xg for 10 min each time.

[0074] Example 4 This embodiment prepares Hf-based nanocomposites using a preparation method including the following steps: (1) Preparation of Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups: IR808, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were mixed evenly in a molar ratio of 1:2.5:5 to prepare an activated IR808 solution with a concentration of 1 mg / mL. 1.2 mg of the Hf-based nanoparticles obtained in Example 1 were mixed with 10 μL of activated IR808 solution and reacted at room temperature in the dark for 6 h. After centrifugation at 15000 xg for 10 min, Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups (denoted as V@Hf-ADP@IR) were obtained.

[0075] (2) Preparation of intermediate nanocomposite materials: Cell-penetrating peptides TAT, EDC, and NHS were mixed evenly in a molar ratio of 1:2.5:5 to prepare an activated TAT solution with a concentration of 1 mg / mL. The Hf-based nanoparticles modified with the above-mentioned carboxyl-containing fluorescent molecules (V@Hf-ADP@IR) were mixed with 100 μL of activated TAT solution and stirred at room temperature for 24 h. After centrifugation at 15000xg for 10 min, the intermediate nanocomposite material (denoted as V@Hf-ADP@IR-T) was obtained.

[0076] (3) Preparation of Hf-based nanocomposites: mPEG2000-COOH, EDC, and NHS were mixed evenly in a molar ratio of 1:2.5:5 to prepare an activated mPEG2000-COOH solution with a concentration of 1 mg / mL. The above intermediate nanocomposite material was mixed with 94 μL of activated mPEG2000-COOH solution and stirred at room temperature for 24 h. After centrifugation at 15000xg for 10 min, Hf-based nanocomposite material (denoted as V@Hf-ADP@IR-TP) was obtained.

[0077] Comparative Example 1 (Hf-ADP) This comparative example provides a method for preparing Hf-based nanoparticles, comprising the following steps: (1) Dissolve 18.7 mmol hafnium tetrachloride in 200 μL of 0.5 M sodium hydroxide aqueous solution to prepare a hafnium tetrachloride solution with a concentration of 0.0935 mmol / μL.

[0078] (2) Dissolve 93.5 mmol of adenosine diphosphate (ADP) in 200 μL of ultrapure water to prepare an ADP solution with a concentration of 0.4675 mmol / μL.

[0079] (3) Prepare an oil phase dispersion by mixing cyclohexane, n-hexanol and Triton X-100 in a volume ratio of 34:9:9.

[0080] (4) Mix 200 μL of hafnium tetrachloride solution with 1 mL of oil phase dispersion and stir for 10 min to obtain water-in-oil microemulsion A.

[0081] (5) Mix 200 μL of ADP solution with 1 mL of oil phase dispersion and stir for 10 min to obtain water-in-oil microemulsion B.

[0082] (6) Mix microemulsion A and microemulsion B to obtain a microemulsion system; (7) Stir the microemulsion system for 10 min, heat it at 120 °C for 15 min, perform pulsed ultrasound after heating, add 20 µL of triethylamine after pulsed ultrasound and stir for 20 min, and perform differential centrifugation with anhydrous ethanol to obtain Hf-based nanoparticles with an average particle size of 40 nm (denoted as Hf-ADP). The pulsed ultrasound has a power of 120 W, a temperature of 25 ℃, and a duration of 1 min, with each pulse cycle consisting of 4 s of running and 2 s of stopping. The differential centrifugation process is as follows: First, centrifuge at 10000 xg for 10 min and collect the supernatant; then, centrifuge the collected supernatant at 15000 xg for 10 min and collect the precipitate; finally, wash the collected precipitate three times at 15000 xg for 10 min each time.

[0083] Comparative Example 2 (V@Hf-ADP@IR) This comparative example prepared Hf-based nanocomposites using a preparation method including the following steps: IR808, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were mixed evenly in a molar ratio of 1:2.5:5 to prepare an activated IR808 solution with a concentration of 1 mg / mL. 1.2 mg of the Hf-based nanoparticles obtained in Example 1 were mixed with 10 μL of activated IR808 solution and reacted at room temperature in the dark for 6 h. After centrifugation at 15000 xg for 10 min, the Hf-based nanocomposite material (denoted as V@Hf-ADP@IR) was obtained.

