A attenuated salmonella loaded adjuvant nano-combination medicine and a preparation method and application thereof

Abstract

The application discloses a kind of attenuated salmonella load adjuvant nano combination medicine, the combination medicine is modified to bacterial surface by means of amino and carboxyl condensation reaction, adamantane is simultaneously modified to adjuvant nano medicine surface using diselenium dynamic bond, then the host-guest interaction of adamantane and cyclodextrin is used to assemble two, and then nano medicine is stably combined on the surface of attenuated salmonella.Diselenium dynamic bond can be broken by ultrasound response, so as to realize the effect of drug release.This combination medicine has the tumor targeting enrichment ability of bacteria and the release characteristics of adjuvant nanoparticles under the action of ultrasonic wave, can realize targeted delivery and deep penetration in tumor tissue, and obtain significant tumor inhibition effect under ultrasonic stimulation.The experimental results show that the combination medicine exhibits good therapeutic effect in inhibiting tumor growth, and has excellent biological safety.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterial preparation and biomedicine, and in particular to a nanocombination drug containing attenuated Salmonella-loaded adjuvant, its preparation method, and its application. Background Technology

[0002] Cancer remains one of the most incurable diseases in the world. Before the 21st century, the main treatments for cancer were surgical resection, radiotherapy, and chemotherapy. However, many cancers have already metastasized before they are discovered, and most surgical resections are radical removals of the entire organ, causing some degree of harm to the patient. While radiotherapy and chemotherapy can kill most tumor cells, many tumor cells still remain as micrometastases, making them difficult to eradicate completely. Tumor immunotherapy, as an emerging treatment approach, shows great potential and unique therapeutic advantages by activating or enhancing the patient's own immune system to recognize and eliminate tumor cells. In particular, the discovery and application of immune checkpoint inhibitors (ICIs) have made breakthrough progress in the treatment of various malignant tumors. Utilizing nanotechnology to improve the efficiency of immunotherapy has been a major research hotspot in recent years. However, there are obstacles to using a single nanoparticle to meet all the requirements in the complex immune activation process. These obstacles include the poor performance of cationic nanoparticles that easily capture antigens during circulation, the slow accumulation of nanoparticles in tumor tissue, and the low accumulation efficiency in tumor draining lymph nodes (TdLNs).

[0003] Some bacteria possess natural lesion targeting ability, induction of innate and adaptive immunotherapeutic activity, ease of genetic modification, and readily modifiable surface chemicals, attracting widespread attention in the field of cancer treatment. Significant progress has been made in developing novel drug delivery systems using engineered bacteria as carriers to achieve targeted and synergistic cancer therapy. However, single-bacterial therapies exhibit dose-dependent toxicity, excessive bacterial proliferation in vivo can lead to potential toxicity, and bacteria are easily cleared by the immune system during transport. Furthermore, the methods and effects of synergistic therapy with multiple therapies still have certain limitations, severely restricting the research and development of engineered bacterial delivery systems. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a nanocombination drug with attenuated Salmonella loaded with adjuvant, its preparation method and application. After being injected into the body, the drug can achieve graded targeting of tumors and lymph nodes, complete multiple steps in sequence, and stimulate a strong anti-tumor immune response in situ.

[0005] To solve the above-mentioned technical problems, the first technical solution adopted by the present invention is: to provide a nanocombination drug of attenuated Salmonella loaded with adjuvant, wherein cyclodextrin is modified onto the surface of attenuated Salmonella by means of the condensation reaction of amino and carboxyl groups; adamantane is modified onto the surface of the adjuvant nanomedicine to form adjuvant nanoparticles by using diselenylene dynamic bonds; and the two are assembled by the host-guest interaction between adamantane and cyclodextrin, so that the nanomedicine is bound to the surface of attenuated Salmonella to form a bacteria-nanoparticle composite system.

[0006] In a preferred embodiment of the present invention, the attenuated Salmonella strain is VNP20009.

[0007] In a preferred embodiment of the present invention, the adjuvant nanoparticles are polyethyleneimine-4-benzimidazole adjuvant nanoparticles, and adamantane is modified onto the surface of the adjuvant nanodrug by means of the condensation reaction of amino and carboxyl groups.

