A nano-diagnostic and therapeutic agent for inducing tumor calcification, and its preparation method and application

Abstract

The present invention discloses a nano-diagnostic and therapeutic agent that induces tumors to form calcification foci, as well as its preparation method and application. The method comprises: coupling FA to the surface of albumin to obtain FA-modified albumin; dissolving the FA-modified albumin in Dulbecco's medium, adding MnCl2 and CaCl2 for mineralization to obtain CMPF nanoparticles; loading CA onto the surface of the CMPF nanoparticles to obtain CMPCF nanoparticles. The CMPCF nanoparticles of the present invention can achieve tumor accumulation and improvement of treatment efficiency through FA receptor targeting strategy and TRPA1 specific activation strategy in cancer cells. After the CMPCF nanoparticles enter the tumor cells, they release Mn in the slightly acidic environment of the tumor. 2+ MRI can be used to diagnose in situ liver cancer with a diameter of less than 1 cm. CMPCF nanoparticles can induce tumor necrosis and form calcification foci by generating strong oxidative stress, which can achieve the diagnosis of in situ liver cancer with a diameter of more than 100 mm. 3 Calcification of solid tumors, and then non-invasive tumor efficacy evaluation through calcium-based CT imaging and tumor bioluminescence-based BL imaging.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nano-diagnostic and therapeutic agent for inducing the formation of calcification foci in tumors, and a preparation method and application thereof. Background Art

[0002] Tumor calcifications form when tumor cells are destroyed and inactivated, leading to the exudation of intracellular tissue fluid and the deposition of calcium salts. Clinical studies have shown that the formation of tumor calcifications indicates an improvement in the malignancy of the tumor and a gradual improvement in prognosis. Computed tomography (CT) can be used to image and locate these calcifications; larger calcification particles indicate a lower degree of malignancy.

[0003] Although after cell necrosis, free Ca 2+ Easily interacts with PO4 in cells 3- Phosphate precipitation forms, but inducing death of solid tumors and producing macroscopic calcification effects remain difficult. The strict limitations on the dosage of nanomaterials and the high osmotic pressure of solid tumors make the dose that enters the tumor tissue to exert drug activity very small.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a nano-diagnostic and therapeutic agent that induces tumor calcification foci, as well as its preparation method and application. The invention aims to achieve non-invasive diagnosis, visual treatment and efficacy evaluation of tumors by killing tumors and forming calcification foci in combination with multimodal molecular imaging.

[0006] The technical solutions of the present invention are as follows:

[0007] A method for preparing a nano-diagnostic and therapeutic agent for inducing the formation of calcification foci in tumors, comprising the following steps:

[0008] Folic acid (denoted as FA) was coupled to the albumin surface via a one-step carbodiimide coupling method to obtain folic acid-modified albumin;

[0009] The folic acid-modified albumin is dissolved in Dulbecco's medium, and MnCl2 and CaCl2 are added for in situ mineralization to obtain CMPF nanoparticles;

[0010] Through the hydrophobic-hydrophobic interaction between the hydrophobic cavity in albumin and cinnamaldehyde (denoted as CA), the cinnamaldehyde is loaded onto the surface of the CMPF nanoparticles to obtain CMPCF nanoparticles, i.e., the nano-diagnostic and therapeutic agent.

[0011] Optionally, the step of coupling folic acid to the albumin surface by a one-step carbodiimide coupling method to obtain folic acid-modified albumin specifically comprises:

[0012] Mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and a folic acid solution, stirring for a first predetermined time at room temperature in the dark, and then adding an N-hydroxysuccinimide solution to carry out a reaction;

[0013] The albumin solution is added to the reaction system, stirred for a second predetermined time, and then ultrafiltered and centrifuged to obtain the folic acid-modified albumin.

[0014] Optionally, in the FA-modified albumin, the feed mass ratio of the FA to the albumin is 0.1-1.

[0015] Optionally, the step of dissolving the FA-modified albumin in Dulbecco's medium, adding MnCl2 and CaCl2 for in situ mineralization to obtain CMPF nanoparticles specifically comprises:

[0016] The FA-modified albumin is dissolved in Dulbecco's medium, and then a MnCl2 solution is added. The mixture is mineralized at 25-37°C for 12-36 hours under sealed conditions. Subsequently, a CaCl2 solution is added, and the mineralization is continued at 25-37°C for 12-36 hours to obtain the CMPF nanoparticles.

