Hafnium-based complex protein nanoparticles for radiotherapy sensitization as well as preparation method and application of hafnium-based complex protein nanoparticles

Abstract

The invention discloses hafnium-based complex protein nanoparticles for radiotherapy sensitization as well as a preparation method and application of the hafnium-based complex protein nanoparticles. In order to break through the restrictions of complex preparation process, high energy consumption, only intratumor injection in clinical application, single administration route and the like in the current synthesis of hafnium-based compounds, especially the only clinical application hafnium oxide, hafnium ions in a solution enter a protein cavity and then generate a complex (precipitate) with ligand ion hexametaphosphate radicals, so that the hafnium-based compounds are synthesized. Biomineralization is carried out (preparation is completed at room temperature), such that protein-coated hafnium-based complex nanoparticles are obtained; when the nanoparticles are subjected to X-ray radiation, the radiotherapy effect can be remarkably enhanced. The hafnium-based complex protein nanoparticle disclosed by the invention is composed of the internal hafnium-based complex and the external protein shell layer, has the capabilities of being simple and convenient to prepare, uniform and controllable in particle size, good in dispersity in water, strong in tumor targeting property and the like, and lays a solid foundation for further developing a high-efficiency sensitizer for tumor radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a hafnium-based complex protein nanoparticle for radiotherapy sensitization, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant tumors are one of the diseases with high incidence and mortality rates globally, seriously threatening human health. As a non-invasive treatment method, radiotherapy plays an important role in the treatment of various tumors. The biological effects of radioactive rays on organisms are mainly direct effects: radioactive rays directly act on DNA molecules in cells, resulting in DNA strand breaks, base damage, etc., thereby affecting the normal functions and division of cells; and indirect effects: radioactive rays ionize water molecules in cells to generate reactive substances such as free radicals, and these reactive substances can further react with DNA molecules to cause DNA damage. However, radiotherapy also causes damage to normal cells, limiting its efficacy and application. In order to improve the radiotherapy effect and reduce side effects, the development of efficient radiotherapy methods has become a research hotspot. The interaction and absorption degree of X-rays with tissues determine the deposition of radiation dose in tissues, and the intervention of high electron density substances can enhance the dose deposition of X-rays in tumor tissues, and this phenomenon is the radiation sensitization effect. After a sensitizing substance contacts water or oxygen in cells, it can absorb the energy of X-rays and generate free radicals to be activated, and can convert the oxygen around it into free radicals, thereby causing irreversible damage to cells and promoting the improvement of the efficacy of tumor treatment. Therefore, it can be used as an adjuvant drug for tumor treatment.

[0003] Hafnium-based nanoparticles have shown potential in tumor radiotherapy sensitization due to the unique physical and chemical properties of hafnium atoms (atomic number 72, high electron density enables amplification of X-ray irradiation dose deposition at the tumor site). Currently, hafnium oxide and hafnium ions are more studied in the field of radiotherapy sensitization. Due to the special role of hafnium, hafnium oxide (HfO2) nanoparticles currently used in clinical applications, such as the commercially available NBTXR3 (an injectable aqueous suspension of 50 nm crystalline HfO2 nanoparticles for the treatment of solid tumors such as non-small cell lung cancer, jointly developed by French company Nanobiotix and US company Janssen), are used for radiotherapy sensitization and show good therapeutic effects. However, the synthesis of HfO2 usually uses hafnium tetrachloride, hafnium disulfide, hafnium boride, hafnium nitride, hafnium carbide, hafnium oxychloride, hafnium sulfate, etc. as raw materials, and is obtained by thermal decomposition, hydrolysis or high-temperature calcination (for example: CN114906874B, CN108815137B). Therefore, there are environmental impacts, as well as requirements and challenges in terms of efficiency, phase control, microstructure control, doping uniformity, cost and thermal stress. And, more importantly, currently used HfO2 in clinical practice is administered by intratumoral injection (only enters the tumor tissue, not into cells), and the therapeutic effect is limited (especially for deep tumors). Although there are reports on the preparation of hafnium oxide + PEG, realizing intravenous administration of hafnium oxide, it still has limitations such as the need for high-temperature reactions during preparation and poor tumor targeting during application.

[0004] Therefore, developing new hafnium-based complex nanodrugs with long circulation and passive targeting effects, especially green and environmentally friendly nanodrugs that can efficiently target deep tumor cells and have high bioavailability, is of great significance for enhancing the effect of tumor radiotherapy and reducing side effects. Summary of the Invention

[0005] Currently, the radiotherapy sensitization achieved by using hafnium oxide in this field has problems such as the need for high-temperature preparation and the use of intratumoral injection for drug delivery with unsatisfactory therapeutic effects. Aiming at the problems of the existing technology, the purpose of the present invention is to provide a hafnium-based complex protein nanoparticle for radiotherapy sensitization, its preparation method and application. Hafnium metaphosphate albumin nanoparticles, namely Hf(PO3)4@HSA, are prepared by biomineralization within the albumin cavity under a very low pH environment. Its preparation process is simple, the particle size is small and uniform, and it has been confirmed by cell and animal experiments to have good tumor targeting and anti-tumor effects.

[0006] The present invention adopts the following technical solutions: A hafnium-based complex protein nanoparticle for radiotherapy sensitization, comprising a hafnium-based complex and a protein.

[0007] Furthermore, the hafnium-based complex is located within the protein, that is, the hafnium-based complex protein nanoparticle of the present invention comprises an internal hafnium-based complex and its outer protein material.

[0008] In the present invention, the hafnium-based complex precipitates and mineralizes within the protein cavity to form a mineralized structure, consisting of the internal hafnium-based complex and its outer protein material, resulting in hafnium-based complex protein nanoparticles. These nanoparticles can be used for radiosensitization, possess the characteristics of biodegradability in vivo and good tumor targeting ability, can promote the enrichment of the hafnium-based complex in tumor tissues and its entry into tumor cells, and when combined with radiotherapy, significantly improve the effect of inhibiting tumor growth.