[0008] To solve the above-mentioned technical problems, the second technical solution adopted by the present invention is: to provide a method for preparing a combination drug as described in any of the preceding claims, comprising the following steps:

[0009] (1) Polyethyleneimine (PEI) with a molecular weight of 10,000, benzimidazole-4-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were reacted in dimethyl sulfoxide (DMSO) at room temperature to prepare polyethyleneimine-4-benzimidazole (PEI-4Blmi) nanoparticles.

[0010] (2) Selenium powder Se and sodium borohydride NaBH4 were reacted in an aqueous solution at 105°C under a nitrogen atmosphere to prepare sodium diselenide Na2Se2; then, 3-bromopropionic acid CPA was added to the solution and reacted at room temperature to prepare 3,3-diselenopropionic acid DSeDPA.

[0011] (3) 1-adamantaneamine, 3,3-diselenopropionic acid, EDC·HCl and NHS were reacted at room temperature to connect adamantaneAd to the carboxyl group at one end of DSeDPA. Then PEI-4Blmi nanoparticles were added to connect PEI-4Blmi to the carboxyl group at the other end of DSeDPA to obtain PEI-4Blmi-Se-Se-Ad nanoparticles.

[0012] (4) Attenuated Salmonella VNP20009, mono-6-amino-6-deoxy-β-cyclodextrin NH2-β-CD, EDC·HCl and NHS were reacted at room temperature to prepare VNP-β-CD;

[0013] (5) The PEI-4Blmi-Se-Se-Ad nanoparticles and VNP-β-CD are assembled using the host-guest interaction between adamantane and cyclodextrin to obtain the combined drug.

[0014] In a preferred embodiment of the present invention, in step (5), the concentration of PEI-4Blmi-Se-Se-Ad nanoparticles is 1 mg / mL, the OD600 value of the attenuated Salmonella suspension in VNP-β-CD is 1.0, and the assembly is carried out at 37°C for 24 hours.

[0015] To solve the above-mentioned technical problems, the third technical solution adopted by the present invention is to provide the application of the combination drug as described in any of the preceding claims in the preparation of antitumor drugs.

[0016] In a preferred embodiment of the present invention, the combined drug is delivered to the tumor site via tail vein injection and released through diselenide bond cleavage under the action of ultrasound.

[0017] Furthermore, the ultrasonic frequency is 1 MHz, the power density is 2 W / cm², the duty cycle is 50%, and the ultrasonic time is 5 minutes.

[0018] The mechanism of the attenuated Salmonella-loaded adjuvant nanoparticle delivery system for tumor treatment described in this invention is as follows: VNP20009, which has hypoxic migration characteristics, can deliver adjuvant nanoparticles to hypoxic tumor sites, allowing the drug to accumulate efficiently at the tumor site. Subsequently, the tumor tissue is subjected to ultrasound. The mechanical action and cavitation effect of ultrasound can break the diselenide bonds, thereby releasing the adjuvant nanoparticles at the tumor site. At the same time, it can kill tumor tissue and release antigens. The drug and antigens can form a complex through electrostatic interaction. This complex can be recognized by the human immune system, taken up by dendritic cells and delivered to lymph nodes, thereby triggering a strong immune response, generating effector T cells and inflammatory factors, and returning them to the tumor tissue to kill the tumor tissue, thus achieving tumor immunotherapy.

[0019] The beneficial effects of this invention are:

[0020] (1) The present invention successfully prepared an adjuvant nanomedicine delivery system by chemical synthesis. This method not only maintains the structural stability and integrity of the adjuvant nanomedicine, but also stably loads the adjuvant nanomedicine onto the surface of VNP20009 through the host-guest interaction between adamantane and β-cyclodextrin.

[0021] (2) This invention utilizes a delivery system mediated by attenuated Salmonella VNP20009 to significantly enhance the tumor targeting and penetration ability of adjuvant nanomedicines into deep tumor tissues; using attenuated Salmonella (VNP20009) as a drug carrier, a targeted and synergistic delivery system for anti-tumor drugs is constructed, ultimately achieving safe and efficient tumor treatment;

[0022] (3) While achieving the tumor-targeting effect of the drug, the present invention utilizes the electrostatic interaction between the drug and the antigen to form a complex, which is then taken up by dendritic cells to achieve lymph node reflux of adjuvant nanomedicine.

[0023] (4) The Salmonella attenuated strain adjuvant nanoparticle delivery system of the present invention is simple to prepare and has mild conditions, and has the potential for industrialization and commercialization.

