An ultrasonic piezoelectric catalytic effect promotes the release of nitric oxide gas nanoprodrug, and a preparation method and application thereof

Abstract

The application discloses a nano prodrug for releasing nitric oxide gas by ultrasonic piezoelectric catalysis effect and a preparation method and application thereof. 2000 The nano prodrug is formed by covalently coupling a nitric oxide precursor poly-L-arginine and a phospholipid DSPE-PEG -NH2 through an amidation reaction to form an amphiphilic prodrug molecule, and then encapsulating a nano piezoelectric material barium titanate to form the nano prodrug, which is accumulated in tumor tissues through enhanced permeation and retention effect, and generates a piezoelectric effect by using ultrasonic waves to stimulate the barium titanate, so that water molecules generate singlet oxygen, hydrogen peroxide and oxygen, and then the polyarginine is oxidized to release nitric oxide; the process is independent of oxygen and can produce oxygen, and is suitable for treatment of hypoxic tumors. The application further discloses application of the nano particles in breast cancer treatment, and the nano particles have good biocompatibility and remarkable effects of inhibiting tumor growth and metastasis, and have an advantage of being not limited by a tumor microenvironment compared with existing NO controlled release technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a nano-prodrug for releasing nitric oxide gas under the effect of ultrasonic piezoelectric catalysis, and a preparation method and application thereof. BACKGROUND

[0002] Cancer has a very high mortality rate and a very low cure rate, and is one of the most serious diseases threatening human health worldwide. Conventional treatment methods for cancer include surgical resection, radiotherapy, chemotherapy, etc. However, surgical resection is difficult to accurately resect tumor tissue, and cannot resect metastatic tumors, often resulting in residual and recurrence. Radiotherapy and chemotherapy have high toxic side effects, leading to a serious decline in the quality of life of patients. Gas therapy is a very promising treatment method with almost no side effects. In recent years, many studies have reported tumor treatment methods based on different gases, such as nitric oxide (NO), hydrogen sulfide, sulfur dioxide, and carbon monoxide. Among them, NO has obvious advantages: high-concentration NO can destroy cell mitochondria, interfere with energy supply, and inhibit cell proliferation; low-concentration NO can act as an inhibitor of P-glycoprotein, overcoming tumor multidrug resistance, and showing great potential in cancer treatment. However, gaseous NO is too active and has a very short half-life, and cannot be effectively delivered to the target tissue. Therefore, controlling the delivery and release of NO to achieve on-demand gas therapy is a great challenge.

[0003] In order to more stably supply NO, some small-molecule NO donors (such as S-nitrosylthiol (RSNO), N-nitroso-diazeniumdionyl (NONOate), etc.) have been used to deliver NO. However, due to the uncontrollable release behavior and rapid clearance of these donors in the body, there are often poor therapeutic effects and systemic toxic side effects, greatly limiting their application. L-arginine (L-Arg) as a natural NO donor can produce NO under the action of endogenous NO synthase, and can also release NO by oxidation with reactive oxygen species (ROS) such as H2O2 and 1 O2). In order to achieve controlled release of NO, researchers have developed different strategies based on L-Arg, such as combining L-Arg with GoX enzyme, converting glucose in the tumor to H2O2 to increase the level of H2O2 in the tumor, or combining photodynamic therapy or sonodynamic therapy to generate 1 O2, using these oxidants to trigger the release of NO from L-Arg, which well avoids the premature release of NO during blood circulation, and shows better controllability and tumor targeting. However, these strategies still have some defects, such as the loss of enzyme activity during preparation, the limited tissue penetration depth of light, and the lack of oxygen required for ROS production in the hypoxic microenvironment of the tumor, which limits the production of NO and reduces the therapeutic effect. Therefore, there is still an urgent need for more effective strategies to achieve spatial, temporal, and dose-controlled release of NO that is not limited by the tumor microenvironment.

[0004] Piezoelectric materials are emerging in biomedical field. Some piezoelectric materials can decompose water to produce H2O2 and O2 through piezocatalysis. 1 Barium titanate is a typical piezoelectric material. Under the action of ultrasound, barium titanate can generate unbalanced charges on the surface and undergo redox reaction with the surrounding water to produce H2O2 and O2. 1 Meanwhile, barium titanate can also decompose water to produce oxygen under the action of ultrasound.

