Preparation method and application of calcium cobaltate piezoelectric sensitizer

Abstract

The invention discloses a preparation method of a calcium cobaltate piezoelectric sensitizer and application of the calcium cobaltate piezoelectric sensitizer in tumor treatment. The preparation method comprises the following steps: dissolving calcium nitrate and cobalt nitrate as raw materials in a citric acid aqueous solution, adding 2% PEG to form a precursor solution, carrying out hydrolytic condensation at 80 DEG C to obtain sol, aging to obtain gel, drying at 120 DEG C for 12 hours, calcining at 800 DEG C for 2 hours to prepare calcium cobaltate nanoparticles, and carrying out PVP surface modification to obtain the calcium cobaltate piezoelectric sensitizer. The sonosensitizer is good in biocompatibility and stability, has excellent piezoelectric response, can form a built-in electric field under ultrasound, promotes separation of electron-hole pairs, inhibits rapid compounding of the electron-hole pairs and improves the yield of active oxygen, and can be used for sonodynamic therapy of tumors. The preparation device is simple and easy to operate, the period is short, mass production is facilitated, and a novel efficient nano material platform is provided for tumor sonodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly discloses a preparation method of a calcium cobaltate piezoelectric acoustic sensitizer and application thereof in tumor treatment. BACKGROUND

[0002] As a global high-incidence malignant disease, the treatment of tumors has long been limited by the inherent defects of traditional therapies: surgical treatment is difficult to completely remove deep or diffuse tumors, and is prone to cause tissue damage and infection after operation; radiotherapy and chemotherapy can act on tumor cells throughout the body or locally, but lack of targeting, and are prone to cause toxic side effects to normal tissues such as bone marrow and gastrointestinal mucosa, resulting in complications such as decreased immunity, nausea and vomiting in patients, and some tumor cells are prone to drug resistance, ultimately leading to difficulty in improving the cure rate.

[0003] To break through the above bottleneck, ultrasound-mediated sonodynamic therapy (SDT) has become a research hotspot in the field of tumor treatment due to its unique advantages: on the one hand, ultrasound has excellent tissue penetration ability, and can be accurately focused on deep tumor tissue to avoid damage to normal tissues on the surface; on the other hand, SDT activates specific acoustic sensitizer by ultrasound, so that the acoustic sensitizer reacts with oxygen in the tumor microenvironment to produce reactive oxygen species (ROS), which can damage the DNA, proteins and lipids of tumor cells through oxidation, achieving the effect of targeted killing of tumor cells, and the treatment process is non-invasive, biologically resistant, and has high clinical transformation potential.

[0004] The curative effect of SDT depends on the performance of the acoustic sensitizer, and the existing acoustic sensitizer mainly includes organic acoustic sensitizer and inorganic acoustic sensitizer: The organic acoustic sensitizer is represented by porphyrin and its derivatives, which has been widely used in early stage, but has obvious defects: poor chemical stability, easy to degrade or aggregate in physiological environment; strong phototoxicity, which may damage normal tissues such as skin under natural light; fast metabolic clearance rate, difficult to effectively enrich in tumor site, ultimately leading to insufficient ROS yield and limited treatment effect.

[0005] With the development of nanomedicine, inorganic acoustic sensitizer based on semiconductor nanomaterials (such as titanium dioxide, zinc oxide, cadmium sulfide, etc.) gradually replaces part of the organic acoustic sensitizer, and its advantages are high chemical stability, low phototoxicity, and performance optimization through regulation of nanostructure. However, the existing inorganic acoustic sensitizer still faces key technical bottlenecks: the semiconductor material is limited by its own energy band structure, and the generated electrons and holes are easily and quickly recombined under ultrasonic excitation, resulting in that the electrons cannot effectively reduce oxygen and the holes cannot efficiently oxidize hydroxyl, and ultimately the ROS yield cannot meet the clinical treatment needs; at the same time, part of the inorganic nanomaterials (such as heavy metal-based semiconductors) have poor biocompatibility, and are easy to accumulate in the body to cause long-term toxicity, which further limits their clinical application.

[0006] In summary, the existing sonosensitizers cannot simultaneously meet the three core requirements of "high ROS yield, good biocompatibility, and stable dispersion", especially the problem of "low electron-hole pair separation efficiency", which has become a key obstacle to the clinical transformation of SDT. Therefore, developing a new type of sonosensitizer that can improve electron-hole separation efficiency through active regulation mechanism and has biocompatibility and stability is a technical problem that needs to be solved in the current field of tumor sonodynamic therapy. SUMMARY

[0007] In view of this, the present application proposes a preparation method of calcium cobaltate piezoelectric sonosensitizer and its application in tumor treatment, which combines the piezoelectric properties of perovskite calcium cobaltate with surface modification technology to achieve a breakthrough in the efficacy of SDT.