[0009] In the hafnium-based complex of the present invention, the ligand ions include anions, including inorganic anions and organic anions, such as one or several of halogen ions, phosphate ions, metaphosphate ions, and organic acid radicals.

[0010] The present invention discloses a method for preparing the above-mentioned hafnium-based complex protein nanoparticles for radiotherapy sensitization, which includes the following steps: precipitating and mineralizing hafnium ions and ligands within the protein to obtain the above-mentioned hafnium-based complex protein nanoparticles for radiotherapy sensitization.

[0011] In the present invention, a hafnium ion solution, a protein solution, and a ligand solution are mixed to achieve the precipitation and mineralization of hafnium ions and ligands within the protein, resulting in the above-mentioned hafnium-based complex protein nanoparticles for radiotherapy sensitization.

[0012] Preferably, the hafnium ion solution and the protein solution are mixed first, and then the ligand solution is added to achieve the precipitation and mineralization of hafnium ions and ligands within the protein, resulting in the above-mentioned hafnium-based complex protein nanoparticles for radiotherapy sensitization.

[0013] In the present invention, all the solutions are aqueous solutions, that is, an aqueous hafnium ion solution, an aqueous protein solution, and an aqueous ligand solution are mixed to achieve the precipitation and mineralization of hafnium ions and ligands within the protein, resulting in the above-mentioned hafnium-based complex protein nanoparticles for radiotherapy sensitization.

[0014] In the present invention, the pH of the aqueous hafnium ion solution is 2 - 3, the pH of the aqueous protein solution is 6 - 7, and the pH of the aqueous ligand solution is 7 - 8.

[0015] In the present invention, after stirring and mixing the hafnium ion solution (pH 2 - 3) and the protein solution (pH 6 - 7) at room temperature, the solution remains clear (pH 2 - 4). After adding the ligand solution (pH 7 - 8) to the mixed solution, the solution becomes turbid. Continuing to add the ligand solution until the solution becomes clear, the hafnium ions and ligand ions precipitate within the protein cavity, namely biomineralization, to obtain the hafnium-based complex protein nanoparticles for radiotherapy sensitization.

[0016] In the present invention, the concentration of the protein solution is 2 - 30 mg / mL; in the mixed solution of the hafnium ion solution and the protein solution, the concentration of hafnium ions is 0.001 - 0.10 mol / L; the molar ratio of hafnium ions to ligands is 1∶(10 - 100); and the solvent is water in all cases.

[0017] In the present invention, the hafnium ion solution prepared at room temperature has a pH of 2 - 3 (such a low pH has not been seen in the metal elements known for preparing protein nanoparticles), and the protein solution has a pH of 6 - 7. After mixing the two, a clear solution (pH 2 - 4) is obtained, and the protein still has a spatial structure that can undergo biomineralization, which is very important. Mixing it with the ligand solution (pH 7 - 8) by stirring enables the precipitation and mineralization of hafnium ions and ligand ions in the protein cavity, obtaining the hafnium-based complex protein nanoparticles for radiotherapy sensitization as described above.

[0018] In the present invention, the preparation temperature is all at room temperature, and the solutions are all aqueous solutions with a certain pH value.

[0019] The present invention uses protein as a molecular reactor, and the reactions are all carried out under room temperature conditions. Its uniqueness lies in that the pH of the hafnium ion solution is very low (pH 2 - 3), and the pH after mixing with the protein solution is also very low (pH 2 - 4), while it is necessary to ensure that the protein does not denature. After adding the ligand solution, the protein cavity can be utilized to make the solution of hafnium-based complex nanoparticles turbid. Continuing to add the ligand solution can adjust the solution to be clear (at this time, the pH is 7 or above), and nanoparticles are prepared. In addition, the preparation method of hafnium oxide requires high temperature conditions (the temperature is above 100 °C, resulting in protein denaturation and making it difficult to obtain protein nanoparticles). The preparation of the hafnium-based albumin nanoparticles in the present invention is all completed at room temperature. Even when the reaction temperature is 55 °C or higher, the nanoparticle system disintegrates. The present invention uses aqueous solutions of relevant substances with different pH values to enable albumin to play a role in a very low pH environment, and prepares hafnium-based complex protein nanoparticles with a high drug loading, breaking through the dilemma that the existing methods cannot obtain protein nanoparticles encapsulating hafnium elements.

[0020] The drug loading of the hafnium-based complex protein nanoparticles obtained in the present invention is 1% - 50%. The drug loading refers to the mass of hafnium-based complex / (the mass of hafnium-based complex + the mass of protein) × 100% in the purified nanoparticle sample.

[0021] The hafnium-based complex protein nanoparticles described in the present invention have good safety. Using PBS as a reference, the cytotoxicity of nanoparticles with different concentrations on normal cells human embryonic kidney cells 293T was investigated, and the experimental results showed that the cell viability exceeded 87%.

[0022] As an example, the present invention provides a method for preparing the above-mentioned hafnium-based complex protein nanoparticles, including the following steps: (1) Dissolve the hafnium compound in water at room temperature to obtain an aqueous solution of hafnium ions (pH 2 - 3), and then stir and mix it with an aqueous solution of protein (pH 6 - 7) to obtain an aqueous solution (pH 2 - 4) in which hafnium ions can freely enter the protein cavity in large quantities; (2) Add an aqueous ligand solution (pH 7 - 8). The solution becomes turbid. Continue to add the ligand solution until the solution becomes clear (at this time, pH ≥ 7). React at room temperature for 0 - 4 hours, preferably 0.5 - 4 hours, to allow the ligand ions to coordinate with hafnium ions inside the protein cavity to form a precipitate, i.e., biomineralization, to obtain hafnium-based complex protein nanoparticles.