[0005] Based on the above considerations, the present application realizes NO controlled release based on the ultrasound-induced piezocatalysis of nanometer piezoelectric material barium titanate, which can effectively trigger the oxidation of L-Arg to generate NO without relying on the tumor microenvironment. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a nano-prodrug for releasing nitric oxide gas by ultrasound piezocatalysis effect, a preparation method and application thereof, which solves the problems of uncontrollable release behavior, low penetration depth and limitation to tumor microenvironment in nitric oxide gas therapy, and has industrial transformation value.

[0007] The technical problem to be solved by the present application is solved by the following technical scheme:

[0008] A nano-prodrug for releasing nitric oxide gas by ultrasound piezocatalysis effect, wherein the nano-prodrug is formed by covalently coupling a nitric oxide precursor poly-L-arginine and a phospholipid DSPE-PEG 2000 -NH2 through amide reaction to form an amphiphilic prodrug molecule, and then encapsulating a nanometer piezoelectric material barium titanate.

[0009] Preferably, in the above technical scheme, the poly-L-arginine is a nitric oxide precursor capable of generating nitric oxide gas in response to reactive oxygen species; the phospholipid DSPE-PEG 2000 -NH2 is an amphiphilic molecule; and the barium titanate is a piezoelectric material capable of catalytically decomposing water to produce reactive oxygen species and oxygen under the action of ultrasound.

[0010] The above nano-prodrug for releasing nitric oxide gas by ultrasound piezocatalysis effect, wherein the structure of the poly-L-arginine is as follows:

[0011]

[0012] wherein n = 2-30, preferably n = 6-10.

[0013] Preferably, in the above technical solution, the nano-prodrug has a particle size of 5-500 nm; the nano-prodrug has a molecular molar ratio of L-arginine to the phospholipid of 1:1; and the mass ratio of the amphiphilic prodrug molecule to the nano-piezoelectric material is 2.2:1.

[0014] A preparation method of a nano-prodrug for releasing nitric oxide gas under the promotion of an ultrasonic piezoelectric catalytic effect, comprising the following steps:

[0015] S1: covalently coupling poly-L-arginine and phospholipid with an amide bond to prepare an amphiphilic prodrug molecule;

[0016] S2: encapsulating the nano-piezoelectric material with the amphiphilic prodrug molecule to form a nano-particle.

[0017] Preferably, in the above technical solution, the S1 step specifically comprises:

[0018] ① dissolving poly-L-arginine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide in a molar ratio of 1:1.3 in N,N-dimethylformamide and stirring at 25°C for 10 minutes;

[0019] ② dissolving the same number of moles of N-hydroxysuccinimide as 1-ethyl-(3-dimethylaminopropyl) carbodiimide in N,N-dimethylformamide, adding the mixture in ①, and continuing to stir for 1 hour;

[0020] ③ dissolving the same number of moles of DSPE-PEG 2000 -NH2 as poly-L-arginine in N,N-dimethylformamide, adding the mixture in ① and ②, and continuing to stir for 24 hours;

[0021] ④ dialyzing the mixture in ③ against deionized water for 2 hours (MWCO 8000-14000 Da) to remove 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, and N,N-dimethylformamide;

[0022] ⑤ freeze-drying the mixed aqueous solution after dialysis in ④ to obtain the amphiphilic prodrug molecule.

[0023] Preferably, in the above technical solution, the S2 step specifically comprises:

[0024] dispersing the amphiphilic prodrug molecule and the nano-piezoelectric material barium titanate with an organic solvent, fully mixing them, injecting them into deionized water under the condition of water bath ultrasonic, and dialyzing (MWCO 8000-14000 Da) against deionized water to remove the organic solvent to obtain the nano-prodrug.

[0025] Preferably, in the technical scheme, the organic solvent is an organic reagent that is miscible with water, and specifically one or more of tetrahydrofuran, anhydrous ethanol, DMF or dimethyl sulfoxide; the mixed volume of the organic solvent and water is 1:(5-10).

[0026] Preferably, in the technical scheme, the nano-prodrug can be prepared into an injection for intravenous injection.

[0027] The application also provides the use of the nano-prodrug in the preparation of a medicament for tumor treatment.

[0028] The application also provides the use of the nano-prodrug in the preparation of a medicament for tumor treatment.

[0029] The technical scheme has the following beneficial effects:

[0030] The application successfully prepares a nano-prodrug for releasing nitric oxide gas under the effect of ultrasonic piezocatalysis, the prepared nano-prodrug is uniformly dispersed in an aqueous solution, has regular morphology, and has obvious targeting property for tumors.

