Combined medicine of attenuated salmonella loaded barium titanate piezoelectric nanoparticles and application

Abstract

The invention relates to the technical field of nano material preparation and biological medicine, in particular to a combined medicine of attenuated salmonella loaded barium titanate piezoelectric nano particles and application. The combined medicine provided by the invention is barium titanate piezoelectric nano-particles which are modified by a silane coupling agent and have the characteristics of good surface charges and the like, and the barium titanate piezoelectric nano-particles are combined on the surface of attenuated salmonella through electrostatic interaction. The invention further provides a preparation method of the combined medicine. The preparation method comprises the following steps: step 1, preparing the barium titanate piezoelectric nano-particles with good surface charge and other characteristics; and step 2, preparing the attenuated salmonella loaded barium titanate piezoelectric nano-particle combined medicine. The invention further provides application of the combined medicine in the anti-tumor aspect. The combined medicine disclosed by the invention has good anti-tumor performance and excellent biological safety, and a new thought is provided for bacterium-nanoparticle combined treatment.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterial preparation and biomedicine, specifically to combination drugs and their applications using attenuated Salmonella-loaded barium titanate piezoelectric nanoparticles. Background Technology

[0002] Malignant tumors are a major threat to human health, with their incidence and mortality rates rising year by year. Sonodynamic therapy, as a non-invasive treatment, represents a new direction in cancer treatment. Sonodynamic therapy utilizes ultrasound to activate a sonosensitive agent, generating reactive oxygen species and cavitation effects, inducing apoptosis and necrosis, thereby killing tumor cells. Due to the excellent tissue penetration and precise focusing capabilities of ultrasound, sonodynamic therapy exhibits significant advantages such as non-invasiveness, targeted therapy, safety, and repeatability.

[0003] Piezoelectric semiconductor nanomaterials are ideal sonosensitizers, among which barium titanate piezoelectric nanoparticles have become widely used sonosensitizers in sonodynamic therapy due to their good biocompatibility, low cytotoxicity, and strong piezoelectric properties. However, barium titanate piezoelectric nanoparticles are unevenly distributed in tumor tissues, making it difficult to achieve deep penetration and uniform distribution, thus limiting therapeutic efficacy. Attenuated Salmonella has natural hypoxia targeting and deep penetration capabilities, making it a potential drug delivery carrier, but its application in cancer treatment still faces challenges in terms of biosafety and delivery efficiency.

[0004] Therefore, there is an urgent need to develop a novel delivery strategy to achieve efficient and specific delivery of barium titanate piezoelectric nanoparticles and ensure that attenuated Salmonella can self-inactivate after drug delivery, ultimately achieving safe and efficient sonodynamic therapy for tumors. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention proposes a combination drug and its application using barium titanate piezoelectric nanoparticles loaded with attenuated Salmonella. By electrostatically adsorbing barium titanate piezoelectric nanoparticles with desirable surface charge and other properties onto the surface of attenuated Salmonella, a combination drug with both highly efficient tumor targeting and deep penetration capabilities is constructed. This system can efficiently generate reactive oxygen species under ultrasound irradiation, achieving precise tumor ablation, and then self-inactivates after treatment, exhibiting good biocompatibility.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a combination drug for attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles. The combination drug refers to barium titanate piezoelectric nanoparticles with good surface charge and other properties after being modified by a silane coupling agent, which are bound to the surface of attenuated Salmonella through electrostatic interaction.

[0008] Furthermore, the barium titanate piezoelectric nanoparticles with good surface charge and other properties are a type of barium titanate piezoelectric nanoparticles with positive surface charge, uniform morphology, and good dispersibility.

[0009] Furthermore, preferably, the barium titanate piezoelectric nanoparticles with good surface charge and other properties have a particle size of 60-100 nm and a tetragonal crystal structure.

[0010] Secondly, the present invention provides a method for preparing a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, which is carried out according to the following steps:

[0011] Step 1: Prepare barium titanate piezoelectric nanoparticles with good surface charge and other properties;

[0012] Step 2: Prepare a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles.

[0013] Furthermore, step 1 includes the following steps:

[0014] Step 1.1: Barium nitrate, sodium hydroxide, oleic acid, oleylamine and tetrabutyl titanate are subjected to a hydrothermal reaction at 135-150 °C to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles.