[0008] The technical scheme of the present application is realized as follows: The present application prepares calcium cobaltate nanoparticles with piezoelectric response ability and good biocompatibility by sol-gel method combined with surface modification technology, and the specific steps are as follows: 1. Preparation of precursor solution: Dissolve calcium nitrate and cobalt nitrate in a molar ratio of n(Ca):n(Co)=3:4 in a citric acid aqueous solution, add polyethylene glycol (PEG) with a volume ratio of 2% to the mixed solution, continuously stir at room temperature for 30 minutes, and form a uniform transparent precursor solution; wherein citric acid as a complexing agent can form a stable complex with calcium and cobalt ions to prevent early precipitation of ions; PEG as a dispersant can inhibit the agglomeration of particles in the subsequent sol-gel process.

[0009] 2. Sol-gel formation: Transfer the above-mentioned precursor solution to a constant temperature water bath, continuously stir at 80℃ for 6 hours, during which the solution undergoes hydrolysis reaction (citric acid complex gradually hydrolyzes to form hydroxyl groups) and condensation reaction (hydroxyl groups dehydrate to form -O- bonds), and gradually forms a viscous sol; after stopping stirring, the sol is aged at room temperature for 12 hours to fully convert the sol into stable gel, and the gel structure can fix the distribution of calcium and cobalt ions, laying a foundation for the formation of uniform calcium cobaltate crystals in the subsequent process.

[0010] 3. Preparation of calcium cobaltate nanoparticles: Transfer the aged gel to a vacuum drying oven and dry at 120℃ for 12 hours to remove water and volatile impurities in the gel, obtaining a dry gel; grind the dry gel into powder and transfer it to a muffle furnace, and calcine at 800℃ for 2 hours; during the calcination process, the citric acid complex decomposes, and calcium and cobalt ions form perovskite calcium cobaltate nanoparticles in a stoichiometric ratio. This structure has excellent piezoelectric properties and can generate polarized charges under mechanical stress (such as ultrasonic vibration).

[0011] 4. PVP Surface Modification: Calcium cobaltate nanoparticles obtained by calcination were dispersed in deionized water to form a suspension with a concentration of 5 mg / mL. Polyvinylpyrrolidone (PVP) powder was added to the suspension and stirred for 24 hours. PVP molecules formed hydrogen bonds with the hydroxyl groups on the surface of calcium cobaltate nanoparticles through hydroxyl groups, achieving tight coating. Subsequently, unbound free PVP was removed by centrifugation and washing with deionized water three times, finally obtaining calcium cobaltate piezoelectric acoustic sensor. PVP modification can significantly improve the biocompatibility of calcium cobaltate, prevent it from being cleared by the body's immune system, and improve its dispersion stability in physiological solutions.

[0012] In some implementations, during surface modification, the centrifugation speed is 8000 rpm for 10 minutes.

[0013] In some implementations, the mass ratio of PVP to calcium cobaltate in the surface modification is 1:5.

[0014] In some embodiments, the calcium cobaltate piezoelectric acoustic sensor prepared by the present invention has the following key characteristics: 1. Piezoelectric response characteristics: The perovskite structure of calcium cobaltate has a non-centrosymmetric lattice. Under mechanical vibration, such as ultrasound, the lattice deforms, generating polarization charges and forming an internal electric field. This electric field can effectively suppress electron-hole recombination, promote electron migration to the surface of calcium cobaltate, and reduce oxygen to generate •O2. - Meanwhile, cavitation holes remain on the surface of calcium cobaltate, oxidizing OH groups in the tumor microenvironment. - •OH is generated; furthermore, the separation of electron-hole pairs can be promoted through energy transfer. 1 The generation of O2 ultimately leads to a significant increase in ROS yield.

[0015] 2. Structural and performance stability: The calcium cobaltate nanoparticles formed by calcination at 800℃ have a stable crystal structure and are not easily degraded in the physiological environment; the surface coating of PVP can form a protective layer, which can prevent calcium cobaltate from non-specifically binding with proteins in the blood, reduce aggregation, and ensure that it can be stably dispersed in the body and reach the tumor site.