[0024] (5) The attenuated Salmonella-loaded adjuvant nanoparticle delivery system of the present invention has good anti-tumor properties, exhibiting good therapeutic effects in inhibiting tumor growth, while possessing excellent biosafety. When a mouse melanoma cancer model was selected for experiments, the system showed significant anti-tumor activity both in vivo and in vitro;

[0025] (6) This invention proposes a novel anti-tumor treatment strategy, providing a new approach for combined bacterial-nanoparticle therapy. Attached Figure Description

[0026] Figure 1 This is a transmission electron microscope image of the adjuvant nanomedicine prepared in Example 1.

[0027] Figure 2 This is a transmission electron microscope image of the attenuated Salmonella strain loaded with adjuvant nanomedicine prepared in Example 2.

[0028] Figure 3 This is a transmission electron microscope image of the attenuated Salmonella strain loaded with adjuvant nanomedicine prepared in Example 2 after ultrasonic treatment.

[0029] Figure 4 This is a Zeta potential diagram of bacteria before and after assembly of bacterial carrier and adjuvant nanomedicine in Example 2.

[0030] Figure 5 This is the ultraviolet spectrum of bacteria before and after assembly of the bacterial carrier and adjuvant nanodrug in Example 2.

[0031] Figure 6 This is the ultrasonic response release curve of the bacterial composite delivery system in Example 3.

[0032] Figure 7 Example 4 describes the cytotoxicity of B16F10 cells after treatment with different drug concentrations and ultrasound.

[0033] Figure 8 This is a quantitative analysis of the fluorescence intensity of tumors in B16F10 tumor-bearing mice within 24 hours of drug injection, as described in Example 5.

[0034] Figure 9 This is a quantitative analysis of the fluorescence intensity of the resected tumor and major organs within 24 hours of drug injection, as described in Example 5.

[0035] Figure 10 This is a quantitative analysis of the lymph node fluorescence intensity 24 and 48 hours after drug injection, as described in Example 6.

[0036] Figure 11 This is the tumor inhibition curve of the drug treatment for tumors in Example 7. Detailed Implementation

[0037] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0038] Example 1:

[0039] (1) Preparation of adjuvant nanoparticles

[0040] First, 648.6 mg of benzimidazole-4-carboxylic acid, 2300.4 mg of EDC·HCl, and 1841.4 mg of NHS were dissolved in 50 mL of dimethyl sulfoxide and reacted with the solution at room temperature for 2 hours. Then, 2 g of polyethyleneimine (molecular weight 10000) was added to the above solution, and the reaction was carried out with the solution at room temperature for 72 hours. Next, 50 mL of diethyl ether was added to the above solution, causing the drug to precipitate and yielding a yellow solid. The obtained yellow solid drug was dialyzed with deionized water to remove the organic solvent. Finally, the resulting aqueous solution was lyophilized to obtain polyethyleneimine-tetrabenzimidazole adjuvant nanoparticles.

[0041] (2) Preparation of 3,3-diselenodipropionic acid

[0042] 2.27 g of sodium borohydride was dissolved in 25 mL of deionized water and stirred in an ice bath until the solution became colorless. 2.37 g of selenium powder was dispersed in 10 mL of deionized water and placed in a round-bottom flask, which was then sealed under nitrogen. The sodium borohydride aqueous solution was then added dropwise to the selenium powder while continuously stirring in an ice bath to obtain a white suspension. Next, the white suspension was placed in a nitrogen atmosphere and reacted in an oil bath at 105°C for 20 minutes to obtain a light red sodium diselenide aqueous solution. After the light red aqueous solution returned to room temperature, 6.5 g of 3-bromopropionic acid was weighed and placed in 15 mL of deionized water. Sodium carbonate powder was added to adjust the pH to 8.0, resulting in a colorless and transparent solution. Under a nitrogen atmosphere, the resulting colorless solution was added dropwise to the sodium diselenide aqueous solution and stirred at room temperature for 18 hours to obtain a yellow solution. Hydrochloric acid was slowly added dropwise to the above yellow solution to adjust the pH to 3-4, at which point a large amount of red and white precipitates formed in the solution. The product was extracted three times with ethyl acetate, and the organic phase was collected to obtain a yellow solution. Anhydrous sodium sulfate was added to this yellow liquid, and the solution was dried overnight. Finally, rotary evaporation was performed to remove the organic solvent, yielding the final product 3,3-diselenodipropionic acid.