[0084] Comparative Example 3 (V@Hf-ADP@IR-T) This embodiment prepares Hf-based nanocomposites using a preparation method including the following steps: (1) Preparation of Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups: IR808, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were mixed evenly in a molar ratio of 1:2.5:5 to prepare an activated IR808 solution with a concentration of 1 mg / mL. 1.2 mg of the Hf-based nanoparticles obtained in Example 1 were mixed with 10 μL of activated IR808 solution and reacted at room temperature in the dark for 6 h. After centrifugation at 15000 xg for 10 min, Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups (denoted as V@Hf-ADP@IR) were obtained.

[0085] (2) Preparation of Hf-based nanocomposites: Cell-penetrating peptides TAT, EDC, and NHS were mixed evenly in a molar ratio of 1:2.5:5 to prepare an activated TAT solution with a concentration of 1 mg / mL. The Hf-based nanoparticles modified with the above-mentioned carboxyl-containing fluorescent molecules (V@Hf-ADP@IR) were mixed with 100 μL of activated TAT solution and stirred at room temperature for 24 h. After centrifugation at 15000xg for 10 min, the intermediate nanocomposite material (denoted as V@Hf-ADP@IR-T) was obtained.

[0086] Results Explanation 1. The Hf-based nanoparticles obtained in Examples 1-3 of this invention were subjected to transmission electron microscopy (TEM) tests, and the results are as follows: Figure 1 As shown.

[0087] Figure 1These are TEM images of the Hf-based nanoparticles obtained in Examples 1-3 of this invention, wherein... Figure 1 Image a is a TEM image of the Hf-based nanoparticles obtained in Example 1. Figure 1 b is a TEM image of the Hf-based nanoparticles obtained in Example 2. Figure 1 c is a TEM image of the Hf-based nanoparticles obtained in Example 3.

[0088] Depend on Figure 1 As can be seen, the Hf-based nanoparticles (V@Hf-ADP) obtained in Examples 1-3 are spherical, with uniform particle morphology and exhibit a highly dispersed state.

[0089] 2. The particle size of the Hf-based nanoparticles obtained in Examples 1-3 of this invention was measured using a nanoparticle size analyzer, and the results are as follows: Figure 2 As shown.

[0090] Figure 2 The particle size test results of the Hf-based nanoparticles obtained in Examples 1-3 of this invention are shown.

[0091] Depend on Figure 2 It can be seen that the average particle size of the Hf-based nanoparticles obtained in Example 1 is 40 nm, the average particle size of the Hf-based nanoparticles obtained in Example 2 is 100 nm, and the average particle size of the Hf-based nanoparticles obtained in Example 3 is 150 nm.

[0092] 3. Fluorescence imaging tests were performed on the Hf-based nanoparticles (V@Hf-ADP) obtained in Comparative Example 1 and the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2 using a fluorescence imager. The results are as follows: Figure 3 As shown.

[0093] Figure 3 The fluorescence imaging test results are for the Hf-based nanoparticles (V@Hf-ADP) obtained in Comparative Example 1 and the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2.

[0094] Depend on Figure 3 As can be seen, V@Hf-ADP did not produce a fluorescence signal, but after the reaction with IR808, the resulting V@Hf-ADP@IR produced a fluorescence signal, indicating that the IR808 was successfully connected.

[0095] 4. The Hf-based nanoparticles (Hf-ADP) obtained in Comparative Example 1, the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, the Hf-based nanocomposite material (V@Hf-ADP@IR-T) obtained in Comparative Example 3, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 were subjected to ultraviolet absorption tests using a UV spectrophotometer. The results are as follows: Figure 4 As shown.

[0096] Figure 4 The ultraviolet absorption test results are for the Hf-based nanoparticles (Hf-ADP) obtained in Comparative Example 1, the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, the Hf-based nanocomposite material (V@Hf-ADP@IR-T) obtained in Comparative Example 3, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4.

[0097] Depend on Figure 4 It can be seen that the UV absorption changed significantly after the cell-penetrating peptide TAT was connected, indicating that the cell-penetrating peptide TAT was successfully connected.