[0016] 3. Good biocompatibility: PVP, as a medical polymer material approved by the FDA, is non-toxic and can reduce the cytotoxicity of calcium cobaltate nanoparticles. Experimental verification shows that even at a concentration as high as 200 μg / mL, the survival rate of normal cells after PVP surface modification of calcium cobaltate still exceeds 80%. In animal in vivo treatment, there was no significant change in the body weight of tumor-bearing mice and no toxic side effects such as abnormal liver and kidney function were observed.

[0017] The application also provides application of the above-mentioned calcium cobaltate piezoelectric acoustic sensitizer in tumor treatment, the calcium cobaltate piezoelectric acoustic sensitizer is delivered to a tumor site by intratumoral injection or intravenous injection, after the acoustic sensitizer is enriched at the tumor site (reaching an enrichment peak 6 hours after intravenous injection), the tumor site is irradiated by ultrasound with a frequency of 1.0-1.5 MHz, a power density of 1.0-2.0 W / cm 2 , and a duty cycle of 40-60%, the irradiation time is 3-5 minutes; the calcium cobaltate generates a piezoelectric effect under the excitation of ultrasound, efficiently produces reactive oxygen species, realizes targeted killing of tumor cells, and can inhibit tumor angiogenesis, prevent tumor metastasis and recurrence, and is especially suitable for treatment of deep solid tumors such as breast cancer, liver cancer and pancreatic cancer.

[0018] The application has the following beneficial effects compared with the prior art: The piezoelectric property of the perovskite calcium cobaltate is combined with SDT for the first time, the built-in electric field under the excitation of ultrasound can improve the separation efficiency of electron-hole pairs by more than 40%, and then the total yield of ROS is improved compared with traditional inorganic acoustic sensitizers; Through surface modification by PVP, the dispersion stability of calcium cobaltate in a physiological solution is improved, and the agglomeration rate is less than 10% after 72 hours of placement; the toxicity problem of some inorganic acoustic sensitizers is completely solved, and safety is provided for clinical application; The sol-gel method and surface modification technology used in the application are mature nanomaterial preparation processes, the required equipment is conventional and easy to obtain, and no special high-end equipment is needed; the raw materials are all commercially available bulk commodities, and the cost is low; the preparation period is short, and the process parameters are easy to control, so that kilogram-level batch production can be realized, meeting the demand of clinical transformation for material scale; Relying on the deep penetration characteristics of ultrasound and the piezoelectric response ability of calcium cobaltate, the application can be used for treating deep tumors that cannot be covered by traditional SDT, breaking through the shallow treatment limitation of organic acoustic sensitizers depending on light excitation; at the same time, calcium cobaltate can be adapted to different administration modes by adjusting the particle size, and is suitable for various solid tumors such as breast cancer, lung cancer and colon cancer, and the application scenarios are flexible and diverse. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description, and obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0020] Figure 1Figure for basic characterization results of the calcium cobaltate piezoelectric sonosensitizer of Example 1 of the present application; wherein (A, B) are transmission electron microscope (TEM) images of the calcium cobaltate piezoelectric sonosensitizer, (C) is a high-resolution transmission electron microscope (HRTEM) image of the calcium cobaltate piezoelectric sonosensitizer, and (D) is a particle size distribution (determined by dynamic light scattering) diagram of the calcium cobaltate piezoelectric sonosensitizer.

[0021] Figure 2 Figure for band structure and piezoelectric performance characterization results of the calcium cobaltate piezoelectric sonosensitizer of Example 1 of the present application; wherein (A) is a Tauc diagram of the ultraviolet-visible diffuse reflectance spectrum of the calcium cobaltate piezoelectric sonosensitizer (for calculating the band gap width), (B) is a Mott-Schottky diagram of the calcium cobaltate piezoelectric sonosensitizer (for calculating the valence band and conduction band potentials), (C) is a piezoelectric force microscope (PFM) amplitude curve diagram of the calcium cobaltate piezoelectric sonosensitizer, and (D) is a piezoelectric force microscope (PFM) phase curve diagram of the calcium cobaltate piezoelectric sonosensitizer.

[0022] Figure 3 Figure for in vitro cytotoxicity of the calcium cobaltate piezoelectric sonosensitizer of Example 2 of the present application on 4T1 breast cancer cells under the action of ultrasound (determined by CCK-8 method to determine the effect of different concentrations of calcium cobaltate on the survival rate of 4T1 cells).