[0023] The hafnium-based complex protein nanoparticles of the present invention can be formulated into conventional preparations, such as injections and their lyophilized products, and can also be formulated into various semi-solid preparations, solid preparations, etc. The preparation of specific preparations is a conventional technique.

[0024] The present invention discloses a freeze-dried powder of hafnium-based protein nanoparticles, which is prepared from the above-mentioned hafnium-based complex protein nanoparticles.

[0025] In the present invention, the aqueous hafnium ion solution is an aqueous solution of a water-soluble hafnium compound, which is prepared from a water-soluble hafnium compound in an aqueous solution; the water-soluble hafnium compound includes one or more of hafnium tetrachloride, hafnium(IV) bis(diethylamino)tetrachloride, hafnium iodide, and hafnium acetylacetonate; the concentration of hafnium ions is 1 - 20 mmol / L.

[0026] In the present invention, the protein includes one or more of human serum albumin, bovine serum albumin, silk fibroin, transferrin, and hemoglobin.

[0027] In the present invention, the ligand includes a compound capable of providing ligand ions that form a hafnium-based complex with hafnium ions, including inorganic compounds and organic compounds, such as halides, phosphoric acid compounds, metaphosphoric acid compounds, oxalic acid compounds, etc. For the hafnium-based complex protein nanoparticles of the present invention, the compound encapsulated in the nanoparticles, i.e., the hafnium-based complex, can have hexametaphosphate provided by sodium hexametaphosphate as the anion as the ligand, and also includes one or more anions provided by sodium chloride, potassium chloride, sodium oxalate, sodium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium metaphosphate, sodium phosphate, etc. as the ligand.

[0028] In the present invention, the concentration of the protein solution is 2 - 30 mg / mL; preferably, the concentration of the protein solution is 10 - 30 mg / mL.

[0029] In the present invention, in the mixed solution of the hafnium ion solution and the protein solution, the concentration of hafnium ions is 0.001 - 0.10 mol / L, preferably 0.01 - 0.02 mol / L.

[0030] In the present invention, the molar ratio of hafnium ions to the ligand is 1∶(10 - 100), limited by the solution turning from turbid to clear.

[0031] The present invention discloses an anti-tumor drug, comprising the above-mentioned hafnium-based complex protein nanoparticles or freeze-dried powder of hafnium-based protein nanoparticles.

[0032] The present invention discloses the application of the above-mentioned hafnium-based complex protein nanoparticles or freeze-dried powder of hafnium-based protein nanoparticles in the preparation of anti-tumor drugs.

[0033] The present invention discloses the application of the above-mentioned hafnium-based complex protein nanoparticles or freeze-dried powder of hafnium-based protein nanoparticles in the preparation of a reagent for improving the anti-tumor treatment effect; the reagent can be an adjuvant drug.

[0034] The present invention discloses the application of the above-mentioned hafnium-based complex protein nanoparticles or freeze-dried powder of hafnium-based protein nanoparticles in the preparation of a reagent for improving the effect of tumor radiotherapy; the reagent can be an adjuvant drug.

[0035] Using water as a solvent and adopting a room-temperature preparation method, the present invention successfully prepares hafnium-based complex protein nanoparticles. The nanoparticles are evenly dispersed in an aqueous solution, and the cytotoxicity experiment on normal human embryonic kidney cells 293T shows that the nanoparticles have good biosafety.

[0036] Currently, it is difficult to achieve passive targeting drug delivery by the intratumoral administration method of hafnium oxide in clinical practice. The present invention solves the problems such as unsatisfactory treatment effect by using the intratumoral injection administration method. The passive targeting drug delivery system can utilize the EPR effect to increase the drug concentration in the target area, improve the drug solubility and stability, has the advantages of long-circulation effect and promoting the accumulation of drugs in tumor tissues and tumor cells, and has become an effective strategy in the field of drug delivery.

[0037] After the hafnium-based complex protein nanoparticles of the present invention were combined with irradiation to treat tumor-bearing mice, the toxicity increased with the increase of irradiation dose, and the toxicity was significantly improved compared with the single irradiation group. The present invention stirred hafnium ions (pH 2-3) into the protein cavity at room temperature, the solution was clear (pH 2-4), and the ligand solution (pH7-8) was added and stirred, mineralization began, the solution was turbid, and the ligand solution was continued to be added and stirred until the solution was clear (pH ≥ 7) to obtain nanoparticles, which solved the problem that hafnium-based drugs had poor lipid solubility and were difficult to effectively encapsulate, and also pioneered the preparation of protein nanoparticles by biomineralization in a low pH environment to maintain the protein cavity structure. The present invention discloses for the first time a method for preparing hafnium protein nanoparticles, realizes the regulation of its particle size, drug release behavior and other physical and chemical properties, and obtains hafnium-based complex protein nanoparticles with no or little release under physiological conditions, tumor targeting and intracellular release. Its significant advantages are ingenious design, green and environmentally friendly preparation process, uniform size, stable particle size, good water dispersibility, good biocompatibility, high tumor targeting, etc., laying the foundation for efficient tumor radiation therapy. Therefore, the present invention solves the problem of cumbersome preparation of hafnium oxide used for sensitization in the prior art, overcomes the technical prejudice of the prior art that hafnium needs to be used in the form of hafnium oxide for tumor radiation (sensitization) therapy, and especially solves the problem that the existing hafnium oxide used for sensitization needs to be injected into the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The electron microscope images of the mixed solution of hafnium-based complex protein nanoparticles and hafnium oxide in Examples 1 to 4 and 6 are shown.