[0098] 5. Fourier transform infrared spectroscopy was used to perform FTIR analysis on the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4. The results are as follows: Figure 5 As shown.

[0099] Figure 5 The results are FTIR analysis of the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4.

[0100] Depend on Figure 5 Therefore, at 550cm -1 The absorption peak at 550 cm⁻¹ originates from the C-Cl stretching vibration of IR808. FTIR results show that V@Hf-ADP has an absorption peak at 550 cm⁻¹. -1 There is no absorption peak at 550 cm⁻¹, while V@Hf-ADP@IR and V@Hf-ADP@IR-TP show absorption peaks at 550 cm⁻¹. -1 Absorption peaks were observed at all locations, indicating successful IR808 bonding. At 1643 cm⁻¹...-1 and 3430cm -1 The point is the stretching vibration of the amide bond, at 1643 cm⁻¹. -1 The point is the stretching vibration of the C=O bond, at 3430 cm⁻¹. -1 The stretching vibration of the NH bond is at 1643 cm⁻¹, V@Hf-ADP@IR-TP. -1 and 3430cm -1 The absorption peak at this location is larger than that at V@Hf-ADP@IR, indicating that the cell-penetrating peptide TAT and mPEG2000-COOH are successfully linked.

[0101] 6. Using nanoparticle size analyzers and Zeta potential meters, the Hf-based nanoparticles (Hf-ADP) obtained in Comparative Example 1, the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, the Hf-based nanocomposite material (V@Hf-ADP@IR-T) obtained in Comparative Example 3, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 were subjected to particle size analysis and Zeta potential analysis, respectively. The results are as follows: Figure 6 As shown.

[0102] Figure 6 The particle size and zeta potential test results are for the Hf-based nanoparticles (Hf-ADP) obtained in Comparative Example 1, the Hf-based nanoparticles (V@Hf-ADP) obtained in Example 1, the Hf-based nanocomposite material (V@Hf-ADP@IR) obtained in Comparative Example 2, the Hf-based nanocomposite material (V@Hf-ADP@IR-T) obtained in Comparative Example 3, and the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention. Figure 6 'a' represents the particle size test result. Figure 6 b represents the result of the Zeta potential test.

[0103] Figure 6The results of a showed that the particle size of the materials modified with IR808 (V@Hf-ADP@IR), cell-penetrating peptide TAT (V@Hf-ADP@IR-T), and mPEG2000-COOH (V@Hf-ADP@IR-TP) changed. The average particle size of V@Hf-ADP was 40 nm. The average particle size of the material modified with IR808 (V@Hf-ADP@IR) increased to 51 nm. The average particle size of the material modified with cell-penetrating peptide TAT (V@Hf-ADP@IR-T) increased to 52 nm. The average particle size of the material modified with mPEG2000-COOH (V@Hf-ADP@IR-TP) increased to 59 nm. The average particle size of Hf-ADP was 37 nm.

[0104] Depend on Figure 6 As shown in b, the Zeta potential of Hf-ADP is -31.6 mV, the Zeta potential of V@Hf-ADP is -32.6 mV, the Zeta potential of V@Hf-ADP@IR is -37.5 mV, the Zeta potential of V@Hf-ADP@IR-T is -25.2 mV, and the Zeta potential of @Hf-ADP@IR-TP is -42.2 mV. The Zeta potential of the material (V@Hf-ADP@IR) modified with IR808 becomes more negative. The Zeta potential of the material (V@Hf-ADP@IR-T) after ligation with the cell-penetrating peptide TAT changes positively. The Zeta potential of the material (V@Hf-ADP@IR-TP) after ligation with mPEG2000-COOH becomes more negative again. The above results indicate that Hf-ADP, V@Hf-ADP, V@Hf-ADP@IR, V@Hf-ADP@IR-T, and V@Hf-ADP@IR-TP were successfully prepared.

[0105] 7. The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was subjected to degradation performance testing.

[0106] ① The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was irradiated with different doses (2Gy, 4Gy, 6Gy, 8Gy) of X-rays. After 24 h of irradiation, it was characterized by transmission electron microscopy (TEM). The results are as follows: Figure 7 As shown.