[0023] Figure 4 Figure for in vitro cellular level reactive oxygen species production evaluation of the calcium cobaltate piezoelectric sonosensitizer of Example 2 of the present application (by DCFH-DA staining method, the green fluorescence intensity of ROS in 4T1 cells in different treatment groups was observed under an inverted fluorescence microscope, and the scale was 100 μm).

[0024] Figure 5 Figure for in vivo treatment effect of the calcium cobaltate piezoelectric sonosensitizer of Example 2 of the present application under the action of ultrasound; wherein (A) is a figure showing the body weight change of 4T1 tumor-bearing mice in each treatment group during treatment, (B) is a figure showing the tumor volume change of 4T1 tumor-bearing mice in each treatment group during treatment, (C) is a figure showing the tumor weight of 4T1 tumor-bearing mice in each treatment group after treatment, and (D) is a figure showing the tumor photos of 4T1 tumor-bearing mice in each treatment group after treatment. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] Example 1 Preparation steps Step 1: Preparation of precursor solution. Dissolve calcium nitrate (Ca(NO3)2·4H2O) and cobalt nitrate (Co(NO3)2·6H2O) in deionized water with a molar ratio of n(Ca):n(Co)=3:4, and use a magnetic stirrer to stir until fully dissolved to form a red transparent solution; weigh citric acid with a molar ratio of metal cation to citric acid of 1:1.2, and add it to the nitrate solution; add 2% (volume ratio) polyethylene glycol (PEG-400), and continue stirring to obtain the precursor solution.

[0027] Step 2: Sol-gel formation. Transfer the precursor solution to a 500 mL three-necked flask, place it in a constant temperature water bath, set the water bath temperature to 80°C, and maintain a stirring speed of 300 rpm for continuous stirring for 6 hours. During this period, the solution gradually changes from transparent to viscous sol; after stopping the stirring, seal the three-necked flask and place it in a room temperature environment for aging for 12 hours to allow the sol to fully convert to a stable light pink gel.

[0028] Step 3: Preparation of calcium cobaltate nanoparticles. Transfer the aged gel to a vacuum drying oven, set the drying temperature to 120°C and the vacuum degree to -0.09 MPa, and dry for 12 hours to remove water and volatile impurities in the gel, obtaining a loose dry gel; grind the dry gel into fine powder in an agate mortar, transfer it to an alumina crucible, and place it in a muffle furnace, heating to 800°C at a rate of 3°C / min, and calcining at this temperature for 2 hours to obtain black calcium cobaltate nanoparticles.

[0029] Step 4: PVP surface modification. Weigh 100 mg of the above calcium cobaltate nanoparticles and disperse them in 20 mL of deionized water, stirring at a speed of 500 rpm for 10 minutes to form a suspension; add 20 mg of polyvinylpyrrolidone (PVP, molecular weight 58000) to the suspension, at this time the mass ratio of calcium cobaltate to PVP is 5:1; place the mixed system in a 30°C constant temperature water bath and continue stirring at a speed of 500 rpm for 24 hours; after stirring, transfer the suspension to a centrifuge tube and centrifuge at a speed of 8000 rpm for 10 minutes, discard the supernatant; repeat the washing of the precipitate with deionized water for 3 times, each time after washing, centrifuge at 8000 rpm for 10 minutes; finally, dry the precipitate in a vacuum drying oven for 6 hours to obtain black powder of calcium cobaltate piezoelectric acoustic sensitizer.

[0030] Performance verification (1) Structure and morphology characterization: Transmission electron microscopy (TEM) was used to observe the morphology of calcium cobaltate at an acceleration voltage of 200 kV; high-resolution transmission electron microscopy (HRTEM) was used to observe the crystal lattice structure; dynamic light scattering (DLS) was used to measure the particle size distribution of calcium cobaltate in physiological saline, the test temperature was 25°C, and each sample was tested 3 times.

[0031] (2) Piezoelectric performance characterization: The piezoelectric response was tested by atomic force microscopy (AFM) in piezo force microscopy (PFM) mode, with an applied electric field range of -10V~+10V and a scanning rate of 1Hz. The diffuse reflectance spectrum of calcium cobaltate was tested by a UV-visible diffuse reflectance spectrometer, with a scanning range of 200~800nm, and the band gap width was calculated by Tauc formula. The Mott-Schottky curve was tested by an electrochemical workstation (model CHI660E), with a reference electrode of Ag / AgCl electrode, a counter electrode of platinum sheet electrode, an electrolyte of 0.1mol / L Na2SO4 solution, a scanning voltage range of 0~2V, and a scanning rate of 50mV / s. The valence band (VB) and conduction band (CB) potentials were calculated according to the curve.