[0043] (3) Preparation of PEI-4Blmi-Se-Se-Ad nanoparticles

[0044] 1.52 g of 3,3-diselenopropionic acid, 1.43 g of EDC·HCl, and 0.86 g of NHS were dissolved in 40 mL of dichloromethane and reacted with the solution at room temperature for 6 hours. 0.68 g of 1-adamantaneamine was dispersed in 10 mL of dichloromethane and added dropwise to the above solution. The mixture was stirred at room temperature for 48 hours to obtain a light green solution. The solution was then washed three times with saturated sodium chloride aqueous solution and once with deionized water to obtain the green oily product HOOC-Se-Se-Ad. 40 mg of HOOC-Se-Se-Ad, 28.9 mg of EDC·HCl, and 18.2 mg of NHS were dissolved in 10 mL of dimethyl sulfoxide and reacted with the solution at room temperature for 3 hours. 200 mg of PEI-4Blmi was dispersed in 20 mL of dimethyl sulfoxide and added dropwise to the above solution. The mixture was stirred at room temperature for 48 hours. After the reaction was complete, the organic solution was dialyzed against deionized water. A large amount of white solid powder precipitated after dialysis. The solid powder was collected and lyophilized to obtain the final product, PEI-4Blmi-Se-Se-Ad nanoparticles. The nanoparticles were dispersed in ethanol, and the transmission electron microscopy results are shown below. Figure 1 As shown.

[0045] Example 2: Preparation of a Salmonella composite delivery system with adjuvant-loaded nanoparticles for attenuated bacteria

[0046] Adjuvant nanoparticles were loaded onto the surface of attenuated Salmonella bacteria via a host-guest interaction between adamantane and cyclodextrin. Attenuated Salmonella strains grown on solid LB agar plates were picked using a 10 μL pipette tip and cultured in liquid LB medium until the OD600 value reached 1.0. Subsequently, the bacteria were washed three times with PBS and then dispersed in an equal volume of PBS. 15 mL of the bacterial suspension was taken, and 20.0 mg of mono-6-amino-6-deoxy-β-cyclodextrin, 3.3 mg of EDC·HCl, and 4.4 mg of NHS were added to the bacterial suspension. The mixture was stirred at 37 °C for 24 hours. After the reaction was complete, the bacteria were washed three times with PBS and resuspended in 15 mL of PBS to obtain the bacterial vector VNP-β-CD.

[0047] Subsequently, 15 mg of PEI-4Blmi-Se-Se-Ad nanoparticles were added to 15 mL of bacterial suspension, and the mixture was stirred at 37°C for 12 hours. The mixture was then centrifuged at 4000 rpm for 5 minutes, the precipitate was collected, and washed three times with PBS. A bacterial-targeted adjuvant nanomedicine system was obtained and dispersed in PBS buffer for storage. Figure 2 and Figure 3 The image shown is a transmission electron microscope (TEM) image of a bacterial-targeted adjuvant nanomedicine system before and after ultrasound. Figure 4 The figure shows the zeta potential changes of attenuated Salmonella before and after loading adjuvanted nanoparticles, demonstrating the assembly process verified by charge conversion. The initial component VNP-β-CD carries a negative surface charge (approximately -18 mV), while the PEI-4BImi-Se-Se-Ad module, containing a large number of amino groups, exhibits a strong positive potential (approximately +38 mV). When the two recombine through host-guest recognition or electrostatic interactions, the potential of the product PEI-4BImi-Se-Se-VNP neutralizes to approximately +28 mV. This positively charged characteristic is beneficial for enhancing the affinity of nanoparticles to cell membranes, thereby increasing cellular uptake efficiency. The assembly process of adamantane and cyclodextrin was determined using UV-Vis absorption spectroscopy. Figure 5 Figure 5 shows the UV spectra of attenuated Salmonella before and after loading adjuvanted nanoparticles. Figure 5 further confirms the optical properties and component stability of the material. Both samples show obvious characteristic absorption peaks at 550 nm, indicating the introduction of specific chromophores (such as a fluorescent dye or metal nanostructure) into the system. Furthermore, this characteristic peak remains even after coupling with the PEI component, with a slight increase in intensity, demonstrating that the assembly process did not damage the core functional structure and also reflecting the good dispersibility of the complex in solution. Such a Se-Se (selenium-selenium bond) system typically indicates that the carrier possesses the ability to release drugs in response to the tumor microenvironment.