[0039] Figure 2 This is the X-ray diffraction pattern of the hafnium-based complex protein nanoparticles in Example 1.

[0040] Figure 3 This is the X-ray photoelectron spectrum of the hafnium-based complex protein nanoparticles in Example 1.

[0041] Figure 4 Observation results of the stability (presence or absence of precipitation) of the hafnium-based complex protein nanoparticle solution and the hafnium oxide protein mixture after dilution with PBS.

[0042] Figure 5 This is a comparison chart of the in vitro ROS generation levels of the hafnium-based complex protein nanoparticles in Example 1, the nanoparticles in the hafnium-based complex protein in Example 2, and the mixed solution in Example 5.

[0043] Figure 6 This is the result of investigating the cytotoxicity of the hafnium-based complex protein nanoparticles combined with X-ray irradiation in Example 1 (MOC 1 cell survival rate graph).

[0044] Figure 7Comparison of the tissue distribution results of hafnium-based complex protein nanoparticles in Example 1 and the hafnium oxide mixture in Example 6 (1) in C57 tumor-bearing mice.

[0045] Figure 8 In Example 1, the tumor inhibition curve and survival rate curve of C57 tumor-bearing mice by hafnium-based complex protein nanoparticles injected intravenously and the hafnium oxide-protein mixture in Example 6 (3).

[0046] Figure 9 This is the experiment to improve protein modification in the reaction system of the present invention.

[0047] Figure 10 This is the experiment of mixing hafnium ions and ligands. Detailed implementation mode

[0048] In the prior art, there is no report on directly encapsulating hafnium-based complexes with proteins. Due to the difficulty in significantly improving the solubility, tumor targeting effect and curative effect of hafnium-based complexes; hafnium oxide cannot be used to prepare protein hafnium oxide nanoparticles. The hafnium-based complex protein nanoparticles provided by the present invention have the characteristics of biodegradability in vivo and good tumor targeting, can promote the enrichment of hafnium-based complexes in tumor tissues and enter tumor cells, and the effect of inhibiting tumor growth is significantly improved when combined with radiotherapy, and the safety is good.

[0049] In the present invention, proteins such as human serum albumin, bovine serum albumin, silk fibroin, transferrin, hemoglobin, etc. are carrier materials with good biocompatibility and can all be used. Usually, proteins are prone to denaturation in a very low pH environment. The proteins in the aqueous solution of the present invention, after being mixed with the aqueous solution of hafnium ions with pH 2-3, can maintain their spatial structure within a certain period of time, and successfully enable the coordination precipitation reaction between hafnium ions and ligand ions in their cavities, obtaining hafnium-based complex protein nanoparticles, thus solving the problem that hafnium-based complexes have poor solubility and are difficult to be effectively encapsulated by proteins. This technological progress overcomes the prejudice of the prior art and is unpredictable.

[0050] The preparation method of the hafnium-based complex protein nanoparticles of the present invention is as follows: (1) At room temperature, hafnium tetrachloride is dissolved in water to obtain an aqueous solution of hafnium ions (pH 2-3), and then it is stirred and mixed with an aqueous solution of protein (pH 6-7). The solution becomes clear (pH 2-4), obtaining an aqueous solution in which hafnium ions can freely enter the protein cavity in large quantities; (2) After adding an aqueous solution of ligand (pH 7-8) under stirring at room temperature, the reaction system becomes turbid. Continue to add the ligand solution until the solution becomes clear (pH≥7), and carry out a conventional stirring reaction to enable the ligand ions and hafnium ions to undergo a coordination reaction in the protein cavity to form a precipitate, obtaining hafnium-based complex protein nanoparticles.

[0051] In the above step (1), the obtained hafnium ion aqueous solution has a pH of 2-3, and the hafnium ion and protein mixture has a pH of 2-4. The pH of the two solutions does not need to be adjusted, and the operation is carried out according to the description to obtain the product. In step (2), a ligand solution (pH 7-8) is added until the solution becomes clear. The reaction temperature is 0-25 °C, and the time is 0.5-4 hours, preferably 0.5-3 hours. The ligand ions and hafnium ions undergo a coordination reaction in the protein cavity to form a precipitate, and hafnium-based complex protein nanoparticles are obtained. Preferably, the ligand ions and hafnium ions undergo a coordination reaction in the protein cavity to form a precipitate, and then the product is purified, for example, by centrifugation in an ultrafiltration tube. Among them, the rotation speed of the centrifugation treatment is 1500-3000 rpm.

[0052] The hafnium-based complex protein nanoparticles of the present invention can be made into an injection, and can also be further freeze-dried into a freeze-dried powder injection, and made into dosage forms such as semi-solid preparations and solid preparations.

[0053] In the present invention, the cryoprotectants used in freeze-drying include one or more of mannitol, glucose, sucrose, lactose, etc.

[0054] The following will further describe in detail the specific embodiments of the present invention with reference to the drawings and examples. The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. The raw materials used in the experiments are all commercially available products, and the specific preparation operations and performance tests are all conventional technologies.

[0055] Example 1 Preparation of Hafnium-Based Complex Protein Nanoparticles The present invention discloses a hafnium-based complex protein nanoparticle for radiotherapy sensitization. The nanoparticle is composed of an internal hafnium-based complex and its outer protein material, with a size of 1-100 nm. The nanoparticle can absorb X-ray energy and generate free radicals after contacting with water or oxygen and be activated, and can convert the oxygen around it into free radicals, thereby causing irreversible damage to cells.

[0056] The preparation method of the hafnium-based complex protein nanoparticle for radiotherapy sensitization of the present invention includes the following steps: after mixing a hafnium compound solution (pH 2-3) and a protein solution (pH 6-7) evenly, a ligand solution (pH 7-8) is added until the solution becomes clear (pH≥7), and ultrafiltration centrifugation is carried out to obtain a solution of hafnium-based complex protein nanoparticles.