[0032] Performance results

[0033] In this embodiment, calcium cobaltate piezoelectric sonosensitizer was successfully prepared by the core process of “sol-gel method + PVP surface modification”. Structure characterization confirmed that it had target perovskite crystal faces (202) and (203) and uniform sheet morphology. DLS results showed that PVP modification effectively improved the dispersion stability. Piezoelectric performance test (PFM, band gap and potential calculation) verified its excellent piezoelectric response ability, which provided a structure and performance basis for charge separation and ROS generation under subsequent ultrasonic excitation.

[0034] Example 2 Preparation steps The same as Example 1, directly using the calcium cobaltate prepared in Example 1 Performance verification (1) In vitro cytotoxicity test (CCK-8 method): 4T1 breast cancer cells in logarithmic growth phase were inoculated in a 96-well plate at a density of 1.5×10 5 μg / mL, and cultured for 24 hours. Then they were divided into four groups: Control group (only fresh culture medium), US only group (only ultrasonic irradiation), calcium cobaltate only group (calcium cobaltate was added, with concentrations of 12.5, 25, 50, 100, and 200μg / mL), and calcium cobaltate + US group (ultrasonic irradiation after adding calcium cobaltate at corresponding concentrations). The ultrasonic parameters were set as follows: frequency 1.0MHz, power density 1.5W / cm², duty cycle 50%, and irradiation time 3 minutes. After continuing to culture for 24 hours, 10μL CCK-8 reagent was added to each well, and incubated at 37℃ for 2 hours. The absorbance at 450nm was measured by an enzyme marker, and the relative survival rate of cells was calculated, with three repeats for each concentration.

[0035] (2) In vitro ROS detection (DCFH-DA staining method): 4T1 cells were inoculated in a 96-well plate at a density of 1.5×10 5The density of 1×104cells / well was inoculated in 6-well plates, and after 24 hours of culture, the above-mentioned 4 groups were treated; after the treatment was completed, the culture was continued for 6 hours, the medium was aspirated, and the cells were washed once with PBS; 1 mL of DCFH-DA working solution (10 μmol / L, diluted with serum-free medium) was added to each well, and incubated at 37°C for 20 minutes in the dark; the working solution was discarded, and the cells were washed 3 times with serum-free medium; green fluorescence was observed under an inverted fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm), and the fluorescence intensity was quantitatively analyzed by ImageJ software, and each group was repeated 3 times.

[0036] (3) In vivo efficacy verification (mouse tumor transplantation model): 4-6-week-old female BALB / c mice were selected, and 1×10 6 2

[0037] Performance results

[0038] This embodiment comprehensively verifies the application effect of calcium cobaltate piezoelectric sonosensitizer in tumor SDT through in vitro and in vivo experiments: the in vitro experiment proves that calcium cobaltate combined with ultrasound can efficiently produce reactive oxygen species (ROS fluorescence intensity is 6.2 times that of the control group), and the killing rate of 4T1 cells is significantly improved (survival rate is only 36.59% at 200 μg / mL); the in vivo experiment shows that this combined treatment can achieve a tumor inhibition rate of 73.2%, and the mice have stable body weight and no obvious toxic side effects during treatment, proving its biological safety.

[0039] Example 3 Preparation steps Referring to the preparation steps of Example 1, only the molar ratio of calcium nitrate to cobalt nitrate in step 1 is changed, and 3 variable groups are set: Variable 3-1: n(Ca):n(Co)=2:4 (0.2 mol of calcium nitrate and 0.4 mol of cobalt nitrate were weighed);​​ Variable 3-2: n(Ca): n(Co) = 3:4 (same as Example 1, reference group); Variable 3-3: n(Ca): n(Co) = 4:4 (0.4 mol of calcium nitrate and 0.4 mol of cobalt nitrate were weighed); The remaining steps (PEG dosage, drying temperature, calcination conditions, PVP modification parameters) were consistent with Example 1, and calcium cobaltate-3-1, calcium cobaltate-3-2, and calcium cobaltate-3-3 were prepared, respectively.

[0040] Performance verification The phase switching angle of the three groups of samples was determined by using the PFM test method of Example 1; the signal intensity of •O2⁻ in ROS was tested by using an electron spin resonance instrument (ESR, model Bruker A300), and the test conditions were as follows: DMPO was used as a spin trapping agent, the concentration of calcium cobaltate was 100 μg / mL, the ultrasonic parameters were the same as those of Example 2, the scanning range was 3460-3560 G, and each sample was tested repeatedly for three times.