[0048] Example 3: Verification of the ultrasonic-responsive release of an adjuvant-loaded, attenuated Salmonella complex delivery system

[0049] The nanomedicine prepared in this invention contains diselenyl bonds, which can be broken through the mechanical action and cavitation effect of ultrasound, thereby achieving drug release. To verify this release process, 10 mg of PEI-4Blmi-Se-Se-Ad was labeled with fluorescein isothiocyanate (FITC) and assembled with 10 mL of VNP-β-CD with an OD value of 1.0. A certain amount of the assembled attenuated Salmonella complex delivery system was then taken and sonicated (frequency 1 MHz, power density 2 W / cm², duty cycle 50%). The bacterial drug was then centrifuged (4000 rpm, 5 minutes), and the fluorescence intensity of the supernatant was measured. Figure 6 As shown, the drug release rate of the bacterial composite delivery system changes over time under two conditions: with and without ultrasound. The ultrasound-responsive drug release effect can be clearly observed.

[0050] Example 4: In vitro cytotoxicity experiment of attenuated Salmonella combined drug delivery system

[0051] B16-F10 tumor cells were seeded in 96-well plates and cultured overnight in a cell culture incubator. After cell attachment, attenuated Salmonella bio-complex drug delivery system (concentrations: 0, 62.5, 125, 250, 500, and 1000 μg / mL) was added and co-incubated for 2 h. The cells were then sonicated (1 MHz, 2 W / cm², duty cycle 50%, 2 min). After this, the culture medium was removed, and the cells were washed three times with PBS. Then, 10 μL of CCK8 solution and 90 μL of 1640 medium were added to each well, and the cells were incubated for 1 h. After color change, the absorbance of each well was measured at 450 nm using a microplate reader. Based on the experimental results, wells without drug were used as the control group, and wells without cells were used as the blank group. Cell viability was calculated using the following formula: Cell viability (%) = (OD sample - OD blank) / (OD control - OD blank) × 100%. Figure 7 As shown, the survival rate of tumor cells decreased significantly with increasing drug concentration, demonstrating that nanomedicines have high cytotoxicity.

[0052] Example 5: Tumor-targeting experiment using a combined attenuated Salmonella drug delivery system

[0053] To evaluate the feasibility of tail vein injection as the delivery route for this system, 10 mL of VNP-β-CD with an OD value of 1.0 was labeled with fluorescein isothiocyanate (FITC) and assembled with 10 mg of PEI-4Blmi-Se-Se-Ad. This example then investigated the accumulation of the attenuated Salmonella nanocomposite drug delivery system at the tumor site in B16F10 tumor-bearing mice after tail vein injection, and monitored using an in vivo imaging system, with fluorescein isothiocyanate fluorescence signal as the tracer. Figure 8 As shown, compared with the tail vein injection of adjuvant nanomedicine alone, the bacterial composite drug delivery group exhibited stronger fluorescence intensity at the tumor site. The fluorescence signal of the bacterial drug delivery group continuously increased over time and remained stable throughout the observation period, highlighting its tumor-targeting ability. These results fully demonstrate that attenuated Salmonella can actively target tumors and efficiently deliver its carried adjuvant nanomedicine particles. Figure 9 As shown, in vitro fluorescence imaging of major organs and tumors 24 hours after injection further validated the above in vivo observations. The fluorescence signal intensity of isolated tumors in mice treated with the bacterial combined drug delivery system was significantly higher than that in mice treated with intravenous adjuvant nanoparticles alone.

[0054] Example 6: Lymph Node Targeting Experiment of Attenuated Salmonella Combined Drug Delivery System

[0055] To verify the lymph node reflux effect of the adjuvant nanomedicine, this example uses the drug preparation method of Example 3, labeling PEI-4Blmi-Se-Se-Ad with fluorescein isothiocyanate (FITC), and then assembling it with VNP-β-CD. Next, the study investigated the drug accumulation at the tumor site in B16F10 tumor-bearing mice after tail vein injection of the attenuated Salmonella nanocomposite drug delivery system. After 8 hours of accumulation, the tumor tissue was sonicated to release the drug, and lymph nodes were removed at 24 and 48 hours later for in vivo imaging to monitor drug accumulation in the lymph nodes. Figure 10 As shown, compared with the single tail vein injection combined drug delivery system group, the ultrasound group showed stronger fluorescence intensity in the lymph nodes, which confirms that the combined drug delivery system can release drugs through ultrasound action, and the released drugs can flow back into the lymph nodes.