[0017] Optionally, based on the system after the FA-modified albumin is dissolved in Dulbecco's medium, the concentration of the FA-modified albumin is 1 to 10 mg mL -1 ;

[0018] The concentration of the MnCl2 solution is 0.05-0.2M, the concentration of the CaCl2 solution is 0.5-2M, and the volumes of the MnCl2 solution and the CaCl2 solution added are the same.

[0019] Optionally, the step of loading the CA onto the surface of the CMPF nanoparticles through the hydrophobic-hydrophobic interaction between the hydrophobic cavity in albumin and CA to obtain CMPCF nanoparticles specifically includes:

[0020] The CMPF nanoparticles are dispersed in a CA solution and stirred at room temperature in the dark for a third predetermined time to obtain the CMPCF nanoparticles.

[0021] Optionally, in the CMPCF nanoparticles, the feeding mass ratio of the CA to the CMPF nanoparticles is 0.1-1.

[0022] Optionally, the drug loading amount of the CA in the CMPCF nanoparticles is 1% to 10.3%.

[0023] A nano-diagnostic and therapeutic agent for inducing tumors to form calcification foci is prepared using the preparation method of the nano-diagnostic and therapeutic agent for inducing tumors to form calcification foci described in the present invention.

[0024] A use of the nano-diagnostic and therapeutic agent for inducing tumor calcification formation according to the present invention in the preparation of drugs for treating tumors.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention utilizes the tumor microenvironment to regulate Mn in CMPCF 2+ The degradation of CMPCF NPs has enabled the diagnosis of in situ liver cancer with a diameter of less than 1 cm through magnetic resonance (MR) imaging. CMPCF NPs can release CA to induce mitochondria to produce a large amount of hydrogen peroxide (H2O2), manipulating the TRPA1 receptor from the "off" state to the "on" state, thereby inducing calcium influx into tumor cells. The rapidly released Ca in CMPCF NPs 2+ and extracellular influx of Ca 2+ This can lead to free Ca in cancer cells 2+ The concentration increases rapidly in a short period of time, forming a calcium overload, and H2O2 can be released by the synchronously released Mn 2+ Utilization, generating more biologically toxic hydroxyl radicals (·OH), thereby exacerbating cellular oxidative stress and necrosis. 2+ 、Mn 2+ and PO4 3- In the microenvironment, FA is used as a nucleating agent to form calcification foci, thereby achieving the goal of treating calcifications with a volume greater than 100 mm 3 Calcification of the tumor.

[0027] 2. The present invention utilizes FA targeting strategy and TRPA1-specific activation strategy in cancer cells to improve tumor accumulation efficiency. Fluorescence (FL) imaging based on CMPCF shows that its tumor enrichment efficiency is significantly improved compared with CMPC without FA modification.

[0028] 3. The present invention can evaluate the calcification treatment effect of CMPCF in real time and non-invasively through CT imaging based on tumor calcification foci and tumor bioluminescence (BL) imaging, where CT imaging is used to calculate the volume of calcification foci within cancer cells, and BL imaging is used to evaluate the distribution and malignancy of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart for the preparation of a nano-diagnostic and therapeutic agent that induces tumor calcification formation in Example 1, a schematic diagram for inducing tumor calcification, and a flow chart for its application in living mice.

[0030] Figure 2 This is a transmission electron microscopy image of CMPCF NPs obtained in Example 1.

[0031] Figure 3 This is the ultraviolet absorption diagram of CMPCF NPs in Example 1.

[0032] Figure 4 The encapsulation efficiency and drug loading of CA in the CMPCF NPs obtained in Example 1.

[0033] Figure 5 Thermogravimetric curves of CMPF and CMPCF NPs obtained in Example 1.

[0034] Figure 6 X-ray photoelectron spectra of Ca, Mn and P elements in CMPCF NPs obtained in Example 1.

[0035] Figure 7 Transmission electron microscopy images of CMPCF NPs obtained in Example 1 at pH 5.0, 6.5 and 7.4.

[0036] Figure 8 The CMPCF NPs obtained in Example 1 were degraded and released Ca at pH 5.0, 6.5 and 7.4. 2+ (a) and Mn 2+ (b) Quantization curve.

[0037] Figure 9 Mn in the CMPCF NPs obtained in Example 1 2+ UV absorption changes of methylene blue (MB) degradation catalyzed by H2O2 to produce ·OH.

[0038] Figure 10 The scanning electron microscope and elemental energy spectrum analysis images of the cancer cell morphology after the cancer cells were treated with CMPCF NPs obtained in Example 1.