[0031] The application uses ultrasonic mediation piezocatalysis to decompose water to produce active oxygen and oxygen, and then effectively triggers the release of nitric oxide gas from poly-L-arginine, so as to realize the accurate on-demand release of nitric oxide at a target position, and the process is not limited by the tumor microenvironment, and the treatment effect of nitric oxide gas is significantly improved.

[0032] The application uses ultrasonic mediation piezocatalysis to decompose water to produce oxygen, which can effectively alleviate the tumor hypoxic microenvironment and inhibit tumor metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0033] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0034] Figure 1 The synthesis route diagram of the amphiphilic prodrug molecule synthesized in Example 1 and the structure schematic diagram of the nano-prodrug of the application;

[0035] Figure 2 The infrared spectrum of the amphiphilic prodrug molecule synthesized in Example 1;

[0036] Figure 3 The mass spectrum of the amphiphilic prodrug molecule synthesized in Example 1;

[0037] Figure 4 The transmission electron microscope (A) and scanning electron microscope (B) of the nano-prodrug for releasing nitric oxide gas under the effect of ultrasonic piezocatalysis synthesized in Example 2;

[0038] Figure 5 In vitro oxygen generation characterization of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 3);

[0039] Figure 6 In vitro singlet oxygen generation characterization of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 4);

[0040] Figure 7 In vitro hydrogen peroxide generation characterization of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 5);

[0041] Figure 8 In vitro nitric oxide generation characterization of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 6);

[0042] Figure 9 Cell live and dead staining pictures of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 7);

[0043] Figure 10 Cell layer oxygen content staining pictures (A) and their quantitative results (B) of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 8);

[0044] Figure 11 Cellular level HIF-1a protein immunoblotting pictures (A) and their quantitative results (B) of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 9);

[0045] Figure 12 Cell invasion and migration Transwell pictures (A) and their quantitative results (B) of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 (Example 10);

[0046] Figure 13 Animal fluorescence pictures of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2. Wherein, A is the in vivo fluorescence picture of mouse 4T1 tumor model, B is the fluorescence intensity quantification picture of tumor site (Example 11);

[0047] Figure 14 Tumor inhibition curve of the ultrasound piezocatalytic effect-triggered nano-prodrug releasing nitric oxide gas synthesized in Example 2 in mouse 4T1 tumor model (Example 12);

[0048] Figure 15Figure 1 1 shows the immunofluorescence staining of HIF-1a in the mouse 4T1 tumor treated with the ultrasound-piezoelectric catalysis effect of the nano-prodrug synthesized in Example 2 to release nitric oxide gas (Example 13).

[0049] Figure 16 Figure 12 shows the H&E staining of the lung metastasis of the mouse 4T1 tumor treated with the ultrasound-piezoelectric catalysis effect of the nano-prodrug synthesized in Example 2 to release nitric oxide gas (Example 14). DETAILED DESCRIPTION

[0050] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions, and numerical values set forth in these embodiments are not limiting to the scope of the present application unless specifically stated otherwise.

[0051] Unless specifically stated otherwise, the reagents and materials used in the following examples are commercially available; the specific preparation procedures and testing methods are conventional methods in the art, which meet the requirements of the laboratory routine.

[0052] Specific examples are as follows:

[0053] Example 1 Preparation of an amphiphilic nano-prodrug (DPA)

[0054] Sixteen milligrams of nona-L-arginine (9-Arg) (10.67 μmol of 9-Arg, 10.67 μmol of carboxyl group) was dissolved in DMF (500 μL) and reacted with 2.6 mg of EDC (13 μmol) for 10 minutes, and then with 1.5 mg of NHS (13 μmol) for 1 hour to activate the carboxyl group; subsequently, the solution was mixed with 20 mg of DSPE-PEG 2000 -NH2in DMF (600 μL) and reacted at 25°C for 24 hours. The reactants were dialyzed against distilled water for 2 hours (MWCO 8000-14000 Da) to remove excess 9-Arg, and finally freeze-dried to obtain DPA powder.

[0055] As shown in Figure 1, the synthesis route of the amphiphilic prodrug molecule is as follows: n = 9. Figure 1 As shown in Figure 1, the synthesis route of the amphiphilic prodrug molecule is as follows: n = 9.