[0015] Step 1.2: Wash the oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles with diethylamine to remove the oleic acid / oleylamine-terminated ligands and obtain barium titanate piezoelectric nanoparticles with a clean surface.

[0016] Step 1.3: Graft a silane coupling agent onto barium titanate piezoelectric nanoparticles with a clean surface to obtain barium titanate piezoelectric nanoparticles with good surface charge and other properties.

[0017] Furthermore, the specific process of step 2 is as follows:

[0018] A solution of barium titanate piezoelectric nanoparticles with good surface charge and other properties was incubated with an equivalent volume of attenuated Salmonella suspension at 36-37°C for 1-4 hours. The precipitate was collected by centrifugation at 800-1200g for 10-15 minutes and washed with PBS at pH 7.4 to obtain a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, which was then dispersed in PBS for storage.

[0019] Further, in step 1.1, the hydrothermal reaction of barium nitrate, sodium hydroxide, oleic acid, oleylamine, and tetrabutyl titanate at 135-150 °C to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles is specifically carried out as follows: 0.4 M barium nitrate solution, 2 M sodium hydroxide solution, oleic acid, oleylamine, and a 2 M tetrabutyl titanate n-butanol solution are added to a high-pressure reactor, stirred, sealed, and subjected to a hydrothermal reaction at 135-150 °C for 16-24 hours; the product is separated by centrifugation at a relative centrifugal force of 5000-8000 g for 10-15 minutes, washed with ethanol, and dispersed in toluene to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles.

[0020] Furthermore, in step 1.2, the barium titanate piezoelectric nanoparticles with clean surfaces are refluxed at 85-95°C for 6-10 hours with 30% hydrogen peroxide, followed by centrifugation, washing with ethanol, and ultrasonic dispersion, so that the barium titanate piezoelectric nanoparticles with clean surfaces are stably dispersed in ethanol.

[0021] Further, in step 1.3, the silane coupling agent is any one of amino, quaternary ammonium salt, guanidinyl, or imidazolyl silane coupling agents.

[0022] Furthermore, the attenuated Salmonella is any one of the attenuated Salmonella strains VNP20009, YB1, SL7207, and BRD509.

[0023] Furthermore, the proportions of the combined drugs are as follows: the concentration of barium titanate piezoelectric nanoparticles with good surface charge and other properties is 550-800 μg / mL, and the OD600 value of the attenuated Salmonella suspension is 0.6-1.0.

[0024] Thirdly, the present invention provides an application of a combination drug containing attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, the combination drug being used to treat tumors and / or inhibit tumor growth.

[0025] Furthermore, the specific steps for applying the combined drug to treat tumors and / or inhibit tumor growth are as follows: The combined drug is delivered into the body, and external ultrasound irradiation is applied to the tumor site to generate reactive oxygen species in the barium titanate piezoelectric nanoparticles, thereby treating or inhibiting the tumor; the frequency of the ultrasound irradiation is 0.1-3.0 MHz, and the sound intensity is 0.1-3.0 W / cm². 2 The duty cycle is 10%-100%, and the action time is 1-30 minutes.

[0026] Furthermore, the tumor is any one of melanoma, breast cancer, colon cancer, pancreatic cancer, liver cancer, kidney cancer, osteosarcoma, glioma, lung cancer, prostate cancer, stomach cancer, esophageal cancer, cervical cancer, ovarian cancer, endometrial cancer, bladder cancer, or thyroid cancer.

[0027] Compared with the prior art, the present invention has the following beneficial technical effects:

[0028] 1. This invention overcomes the dual technical bottlenecks of poor targeting and low biocompatibility of traditional barium titanate piezoelectric nanoparticles in sonodynamic therapy by constructing a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles using barium titanate piezoelectric nanoparticles with excellent surface charge and other properties. The system cleverly utilizes the inherent hypoxic chemotaxis of bacteria to achieve efficient enrichment and deep penetration of the sonosensitive agent in tumor tissue, significantly improving targeting efficiency. Crucially, this invention employs an ultrasound-triggered programmed suicide mechanism, which promptly lyses and eliminates the bacterial carrier after drug delivery, fundamentally eliminating the potential infection risk caused by excessive proliferation and ensuring high biocompatibility. This combination drug provides a safe, efficient, and controllable new platform for cancer treatment.