[0039] Figure 11 This is a distribution diagram of calcification foci on the surface of the three-dimensional cell spheroids after the three-dimensional cell spheroids were treated with CMPCF NPs obtained in Example 1.

[0040] Figure 12 This is an analysis chart of the migration ability and cell number of cancer cells after the CMPCF NPs obtained in Example 1 treated cancer cells.

[0041] Figure 13 This is a metabolic analysis diagram of cellular oxygen consumption after cancer cells were treated with different nanoparticle groups obtained in Example 1 and Comparative Example 1.

[0042] Figure 14 These are the fluorescence semi-quantitative values ​​of the IR-800 probe enrichment efficiency of the different nanoparticle groups obtained in Example 1 and Comparative Example 1 in the treatment of Huh-7 tumors in vivo.

[0043] Figure 15 (a) Mouse MR imaging and (b) corresponding Mn images obtained by different nanoparticle groups obtained in Example 1 and Comparative Example 1 in the treatment of Huh-7 tumors in vivo 2+ T1 contrast gray value.

[0044] Figure 16 (a) Mouse BL imaging images and (b) corresponding chemiluminescence intensity semi-quantitative values ​​obtained by different nanoparticle groups obtained in Example 1 and Comparative Example 1 in the treatment of Huh-7 tumors in vivo.

[0045] Figure 17 (a) Mouse CT imaging and (b) corresponding calcification volume quantitative values ​​obtained by different nanoparticle groups obtained in Example 1 and Comparative Example 1 in the treatment of Huh-7 tumors in vivo.

[0046] Figure 18 This is a graph showing the tumor volume inhibition curves obtained by using different nanoparticle groups obtained in Example 1 and the comparative example in the treatment of Huh-7 tumor calcification in vivo.

[0047] Figure 19 This is a graph showing the survival curves of mice obtained by using different nanoparticle groups obtained in Example 1 and the comparative example in the treatment of Huh-7 tumor calcification in vivo. DETAILED DESCRIPTION

[0048] The present invention provides nanoparticles that induce tumor calcification, as well as methods for preparing and using the nanoparticles. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0049] After cell necrosis, free Ca 2+ Easily interacts with PO4 in cells 3- Phosphate is formed by precipitation, but it is still difficult to induce death of solid tumors and produce macroscopic calcification effects. The strict limitation of the dosage of nanomaterials and the high osmotic pressure of solid tumors make the dosage that enters the tumor tissue to exert drug activity very small. Therefore, it is necessary to take advantage of the characteristics of tumor cell metabolism to increase the components that form calcification foci. Folic acid (FA) and Ca 2+ FA is an abundant metabolic substrate in tumors, and the carboxyl groups on its surface are crucial for calcium salt nucleation. Therefore, this study leverages the metabolic characteristics of tumor-overexpressed neuronal redox-sensing calcium channels, TRPA1, and FA receptors to construct companion tumor nanotheranostics (CMPCF NPs) for multimodal molecular imaging-guided tumor diagnosis, visual therapy, and efficacy assessment.

[0050] Specifically, an embodiment of the present invention provides a method for preparing a nano-diagnostic and therapeutic agent that induces the formation of calcification foci in tumors, which comprises the following steps:

[0051] S1. Folic acid was coupled to the albumin surface by a one-step carbodiimide coupling method to obtain FA-modified albumin;

[0052] S2, dissolving FA-modified albumin (as a template) in Dulbecco's medium, adding MnCl2 and CaCl2 for in situ mineralization to obtain CMPF nanoparticles (denoted as CMPF NPs);

[0053] S3. CA is loaded onto the surface of the CMPF nanoparticles through the hydrophobic-hydrophobic interaction between the hydrophobic cavity in albumin and the TRPA1 activator cinnamaldehyde to obtain CMPCF nanoparticles (denoted as CMPCF NPs, i.e., the nano-diagnostic and therapeutic agent).

[0054] In this embodiment, the CMPCF NPs have the ability to target tumor FA and specifically activate TRPA1 in cancer cells, and have good degradation ability at weakly acidic pH (5.0-7.4). 2+ It can catalyze H2O2 in tumors to produce hydroxyl radicals (·OH). The CMPCF NPs can enter cells through tumor FA targeting, promote mitochondrial production of H2O2 by releasing CA, and thus specifically activate the tumor-overexpressed TRPA1 redox sensing channel from inside the cell, inducing the confluence of calcium ions inside and outside the cell, forming severe cytoplasmic calcium overload, causing rapid cancer cell death and calcification. In addition, Mn 2+ Release provides a time window for early diagnosis of cancer by MR imaging, which is caused by Ca 2+ Tumor calcification caused by overload can be used to determine the treatment effect through CT imaging and BL imaging, thereby achieving accurate accompanying diagnosis and treatment of tumors.