[0057] Among them, the hafnium compounds used include but are not limited to hafnium tetrachloride, hafnium(IV) tetra(diethylamino), hafnium iodide, and hafnium acetylacetonate, and their solubility is 1-20 mmol / L.

[0058] Among them, the proteins used include but are not limited to human serum albumin, bovine serum albumin, silk fibroin, transferrin, and hemoglobin, and their concentration is 2-30 mg / mL.

[0059] Among them, for the compound encapsulated in the nanoparticles, the hexametaphosphate provided by sodium hexametaphosphate can be used as the anion (as a ligand), and it also includes, but is not limited to, anions provided by sodium chloride, potassium chloride, sodium oxalate, sodium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium metaphosphate, sodium phosphate, etc., and the concentration thereof is 0.5 - 1 mol / L.

[0060] Among them, the molar ratio of the hafnium compound solution to the ligand solution is 1:(10 - 100), based on the clarity of the solution.

[0061] Among them, the temperature of the reaction is 0 - 25 °C, and the reaction time is 0.4 - 4 hr.

[0062] Among them, during ultrafiltration centrifugation, the molecular weight cut-off is 3.5 - 50 kD, the rotation speed of ultrafiltration centrifugation is 1500 - 3000 r / min, and the number of ultrafiltration centrifugation times is 1 - 10 times.

[0063] The hafnium-based complex protein nanoparticles prepared by the present invention can be made into injection solutions or freeze-dried powder injections, and further made into dosage forms such as semi-solid preparations and solid preparations. They can be used in tumor treatment methods such as intratumoral injection and tail vein injection.

[0064] In particular, the hafnium-based complex protein nanoparticles obtained by the present invention have a radiotherapy sensitization effect and can be applied in tumor radiotherapy.

[0065] The specific steps are as follows: (1) Preparation of hafnium ion aqueous solution: At room temperature, weigh hafnium tetrachloride solid powder and dissolve it in water to prepare a hafnium ion aqueous solution with a concentration of 10 mM (pH 3); (2) Preparation of hafnium-based protein nanoparticles: At room temperature, take 10 mM hafnium ion aqueous solution and 10 mg / mL human serum albumin (HSA) aqueous solution (pH 7), mix and stir them in a volume ratio of 1:1. After the solution becomes clear (pH 3), add 0.5 M sodium hexametaphosphate aqueous solution (pH 7) to start mineralization. The solution becomes turbid. Continue to add the ligand solution and stir until the solution becomes clear (measured pH = 7). Continue to stir and react at 25 °C for 1 hr, and the solution remains clear. Add the obtained solution into an ultrafiltration tube, centrifuge at 2500 rpm, 10 min / time, for a total of 3 times, to remove impurities such as hafnium ions, sodium ions, and chloride ions, and obtain a solution of hafnium-based complex protein nanoparticles of the present invention, that is, Hf(PO3)4@HSA.

[0066] Perform X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analyses on the prepared hafnium-based protein nanoparticles Hf(PO3)4@HSA. The XRD results are as Figure 2, it can be seen that the peak shapes of the components of hafnium-based protein nanoparticles mainly show characteristics such as no obvious diffraction peak, broad peak (bread-like peak), non-orientation, and low peak intensity, all of which are amorphous structures, proving the rapid reaction of hafnium-based complexes within the protein cavity to form precipitates. As a control, when the reaction temperature is 55 °C or higher, the nanoparticle system disintegrates to form a gel-like precipitate, and Hf(PO3)4@HSA nanoparticles cannot be obtained.

[0067] The XPS results are as Figure 3 . It can be seen that compared with the Hf(PO3)4 precipitate without protein, the valence states of hafnium element and phosphorus element forming complexes with it in Hf(PO3)4@HSA nanoparticles have not changed, proving that the reaction conditions are mild during the preparation of nanoparticles and the element valence states have not been altered.

[0068] Using mannitol as a protective agent, the solution of hafnium-based complex protein nanoparticles was freeze-dried to obtain the freeze-dried powder of hafnium-based protein nanoparticles. The specific method is as follows: The sample solution was pre-frozen at -80 °C for 10 hr, transferred to a freeze-dryer with a cold trap temperature reduced to -20 °C, with a vacuum degree of 10 Pa, dried for 12 hr, and then gradually heated to 30 °C and continued to be dried for 2 hr to obtain the product.

[0069] The solution of hafnium-based complex protein nanoparticles was photographed by transmission electron microscopy, and the results are as Figure 1 a. It can be seen that the prepared nanoparticles are uniformly dispersed and have a uniform particle size. The average particle size obtained from TEM image statistics is (11.94 ± 3.28) nm, the hydrated particle size measured by a particle size analyzer is 20.78 ± 7.07 nm, and the PDI is 0.112. In addition, the drug loading of the nanoparticles was measured to be 10.73%.

[0070] Example 2 In this example, the reaction time for preparing nanoparticles is different from 1 hr in Example 1, and the other conditions are the same. The specific reaction times are 0 min, 30 min, and 4 hr respectively. The prepared nanoparticles were photographed by transmission electron microscopy, and the results are shown in Table 1, Figure 1 b: When the reaction just becomes clear (0 min), the mineralization reaction is nearly completed, there is no precipitate, but the nanoparticle shape is not uniform; when the reaction time is 4 hours, uniform nanoparticles can be formed, but there will be a precipitate after the reaction is completed, presenting a flocculent shape.

[0071] Table 1 Results of preparing hafnium-based complex protein nanoparticles at different reaction times

[0072] Example 3 The protein used to prepare the nanoparticles in this example is different from that in Example 1 (using HSA), and the other conditions are the same. The proteins are: silk fibroin, bovine serum albumin, and transferrin. The prepared nanoparticles were photographed by transmission electron microscopy, and the results are shown in Table 2, Figure 1 c, indicating that different types of proteins can be used as carriers to encapsulate hafnium-based complexes, with wide applicability.