[0029] 2. The combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles of the present invention has good anti-tumor properties. As can be seen from the mouse experiments in the specific embodiments, the combination drug exhibits significant anti-tumor ability both in vivo and in vitro.

[0030] 3. This invention successfully prepared barium titanate piezoelectric nanoparticles with good surface charge and other properties using hydrothermal synthesis, ligand exchange, and silane coupling agent surface modification. This method not only maintains the piezoelectric catalytic activity of barium titanate nanoparticles, but also achieves stable electrostatic binding with attenuated Salmonella through good surface charge and other properties.

[0031] 4. The preparation process of the attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles of the present invention is simple and the conditions are mild, which has the potential for industrialization and commercialization.

[0032] 5. This invention utilizes attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles possessing excellent surface charge and other properties to form a combination drug. Compared to carriers such as bacterial outer membrane vesicles or exosomes, this combination drug significantly enhances the tumor targeting and deep tissue penetration capabilities of the piezoelectric nanoparticles. Compared to natural Escherichia coli, Helicobacter pylori, and other bacterial flora, the genetically engineered attenuated Salmonella carrier used in this invention retains excellent tumor targeting capabilities while fundamentally improving its biosafety. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the preparation process of the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to the present invention.

[0034] Figure 2 This is a transmission electron microscope image of the barium titanate piezoelectric nanoparticles prepared in Example 1;

[0035] Figure 3 The diagram shows the zeta potential of barium titanate piezoelectric nanoparticles before and after modification with the silane coupling agent in Example 1.

[0036] Figure 4 The infrared spectra of barium titanate piezoelectric nanoparticles before and after modification with the silane coupling agent in Example 1 are shown.

[0037] Figure 5 This is a transmission electron microscope image of the attenuated Salmonella-loaded barium titanate piezoelectric nanoparticle combination drug prepared in Example 4 without ultrasonic irradiation.

[0038] Figure 6 This is a transmission electron microscope image of the attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles prepared in Example 4 after ultrasonic irradiation.

[0039] Figure 7 This is a zeta potential diagram of the attenuated Salmonella strain in Example 4 before and after loading barium titanate piezoelectric nanoparticles with good surface charge and other properties.

[0040] Figure 8 The activity of the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles with different delivery ratios in Example 6 before and after ultrasonic irradiation;

[0041] Figure 9 This is a graph showing the toxicity of different drug concentrations and pre- and post-ultrasound treatments to B16F10 cells in Example 7.

[0042] Figure 10 This is a distribution diagram of barium titanate piezoelectric nanoparticles with good surface charge and other properties in a tumor section 12 hours after the injection of the combined drug in Example 9.

[0043] Figure 11 This is a quantitative analysis of the in vivo fluorescence intensity in B16F10 tumor-bearing mice within 12 hours of drug injection in Example 9;

[0044] Figure 12 This is a quantitative analysis graph of the fluorescence intensity of the resected tumor and major organs within 12 hours after drug injection in Example 9;

[0045] Figure 13 This is a diagram showing the effect of drug treatment on tumors in Example 10. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] The mechanism by which the attenuated Salmonella-loaded barium titanate piezoelectric nanoparticle combination drug of the present invention is used for tumor sonodynamic therapy is as follows:

[0049] Attenuated Salmonella bacteria can utilize their natural anaerobic tendency to specifically deliver and efficiently accumulate piezoelectric nanoparticles (such as barium titanate piezoelectric nanoparticles) in hypoxic regions of tumors. Barium titanate, a typical piezoelectric material, generates a periodically changing electric field within itself due to the piezoelectric effect when exposed to external ultrasound irradiation. This promotes charge separation and catalyzes electrochemical reactions of water or oxygen molecules in the surrounding environment, generating a large amount of reactive oxygen species (such as singlet oxygen and hydroxyl radicals). This effectively initiates sonodynamic therapy, killing tumor cells. Simultaneously, the high levels of reactive oxygen species generated during sonodynamic therapy not only directly damage tumor cells but also rapidly trigger the lysis of attenuated Salmonella, allowing it to quickly self-eliminate after completing drug delivery and accumulation. This effectively avoids systemic inflammatory responses that may be caused by persistent or excessive bacterial proliferation, improving the safety of treatment. Specific Implementation Method 1

[0051] This invention provides a combination drug for attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles. The combination drug refers to barium titanate piezoelectric nanoparticles with good surface charge and other properties after being modified with a silane coupling agent, which are bound to the surface of attenuated Salmonella through electrostatic interaction.