[0041] Performance results

[0042] This example verified the key role of the Ca / Co molar ratio through a variable experiment: only when n(Ca): n(Co) = 3:4, calcium cobaltate can form a complete perovskite structure (clearly detect (202) / (203) crystal face), and then exhibit excellent piezoelectric response (phase switching angle 180°) and ROS generation ability (•O2 - signal intensity 1.00); when deviating from this ratio, the crystal structure is incomplete, and the piezoelectric performance and ROS yield decrease significantly.

[0043] Example 4 Preparation steps Referring to the preparation steps of Example 1, only the calcination temperature in step 3 was changed, and three variable groups were set: Variable 4-1: calcination at 700°C for 2 hours (heating rate 3°C / min, holding for 2 hours); Variable 4-2: calcination at 800°C for 2 hours (same as Example 1, reference group); Variable 4-3: calcination at 900°C for 2 hours (heating rate 3°C / min, holding for 2 hours); The remaining steps (raw material ratio, drying conditions, PVP modification parameters) were consistent with Example 1, and calcium cobaltate-4-1, calcium cobaltate-4-2, and calcium cobaltate-4-3 were prepared, respectively.

[0044] Performance verification The crystal face structure of the three groups of samples was observed by HRTEM of Example 1; the amplitude peak value (piezoelectric response strength) was tested by PFM; the in vitro 4T1 cell killing rate was tested by CCK-8 method of Example 2 (calcium cobaltate concentration 200 μg / mL, ultrasonic conditions same as Example 2), each sample was repeated 3 times.

[0045] Performance results

[0046] This example demonstrates that the calcination temperature has a decisive influence on the performance of calcium cobaltate: calcination at 800°C can make calcium cobaltate form clear (202) / (203) characteristic crystal faces, with the strongest piezoelectric response (amplitude peak value 85 mV), and thus achieve the optimal cell killing effect (63.4%); calcination at 700°C cannot form the target crystal faces due to insufficient crystallization, and the piezoelectric and killing performances are insufficient; calcination at 900°C leads to the collapse of the crystal structure, and the performance is further reduced.

[0047] Example 5 Preparation steps Referring to the preparation steps of Example 1, only the amount of PVP added in step 4 is changed, and three variable groups are set: Variable 5-1: calcium cobaltate:PVP=10:1 (weigh 100 mg calcium cobaltate, 10 mg PVP); Variable 5-2: calcium cobaltate:PVP=5:1 (same as Example 1, reference group); Variable 5-3: calcium cobaltate:PVP=2:1 (weigh 100 mg calcium cobaltate, 50 mg PVP); The remaining steps (raw material ratio, drying temperature, calcination conditions) are consistent with Example 1, and calcium cobaltate-5-1, calcium cobaltate-5-2, and calcium cobaltate-5-3 are prepared respectively.

[0048] Performance verification The three groups of samples were dispersed in physiological saline (concentration 100 μg / mL), and after 72 hours of room temperature standing, the agglomeration rate was measured by DLS (agglomeration rate=(particle size after standing for 72 h-initial particle size) / initial particle size x 100%); BALB / c mice (n=3 / group) were injected with 10 mg / kg of calcium cobaltate suspension via the tail vein, and 72 hours later, the mouse serum was collected, and the alanine aminotransferase (ALT, reflecting liver function) and serum creatinine (Scr, reflecting kidney function) were detected by a fully automatic biochemical analyzer (model Hitachi 7600); the phase switching angle was tested by PFM of Example 1, each sample was repeated 3 times.

[0049] Performance results

[0050] The embodiment determines the optimal amount of PVP modification through a variable experiment: only when the calcium cobaltate:PVP = 5:1, the balance of “low agglomeration rate (7.8%), good biocompatibility (normal liver and kidney function), excellent piezoelectric performance (phase switching 180°)” can be achieved; the PVP amount is too low (10:1), which leads to high agglomeration rate and liver damage, and the PVP amount is too high (2:1), which leads to too thick coating and weak piezoelectric response.

[0051] Example 6 Preparation step The control group (TiO2): 100 mg of commercial anatase TiO2 nanoparticles (particle size about 50 nm) was weighed and dispersed in 20 mL of deionized water, 20 mg of PVP (TiO2:PVP = 5:1) was added, stirred at 30°C for 10 hours, centrifuged at 8000 rpm for 12 minutes, washed for 3 times, and dried at 60°C for 6 hours to obtain TiO2; The experimental group (calcium cobaltate): the calcium cobaltate prepared in Example 1.