[0055] In step S1, in one embodiment, the albumin can be a protein such as human serum albumin (HSA) or bovine serum albumin (BSA).

[0056] In one embodiment, the step of coupling FA to the albumin surface by a one-step carbodiimide coupling method to obtain FA-modified albumin specifically comprises:

[0057] Mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (denoted as EDC·HCl solution, the solvent may be dimethyl sulfoxide) and a folic acid solution (the concentration may be 0.1 to 1 mg / mL), stirring for a first predetermined time (1 to 2 hours) at room temperature (15 to 25° C.) in the dark, and then adding an N-hydroxysuccinimide solution (denoted as NHS solution, the solvent may be dimethyl sulfoxide) to carry out a reaction (the reaction time may be 10 to 30 minutes, such as 20 minutes);

[0058] An albumin solution (at a concentration of about 0.1 mg / mL, and the solvent can be deionized water) is added to the reaction system, and after stirring for a second predetermined time (the time can be 5 to 7, such as 6 hours), free FA is removed by ultrafiltration centrifugation several times (such as three times) (the time can be 5 to 10 minutes, such as 10 minutes), and the supernatant is obtained to obtain the FA-modified albumin (as a template).

[0059] In one embodiment, in the FA-modified albumin, the feed mass ratio of the FA to the albumin is 0.1-1.

[0060] In step S2, in one embodiment, the step of dissolving the FA-modified albumin (as a template) in Dulbecco's medium, adding MnCl2 and CaCl2 for in situ mineralization to obtain CMPF nanoparticles specifically includes:

[0061] The FA-modified albumin was dissolved in a sugar-free Dulbecco's medium, and then a MnCl2 solution (the solvent was deionized water) was added. The mixture was mineralized at 25-37°C for 12-36 hours under sealed conditions. Subsequently, a CaCl2 solution (the solvent was deionized water) was added, and the mineralization was continued at 25-37°C for 12-36 hours to obtain CMPF nanoparticles.

[0062] In this example, negatively charged albumin tends to concentrate on adsorbing Ca 2+ and Mn 2+ , causing local ion supersaturation and providing nucleation sites, thereby achieving in situ mineralization of CMPF nanoparticles.

[0063] In one embodiment, the concentration of the FA-modified albumin is 1 to 10 mg mL-1 based on the system after the FA-modified albumin is dissolved in Dulbecco's medium. -1 .

[0064] In one embodiment, the concentration of the MnCl2 solution is 0.05-0.2 M, the concentration of the CaCl2 solution is 0.5-2 M, and the volumes of the MnCl2 solution and the CaCl2 solution added are the same.

[0065] In step S3, in one embodiment, the step of loading CA onto the surface of the CMPF nanoparticles through the hydrophobic-hydrophobic interaction between the hydrophobic cavity in albumin and CA to obtain CMPCF nanoparticles specifically includes:

[0066] Dispersing the CMPF nanoparticles in a buffer solution (such as PBS buffer solution, with the pH adjusted to about 7.4) to obtain a CMPF nanoparticle solution;

[0067] The CMPF nanoparticles are dispersed in a CA solution (CA is first dispersed in dimethyl sulfoxide and then dissolved in a 0.1 mM PBS solution, and the pH is adjusted to about 7.0), and stirred at room temperature and in the dark for a third predetermined time (the time can be 1 to 3 hours, such as 2 hours) to obtain the CMPCF nanoparticles.

[0068] In one embodiment, in the CMPCF nanoparticles, the feed mass ratio of the CA to the CMPF nanoparticles is 0.1-1.

[0069] In one embodiment, the drug loading amount of CA in the CMPCF nanoparticles is 1 to 10.3%.

[0070] An embodiment of the present invention provides a nano-diagnostic and therapeutic agent that induces tumors to form calcification foci, wherein the nano-diagnostic and therapeutic agent is prepared using the preparation method of the nano-diagnostic and therapeutic agent that induces tumors to form calcification foci described in the embodiment of the present invention.