[0052] The barium titanate piezoelectric nanoparticles with good surface charge and other properties are a class of barium titanate piezoelectric nanoparticles with positive surface charge, uniform morphology and good dispersibility.

[0053] Preferably, the barium titanate piezoelectric nanoparticles with good surface charge and other properties have a particle size of 60-100 nm and a tetragonal crystal structure. Specific Implementation Method Two

[0055] This invention also provides a method for preparing a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, see [link to relevant documentation]. Figure 1 It is done in the following steps:

[0056] Step 1: Prepare barium titanate piezoelectric nanoparticles with good surface charge and other properties.

[0057] Step 1.1: Barium nitrate, sodium hydroxide, oleic acid, oleylamine, and tetrabutyl titanate are subjected to a hydrothermal reaction at 135-150 °C to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles.

[0058] Specifically, a 0.4 M barium nitrate solution, a 2 M sodium hydroxide solution, oleic acid, oleylamine, and a 2 M tetrabutyl titanate solution in n-butanol were added to an autoclave. After stirring, the autoclave was sealed and subjected to a hydrothermal reaction at 135-150°C for 16-24 hours. The product was separated by centrifugation at a relative centrifugal force of 5000-8000 g for 10-15 minutes, washed with ethanol, and dispersed in toluene to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles.

[0059] Step 1.2: Wash the oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles with diethylamine to remove the oleic acid / oleylamine-terminated ligands and obtain barium titanate piezoelectric nanoparticles with a clean surface.

[0060] To improve the hydrophilicity of barium titanate piezoelectric nanoparticles with clean surfaces, they can be refluxed at 85-95℃ with 30% hydrogen peroxide for 6-10 hours, followed by centrifugation, washing with ethanol, and ultrasonic dispersion, so that the barium titanate piezoelectric nanoparticles with clean surfaces are stably dispersed in ethanol.

[0061] Step 1.3: Graft a silane coupling agent onto barium titanate piezoelectric nanoparticles with a clean surface to obtain barium titanate piezoelectric nanoparticles with good surface charge and other properties.

[0062] The silane coupling agent is any one of amino, quaternary ammonium salt, guanidinyl or imidazole silane coupling agents.

[0063] Step 2: Preparation of a combination drug using attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles. The specific process is as follows:

[0064] A solution of barium titanate piezoelectric nanoparticles with good surface charge and other properties was incubated with an equivalent volume of attenuated Salmonella suspension at 36-37°C for 1-4 hours. The precipitate was collected by centrifugation at 800-1200g for 10-15 minutes and washed with phosphate-buffered saline (PBS) at pH 7.4 to obtain a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, which was then dispersed in PBS for storage.

[0065] The proportions of the combined drugs are as follows: the concentration of barium titanate piezoelectric nanoparticles with good surface charge and other properties is 550-800 μg / mL, and the OD600 value of the attenuated Salmonella suspension is 0.6-1.0.

[0066] The attenuated Salmonella is any one of the attenuated Salmonella strains such as VNP20009, YB1, SL7207, or BRD509. Specific Implementation Method 3

[0068] This invention also provides an application of a combination drug containing attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, which can be used to treat tumors and / or inhibit tumor growth.

[0069] This application delivers a combination drug of attenuated Salmonella bacteria loaded with barium titanate piezoelectric nanoparticles into the body via intravenous injection. Leveraging the tumor-targeting properties of attenuated Salmonella, piezoelectric nanoparticles with favorable surface charge and other characteristics are delivered to the tumor site. Subsequently, external ultrasound irradiation is applied to the tumor site, utilizing the mechanical stress of the ultrasound to activate the piezoelectric effect of the barium titanate nanoparticles. This catalyzes water and oxygen molecules in the surrounding environment, generating a large amount of highly cytotoxic reactive oxygen species (ROS). These ROS attack key biomolecules such as lipids, proteins, and DNA in tumor cells, inducing irreversible oxidative damage. Ultimately, by activating apoptosis and necrosis pathways, precise tumor treatment and inhibition effects are achieved.