[0107] Figure 7 The images shown are TEM images of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention after X-ray irradiation with different doses (2Gy, 4Gy, 6Gy, 8Gy). Figure 7Image a is a TEM image of V@Hf-ADP@IR-TP after 2Gy X-ray irradiation. Figure 7 b is a TEM image of V@Hf-ADP@IR-TP after 4Gy X-ray irradiation. Figure 7 c is a TEM image of V@Hf-ADP@IR-TP after 6 Gy X-ray irradiation. Figure 7 d is a TEM image of V@Hf-ADP@IR-TP after 8Gy X-ray irradiation.

[0108] Depend on Figure 7 It can be seen that as the X-ray irradiation dose increases, the particle size of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) gradually increases.

[0109] ② The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was irradiated with different doses (2Gy, 4Gy, 6Gy, 8Gy) of X-rays. After 24 h of irradiation, it was characterized by nanoparticle size and Zeta potential. The results are as follows: Figure 8 As shown.

[0110] Figure 8 The figures show the particle size and zeta potential test results of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention after irradiation with different doses (2Gy, 4Gy, 6Gy, 8Gy) of X-rays. Figure 8 'a' represents the particle size test result. Figure 6 b represents the result of the Zeta potential test.

[0111] Depend on Figure 8 As shown in section a, the average particle size of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) after 2 Gy X-ray irradiation is 92 nm; the average particle size after 4 Gy X-ray irradiation is 200 nm; the average particle size after 6 Gy X-ray irradiation is 345 nm; and the average particle size after 8 Gy X-ray irradiation is 477 nm. The results indicate that the particle size of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) gradually increases with increasing X-ray irradiation dose. This is because the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) degrades after X-ray irradiation, and the degraded substances reorganize, leading to the increase in particle size.

[0112] Depend on Figure 8As shown in b, with increasing X-ray irradiation dose, the Zeta potential of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) gradually increases and tends to 0. This also indicates that the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) degrades after X-ray irradiation, and the degraded substances recombine, leading to an increase in the Zeta potential. The above results demonstrate that the Hf-based nanocomposite material provided in the embodiments of this invention has degradable properties.

[0113] 8. Cell viability test was performed on the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of the present invention.

[0114] Hf-based nanocomposite material (V@Hf-ADP@IR-TP) was co-incubated with breast cancer 4T1 cells at concentrations of 0, 50, 150, 200, and 300 μg / mL for 24 h. Cell viability was then detected using an enzyme-linked immunosorbent assay (ELISA) reader. The results are as follows: Figure 9 As shown.

[0115] Figure 9 The results show the cell survival rate test results for each group in this invention.

[0116] Depend on Figure 9 The results showed that the cell viability of the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) was 96% at a concentration of 50 μg / mL, 92% at 150 μg / mL, 94% at 200 μg / mL, and 86% at 300 μg / mL. These results indicate that the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) exhibits low toxicity and good biocompatibility.

[0117] 9. The effects of different treatments on the content of reactive oxygen species (ROS) were investigated, and the groups were divided into the following 6 groups: 300 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 300 μg / mL for 24 h, then irradiated with 6 Gy X-rays for 6 h, and then co-incubated with ROS probe DCFH-DA for 20 min. The cell nuclei were then stained with Hoechst dye, and the fluorescence intensity was detected by fluorescence microscopy. 200 μg / mL Hf-based nanocomposite material + X-ray group (200 μg / mL + X-ray(+)): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 200 μg / mL for 24 h, then irradiated with 6 Gy X-rays for 6 h, and then co-incubated with ROS probe DCFH-DA for 20 min. The cell nuclei were then stained with Hoechst dye, and the fluorescence intensity was detected by fluorescence microscopy. 150 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 150 μg / mL for 24 h, then irradiated with 6 Gy X-rays for 6 h, and then co-incubated with ROS probe DCFH-DA for 20 min. The cell nuclei were then stained with Hoechst dye, and the fluorescence intensity was detected by fluorescence microscopy. 50 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 50 μg / mL for 24 h, then irradiated with 6 Gy X-rays for 6 h, and then co-incubated with ROS probe DCFH-DA for 20 min. The cell nuclei were then stained with Hoechst dye, and the fluorescence intensity was detected by fluorescence microscopy. X-ray group (0 μg / mL+X-ray(+)): 4T1 breast cancer cells were incubated for 24 h, then irradiated with 6 Gy X-rays for 6 h, and then co-incubated with ROS probe DCFH-DA for 20 min. The cell nuclei were stained with Hoechst dye and the fluorescence intensity was detected by fluorescence microscopy. Blank control group (0 μg / mL + X-ray(-)): Breast cancer 4T1 cells were incubated for 24 h, then irradiated with 6 Gy X-rays and incubated for 6 h. The ROS probe DCFH-DA was added and co-incubated for 20 min. The cell nuclei were stained with Hoechst dye and the fluorescence intensity was detected by fluorescence microscopy.