[0071] In this embodiment, CMPCF nanoparticles are mainly composed of CA and CMPF NPs with tumor acid degradation and FA targeting. The tumor accumulation efficiency of CMPCF nanoparticles can be improved through the FA receptor targeting strategy and the TRPA1 specific activation strategy in cancer cells. After CMPCF nanoparticles enter the tumor cells, they release Mn in the micro-acid environment of the tumor. 2+ , MRI has enabled the diagnosis of in situ liver cancer with a diameter of less than 1 cm. In addition, CMPCF nanoparticles can induce tumor necrosis and form calcification foci by generating strong oxidative stress, which can achieve the diagnosis of in situ liver cancer with a diameter of more than 100 mm. 3 Calcification of solid tumors, and then non-invasive tumor efficacy evaluation through calcium-based CT imaging and tumor bioluminescence-based BL imaging.

[0072] An embodiment of the present invention provides a use of the nano-diagnostic and therapeutic agent for inducing the formation of calcification foci in tumors as described above in the preparation of drugs for treating tumors.

[0073] The present invention will be further described below with reference to specific examples.

[0074] Example 1

[0075] The preparation steps of a nano-diagnostic and therapeutic agent for inducing tumor calcification in this embodiment are as follows:

[0076] Step 1: Dissolve 20 mg of EDC·HCl directly in 1 mL of dimethyl sulfoxide and add to 0.1 mg·mL -1 After stirring and activating for 1 hour at room temperature and in the dark, 20 mg of NHS was dissolved in 1 mL of dimethyl sulfoxide and added to the carboxyl-activated FA solution to react for 20 minutes. Then, HSA solution (0.1 mg mL -1 ) and stirred for 6 h. The resulting product was subjected to three ultrafiltration centrifugations (12,000 rpm, 10 min) to remove free FA, and FA-modified HSA was separated and collected.

[0077] Step 2: 100 mg of FA-modified HSA was dissolved in 10 mL of Dulbecco's medium without glucose, and then 100 μL of MnCl2 solution (0.1 M in deionized water) was added. The system was sealed and mineralized at 37°C for 24 h. Subsequently, 100 μL of CaCl2 solution (1 M in deionized water) was added to the system and mineralization continued at 37°C for 24 h. Afterwards, CMPF NPs were isolated by centrifugation (12,000 rpm for 10 minutes).

[0078] Step 3: 1 mg of CMPF NPs was dispersed in 2 mL of a 0.5 mg / mL CA solution (CA was first dispersed in dimethyl sulfoxide and then dissolved in 0.1 mM PBS, pH adjusted to approximately 7.0). The mixture was then stirred at room temperature in the dark for 2 h. Subsequently, the CMPF NPs (i.e., nanotheranostic agents) were separated and collected by centrifugation (12,000 rpm, 10 min).

[0079] Comparative Example 1: Under other identical conditions, only steps 2 and 3 were taken to obtain CMPC NPs.

[0080] Comparative Example 2: Under the same conditions, only step 2 was adopted to obtain CMP NPs.

[0081] Figure 1 The following is a flow chart of the preparation of nanoparticles that induce tumor calcification, a schematic diagram of inducing tumor calcification, and a flow chart of the application in living mice in Example 1 of the present invention. Figure 1 Figure a is a schematic diagram of the preparation of CMPCF NPs. Figure 1Figure b is a schematic diagram of CMPCF NPs-induced tumor calcification, which specifically includes CMPCF NPs inducing cells to produce H2O2 by releasing CA, manipulating the TRPA1 receptor from the "off" state to the "on" state, thereby inducing calcium influx into tumor cells. The rapidly released Ca in CMPCF NPs 2+ and extracellular Ca influx 2+ This can lead to free Ca in cancer cells 2+ The concentration increases rapidly in a short period of time, forming a calcium overload, and H2O2 can be released by the synchronously released Mn 2+ Utilization, generating more biologically toxic ·OH, thereby exacerbating cellular oxidative stress and necrosis. 2+ 、Mn 2+ and PO4 3- In the microenvironment, calcification foci are formed with FA as the nucleating agent. Figure 1 Figure (c) is a schematic diagram of the application of CMPCF NPs. The specific application steps are as follows: the prepared CMPCF NPs are injected into mice through the tail vein, and the FL / BL signals, MR signals, and CT signals of the tumor site are collected in real time using the IVIS fluorescence imaging spectroscopy system, UIHuPMR 790 magnetic resonance imaging, and Quantum GX small animal Micro-CT imaging system.