[0070] The specific steps are as follows: The combined drug is delivered into the body, and external ultrasound irradiation is applied to the tumor site to induce reactive oxygen species in the barium titanate piezoelectric nanoparticles, thereby treating or inhibiting the tumor; the frequency of the ultrasound irradiation is 0.1-3.0 MHz, and the sound intensity is 0.1-3.0 W / cm². 2 The duty cycle is 10%-100%, and the action time is 1-30 minutes.

[0071] The tumor is any one of the following: melanoma, breast cancer, colon cancer, pancreatic cancer, liver cancer, kidney cancer, osteosarcoma, glioma, lung cancer, prostate cancer, stomach cancer, esophageal cancer, cervical cancer, ovarian cancer, endometrial cancer, bladder cancer, or thyroid cancer.

[0072] Example:

[0073] The following examples further illustrate the specific implementation methods and verify their effects.

[0074] Example 1: Preparation of barium titanate piezoelectric nanoparticles with good surface charge and other properties.

[0075] 5 mL of barium nitrate solution (0.4 M), 5 mL of sodium hydroxide solution (2 M), 3.6 mL of oleic acid, 1.8 mL of oleylamine, and 20 mL of n-butanol containing 2 mmol of tetrabutyl titanate were sequentially added to a 50 mL autoclave. After stirring for 30 minutes, the autoclave was sealed and reacted at 135 °C for 18 hours. The product was separated by centrifugation at 6255 g for 10 minutes, washed three times with ethanol, and dispersed in toluene to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles.

[0076] To remove the oleic acid / oleylamine-terminated ligands and obtain a clean surface, the nanoparticles were washed three times with diethylamine. To improve hydrophilicity, the nanoparticles were treated with 100 mL of 30% hydrogen peroxide and refluxed at 85 °C for 8 hours with stirring. The piezoelectric nanoparticles were then collected by centrifugation at 6255 g for 10 minutes, washed with ethanol, and dispersed in ethanol using ultrasonication to obtain barium titanate piezoelectric nanoparticles with a clean surface. Transmission electron microscopy results are shown below. Figure 2 As shown, the barium titanate piezoelectric nanoparticles have uniform morphology, clean surfaces, and good dispersibility.

[0077] Finally, an appropriate amount of 3-aminopropyltriethoxysilane was added to the ethanol solution of barium titanate piezoelectric nanoparticles to adjust the pH to 3-4, and the reaction was carried out overnight at room temperature under nitrogen protection. After the reaction was completed, the solution was centrifuged at 6255 g for 10 minutes to collect the barium titanate piezoelectric nanoparticles, which were then washed with ethanol to finally obtain barium titanate piezoelectric nanoparticles with good surface charge and other properties. Figure 3 As shown, barium titanate piezoelectric nanoparticles with good surface charge and other properties exhibit a positive charge. Figure 4 As shown in the Fourier transform infrared spectrum, barium titanate piezoelectric nanoparticles with good surface charge and other properties... , and The absorption peaks appearing at these locations correspond to 3-aminopropyltriethoxysilane, respectively. Stretching and stretching vibrations of the key Symmetric and asymmetric stretching vibrations and The bending vibrations were observed. These results confirm that 3-aminopropyltriethoxysilane has been successfully modified onto the surface of barium titanate piezoelectric nanoparticles.

[0078] Example 2: The other processes are the same as in Example 1, except that γ-ureidopropyltrimethoxysilane is used as the silane coupling agent.

[0079] Example 3: The other processes are the same as in Example 1, except that the temperature of the hydrothermal reaction is 150°C.

[0080] Example 4: Preparation of a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles.