[0118] Figure 10 This invention illustrates the effect of each treatment group on the content of reactive oxygen species (ROS).

[0119] Depend on Figure 10It was found that, compared with the 200 μg / mL Hf-based nanocomposite + X-ray group (200 μg / mL + X-ray(+)), 150 μg / mL Hf-based nanocomposite + X-ray group (300 μg / mL + X-ray(+)), 50 μg / mL Hf-based nanocomposite + X-ray group (300 μg / mL + X-ray(+)), and X-ray group (0 μg / mL + X-ray(+)), the 300 μg / mL Hf-based nanocomposite + X-ray group (300 μg / mL + X-ray(+)) had the highest content of reactive oxygen species (ROS) in 4T1 cells, significantly increasing the ROS content in 4T1 cells and effectively improving anti-tumor function. By comparing the Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)), 200 μg / mL Hf-based nanocomposite material + X-ray group (200 μg / mL + X-ray(+)), 150 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)), and 50 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)), it can be seen that as the concentration of Hf-based nanocomposite material (V@Hf-ADP@IR-TP) gradually decreases, the ROS content gradually decreases, indicating that Hf-based nanocomposite material (V@Hf-ADP@IR-TP) has a radiosensitizing effect under X-ray irradiation.

[0120] 10. The effects of different treatments on DNA damage were investigated, and the groups were divided into the following four groups: 300 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 300 μg / mL for 24 h, then irradiated with 6 Gy X-rays and incubated for 6 h, γ-H2AX was added for staining, and the cell nuclei were stained with Hoechst dye for staining. The fluorescence intensity was detected by fluorescence microscopy. X-ray group (0 μg / mL+X-ray(+)): 4T1 breast cancer cells were incubated for 24 h, then irradiated with 6 Gy X-rays and incubated for 6 h. γ-H2AX was added for staining, and the cell nuclei were stained with Hoechst dye. The fluorescence intensity was detected by fluorescence microscopy. Blank control group (0 μg / mL + X-ray(-)): After incubating breast cancer 4T1 cells for 24 h, γ-H2AX was added for staining, and the cell nuclei were stained with Hoechst dye. The fluorescence intensity was detected by fluorescence microscopy. 300 μg / mL Hf-based nanocomposite material (300 μg / mL + X-ray(-)): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 300 μg / mL for 24 h, then γ-H2AX was added for staining, and the cell nuclei were stained with Hoechst dye. The fluorescence intensity was detected by fluorescence microscopy.

[0121] Figure 11 This invention illustrates the effects of various treatments on DNA damage.

[0122] Depend on Figure 12 It can be seen that, compared with the X-ray group (0 μg / mL + X-ray(+)), the blank control group (0 μg / mL + X-ray(-)) and the 300 μg / mL Hf-based nanocomposite material (300 μg / mL + X-ray(-)), the 300 μg / mL Hf-based nanocomposite material + X-ray group (300 μg / mL + X-ray(+)) has a better DNA damage ability against 4T1 cells, significantly increases the accumulation of DNA damage in 4T1 cells, and effectively improves anti-tumor function.