[0073] Table 2 Results of preparing hafnium-based complex protein nanoparticles with different proteins

[0074] Example 4 The concentration of HSA used to prepare the nanoparticles in this example is different from that in Example 1 (10 mg / mL), and the other conditions are the same. The specific concentrations are 3 mg / mL, 5 mg / mL, 20 mg / mL, and 30 mg / mL respectively. The prepared nanoparticles were photographed by transmission electron microscopy, and the results are shown in Table 3, Figure 1 d. As the protein concentration increases, the difficulty of ultrafiltration treatment of the prepared nanoparticle solution increases (difficult to ultrafilter), but it has little effect on the actual size of the nanoparticles. In addition, when the concentration is low (3 mg / mL), the reaction system is clear when the feeding is completed, and a large amount of precipitation appears in the system after 10 minutes of stirring, and nanoparticles cannot be formed. When the protein concentration is 5 mg / mL, nanoparticles can be formed, but there will be a small amount of precipitation after the reaction is completed. When higher protein concentrations are used in the experiment, nanoparticles can be prepared. The present invention preferably uses a 10 mg / mL protein solution that is economical and easy to operate (already presented in Example 1).

[0075] Table 3 Results of preparing hafnium-based complex protein nanoparticles with different concentrations of HSA

[0076] Preparation and characterization of physically adsorbed hafnium oxide-protein mixture and characterization of commercially available hafnium oxide After diluting commercially available hafnium oxide with deionized water, its size was measured to be about 109.62 ± 18.67 nm by transmission electron microscopy, as shown in Figure 1 f.

[0077] Since free Hf 4+ cannot exist in a normal physiological environment and is prone to forming hafnium hydroxide precipitation, it cannot be used as a reference for experiments. Therefore, a traditional physical adsorption method was used to prepare a hafnium oxide-protein mixture (HfO2-HAS, for comparison), and the specific steps are as follows: 1 mg of hafnium oxide was dispersed in water and mixed with an aqueous solution of 10 mg / mL human serum albumin (HSA) in a volume ratio of 1:1. After stirring for 1 hour, it remained a precipitate. It was washed with deionized water to obtain a physically adsorbed hafnium oxide - protein mixture. However, the hafnium oxide - protein mixture was unstable and prone to precipitation in the experiment. The particle size was obtained as 146.43 ± 20.87 nm by TEM image statistics. The electron microscope size is shown in Figure 1 e. After diluting the hafnium oxide - protein mixture with PBS, it was used as a positive control sample for in - vivo distribution in mice and intravenous injection experiments in mouse tail veins.

[0078] Example 5 Investigation of the stability and ROS - generating ability of hafnium - based complex protein nanoparticles 1. For the Hf(PO3)4@HSA nanoparticles prepared in Example 1 and the HfO2 - HSA mixture prepared in the comparative example, an in - vitro stability comparison was carried out as follows: The nanoparticles in Example 1 and the mixture prepared in the comparative example were respectively diluted with deionized water to 1 mg / mL (calculated based on the Hf 4+ concentration). Equal volumes of PBS (pH = 7) were added respectively, and after mixing, they were centrifuged (3000 r / min, 3 min) to observe whether there was precipitation in both. The results are as Figure 4 shown. It can be seen that there was no precipitation in the nanoparticle solution prepared in Example 1 (good stability). However, the HfO2 - HSA mixture precipitated in PBS.

[0079] 2. For the Hf(PO3)4@HSA nanoparticles prepared in Example 1 and the Hf(PO3)4@HSA nanoparticles prepared at different reaction times in Example 2, an in - vitro ROS generation comparison was carried out as follows: The nanoparticles in Example 1 and Example 2 were respectively diluted with deionized water to 1 mg / mL (calculated based on the Hf 4+ concentration). Respectively, 4.95 mL of hafnium - based protein nanoparticles and the mixture were taken, and 0.05 mL of a ROS probe (1,3 - diphenylbenzofuran, DPBF) with a concentration of 3 mM was added thereto in the dark. They were divided into two groups for comparison before and after irradiation. The irradiation group was irradiated with 6 Gy of X - rays, and the whole process was carried out in the dark. After the irradiation was completed, the absorbance of each group of nanoparticles at a UV wavelength of 420 nm was recorded. The results are shown in Table 4, Figure 5 .

[0080] Table 4 Comparison results of in - vitro ROS generation of nanoparticles in Example 1 and Example 2

[0081] It can be seen that the ROS yields of the nanoparticles prepared with different preparation reaction times are different, resulting in different degrees of DPBF reduction. Among them, the degree of DPBF reduction of the hafnium-based protein nanoparticles prepared with a reaction time of 60 min in Example 1 is the largest (41.88%), indicating that its ROS production is the largest.

[0082] Example 6 Investigation of the cytotoxicity of the hafnium-based protein nanoparticles in Example 1 against normal cells Take human embryonic kidney cells 293 T cells in the logarithmic growth phase and seed them in each well of a 96-well plate at an inoculation density of 5×10 3 / mL, 100 μL per well. Incubate in a cell culture incubator at a constant temperature for 12 h. After ensuring that the cells are adherent, pour out the culture medium, wash 1-2 times with PBS (pH 7), and add the hafnium-based protein nanoparticle solution prepared with the culture medium, 100 μL per well. The nanoparticle concentrations (calculated based on hafnium element) are 0 (PBS, reference), 0.2, 0.4, 0.6, 0.8, 1.0 mM respectively, with 5 replicate wells for each concentration; after culturing in the incubator for 12 h, change the culture medium; add 100 μL of the culture medium solution containing 5 mg / mL MTT, discard the culture medium after 4 h, add 100 μL of DMSO, shake for 10 minutes, and measure the absorbance value at 490 nm with an enzyme-linked immunosorbent assay reader. The results are shown in Table 5.