[0081] A certain amount of barium titanate piezoelectric nanoparticles with good surface charge and other properties were weighed and dispersed in PBS to prepare a stock solution of 1000 μg / mL. Attenuated Salmonella bacteria (Strain VNP20009) growing on LB agar plates were picked up using a 10 μL pipette tip and placed in liquid culture medium. The culture was continued until the OD600 value reached 0.8. The plates were washed three times with PBS and dispersed in an equal volume of PBS (pH 7.4) to obtain an attenuated Salmonella suspension. Barium titanate piezoelectric nanoparticles with good surface charge and other properties were added to an equal volume of the attenuated Salmonella suspension at concentration gradients of 0 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, and 1000 μg / mL. The mixture was incubated in a dark incubator (37℃, 180 rpm) for 2 hours. Subsequently, the sample was centrifuged for 10 minutes at a relative centrifugal force of 1000g, the precipitate was collected, and washed three times with PBS. A combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles was obtained and dispersed in PBS for storage.

[0082] Figure 5 and Figure 6 The transmission electron microscopy (TEM) images of the combination drug loaded with barium titanate piezoelectric nanoparticles for attenuated Salmonella before and after ultrasound are shown in the figure. The barium titanate piezoelectric nanoparticles, possessing good surface charge and other properties, are loaded onto the surface of rod-shaped attenuated Salmonella bacteria. After 5 minutes of ultrasound irradiation, the barium titanate piezoelectric nanoparticles are gradually released, and the released reactive oxygen species further disrupt the bacterial structure, leading to a large release of bacterial antigens. Figure 7 The figure shows the change in Zeta potential before and after attenuated Salmonella was loaded with barium titanate piezoelectric nanoparticles with good surface charge and other properties, indicating that barium titanate piezoelectric nanoparticles with good surface charge and other properties were successfully loaded onto the surface of attenuated Salmonella.

[0083] Example 5: The other processes are the same as in Example 4, except that the attenuated Salmonella is strain YB1.

[0084] Example 6: Screening for the proportion of the combined drugs of the present invention that achieve a balance between drug delivery and bacterial lysis.

[0085] This study investigated the activity of attenuated Salmonella strains loaded with barium titanate piezoelectric nanoparticles at different delivery ratios before and after ultrasonic irradiation. First, 10 μL of the combined drug mixtures of attenuated Salmonella strains loaded with barium titanate piezoelectric nanoparticles at different concentration gradients synthesized in Example 4 were added to liquid culture medium. After incubation in a dark incubator (37℃, 180 rpm) for 2 h, the OD value was measured and recorded as the pre-ultrasound group. Then, the combined drug mixtures of attenuated Salmonella strains loaded with barium titanate piezoelectric nanoparticles at different concentrations synthesized in Example 4 were ultrasonicated for 5 minutes, then added back to the culture medium. After incubation for 24 h, the OD value was measured and recorded as the post-ultrasound group.

[0086] like Figure 8 As shown, the combination drug with barium titanate piezoelectric nanoparticles loaded with attenuated Salmonella in the range of 550-800 μg / mL had the most ideal effect. That is, barium titanate piezoelectric nanoparticles with good surface charge and other properties before ultrasonic irradiation had the least impact on the activity of attenuated Salmonella, and more attenuated Salmonella could be inactivated after 5 minutes of ultrasonic irradiation.

[0087] Example 7: In vitro cytotoxicity experiment of the combination drug of the present invention

[0088] B16F10 cells were seeded in 96-well plates and cultured overnight in a cell culture incubator. After cell adhesion, a combination of drugs (concentrations of 0, 12.5, 25, 50, 75, and 100 μg / mL) loaded with attenuated Salmonella titanate piezoelectric nanoparticles was added and incubated for 2–4 h. Subsequently, the cells were incubated in the dark or sonicated (1 MHz, 1.5 W / cm², 50% duty cycle, 5 min). Then, 10 μL of CCK8 solution was added to each well, and the cells were incubated in the dark for 4 h. After removing the culture medium, DMSO was added, and the absorbance of each well was measured at 450 nm using a microplate reader. Based on the results, the control group (no drug added) and the blank group (no cell addition) were used. Cell viability was calculated using the following formula:

[0089] like Figure 9 As shown, with increasing drug concentration, the survival rate of tumor cells in the post-ultrasound group decreased significantly, while the survival rate of tumor cells in the pre-ultrasound group did not decrease significantly.

