Ultrasonic response type bacterial composite drug loading system as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic response type bacterial composite drug-loading system as well as a preparation method and application thereof. The system is formed by taking engineering bacteria as a carrier, grafting chemotherapy prodrug molecules on the surface of the engineering bacteria through atom transfer radical polymerization and then loading a sound-sensitive agent. Compared with the prior art, by optimizing ATRP reaction conditions, efficient grafting of the medicine on the surfaces of bacteria is achieved, and the activity of the bacteria is kept. After intravenous injection, the system is enriched at a tumor site by virtue of a tumor hypoxia targeting carrying drug of attenuated salmonella, and ultrasonic irradiation activates a sound-sensitive agent to generate active oxygen: on one hand, the active oxygen splits a TK bond in a prodrug to release a chemotherapeutic drug, and on the other hand, the active oxygen mediates sonodynamic therapy. The system solves the problems that a traditional drug delivery system is poor in targeting property, uncontrollable in drug release and limited in single treatment effect, and the ATRP technology is mild in preparation process and good in biocompatibility and has wide application prospects in the field of deep tumor targeted therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of synthetic biology, nanomaterials and biological medicine, in particular to an ultrasound-responsive bacterial composite drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Malignant tumors, especially deep solid tumors (such as pancreatic cancer), have complex tumor microenvironments, insufficient blood supply, and poor tissue penetration. Traditional treatment methods (surgery, chemotherapy, and radiotherapy) have strong toxic side effects, poor targeting, and drug resistance. Engineered bacteria have natural tumor hypoxia targeting and deep penetration ability, and are ideal drug delivery carriers. Through genetic engineering or chemical modification, the engineered bacteria can be endowed with intelligent response ability to external physical signals such as light, magnetic field, and ultrasound. However, existing surface modification techniques for engineered bacteria mostly use inorganic nanomaterials or linear polymers, which have poor biocompatibility and insufficient functional regulation. In addition, existing drug delivery systems mostly rely on a single treatment mechanism, which cannot cope with the heterogeneity of tumors. Moreover, precise control of deep tumor release and immune microenvironment remodeling are still technical bottlenecks.

[0003] Therefore, there is an urgent need to develop a new type of engineered bacteria composite drug delivery system for efficient and synergistic delivery of multiple therapeutic agents, ultimately achieving safe and efficient tumor treatment. SUMMARY

[0004] The technical problem to be solved by the present application is to provide an ultrasound-responsive engineered bacteria composite drug delivery system, which solves the problems of targeting, controllability, and synergy in deep tumor treatment by optimizing the ATRP technology to graft prodrugs on the surface of bacteria and then loading a photosensitizer.

[0005] To solve the above technical problems, the first technical solution adopted by the present application is to provide an ultrasound-responsive bacterial composite drug delivery system, which uses engineered bacteria as a carrier, grafts chemotherapeutic prodrug molecules on the surface of the bacteria through atom transfer radical polymerization (ATRP), and then loads a photosensitizer.

[0006] In a preferred embodiment of the present application, the chemotherapeutic prodrug molecules use one or more of the following: prodrug molecule TK5-Fu, gemcitabine prodrug molecule, and doxorubicin. The molecular structure of the prodrug molecule TK5-Fu is as follows:

[0007]

[0008] In a preferred embodiment of the present application, the engineered bacteria use one or more of attenuated Salmonella VNP20009 and Escherichia coli.

[0009] In a preferred embodiment of the present application, the sonosensitizer is an inorganic sonosensitizer, including manganese carbonate nanoparticles and titanium dioxide nanoparticles.

[0010] To solve the above technical problems, the second technical solution adopted by the present application is to provide a preparation method of the ultrasound-responsive bacterial composite drug delivery system as described in any one of the above, comprising the following steps:

[0011] Step one: synthesis and preparation of a chemotherapy prodrug molecule;

[0012] Step two: preparation of a bacterial ATRP polymer-modified prodrug;

[0013] Step three: preparation of a synthetic sonosensitizer;

[0014] Step four: resuspend the bacterial ATRP polymer-modified prodrug in PBS, incubate with an equal volume of sonosensitizer solution, centrifuge, and wash several times to obtain the ultrasound-responsive bacterial composite drug delivery system.

[0015] In a preferred embodiment of the present application, in step one, the steps for synthesizing and preparing the prodrug molecule TK5-Fu include:

[0016] TK-1 is used as the starting material, acyl chloride substitution reaction is performed to generate TK-2, then TK-3 is generated by reaction with p-nitrophenyl chloroformate NPC, and finally, 5-fluorouracil is reacted under the catalysis of triethylamine TEA and 4-dimethylaminopyridine DMAP, and TK5-Fu is obtained by silica gel column chromatography purification (n-hexane / ethyl acetate = 2:1).