[0056] Example 7: In vivo antitumor experiment of attenuated Salmonella combined drug delivery system

[0057] To evaluate the in vivo antitumor effects of this composite drug delivery system, this example utilizes the bacterial nanodrug composite delivery system prepared in Example 2 to treat B16F10 melanoma. Twenty-eight C57BL / 6 mice (weighing approximately 18-20g) were randomly divided into four groups: a blank control group, an ultrasound group, a bacterial drug delivery group, and an ultrasound + bacterial drug delivery group. 1.5 × 10⁻⁶ ppm was subcutaneously injected into the left abdomen of each mouse. 6 Eight days after administering B16F10 cells, the drug was injected into mice via tail vein injection, and tumor volume and treatment efficacy were continuously observed. Figure 11 As shown in the figure. The results showed that the treatment effect in the blank control group and the ultrasound group was very poor, the bacterial delivery drug group had a certain treatment effect, and the ultrasound + bacterial delivery drug group showed a highly effective treatment effect.

[0058] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A nanocombination drug containing attenuated Salmonella adjuvant, characterized in that, Cyclodextrin was modified onto the surface of attenuated Salmonella; adamantane was modified onto the surface of adjuvant nanoparticles using diselenylene dynamic bonds to form adjuvant nanoparticles; the two were assembled through the host-guest interaction between adamantane and cyclodextrin, so that the nanoparticles were bound to the surface of attenuated Salmonella, forming a bacteria-nanoparticle composite system.

2. The attenuated Salmonella-loaded adjuvant nanocombination drug according to claim 1, characterized in that, The attenuated Salmonella strain used is VNP20009.

3. The attenuated Salmonella-loaded adjuvant nanocombination drug according to claim 1, characterized in that, The adjuvant nanoparticles are polyethyleneimine-4-benzimidazole adjuvant nanoparticles, and adamantane is modified onto the surface of the adjuvant nanodrug by means of the condensation reaction of amino and carboxyl groups.

4. A method for preparing a combination drug as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Polyethyleneimine (PEI) with a molecular weight of 10,000, benzimidazole-4-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were reacted in dimethyl sulfoxide (DMSO) at room temperature to prepare polyethyleneimine-4-benzimidazole (PEI-4Blmi) nanoparticles. (2) Selenium powder Se and sodium borohydride NaBH4 were reacted in an aqueous solution at 105°C under a nitrogen atmosphere to prepare sodium diselenide Na2Se2; then, 3-bromopropionic acid CPA was added to the solution and reacted at room temperature to prepare 3,3-diselenopropionic acid DSeDPA. (3) 1-adamantaneamine, 3,3-diselenopropionic acid, EDC·HCl and NHS were reacted at room temperature to connect adamantaneAd to the carboxyl group at one end of DSeDPA. Then PEI-4Blmi nanoparticles were added to connect PEI-4Blmi to the carboxyl group at the other end of DSeDPA to obtain PEI-4Blmi-Se-Se-Ad nanoparticles. (4) Attenuated Salmonella VNP20009, mono-6-amino-6-deoxy-β-cyclodextrin NH2-β-CD, EDC·HCl and NHS were reacted at room temperature to prepare VNP-β-CD; (5) The PEI-4Blmi-Se-Se-Ad nanoparticles and VNP-β-CD are assembled using the host-guest interaction between adamantane and cyclodextrin to obtain the combined drug.

5. The method for preparing the combined drug according to claim 4, characterized in that, In step (5), the concentration of PEI-4Blmi-Se-Se-Ad nanoparticles was 1 mg / mL, the OD600 value of the attenuated Salmonella suspension in VNP-β-CD was 1.0, and the assembly was carried out at 37°C for 24 hours.

6. The use of a combination drug as described in any one of claims 1 to 3 in the preparation of an antitumor drug.

7. The application according to claim 6, characterized in that, The combined drug is delivered to the tumor site via tail vein injection and released through diselenide bond breakage under ultrasound.

8. The application according to claim 7, characterized in that, The ultrasonic frequency was 1 MHz, the power density was 2 W / cm², the duty cycle was 50%, and the ultrasonic duration was 5 minutes.

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