[0082] Figure 2 Figure 1 is a transmission electron micrograph of the CMPCF NPs, from which it can be seen that the CMPCF NPs have good dispersibility. Figure 3 Comparing the changes in UV absorption during the preparation of CMPCF NPs, it can be seen that after coupling FA and loading CA, CMPCF NPs have specific absorption peaks at 285nm and 360nm, respectively, compared with CMP NPs.

[0083] Then some basic properties of CMPCF NPs were studied. Figure 4 The CA encapsulation efficiency and drug loading in CMPCF NPs are shown in Table 1. When the CA to CMPF NPs mass ratio is between 0.1 and 1, the CA drug loading ranges from 1% to 10.3%. The drug loading increases with increasing CA to CMPF NP ratio. Figure 5 The thermogravimetric curves of CMP, CMPF, and CMPCF NPs were obtained, and the weights of CA and FA in CMPCF NPs were found to be 10.3% and 11.1% of the total weight of the nanoparticles, respectively. Then, X-ray photoelectron spectroscopy was performed to analyze the elements in CMPCF NPs. Figure 6 The Ca content in CMPCF NPs was verified in 2+ 、Mn 2+ and PO4 3-Furthermore, 11.7% Mn and 23.4% Ca were detected in CMPCF NPs by inductively coupled plasma atomic emission spectroscopy (ICP-AES).

[0084] Then, the acid-responsive degradation ability of CMPCF NPs was verified. 1 mL of CMPCF NPs solution (concentration 1 mg mL -1 , the solvent was deionized water) was placed in a dialysis bag (MWCO = 3.5kDa) and immersed in 9mL PBS with pH values ​​of 5.0, 6.5, and 7.4. The above mixture was placed on a shaker at 37°C to simulate different internal environments. 1mL of solution was taken for detection at regular intervals, and 1mL of PBS with the corresponding pH value was added to the buffer solution at the same time. Further, Mn was determined by ICP-AES analysis. 2+ and Ca 2+ The release amount of CMPCF NPs was determined, and the transmission electron microscopy images of CMPCF NPs at pH 5.0, 6.5 and 7.4 were obtained by tungsten filament transmission electron microscopy. Figure 7 The transmission electron micrographs of CMPCF NPs after degradation for 12 hours under different pH conditions show that the mineralized CMPCF NPs have extremely sensitive acidic pH response degradation ability. After degradation for 12 hours in PBS at pH = 5.0, CMPCF NPs can be decomposed into small fragments, thus providing conditions for the release of CA in the weakly acidic environment of the tumor. Figure 8 As shown in the figure, after 48 h of degradation at pH 7.4, the release of the two ions were 34.8% (Ca 2+ ) and 18.7% (Mn 2 + ), while at pH 5.0, 91.3% of Ca 2+ and 70.3% Mn 2+ The results showed that CMPCF NPs showed better Ca release under weak acid conditions of pH 5.0. 2+ and Mn 2+ Release effect.

[0085] Then, the Mn in CMPCF NPs 2+ The ability to catalyze H2O2 to produce ·OH was verified, such as Figure 9 As shown, CMPCFNPs can degrade MB (20 μM) within 4 h in the presence of H2O2 (1 mM), demonstrating that CMPCF NPs have the ability to produce biotoxic ·OH.

[0086] Then the cell calcification ability of CMPCF NPs was studied. To prove that cancer cells were calcified, cancer cells were seeded on the silicon wafer surface for 24 hours and blank culture medium (Ca 2+ =2 mM), CMP, CMPC, and CMPCF NPs (160 μg mL -1 ), and then incubated in an incubator for 12 hours, fixed with electron microscope fixative (25% glutaraldehyde), and finally dehydrated with gradient ethanol solution and sprayed with gold, and image analysis and element characterization were performed using SEM-EDS. Figure 10 As shown in the figure, there is no presence of Ca and Mn on the cell surface treated with blank culture medium, CMP and CMPCNPs. Although the surface of cells treated with CMPC becomes rougher, no calcium phosphate precipitation is formed on the surface, indicating that FA modification promotes the calcification ability of CMPCF NPs on cancer cells. Furthermore, in order to verify that the tumor cell permeability brought about by the FA active targeting ability of CMPCF NPs can calcify the three-dimensional cell spheroids, free calcein was used to deposit Ca on the surface of the three-dimensional cell spheroids. 2+ For dyeing. Figure 11 As shown in the figure, as the incubation time of CMPC NPs was extended from 6h to 12h, the distribution of the calcified layer on the surface of the three-dimensional cancer cell spheres gradually increased, which further demonstrated the permeability and calcification ability of CMPCF NPs. Finally, in order to explore the inhibitory effect of CMPCF NPs on cell calcification on tumor metastasis, the cell migration ability was tracked using the cell automatic tracking mode of a high-content fluorescence microscope (Operetta, PerkinElmer, USA). Figure 12 As shown in the figure, with the increase of CMPCF NPs concentration, the total migration distance of cancer cells and the number of traceable cells gradually decreased, indicating that CMPCF NPs can significantly reduce tumor migration ability and cell activity. In order to further verify the real-time dynamic effect of CMPCF NPs on reducing cell metabolic activity at the cellular metabolic level, Agilent Seahorse XFe24 was used to perform extracellular oxygen consumption (OCR) metabolic analysis on cancer cells. Figure 13 As shown, the CMPCF NPs (40 μg mL -1 ) were treated with glycolysis stress test for 6 hours, and the energy metabolism process of cancer cells was significantly inhibited.