[0056] As shown in Figure 2, the infrared spectrum and mass spectrum of the amphiphilic prodrug molecule are as follows, which verify the successful synthesis of DPA. Figure 2 As shown in Figure 2, the infrared spectrum and mass spectrum of the amphiphilic prodrug molecule are as follows, which verify the successful synthesis of DPA. Figure 3 Example 2 Preparation of a nano-prodrug for ultrasound-mediated piezoelectric catalysis to enhance the release of nitric oxide

[0057]

[0058] ​O-BTO and DPA were dissolved in 50 μL of tetrahydrofuran at a ratio of 1:3 (w / w) and were fully mixed and uniformly dispersed by water bath ultrasonic treatment for 2 minutes; then the tetrahydrofuran mixed solution of O-BTO and DPA was injected into 1 mL of ultrapure water at a constant speed under the condition of water bath ultrasonic treatment, and was self-assembled into particles by continuing ultrasonic treatment for 2 minutes; then the BTO@DPA aqueous solution was dialyzed in ultrapure water for 2 hours (MWCO 8000-14000 Da) to remove tetrahydrofuran; finally, the excess DPA was removed by centrifugation at a speed of 4000 rpm for 10 minutes, and a schematic diagram of the nano-prodrug assembly structure is shown in Figure 1 .

[0059] As shown in Figure 4 , the scanning electron microscope and transmission electron microscope images of the ultrasound-mediated piezocatalytic synergistic nitric oxide-releasing nano-prodrug obtained in the present embodiment are shown, the nanoparticles are uniform spherical, the size is about 160 nm, and the ultra-small barium titanate tetragonal particles can be clearly observed inside.

[0060] Example 3: In vitro oxygen production capacity test

[0061] In order to investigate whether the ultrasound-mediated piezocatalytic synergistic nitric oxide-releasing nano-prodrug obtained in Example 2 has the ability to produce oxygen. The oxygen dissolved in the solution was measured by using a dissolved oxygen meter (AZ8403, China) to evaluate the ability of BTO@DPA to produce oxygen under the action of ultrasound-mediated piezocatalysis. The solution was deoxygenated by using nitrogen and sealed, and the nanoparticles of different concentrations (100, 200, 400 μg / mL BTO) were treated with the same ultrasonic intensity (1 MHz, 1.5 w / cm 2 , 20% duty cycle), and the oxygen concentration was measured every 5 minutes until 30 minutes.

[0062] As shown in Figure 5 , ultrasound can mediate the production of oxygen by particles containing ultra-small barium titanate, and the amount of oxygen produced is more with the extension of ultrasonic time. Under the same ultrasonic conditions and at the same time point, the amount of oxygen produced gradually increases with the increase of the concentration of BTO@DPA nanoparticles.

[0063] Example 4: In vitro singlet oxygen production capacity test

[0064] In order to investigate whether the ultrasound-mediated piezocatalytic synergistic nitric oxide-releasing nano-prodrug obtained in Example 2 has the ability to produce singlet oxygen. The ability of the nanoparticles to produce singlet oxygen was evaluated by using the SOSG probe (Biyun Tian). Nanoparticles of different concentrations (25, 50, 100 μg / mL BTO) were mixed with the SOSG probe, and were treated with the same intensity of ultrasound (1 MHz, 1.0 w / cm 2 , 20% duty cycle), and the fluorescence intensity of SOSG was measured every 3 minutes by taking part of the solution.

[0065] As shown in Figure 6 , ultrasound can mediate the generation of singlet oxygen by BTO@DPA nanoparticles, and the amount of singlet oxygen generated is more as the ultrasound time is prolonged. Under the same ultrasound conditions and at the same time point, the amount of singlet oxygen generated gradually increases as the concentration of BTO@DPA nanoparticles increases.

[0066] Example 5 Test of in vitro hydrogen peroxide production ability

[0067] To investigate the hydrogen peroxide production ability of the ultrasound-mediated piezocatalytic synergistic nitric oxide release nano-prodrug obtained in Example 2. The hydrogen peroxide detection kit (Ribo) was used to evaluate the hydrogen peroxide production ability of the nanoparticles under the action of ultrasound. Nanoparticles of different concentrations (50, 100, 200 μg / mL BTO) were mixed with the hydrogen peroxide detection kit respectively, and treated with ultrasound of the same intensity (1 MHz, 1.0 w / cm 2 , 20% duty cycle). Every 3 minutes, part of the solution was taken to measure the ultraviolet absorption intensity until 24 minutes.