[0090] Example 8: The other processes are the same as in Example 7, except that the tumor cells are 4T1 cells.

[0091] Example 9: Tumor-targeting experiment of the combination drug of the present invention

[0092] To evaluate the feasibility of intravenous administration as the delivery route for this system, we first labeled piezoelectric nanoparticles with good surface charge and other properties with fluorescein isothiocyanate. 1 g of piezoelectric nanoparticles with good surface charge and other properties were dispersed in a fluorescein isothiocyanate solution, stirred in the dark for 24 hours, and then centrifuged at 6255 g for 10 minutes to collect the fluorescein-labeled piezoelectric nanoparticles with good surface charge and other properties. A combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles was then prepared according to Example 4. Next, we investigated the accumulation of the combination drug at the tumor site in B16F10 tumor-bearing mice after tail vein injection. Figure 10As shown, we sectioned the tumor tissue and took confocal images. The combined drug showed significant fluorescence signals throughout the tumor sections, indicating its ability to penetrate deep tumors, which is attributed to the inherent motility and chemotaxis of the attenuated Salmonella in the system.

[0093] To verify the tumor-targeting ability of the combined drug, we subsequently used an in vivo imaging system for monitoring, and used the fluorescence signal of fluorescein isothiocyanate as a tracer. Figure 11 As shown, compared to intravenously injected barium titanate piezoelectric nanoparticles with good surface charge and other properties, the combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles exhibited significantly stronger fluorescence intensity at the tumor site. The fluorescence signal of the combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles continuously increased over time and remained stable throughout the observation period, highlighting the tumor-targeting efficacy of the combination drug of the present invention. In stark contrast, only a weak fluorescence signal was detected in tumor tissue within 12 hours after intravenous injection of barium titanate piezoelectric nanoparticles with good surface charge and other properties. These results fully demonstrate that attenuated Salmonella can actively target tumors and efficiently deliver its carried barium titanate piezoelectric nanoparticles. Figure 12 As shown, in vitro fluorescence imaging of major organs and tumors 12 hours after injection further validated the above in vivo observations. The fluorescence signal intensity of the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles in mouse isolated tumors was 7 times that of the intravenously injected group with piezoelectric nanoparticles with good surface charge and other properties.

[0094] Example 10: In vivo antitumor experiment of the combination drug of the present invention

[0095] The experimental group was divided into a blank control group, an ultrasound group, a combination drug group, and a combination drug + ultrasound group according to different administration methods. Each mouse in the blank control group was given an equal volume of PBS. Each mouse in the ultrasound group was given an equal volume of PBS and then irradiated with ultrasound (1 MHz, 1.5 W / cm², 50% duty cycle, 5 minutes). Each mouse in the combination drug group was given an equal volume of the combination drug of the present invention. Each mouse in the combination drug + ultrasound group was given an equal volume of the combination drug of the present invention and then irradiated with ultrasound (1 MHz, 1.5 W / cm², 50% duty cycle, 5 minutes).

[0096] A combination drug of attenuated Salmonella strains loaded with barium titanate piezoelectric nanoparticles, prepared in Example 4, was used to treat B16F10 melanoma. C57BL / 6 mice (body weight...) Approximately 20 mice were randomly divided into four groups: a blank control group, an ultrasound group, a combination drug group, and a combination drug + ultrasound group, with 5 mice in each group. The mice were administered the drugs subcutaneously via the abdomen. Seven days after administering the drug to mice via intravenous injection, B16F10 cells were collected, and tumor volume was continuously observed. Treatment efficacy was as follows: Figure 13 As shown, the results indicate that the treatment effects of the blank control group, the ultrasound group, and the combination drug group were very poor, while the combination drug + ultrasound group showed a highly effective treatment effect.

[0097] Example 11: The other procedures are the same as in Example 10, except that the combination drug of the present invention is used to treat 4T1 breast cancer.

Claims

1. A combination drug for attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, characterized in that, The combined drug refers to barium titanate piezoelectric nanoparticles with good surface charge and other properties after being modified with a silane coupling agent, which are bound to the surface of attenuated Salmonella through electrostatic interaction.