[0087] Figure 14In the treatment of a Huh-7 subcutaneous tumor model in which TRPA1 is overexpressed, the uptake of CMPC and CMPCF NPs by Huh-7 tumors was characterized using an IVIS Spectrum imaging system. CMPC and CMPCF NPs were labeled with carboxyl-activated IR-800 and administered via tail vein injection (2 mg kg -1 ).like Figure 14 As shown in the data, 12 hours after the administration of CMPC and CMPCF NPs, the FL signal in the tumor reached its maximum value. The tumor retention time of CMPCF NPs was significantly extended to 96 hours compared with that of CMPC NPs. Moreover, its FL intensity at the maximum enrichment time point was 1.74 times that of the tumor after treatment with CMPC NPs, indicating that CMPCF NPs have the ability to actively target cancer cells.

[0088] Figure 15 In the treatment of Huh-7 subcutaneous tumor model in mice overexpressing TRPA1, the nanoparticles that induce tumor calcification in Example 1 were used to verify the diagnostic function of CMPCF NPs for in situ Huh-7 tumors using the UIH-uPMR-790 MRI system. 21 days after Huh-7 tumor implantation, Huh-7 orthotopic tumor mice were randomly divided into CMPC and CMPCF groups. All mice were subjected to MR imaging at the following time points (0, 1, 2, 4, 8, 24 hours). Figure 15 As shown in Figure 2, with the increase of administration time, the Mn content in the tumor of mice in the CMPCF NPs treatment group increased. 2+ The T1 images gradually enhanced. After 8 h of administration, the Mn released by CMPCF NPs 2+ The T1 grayscale value of the Huh-7 orthotopic tumor can reach 191.2% of the initial value, while the maximum signal intensity of the CMPC group recorded at 4 h is only 118.5% of the initial value, which indicates that FA modification can greatly increase the uptake efficiency of CMPCF NPs in Huh-7 orthotopic tumors.

[0089] Figure 16 This is the evaluation data of the decrease in cell viability and proliferation induced by CMPCF NPs in the treatment of Huh-7 subcutaneous tumor model overexpressing TRPA1 in Example 1. 3 Huh-7 subcutaneous tumor-bearing mice were randomly divided into three groups (n=3): (1) Control, (2) CMPC, and (3) CMPCF. All groups were given an equivalent dose of NPs (2 mg kg -1 ), the drug was given every 8 days, and CT and BL images of the mouse tumor site were recorded every 8 days. Figure 16 As shown in the figure, after 8 days of administration, tumor calcifications appeared in the CMPCF NPs group, but the BL signal remained high, indicating that Huh-7 tumors still maintained a high metabolic rate. Although CMPC NPs reduced the BL signal of Huh-7 tumors after 3 doses, its BL intensity was still 12.6% of the control group, while the BL intensity of the CMPCF-treated group was only 0.05% of the control group.

[0090] Figure 17 This is the evaluation data of the ability of CMPCF NPs to induce tumor calcification in the treatment of Huh-7 subcutaneous tumor model overexpressing TRPA1 in Example 1. The tumor calcification volume of the CMPCNPs-treated group was 0.49 mm 3 , while the volume of calcification in the CMPCF treatment group was 2.05mm 3 This indicates that FA modification can significantly enhance the tumor calcification ability of CMPCF NPs.