[0068] As shown in Figure 7 , ultrasound can mediate the generation of hydrogen peroxide by BTO@DPA nanoparticles, and the amount of hydrogen peroxide generated is more as the ultrasound time is prolonged. Under the same ultrasound conditions and at the same time point, the amount of hydrogen peroxide generated gradually increases as the concentration of BTO@DPA nanoparticles increases.

[0069] Example 6 Test of in vitro nitric oxide production ability

[0070] To investigate the nitric oxide production ability of the ultrasound-mediated piezocatalytic synergistic nitric oxide release nano-prodrug obtained in Example 2. The DAF-2 kit (Macklin) was used to evaluate the NO production ability of the nanoparticles under the action of ultrasound. Nanoparticles of different concentrations (50, 100, 200 μg / mL BTO, 110, 220, 440 μg / mL DPA) were mixed with the DAF-2 kit respectively, and treated with ultrasound of the same intensity (1 MHz, 1.0 w / cm 2 , 20% duty cycle). The fluorescence intensity was measured every 2 minutes for 20 minutes.

[0071] As shown in Figure 8 , ultrasound can mediate the generation of nitric oxide by BTO@DPA nanoparticles, and the amount of nitric oxide generated is more as the ultrasound time is prolonged. Under the same ultrasound conditions and at the same time point, the amount of nitric oxide generated gradually increases as the concentration of BTO@DPA nanoparticles increases.

[0072] Example 7 Verification of tumor killing by nitric oxide released by nano-prodrug

[0073] 4T1 cells were stored at 8 × 10⁸ cells per well. 4 Cells were seeded at a density of 1000 μg / mL into 24-well cell culture plates and incubated overnight in either normoxic (21% oxygen) or hypoxic (1% oxygen) incubators to allow cell adhesion. Cells were then divided into the following groups for corresponding treatments: 1) Control group; 2) Sonication group; 3) BTO@DPA group; 4) DPA + Sonication group; 5) BTO@DPA + Sonication group; 6) BTO@DPA + Sonication group (normoxic conditions); 7) BTO@DPA + Sonication group (hypoxic conditions). The concentration of BTO in each group was 200 μg / mL, and the concentration of DPA was 440 μg / mL. After adding the corresponding nanoparticles to each group, incubation was performed for 4 hours. Unabsorbed nanoparticles were washed away. Sonication (1 MHz, 1.5 W / cm²) was then applied to each group under either normoxic or hypoxic conditions (with or without). 2 (20% duty cycle, 3 minutes). After treatment, continue culturing for 18 hours, add Calcein-AM and PI fluorescence staining detection kit and incubate for 30 minutes. Finally, wash 3 times with PBS, observe and photograph under an inverted fluorescence microscope, where green fluorescence represents live cells and red fluorescence represents dead cells.

[0074] like Figure 9 As shown, no significant red fluorescence was observed in the control group, the ultrasound-only group, or the BTO@DPA nanoparticle group, indicating no cytotoxicity. A small amount of red fluorescence, representing cell death, was observed in the DPA+US and BTO@DP+US groups, but a large number of viable cells (green fluorescence) remained, indicating limited therapeutic efficacy in these two groups. In stark contrast, under normoxic and hypoxic conditions, the red fluorescence detected in the BTO@DP+US group covered the entire area, with almost no green fluorescence, indicating that the nitric oxide released by the ultrasound-mediated piezoelectric catalytic effect has a good tumor-killing effect and is independent of oxygen.

[0075] Example 8: Validation of nano-prodrugs alleviating hypoxia in tumor cells

[0076] The ability of BTO@DPA nanoparticles to produce oxygen at the cellular level was detected using a [Ru(dpp)3]Cl2 (Sigma-Aldrich) fluorescent probe. First, 4T1 cells were cultured in deoxygenated medium at 8 × 10⁶ cells per well. 4The cells were seeded in 24-well plates at a density of 3 x 105cells per well and cultured overnight in a hypoxic incubator to allow cell attachment. The cells were divided into the following groups for corresponding treatment: 1) control group; 2) ultrasound group; 3) BTO@DPA group; 4) DPA + ultrasound group; 5) BTO@DP + ultrasound group; 6) BTO@DPA + ultrasound group; 7) BTO@DPA + ultrasound group (normoxic condition). The concentration of nanoparticles and the ultrasound parameters in each group were the same as those in the cell level reactive oxygen species experiment, and the nanoparticles in the hypoxic condition group were diluted with deoxygenated medium. After adding the corresponding nanoparticles in different groups for 4 hours of incubation, the nanoparticles that were not taken up were washed away with deoxygenated PBS, and each group was subjected to ultrasound treatment or not. After 30 minutes of continued incubation, deoxygenated medium-diluted [Ru(dpp)3]Cl2fluorescent probe (10 μmol / L) was added, and incubated for 8 hours under hypoxic conditions. Finally, deoxygenated PBS was used to wash twice, and observed and photographed under an inverted fluorescence microscope, where red fluorescence represents the hypoxic state of the cells, and the stronger the fluorescence, the more severe the hypoxic state of the cells (λ ex = 450 nm, λ em = 610 nm).