[0123] 12. The effects of different treatments on ATP production were investigated, and the groups were divided into the following 5 groups: Blank group (PBS): After incubating breast cancer 4T1 cells for 24 h, the ATP release level of the cells was measured according to the instructions of the ATP assay kit. Hf-based nanocomposite material group (V@Hf-ADP@IR-TP): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 300 μg / mL for 24 h, and the ATP release level of the cells was measured according to the instructions of the ATP detection kit. X-ray group (6Gy): After incubating breast cancer 4T1 cells for 24 hours, they were irradiated with 6 Gy X-rays and then incubated for 6 hours. The ATP release level of the cells was measured according to the instructions of the ATP detection kit. Hf-based nanocomposite material + 2Gy X-ray group (2Gy + V@Hf-ADP@IR-TP): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 300 μg / mL for 24 h, then irradiated with 2Gy X-rays and incubated for 6 h. The ATP release level of the cells was measured according to the instructions of the ATP detection kit. Hf-based nanocomposite material + 6Gy X-ray group (6Gy + V@Hf-ADP@IR-TP): The Hf-based nanocomposite material (V@Hf-ADP@IR-TP) obtained in Example 4 of this invention was co-incubated with breast cancer 4T1 cells at 300 μg / mL for 24 h, then irradiated with 6Gy X-rays and incubated for 6 h. The ATP release level of the cells was measured according to the instructions of the ATP detection kit.

[0124] Figure 12 This invention illustrates the effect of each treatment group on the ATP production level.

[0125] Depend on Figure 12 It was found that, compared with the blank group (PBS), the Hf-based nanocomposite material group (V@Hf-ADP@IR-TP), the X-ray group (6Gy), and the Hf-based nanocomposite material + 2Gy X-ray group (2Gy+V@Hf-ADP@IR-TP), the Hf-based nanocomposite material + 6Gy X-ray group (6Gy+V@Hf-ADP@IR-TP) produced the highest ATP content. This indicates that the Hf-based nanocomposite material (V@Hf-ADP@IR-TP) can induce 4T1 cells to produce a large amount of ATP under 6Gy X-ray irradiation, thus significantly increasing the ATP content. This demonstrates that the Hf-based nanocomposite material provided in this embodiment of the invention has an immunomodulatory effect.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An Hf-based nanocomposite material, characterized in that, The Hf-based nanocomposite material is prepared by modifying Hf-based nanoparticles with fluorescent molecules containing carboxyl groups and then connecting them with cell-penetrating peptides and carboxylic acid-functionalized polyethylene glycol. The Hf-based nanoparticles are obtained by reverse microemulsion method using hafnium tetrachloride, adenosine diphosphate, and A2AR antagonist.

2. The Hf-based nanocomposite material according to claim 1, characterized in that, The average particle size of the Hf-based nanoparticles is 40–150 nm.

3. The Hf-based nanocomposite material according to claim 1, characterized in that, The A2AR antagonist is one of vipadenant, imaradenant, ciforadenant, and SCH442416; And / or, the fluorescent molecule with the carboxyl group is one of IR808, IR806, and Cy5; And / or, the cell-penetrating peptide is one of cell-penetrating peptide TAT, cell-penetrating peptide R8, and cell-penetrating peptide R9; And / or, in the carboxylic acid-functionalized polyethylene glycol, the weight-average molecular weight of the polyethylene glycol is 700 to 20,000.

4. A method for preparing the Hf-based nanocomposite material according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Hafnium tetrachloride, adenosine diphosphate and A2AR antagonist were mixed using the reverse microemulsion method to obtain a microemulsion system; The microemulsion system was heated, subjected to pulsed ultrasound, and then stirred with triethylamine solution. The mixture was then centrifuged at a differential speed to obtain Hf-based nanoparticles. S2. The Hf-based nanoparticles are mixed with fluorescent molecules containing carboxyl groups and subjected to a light-shielded reaction. After a first centrifugation treatment, Hf-based nanoparticles modified with fluorescent molecules containing carboxyl groups are obtained. S3. The Hf-based nanoparticles modified with fluorescent molecules bearing carboxyl groups are mixed with cell membrane-penetrating peptides and subjected to a first stirring reaction, followed by a second centrifugation treatment to obtain an intermediate nanocomposite material. S4. The intermediate nanocomposite material is mixed with carboxylic acid-functionalized polyethylene glycol and subjected to a second stirring reaction. After a third centrifugation treatment, Hf-based nanocomposite material is obtained.