2. The combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 1, characterized in that, The barium titanate piezoelectric nanoparticles with good surface charge and other properties are a class of barium titanate piezoelectric nanoparticles with positive surface charge, uniform morphology and good dispersibility.

3. The combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 1, characterized in that, Preferably, the barium titanate piezoelectric nanoparticles with good surface charge and other properties have a particle size of 60-100 nm and a tetragonal crystal structure.

4. A method for preparing a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, characterized in that: Follow these steps: Step 1: Prepare barium titanate piezoelectric nanoparticles with good surface charge and other properties; Step 2: Prepare a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles.

5. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 4, characterized in that, Step 1 includes the following steps: Step 1.1: Barium nitrate, sodium hydroxide, oleic acid, oleylamine and tetrabutyl titanate are subjected to a hydrothermal reaction at 135-150℃ to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles. Step 1.2: Wash the oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles with diethylamine to remove the oleic acid / oleylamine-terminated ligands and obtain barium titanate piezoelectric nanoparticles with a clean surface. Step 1.3: Graft a silane coupling agent onto barium titanate piezoelectric nanoparticles with a clean surface to obtain barium titanate piezoelectric nanoparticles with good surface charge and other properties.

6. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 4, characterized in that, The specific process for step 2 is as follows: A solution of barium titanate piezoelectric nanoparticles with good surface charge and other properties was incubated with an equivalent volume of attenuated Salmonella suspension at 36-37°C for 1-4 hours. The precipitate was collected by centrifugation at 800-1200g for 10-15 minutes and washed with PBS at pH 7.4 to obtain a combination drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, which was then dispersed in PBS for storage.

7. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 5, characterized in that, In step 1.1, the process of hydrothermally reacting barium nitrate, sodium hydroxide, oleic acid, oleylamine, and tetrabutyl titanate at 135-150℃ to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles is specifically as follows: 0.4M barium nitrate solution, 2M sodium hydroxide solution, oleic acid, oleylamine, and a 2M tetrabutyl titanate n-butanol solution are added to a high-pressure reactor, stirred, sealed, and subjected to a hydrothermal reaction at 135-150℃ for 16-24 hours; the product is separated by centrifugation at a relative centrifugal force of 5000-8000g for 10-15 minutes, washed with ethanol, and dispersed in toluene to obtain oleic acid / oleylamine-terminated barium titanate piezoelectric nanoparticles.

8. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 5, characterized in that, In step 1.2, the barium titanate piezoelectric nanoparticles with clean surfaces are refluxed at 85-95℃ for 6-10 hours with 30% hydrogen peroxide, followed by centrifugation, washing with ethanol and ultrasonic dispersion, so that the barium titanate piezoelectric nanoparticles with clean surfaces are stably dispersed in ethanol.

9. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 5, characterized in that, In step 1.3, the silane coupling agent is any one of amino, quaternary ammonium salt, guanidinyl, or imidazolyl silane coupling agents.

10. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 6, characterized in that, The attenuated Salmonella strain is any one of the attenuated Salmonella strains VNP20009, YB1, SL7207, or BRD509.

11. The method for preparing the combined drug of attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 6, characterized in that, The proportions of the combined drugs are as follows: the concentration of barium titanate piezoelectric nanoparticles with good surface charge and other properties is 550-800 μg / mL, and the OD600 value of the attenuated Salmonella suspension is 0.6-1.

0.

12. The application of a combination drug containing attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles, characterized in that, The combination drugs are used to treat tumors and / or inhibit tumor growth.

13. The application of the combination drug containing attenuated Salmonella loaded with barium titanate piezoelectric nanoparticles according to claim 11, characterized in that, The specific steps for applying the combined drug to treat tumors and / or inhibit tumor growth are as follows: The combined drug is delivered into the body, and external ultrasound irradiation is applied to the tumor site to induce reactive oxygen species in the barium titanate piezoelectric nanoparticles, thereby treating or inhibiting the tumor; the frequency of the ultrasound irradiation is 0.1-3.0 MHz, and the sound intensity is 0.1-3.0 W / cm². 2 The duty cycle is 10%-100%, and the action time is 1-30 minutes.

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