[0077] As shown in Figure 10 , the control group, ultrasound group, and BTO@DPA group all showed strong red fluorescence, indicating that the tumor cells in these groups were in a state of obvious hypoxia; however, the red fluorescence of the BTO@DP + US group and the BTO@DPA + US group was significantly weakened, similar to the cells cultured under normoxic conditions, indicating that the oxygen generated based on the ultrasound-mediated BTO piezoelectric catalytic effect can sufficiently alleviate the hypoxic environment of tumor cells.

[0078] Example 9 Verification of Nanoparticle Prodrugs in Down-regulating HIF-1α at the Cell Level

[0079] Western blotting was used to detect the inhibition of HIF-1α expression by nanoparticles at the cell level. First, 4T1 cells were seeded in 6-well plates at a density of 3 x 105 5 cells per well using deoxygenated medium, and cultured in a hypoxic incubator (1% oxygen) for 24 hours to induce the expression of HIF-1α. The cells were divided into the following groups: 1) control group; 2) ultrasound group; 3) BTO@DPA group; 4) BTO@DP + ultrasound group; 5) BTO@DPA + ultrasound group. The concentration of BTO in each group was 100 μg / mL, and the concentration of DPA was 220 μg / mL. Each group of nanoparticles was incubated for 4 hours, and then subjected to ultrasound treatment (1 MHz, 1.5 w / cm 2, 20% duty cycle, 3 min), the nanoparticles were washed away with deoxygenated PBS, fresh deoxygenated medium was added to continue the culture under hypoxic condition for 18 hours, then the cells were digested and collected by centrifugation. The steps of Western blotting were as follows: first, the cell pellet was obtained by centrifugation at 1000 rpm for 5 min, 100 μL of protein lysis buffer (RIPA) containing 1% protease inhibitor was added for cell lysis on ice; then the protein concentration of each group was measured using a BCA protein quantification kit, 5x SDS protein loading buffer was added at a ratio of 1:4, and the mixture was boiled at 95°C for 5 min, and then centrifuged at 12000 rpm for 1 min before electrophoresis. The proteins were transferred to the NC membrane at 100 V, and the membrane was cut according to the positions of the Actin and HIF-1a protein bands, and then blocked with 5% milk for 1 hour, and then the membrane containing the Actin and HIF-1a bands was incubated with Actin and HIF-1a antibodies, respectively, at 4°C overnight on a shaker, and then washed with TBST and then incubated with the corresponding secondary antibodies for 1 hour. Finally, the membrane was exposed to light, and the image was taken on a chemiluminescence imaging system.

[0080] As shown in FIG. 9B, compared with other groups, the BTO@DP+US group and the BTO@DPA+US group showed only about 25% expression of HIF-1a, demonstrating that the groups containing ultra-small barium titanate can produce oxygen under ultrasonic conditions, alleviate the hypoxic state of tumor cells, and further down-regulate the expression of HIF-1a. Figure 11

[0081] Example 10 Verification of Nanoparticle Prodrugs Inhibiting Tumor Cell Invasion and Migration

[0082] The Transwell invasion experiment was used to evaluate the inhibitory ability of BTO@DPA nanoparticles on the invasion and migration of 4T1 cells. First, 4T1 cells were plated in a 6-well cell culture plate at a density of 3x10 5 cells per well and cultured under hypoxic or normoxic conditions overnight. The cells under hypoxic conditions were divided into the following groups: 1) control group; 2) ultrasonic group; 3) BTO@DPA group; 4) BTO@DP+ultrasonic group; 5) BTO@DPA+ultrasonic group. The particle concentration and treatment process of each group were the same as in Example 9. After treatment, fresh medium was added to continue the culture for 24 hours. After the cells were digested, 1x10 3 ​Cells were added at a density of [number] cells to the upper part of a Transwell chamber pre-coated with matrix gel. FBS-free medium was added to the upper part, and medium containing 10% FBS was added to the lower part. The chambers were incubated for 24 hours. Uninvaded cells in the upper part of the chamber were wiped away with a cotton swab. Migrating cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The cells were observed and photographed under an inverted fluorescence microscope. To quantify cell invasion and migration, the crystal violet on the chambers was dissolved in 3% acetic acid, and the absorbance at 570 nm was measured using a microplate reader.