[0017] In a preferred embodiment of the present application, in step two, the steps for preparing the attenuated Salmonella ATRP polymer-modified prodrug include:

[0018] The attenuated Salmonella VNP20009 is cultured to OD600=0.8, centrifuged and resuspended in PBS to a final concentration of OD600=3, the initiator is added to a final concentration of 1mM, and the culture is incubated on a shaker; after centrifugal washing, it is transferred to an ATRP reaction solution containing TK5-Fu 12mM, CuBr2 1mM, TPMA 2mM, and ascorbic acid reducing agent, and incubated in the dark, and then centrifuged and washed to obtain VNP@PTK5-Fu.

[0019] Further, the monomer conversion rate of the ATRP reaction is not less than 70%, and the growth ability of VNP@PTK5-Fu is only slightly decreased compared with the original VNP20009.

[0020] To solve the above technical problems, the third technical solution adopted by the present application is to provide an application of the ultrasound-responsive bacterial composite drug delivery system as described in any one of the above in the preparation of an antitumor drug.

[0021] In a preferred embodiment of the present application, the tumor is a deep solid tumor, including pancreatic cancer; the administration mode is intravenous injection, and the therapeutic effect is activated by applying ultrasound irradiation (1.0 MHz, 1.5 W / cm², duty cycle 50%, 5 minutes) to the tumor site 8 hours after administration.

[0022] The ultrasound-responsive bacterial composite drug delivery system described in the present application is enriched in the tumor site by virtue of the tumor hypoxia targeting of attenuated Salmonella, and the active oxygen is activated by ultrasound irradiation (1.0 MHz, 1.5 W / cm 2 , duty cycle 50%, 5 minutes) to activate the sonodynamic agent manganese carbonate to produce reactive oxygen species: on the one hand, ROS cleaves the prodrug to release 5-fluorouracil to achieve chemotherapy, and on the other hand, active oxygen mediates sonodynamic therapy; at the same time, the degradation of manganese carbonate nanoparticles releases Mn 2+ activates the cGAS-STING innate immune pathway, promotes the maturation of dendritic cells and CD8 + T cell infiltration, and achieves immunotherapy.

[0023] The present application has the following beneficial effects:

[0024] (1) Strong targeting: by virtue of the tumor hypoxia targeting of attenuated Salmonella VNP20009, the system achieves efficient enrichment in deep tumors (such as pancreatic cancer);

[0025] (2) Controlled drug release: precise release of 5-Fu is achieved only under ultrasound irradiation, and the release amount is <5% without ultrasound, avoiding toxic side effects on normal tissues;

[0026] (3) Synergistic therapy: integration of "sonodynamic-chemical-immune" triple mechanism significantly improves the anti-tumor effect, and in the in situ pancreatic cancer mouse model, the tumor growth inhibition rate of the ultrasound-responsive bacterial composite drug delivery system group under ultrasound activation is >70%;

[0027] (4) Biological safety: the ATRP reaction conditions are mild, and the bacterial survival rate is >70%; after administration of the system, the blood routine, liver and kidney function of the mice are normal, and there is no tissue damage to the main organs;

[0028] (5) Feasible preparation: the process steps are clear, the reagents used are easy to obtain, the ATRP polymerization conversion rate is high, and it has industrialization potential. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the ¹H NMR spectrum of the TK-5Fu prodrug prepared in Example 1 (d6-DMSO);

[0030] Figure 2 is the FITR graph after ATRP polymerization modification of VNP20009 in Example 2;

[0031] Figure 3 SEM image of the bacteria ATRP polymerization modified prodrug prepared in Example 2;

[0032] Figure 4 EDX element mapping image of the bacteria ATRP polymerization modified prodrug prepared in Example 2;

[0033] Figure 5 Zeta potential image of the simple bacteria, ATRP polymerization modified prodrug, manganese carbonate, ultrasound-responsive engineered bacteria composite drug prepared in Example 3;

[0034] Figure 6 5-Fu release curve of VNP@PTK5-Fu@MnCO3 with or without ultrasound prepared in Example 4;

[0035] Figure 7 KPC-LUC cell survival rate column chart of different treatment groups in Example 5;

[0036] Figure 8 Quantitative analysis curve of KPC-LUC tumor-bearing mice at different time points after injection of drug delivery system in Example 6;

[0037] Figure 9 Quantitative analysis diagram of ex vivo organ fluorescence intensity of KPC-LUC tumor-bearing mice at different time points after injection of drug delivery system in Example 6;

[0038] Figure 10 Effect diagram of drug treatment of tumor in Example 7. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present application are described in detail below with reference to the accompanying drawings, so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the protection scope of the present application is more clearly and definitely defined.