5. The method for preparing Hf-based nanocomposite materials according to claim 4, characterized in that, For every 1.2 mg of the Hf-based nanoparticles used, 0.005–0.02 mg of the carboxyl-containing fluorescent molecule is required. And / or, for every 1.2 mg of the Hf-based nanoparticles used, 0.05–0.5 mg of the cell-penetrating peptide is required; And / or, for every 1.2 mg of the Hf-based nanoparticles used, 0.009 to 0.2 mg of the carboxylic acid-functionalized polyethylene glycol is required.

6. The method for preparing Hf-based nanocomposite materials according to claim 4, characterized in that, In step S1, the heating temperature is 100-130 °C and the heating time is 10-20 min; And / or, in step S1, the power of the pulsed ultrasound is 30-120 W, the temperature is 20-25 ℃, the total ultrasound time is more than 1 min, and each pulse cycle includes 3-5 s of running and 1-3 s of stopping; And / or, in step S1, the differential centrifugation process is as follows: first, centrifuge at a centrifugal force of 4000-10000 xg for 8-15 min and collect the supernatant; then, centrifuge the collected supernatant at a centrifugal force of 7000-15000 xg for 8-15 min and collect the precipitate. Finally, the collected precipitate was washed 2 to 5 times under centrifugal force of 7000 to 15000 xg, each time for 8 to 15 min; And / or, in step S2, the light-protected reaction is: a light-protected reaction at room temperature for 4 to 10 hours; And / or, in step S2, the first centrifugation treatment is: centrifugation at 12000-16000 xg for 8-15 min.

7. The method for preparing Hf-based nanocomposite materials according to claim 4, characterized in that, In step S3, the first stirring reaction is: stirring reaction at room temperature for 12-24 h; And / or, in step S3, the second centrifugation treatment is: centrifugation at 12000-16000 xg for 8-15 min; And / or, in step S4, the second stirring reaction is: a stirring reaction at room temperature for 12 to 24 hours; And / or, in step S4, the third centrifugation treatment is: centrifugation at 12000-16000 xg for 8-15 min.

8. The method for preparing Hf-based nanocomposite materials according to claim 4, characterized in that, In step S1, the reverse microemulsion method is used to mix hafnium tetrachloride, adenosine diphosphate, and the A2AR antagonist to obtain a microemulsion system. Specific steps include: (1) Hafnium tetrachloride solution was prepared by dissolving hafnium tetrachloride in an aqueous sodium hydroxide solution; (2) Dissolve adenosine diphosphate in water to prepare an ADP solution; (3) Dissolve the A2AR antagonist in dimethyl sulfoxide to prepare an A2AR antagonist solution; (4) Mix cyclohexane, n-hexanol and Triton X-100 to prepare an oil phase dispersion; (5) The hafnium tetrachloride solution is mixed with the A2AR antagonist solution, and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion A; (6) The ADP solution is mixed with the A2AR antagonist solution and then mixed with the oil phase dispersion to obtain a water-in-oil microemulsion B; (7) Mix the microemulsion A and the microemulsion B to obtain a microemulsion system.

9. The method for preparing Hf-based nanocomposite materials according to claim 8, characterized in that, In step (1), the concentration of the hafnium tetrachloride solution is 0.09–0.1 mmol / μL; In step (2), the concentration of the ADP solution is 0.4–0.5 mmol / μL; In step (3), the concentration of the A2AR antagonist solution is 25–40 mg / ml; In step (4), the volume ratio of the cyclohexane, the n-hexanol, and the Triton X-100 is (32-36):(8-12):(8-12). In step (5), the ratio of the amount of hafnium tetrachloride solution, the A2AR antagonist solution, and the oil phase dispersion is (150-300 μL): (1-5 μL): (1-1.5 mL). In step (6), the ratio of the ADP solution, the A2AR antagonist, and the oil phase dispersion is (150-300 μL): (1-5 μL): (1-3 mL).

10. The application of an Hf-based nanocomposite material in the preparation of tumor therapeutic drugs, characterized in that, The Hf-based nanocomposite material was used in conjunction with X-ray irradiation; The Hf-based nanocomposite material is the Hf-based nanocomposite material according to any one of claims 1 to 3 or the Hf-based nanocomposite material prepared by the preparation method according to any one of claims 4 to 8.

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