[0083] like Figure 12 As shown, tumor cells treated with BTO@DP+US exhibited a significantly lower cell density (approximately 41%) compared to the control group, ultrasound group, and BTO@DPA group. This is attributed to the oxygen generated by BTO under ultrasound conditions alleviating tumor cell hypoxia, thereby reducing tumor cell invasiveness. Simultaneously, tumor cells treated with BTO@DPA+US showed an even lower cell density (approximately 24%), possibly indicating that while alleviating tumor hypoxia, it also exerts a killing effect on tumor cells, further reducing their invasiveness. In conclusion, ultrasound-mediated BTO piezoelectric catalysis to generate oxygen can effectively alleviate tumor cell hypoxia, reduce HIF-1α expression, and inhibit tumor cell invasiveness.

[0084] Example 11 Biodistribution in mice

[0085] Cy5.5-BTO@DPA nanoparticles were intravenously injected into 4T1 tumor-bearing mice at a dose of 10 mg / kg BTO. Fluorescence imaging of the mice was performed using the IVIS Spectrum small animal in vivo fluorescence imaging system before injection and at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after injection. ex =680nm,λ em =710nm).

[0086] like Figure 13 As shown, a significant fluorescence signal rapidly appeared at the tumor site 0.5 hours after injection, and the fluorescence signal gradually increased over time, reaching a maximum value of (28.68±3.13)×10⁻⁶ at 4 hours. 9 The effect then gradually diminished over the next 20 hours, remaining at (19.13±3.24)×10⁻⁴ mmol / L for up to 24 hours post-injection. 9 The high fluorescence intensity indicates that BTO@DPA nanoparticles have the characteristics of rapid accumulation and long retention time at the tumor site after in vivo injection, making them very suitable for subsequent tumor treatment.

[0087] Implementation of 12 tumor growth inhibition measures

[0088] When the tumor volume of 4T1 tumor-bearing mice reached about 100 mm 3 When the tumor volume of 4T1 tumor-bearing mice reached about 100 mm 2 , the mice were randomly divided into 6 groups, 4 in each group: 1) control group; 2) ultrasound group; 3) BTO@DPA group; 4) DPA+ultrasound group; 5) BTO@DP+ultrasound group; 6) BTO@DPA+ultrasound group, and the corresponding materials (10 mg / kg BTO, 22 mg / kg DPA) were injected intravenously into the mice in each group on the 1st, 3rd and 5th day. For the ultrasound-treated groups, the tumor site was treated with ultrasound (1 MHz, 1.5 w / cm 2 , 20% duty cycle, 10 minutes) according to the maximum enrichment time point of nanoparticles in the tumor site in the biodistribution (i.e. 4 hours). The tumor volume was recorded every two days during the treatment.

[0089] As shown in Figure 14 , the tumors of the mice in the control group, ultrasound group and BTO@DPA group grew rapidly during the treatment, and the tumor volume reached about 1500 mm 3 at the end of the 15th day treatment, indicating that ultrasound or BTO@DPA alone had no therapeutic effect on the tumor; in contrast, a certain inhibition of tumor growth was observed in the DPA+US group and the BTO@DP+US group; while the BTO@DPA+US group showed the best therapeutic effect, with an inhibition rate of 70.27% on the tumor.

[0090] Verification of relieving tumor hypoxia at animal level in Example 13

[0091] After the end of the treatment on the 5th day of the treatment experiment in Example 12, the mice were sacrificed and the tumors were dissected out, fixed with 4% tissue fixative, dehydrated, paraffin-embedded, sectioned, and the tissue sections were blocked with protein blocking solution for 1 h, then incubated with HIF-1α primary antibody at 4°C overnight. After washing with PBS, fluorescently labeled secondary antibody was added and incubated with the tissue sections for 2 hours, the sections were washed again and mounted, and finally the sections were scanned.