[0083] It can be seen that under non-irradiated conditions, when the concentration of the hafnium-based protein nanoparticles is ≤0.20 mM, it has no effect on the cell viability, that is, it has good biosafety.

[0084] Table 5 Effects of nanoparticles at different concentrations on the viability of 293 T cells (n = 5)

[0085] Example 7 Investigation of the cytotoxicity of the hafnium-based protein nanoparticles prepared in Example 1 combined with X-ray irradiation against tumor cells Take mouse oral squamous carcinoma MOC 1 cells in the logarithmic growth phase and seed them in each well of a 48-well plate at an inoculation density of 1×10 4 / mL, 200 μL per well. Incubate in a cell culture incubator at a constant temperature for 12 hr. After ensuring cell adhesion, discard the culture medium, wash once with PBS, and add the hafnium-based protein nanoparticle solution prepared with the culture medium, 200 μL per well. The concentration of the nanoparticles (calculated based on hafnium element) is 0.10 mM for all (lower than the safe concentration of 0.20 mM for normal cytotoxicity investigation); after incubating in the incubator for 12 hr, change the culture medium and irradiate the cells. The irradiation doses are 0, 2, 4, 6, 8, and 12 Gy respectively; there are 4 replicate wells for each irradiation intensity. Incubate for 12 hr after irradiation; add 100 μL of the culture medium solution containing MTT at a concentration of 5 mg / mL, discard the culture medium after 4 hr, add 100 μL of DMSO, shake for 10 minutes, and measure the absorbance value at 490 nm with an enzyme-linked immunosorbent assay reader. The results are as Figure 6 , Table 6.

[0086] It can be seen that under non-irradiated conditions, the toxicity of these nanoparticles to the MOC 1 cell line is relatively low (the cell survival rate is 93%, with the cell survival rate of PBS as the benchmark). Under different irradiation conditions, the cell survival rate decreases as the irradiation intensity increases. For nanoparticles with the same concentration, the irradiation lethality is improved synchronously. In the experimental irradiation intensity range of 2 - 12 Gy, nanoparticles with the same concentration can increase the irradiation-induced cell lethality by 3.2 - 29.8%.

[0087] Table 6 MOC 1 cell survival rate under different conditions and the percentage of improvement in irradiation lethality by nanoparticles (n = 4)

[0088] Note: (1) The concentration of the hafnium-based protein nanoparticles in the above item ② is 0.1 mM for all; (2) In the prior art, the irradiation dose for cells is generally 2 - 8 Gy.

[0089] Example Eight Investigation of the in vivo distribution of the hafnium-based complex protein nanoparticles and hafnium oxide mixture (HfO2-HSA) in Example One Perform an in vivo tissue distribution experiment on mice with the hafnium-based complex protein nanoparticles and hafnium oxide mixture prepared in Example One. The specific steps are as follows: (1) Establishment of a tumor model: Culture MOC 1 tumor cells. When they are in the logarithmic growth phase, digest them with trypsin and dilute them with PBS to prepare a cell suspension of 1×10 7 cells / mL, ensuring uniform cell dispersion. Inoculate the C57 mice in the right hind leg with this cell suspension, 50 μL subcutaneously injected into each mouse, and observe the tumor volume size every day. Tumor volume formula: Tumor volume = (length × width 2 ) / 2; (2)The hafnium-based complex protein nanoparticles and hafnium oxide mixture in Comparative Example 1 were injected into the tail veins of two groups of tumor-bearing C57 mice (the dose of hafnium element was 10 mg / Kg, diluted with normal saline). After injection, at 0, 2, 6, 12, 24, and 48 h time points, the tumor sites of the mice were removed, and high-temperature digestion (300 °C) was carried out with aqua regia. After the tissue digestion (the tissue solution became clear), it was fixed to 8.0 mL, filtered through a 0.22-μm microporous filter membrane, and the hafnium element in each tissue was quantified by ICP-OES. The distribution results of the hafnium content in each tissue of the mice obtained by the above measurement method are as Figure 7 , Table 7.

[0090] As can be seen from Table 7, for the distribution of hafnium element, both the hafnium-based complex protein nanoparticles and the hafnium oxide protein mixture had the highest levels at 12 h at the tumor site, which were 4.87% ID g -1 and 1.67% ID g -1 respectively. The drug accumulation at the tumor site was relatively increased by 65%. This indicates that the hafnium-based complex protein nanoparticles have better tumor targeting than the hafnium oxide protein mixture.

[0091] Table 7 Distribution rate of hafnium element at different time points at the tumor site (ID g -1 )

[0092] Example 9 Investigation of the antitumor effect of tail vein injection of hafnium-based complex protein nanoparticles and hafnium oxide mixture (HfO2-HSA) in mice in Example 1 The antitumor effect of the hafnium-based protein nanoparticles described in Example 1 on oral squamous cell carcinoma in C57 mice was investigated in the following way: A C57 mouse tumor-bearing model was constructed by the method in Example 8. When the tumor grew to a volume of 50-100 mm 3 , tail vein injection was administered according to the following design (the dose of hafnium element was 20 mg / Kg in all cases, and the irradiation intensity was 6 Gy): 1) normal saline group; 2) X-ray irradiation group; 3) hafnium oxide protein mixture group; 4) hafnium-based protein nanoparticle group in Example 1; 5) hafnium oxide protein mixture + X-ray irradiation group; 6) hafnium-based protein nanoparticle + X-ray irradiation group in Example 1; among them, 6) was the experimental group, and the others were control groups. There were 5 tumor-bearing mice in each group. X-ray irradiation (one treatment) was carried out immediately after tail vein injection of the nanoparticles or mixture, and the treatment was carried out once every two days for a total of 3 times. The results are as Figure 8 .