[0052] Performance verification The •O2 - , •OH, 1 signal intensity of O2 (calcium cobaltate / TiO2 concentration 100 μg / mL, ultrasonic conditions same as Example 2) of the two groups of samples was tested by the ESR method of Example 3; the in vitro 4T1 cell killing rate (concentration 200 μg / mL) was tested by the CCK-8 method of Example 2; and the tumor inhibition rate (dose 10 mg / kg) was tested by the in vivo model of Example 2, each sample was repeated for 3 times.

[0053] Performance results

[0054] Through comparison with the existing inorganic sound sensitizer TiO2@P, the core advantage of the present application is highlighted: the ROS yield, cell killing rate and in vivo tumor inhibition rate of calcium cobaltate are 2-3 times of TiO2@P, which proves that “piezoelectric effect” is the key innovation point to improve the efficacy of SDT, rather than simply replacing the inorganic sound sensitizer substrate.

[0055] Example 7 Preparation step The control group (HpD): commercial hematoporphyrin (HpD) was directly prepared into a solution with a concentration of 10 mg / kg by physiological saline; The experimental group (calcium cobaltate): the calcium cobaltate prepared in Example 1 (10 mg / kg suspension prepared by physiological saline).

[0056] Performance verification Phototoxicity test: BALB / c mice (n=3 / group) were selected, 100 μL samples (calcium cobaltate / HpD concentration 10 mg / kg) were applied on the back skin, and after 24 hours of natural light irradiation, the scores were given according to "no redness = 1 point, slight redness = 2 points, obvious redness = 3 points, redness and ulceration = 4 points"; deep efficacy test: 4T1 tumor transplantation model was established (tumor depth 1.5 cm), and the tumor inhibition rate was tested according to the grouping method of Example 2; 72-hour in vivo clearance rate: 72 hours after the mice were injected with samples via the tail vein, the drug residue in the liver was determined (high-performance liquid chromatography, model Agilent 1260), and each sample was repeated 3 times.

[0057] Performance results

[0058] This example proves the significant advantages of the present application in "safety and deep efficacy" by comparing with the traditional organic photosensitizer HpD: calcium cobaltate has no obvious phototoxicity (redness score 1.0), the deep tumor inhibition rate (73.2%) is 2.2 times that of HpD, and the in vivo clearance rate is higher (82.5%), without accumulation risk, solving the inherent defects of strong phototoxicity and poor deep efficacy of organic photosensitizers.

[0059] Example 8 Preparation steps Control group (pure calcium cobaltate): calcium cobaltate nanoparticles were prepared according to steps 1-3 of Reference Example 1, and step 4 of PVP modification was omitted; Experimental group (calcium cobaltate): calcium cobaltate prepared according to Example 1.

[0060] Performance verification BALB / c mice (n=3 / group) were selected, and 10 mg / kg of samples (pure calcium cobaltate / calcium cobaltate) were injected via the tail vein, and the weight was measured every other day within 7 days; after 7 days, the blood of the mice was collected, and the white blood cell count (WBC) was determined using a blood cell analyzer (model Sysmex XN-1000); the tumor inhibition rate was tested using the in vivo model of Example 2, and each sample was repeated 3 times.

[0061] Performance results

[0062] This example proves the necessity of PVP modification by comparing with pure calcium cobaltate without PVP modification: pure calcium cobaltate has poor dispersibility (72h aggregation rate 68.5%) and strong biological toxicity (weight loss, leukopenia), and the tumor inhibition rate is only 35.6%; after PVP modification, the dispersion stability and biocompatibility of calcium cobaltate are significantly improved, and the tumor inhibition rate reaches 73.2%.

[0063] Comparative Example 1 Preparation step Preparation step as the control group of Example 8 (pure calcium cobaltate), omitting PVP modification.

[0064] Performance verification Performance verification step (body weight, WBC, tumor inhibition rate, agglomeration rate) as Example 8.

[0065] Performance results

[0066] This comparative example verifies the decisive influence of the key feature of "PVP modification" on the effect of the invention: pure calcium cobaltate without PVP modification has serious dispersion defects and biological toxicity, and cannot achieve effective tumor treatment, which is in sharp contrast to the excellent performance of calcium cobaltate.

[0067] Comparative Example 2 Preparation step Preparation step as the control group of Example 6 (TiO2).

[0068] Performance verification Performance verification step (ROS signal, cell killing rate, tumor inhibition rate) as Example 6.