[0092] As shown in Figure 15 , strong red fluorescent signals reflecting the expression of HIF-1α were detected in the control group, US, BTO@DPA and DPA+US treatment groups, indicating that these tumors were in a severe hypoxic state; while the BTO@DP+US and BTO@DPA+US treatment groups showed very weak red fluorescence, indicating low expression of HIF-1α, which was due to the effective relief of tumor hypoxia by oxygen generated by ultrasound-mediated BTO piezoelectric catalytic decomposition of water, and the down-regulation of HIF-1α expression.

[0093] Inhibition of tumor metastasis in Example 14

[0094] After the end of the treatment experiment of Example 12, the mice were killed and the lung tissues of each group were dissected, fixed with 4% tissue fixative, dehydrated, paraffin-embedded, sectioned and H&E stained, and finally scanned.

[0095] As shown in Figure 16 , the control group and the simple US or BTO@DPA group all observed obvious tumor metastasis in the lung tissue, the DPA+US group showed relatively less metastasis, due to the small amount of NO and ONOO - produced by DPA+US, which inhibited the tumor; while the BTO@DP+US and BTO@DPA+US treatment groups did not show obvious lung metastasis, due to the fact that BTO can effectively kill tumor cells under the action of ultrasound and also produce oxygen to relieve tumor hypoxia, thereby achieving the best metastasis inhibition effect.

[0096] Although the present application has been disclosed as above with examples, it is not intended to limit the present application, and any person skilled in the art can make various selections and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application is defined by the claims and their equivalent forms.

Claims

1. An ultrasonic piezoelectric catalytic effect induced release of nitric oxide gas nanoprodrug, characterized in that, The nano-prodrug is formed by covalently coupling poly-L-arginine and phospholipid DSPE-PEG 2000 -NH2 by amidation reaction to form an amphiphilic prodrug molecule, and then encapsulating nano-piezoelectric material barium titanate to form a nano-prodrug, and using ultrasonic stimulation to generate piezoelectric effect of the barium titanate to catalyze water molecules to generate singlet oxygen, hydrogen peroxide and oxygen, and then oxidize polyarginine to release nitric oxide, wherein the poly-L-arginine has the following structural formula: Wherein, n = 6-10.

2. The preparation method of the nano-prodrug for promoting the release of nitric oxide gas by ultrasonic piezoelectric catalysis effect according to claim 1, specifically comprising the following steps: S1 : Covalently couple the poly-L-arginine and phospholipid DSPE-PEG 2000 -NH2 with an amide bond to produce an amphiphilic prodrug molecule; S2: The amphiphilic prodrug molecules obtained in the step S1 are used to encapsulate the nano piezoelectric material barium titanate to form nano prodrug particles.

3. The method of claim 2, wherein the method is characterized by: The molecular molar ratio of the poly-L-arginine to the phospholipid is 1:1; the mass ratio of the amphiphilic prodrug molecules to the nano piezoelectric material barium titanate is 2.2:

1.

4. The method of claim 2, wherein the method is characterized by: The step S1 specifically comprises: ① Poly-L-arginine and 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) are dissolved in N,N-dimethylformamide (DMF) at a molar ratio of 1:1.3, and stirred at 25°C for 10 minutes; ② The same molar number of N-hydroxysuccinimide (NHS) as EDC is dissolved in DMF and added to the mixture obtained in step ①, and stirring is continued for 1 hour; iii. The same molar amount of DSPE-PEG 2000 -NH2in DMF was added to the mixture from step ii. and stirring was continued for 24 hours. ④ The mixture obtained in step ③ is dialyzed with deionized water for 2 hours to remove EDC, NHS, DMF, wherein the molecular weight cut-off MWCO of dialysis is 8000-14000 Da; ⑤ The mixed aqueous solution dialyzed in step ④ is freeze-dried to obtain amphiphilic prodrug molecules.

5. The method of claim 2, wherein the method is characterized by: The step S2 specifically comprises: The amphiphilic prodrug molecules and the nano piezoelectric material barium titanate are dispersed and mixed with an organic solvent, and then injected into deionized water under the condition of water bath ultrasonic, and then dialyzed with deionized water for 2 hours to remove the organic solvent to obtain nano prodrug, wherein the molecular weight cut-off MWCO of dialysis is 8000-14000 Da.

6. The method of claim 5, wherein the method is characterized by: The organic solvent is a water-miscible organic reagent, specifically one or more of tetrahydrofuran, anhydrous ethanol, DMF or dimethyl sulfoxide; the mixed volume of organic solvent and water is 1:(5-10).

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