[0093] The observed changes in the mouse tumors were as follows: When using normal saline as a benchmark for the growth of tumors in the MOC 1 tumor model, A) neither hafnium-based protein nanoparticles nor hafnium oxide protein mixture had an impact on tumor growth; B) compared with the hafnium-based protein nanoparticles + X-ray irradiation group and the hafnium oxide protein mixture + X-ray irradiation group, hafnium-based protein nanoparticles + X-ray irradiation had a more significant inhibitory effect on tumor growth. The specific tumor inhibition rates were: hafnium-based complex protein nanoparticles + X-ray irradiation, 70%; hafnium oxide protein mixture + X-ray irradiation, 31%. Moreover, during the 30-day observation period, there was little impact on the survival status of the mice (no deaths occurred), indicating that the hafnium-based protein nanoparticles described in the present invention combined with X-rays can effectively inhibit the growth of MOC 1 tumors, and the tumor inhibition effect is 2.25 times that of the hafnium oxide protein mixture combined with X-rays. This confirmed that the present application achieved the design goal of long-circulating nanoparticles with passive targeting, laying a foundation for further developing them into radiotherapy-assisted nano-drugs with high efficiency in targeting deep tumor cells and high bioavailability.

[0094] Control Example Take two reagent bottles, add 10 mg / mL of HSA, 5 mL respectively, stir, and measure the pH to be 7; in the control group, add HCl, the solution becomes turbid, and the measured pH is 3. In the experimental group, add a Hf ion solution (10 mM, hafnium tetrachloride), the solution is clear, and the measured pH is 3; start adding sodium hexametaphosphate to both bottles. The system in the experimental group starts to become turbid. As the pH gradually changes from acidic to neutral, the system becomes clear, while the control remains turbid. See Figure 9 。

[0095] Take a reagent bottle, add 5 mL of Hf ion solution (10 mM), stir, add sodium hexametaphosphate until the pH is 7, and the system remains turbid. See Figure 10 。

[0096] The prior art believes that hafnium ions cannot exist stably, especially under physiological conditions, they are prone to form hafnium hydroxide, which is an obstacle to the mineralization preparation of protein nanoparticles. Therefore, the prior art uses hafnium oxide or its preparations as auxiliary drugs for radiotherapy sensitization. The present invention adopts a new technical idea, and under room temperature conditions, hafnium ions (pH 2-3) are stirred into the protein cavity, the solution is clarified (pH 2-4), a ligand solution (pH 7-8) is added and stirred, the solution becomes turbid, and the ligand solution is continuously added and stirred until the solution is clarified to obtain nanoparticles, which solves the problem that hafnium-based drugs have poor lipid solubility and are difficult to be effectively encapsulated, and also creates a precedent for preparing protein nanoparticles by biomineralization in a low pH environment while maintaining the protein cavity structure, and overcomes the technical prejudice of the prior art that proteins are easily denatured in a very low pH environment and are difficult to be used as cavities; in particular, it also avoids the limitations of high temperature reaction preparation and poor targeting required for hafnium oxide, and in particular, the hafnium-based complex protein nanoparticles prepared by the present invention have better tumor targeting than the best hafnium oxide currently used, and in particular, the present invention can be intravenously administered, and effectively solves the defect that the prior art hafnium oxide needs to be administered intratumorally.

Claims

1. A hafnium-based complex protein nanoparticle for radiotherapy sensitization, characterized in that: Includes hafnium-based complexes and proteins.

2. The hafnium-based complex protein nanoparticles for radiotherapy sensitization according to claim 1, characterized in that: The hafnium-based complex precipitates in the protein cavity to form mineralization; in the hafnium-based complex, the ligand ions include anions.

3. The hafnium-based complex protein nanoparticles for radiotherapy sensitization according to claim 2, characterized in that: The anions include one or more of inorganic anions and organic anions.

4. The method for preparing hafnium-based complex protein nanoparticles for radiotherapy sensitization according to claim 1, characterized in that: The method comprises the following steps: precipitating and mineralizing hafnium ions and ligands in proteins to obtain hafnium-based complex protein nanoparticles for radiotherapy sensitization.

5. The method for preparing hafnium-based complex protein nanoparticles for radiotherapy sensitization according to claim 4, characterized in that: The hafnium ion solution, protein solution and ligand solution are mixed to achieve precipitation and mineralization of hafnium ions and ligands in the protein, thereby obtaining hafnium-based complex protein nanoparticles for radiotherapy sensitization.

6. The method for preparing hafnium-based complex protein nanoparticles for radiotherapy sensitization according to claim 5, characterized in that: The concentration of the protein solution is 2-30 mg / mL; in the mixed solution of the hafnium ion solution and the protein solution, the concentration of the hafnium ion is 0.001-0.10 mol / L; the molar ratio of the hafnium ion to the ligand is 1:(10-100); in the solution, the solvent is water.

7. A hafnium-based protein nanoparticle freeze-dried powder prepared from the hafnium-based complex protein nanoparticles according to claim 1.

8. An anti-tumor drug comprising the hafnium-based complex protein nanoparticles or hafnium-based protein nanoparticle freeze-dried powder according to claim 1.

9. Use of the hafnium-based complex protein nanoparticles according to claim 1 or the hafnium-based protein nanoparticle freeze-dried powder according to claim 7 in the preparation of tumor therapeutic drugs.

10. Use of the hafnium-based complex protein nanoparticles according to claim 1 or the hafnium-based protein nanoparticle freeze-dried powder according to claim 7 in the preparation of an agent for improving the therapeutic effect of tumors.

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