[0069] Performance results

[0070] This comparative example verifies the core innovative value of "piezoelectric effect" by using "inorganic sonosensitizer TiO2@P without piezoelectric performance": TiO2@P lacks piezoelectric response, and its ROS yield, cell killing rate, and tumor inhibition rate are much lower than those of calcium cobaltate, proving that relying only on conventional inorganic sonosensitizers cannot achieve the high-efficiency treatment effect of the invention.

[0071] Comparative Example 3 Preparation step Preparation step as the control group of Example 7 (HpD) (direct dissolution).

[0072] Performance verification Performance verification step (phototoxicity, deep inhibition rate, clearance rate) as Example 7.

[0073] Performance results

[0074] This comparative example highlights the irreplaceability of the invention in "safety and deep applicability" by using traditional organic sonosensitizer HpD: HpD has serious phototoxicity, poor deep efficacy, slow in vivo clearance, and other inherent defects, while calcium cobaltate can completely avoid these problems.

[0075] Comparative Example 4 Preparation Step Preparation Step with variable 3-1 (n(Ca):n(Co)=2:4) of Example 3.

[0076] Performance Verification Performance Verification Step (PFM phase, •O2 - signal, crystal face) of Example 3.

[0077] Performance Result

[0078] This comparative example verifies the necessity of the parameter of “Ca / Co=3:4” by deviating from the optimal molar ratio: when Ca / Co=2:4, calcium cobaltate cannot form a complete perovskite structure, and the piezoelectric response and ROS yield decrease significantly, which cannot achieve efficient treatment.

[0079] Comparative Example 5 Preparation Step Preparation Step with variable 4-1 (700℃ calcination) of Example 4.

[0080] Performance Verification Performance Verification Step (crystal face, amplitude peak, cell killing rate) of Example 4.

[0081] Performance Result

[0082] This comparative example verifies the necessity of the process parameter of “800℃” by calcination temperature lower than “800℃”: 700℃ calcination results in insufficient crystallization of calcium cobaltate, which cannot form the target crystal face, and the piezoelectric performance and cell killing effect decrease significantly.

[0083] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a calcium cobaltate piezoelectric acoustic sensor, characterized in that, Includes the following steps: (1) Dissolve calcium nitrate and cobalt nitrate in a citric acid aqueous solution at a specific molar ratio, and add polyethylene glycol and stir to form a precursor solution; (2) The precursor solution is stirred at a preset temperature and hydrolyzed and condensed to form a sol. The sol is aged to obtain a gel. (3) After drying the gel, it is calcined at high temperature to obtain calcium cobaltate nanoparticles; (4) The surface of the calcium cobaltate nanoparticles is modified with polyvinylpyrrolidone to obtain a calcium cobaltate piezoelectric acoustic sensor.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of calcium nitrate to cobalt nitrate is n(Ca):n(Co) = 3:

4.

3. The preparation method according to claim 1, characterized in that, In step (1), the amount of PEG added is 2% vol of the volume of the citric acid aqueous solution.

4. The preparation method according to claim 1, characterized in that, The preset temperature in step (2) is 80℃; the drying conditions in step (3) are drying at 120℃ for 12 hours and calcination conditions are calcination at 800℃ for 2 hours.

5. A calcium cobaltate piezoelectric acoustic sensor, characterized in that, The acoustic sensitizer is prepared by any one of claims 1-4. The acoustic sensitizer has piezoelectric response capability and can form a built-in electric field under ultrasonic action, which promotes electron-hole pair separation and improves the efficiency of reactive oxygen generation.

6. The calcium cobaltate piezoelectric acoustic sensor according to claim 5, characterized in that, The sound-sensitive agent has a (202) crystal plane and a (203) crystal plane, and the lattice spacing of the (202) crystal plane is 0.1949 nm and the lattice spacing of the (203) crystal plane is 0.1877 nm.

7. The calcium cobaltate piezoelectric acoustic sensor according to claim 5, characterized in that, The sound-sensitive agent has a particle size of 280-300 nm and a band gap of 2.10-2.20 eV.

8. The use of calcium cobaltate piezoelectric sonosensitive agent according to any one of claims 5-7 in tumor sonodynamic therapy.

9. The application according to claim 8, characterized in that, The tumor is a triple-negative breast cancer.

10. The application according to claim 8, characterized in that, The ultrasound conditions used in the sonodynamic therapy are: frequency 1.0-1.5MHz, power density 1.0-2.0W / cm². 2 The duty cycle is 40-60%, and the ultrasound irradiation time is 3-5 minutes.

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