[0091] Figure 18 In this Example 1, the anti-tumor ability of CMPCF NPs was evaluated by tumor volume in the treatment of Huh-7 subcutaneous tumor model overexpressing TRPA1 in mice. The specific characterization method was as follows: 35 tumors with a size of 100 mm 3 Huh-7 tumor model mice were divided into five experimental groups (n=6), namely Control, CA, CMP, CMPC, and CMPCF groups. Figure 18 As shown, after 32 days of treatment, the tumor volume of mice in the CMPCF NPs-treated group was the smallest, and the tumor inhibition rate reached 95.5%.

[0092] Figure 19 The data of the ability of CMPCF NPs to inhibit tumor metastasis is evaluated by the survival rate of mice in the treatment of Huh-7 subcutaneous tumor model overexpressing TRPA1 using a nanoparticle that induces tumor calcification in Example 1. Figure 19 All Huh-7 tumor-bearing mice in the control and CA groups died after 32 and 40 days of treatment, respectively, while mice in the CMPCF NPs group maintained an 80% survival rate after 60 days of treatment, indicating that CMPCF NPs can prolong the survival of mice by calcifying tumor cells.

[0093] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a nano-diagnostic and therapeutic agent that induces tumor calcification, characterized in that: The steps include: Folic acid was coupled to the albumin surface via a one-step carbodiimide coupling method to obtain folic acid-modified albumin; The folic acid-modified albumin is dissolved in Dulbecco's medium, and MnCl2 and CaCl2 are added for in situ mineralization to obtain CMPF nanoparticles; Through the hydrophobic-hydrophobic interaction between the hydrophobic cavity in albumin and cinnamaldehyde, the cinnamaldehyde is loaded onto the surface of the CMPF nanoparticles to obtain CMPCF nanoparticles, i.e., the nano-diagnostic and therapeutic agent.

2. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 1, characterized in that: The step of coupling folic acid to the albumin surface by a one-step carbodiimide coupling method to obtain folic acid-modified albumin specifically comprises: Mixing a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and a folic acid solution, stirring for a first predetermined time at room temperature in the dark, and then adding an N-hydroxysuccinimide solution to carry out a reaction; The albumin solution is added to the reaction system, stirred for a second predetermined time, and then ultrafiltered and centrifuged to obtain the folic acid-modified albumin.

3. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 1, characterized in that: In the folic acid-modified albumin, the feeding mass ratio of the folic acid to the albumin is 0.1-1.

4. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 1, characterized in that: The step of dissolving the folic acid-modified albumin in Dulbecco's medium, adding MnCl2 and CaCl2 for in situ mineralization to obtain CMPF nanoparticles specifically includes: The folic acid-modified albumin is dissolved in Dulbecco's medium, and then a MnCl2 solution is added. The mixture is mineralized at 25-37°C for 12-36 hours under sealed conditions. Subsequently, a CaCl2 solution is added, and the mineralization is continued at 25-37°C for 12-36 hours to obtain the CMPF nanoparticles.

5. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 4, characterized in that: Based on the system after the folic acid-modified albumin is dissolved in Dulbecco's medium, the concentration of the folic acid-modified albumin is 1 to 10 mg mL -1 ; The concentration of the MnCl2 solution is 0.05-0.2M, the concentration of the CaCl2 solution is 0.5-2M, and the volumes of the MnCl2 solution and the CaCl2 solution added are the same.

6. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 1, characterized in that: The step of loading the cinnamaldehyde onto the surface of the CMPF nanoparticles through the hydrophobic-hydrophobic interaction between the hydrophobic cavity in the albumin and the cinnamaldehyde to obtain the CMPCF nanoparticles specifically comprises: The CMPF nanoparticles are dispersed in a cinnamaldehyde solution and stirred at room temperature in the dark for a third predetermined time to obtain the CMPCF nanoparticles.

7. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 1, characterized in that: In the CMPCF nanoparticles, the mass ratio of the cinnamaldehyde to the CMPF nanoparticles is 0.1-1.

8. The method for preparing the nano-diagnostic and therapeutic agent for inducing tumor calcification according to claim 1, characterized in that: The drug loading amount of the cinnamaldehyde in the CMPCF nanoparticles is 1% to 10.3%.

9. A nano-diagnostic and therapeutic agent that induces tumor calcification, characterized in that: The nano-diagnostic and therapeutic agent for inducing tumor calcification formation is prepared by the preparation method of any one of claims 1 to 8.

10. Use of the nano-diagnostic and therapeutic agent for inducing tumor calcification formation according to claim 9 in the preparation of drugs for treating tumors.

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