[0040] Example 1: Synthesis of prodrug TK-5Fu

[0041] TK-1 (931.8 mg, 4.746 mmol) was dissolved in THF, TEA (689 μL, 7.12 mmol) was added, 160 μL of acyl chloride was added dropwise under ice bath, and stirred at room temperature for 6 h; after dilution with ethyl acetate, it was washed with saturated ammonium chloride, dried with sodium sulfate, and purified by silica gel column (n-hexane / ethyl acetate = 2:1) to obtain TK-2 (36.3% yield) as colorless oil; TK-2 (455.7 mg, 1.724 mmol) was dissolved in THF, TEA (472 μL, 3.447 mmol) was added, and NPC (416.82 mg, 2.068 mmol) was added under ice bath, and stirred at room temperature for 6 h; purified by silica gel column (n-hexane / ethyl acetate = 2:1) to obtain TK-3 (89.5% yield) as brown oil; TK-3 (664.7 mg, 1.547 mmol) was dissolved in DMF, TEA (636 μL, 4.643 mmol) and DMAP (5 mg) were added, 5-fluorouracil (172.19 mg, 1.702 mmol) was added under ice bath, and stirred at room temperature for 8 h; washed with saturated brine, and purified by silica gel column (n-hexane / ethyl acetate = 2:1) to obtain TK5-Fu (65.3% yield) as yellow oil, and its molecular structural formula is as follows:

[0042]

[0043] It was characterized by ¹H NMR (d6-DMSO) to confirm its structure, as shown in Figure 1 .

[0044] Example 2: Preparation of a bacterial ATRP polymer-modified prodrug

[0045] Single colonies of attenuated Salmonella VNP20009 grown on LB agar plates were picked and inoculated into 5 mL of LB liquid medium. The culture was incubated at 37°C and 180 rpm for 12 h using a shaker. 1 mL of the bacterial culture was transferred to 100 mL of LB medium and cultured until OD600 = 0.8. The bacterial culture was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The bacterial sludge was washed twice with PBS and resuspended in 50 mL of PBS (final concentration OD600 = 3). Initiator (final concentration 1 mM) was added, and the culture was incubated at 37°C and 180 rpm for 2 h using a shaker. The culture was then centrifuged at 5000 rpm for 5 min, and washed three times with PBS to remove unbound initiator. The initiator was NHS-Intiator with the molecular formula C9H8BrNO7. The bacteria modified with the initiator were resuspended in 50 mL of ATRP reaction solution (containing 12 mM TK-5Fu, 1 mM CuBr2, 2 mM TPMA, and 0.5 mM ascorbic acid) and incubated at 37°C and 180 rpm in the dark for 1.5 h. Samples were taken during the reaction, and the monomer conversion rate (approximately 72%) was calculated using the TK-5Fu standard curve. After the reaction, the sample was centrifuged at 1000 rpm for 10 min, washed three times with PBS, and VNP@PTK5-Fu was obtained. FTIR characterization showed a C=O stretching vibration peak at 1654 cm⁻¹, confirming successful TK5-Fu grafting. Figure 2 As shown.

[0046] Example 3: Preparation of an ultrasound-responsive bacterial composite drug delivery system

[0047] Manganese carbonate nanoparticles were synthesized using a reverse emulsion microphase method. VNP@PTK5-Fu was resuspended in PBS (OD600 = 0.8), and the manganese carbonate nanoparticles were dissolved in PBS (concentration 1 mg / mL). Equal volumes of the two solutions were mixed and incubated at 37°C and 100 rpm for 1 h. The mixture was then centrifuged at 1000 rpm for 10 min, and washed three times with PBS to remove unbound manganese carbonate nanoparticles. The nanoparticles were then resuspended in PBS (concentration 10...). 6 (CFU / mL) to obtain an ultrasound-responsive bacterial composite drug delivery system, VNP@PTK5-Fu@MnCO3 with a survival rate of not less than 70%, and capable of releasing the chemotherapy drug 5-Fu under ultrasound irradiation.

[0048] SEM observation revealed that the VFM surface was uniformly covered with nanoparticles, such as Figure 3 As shown; EDX mapping shows that Fu (from TK-5Fu) and Mn (from manganese carbonate) are uniformly distributed, as... Figure 4 As shown, from left to right, the images are the electronic image of VFM, C element, Mn element, O element, and Fu element; the Zeta potential was measured to be -15mV, confirming successful electrostatic adsorption. Figure 5 As shown.

[0049] Example 4: Performance detection of the ultrasound-responsive bacterial composite drug delivery system

[0050] The system (5Fu equivalent concentration 100 pg / mL) was divided into three groups: ① no ultrasound group; ② intermittent ultrasound group (1.0 MHz, 1.5 W / cm2, 1 min / once, interval 1 min); ③ continuous ultrasound group (1.0 MHz, 1.5 W / cm2, 5 min). The amount of 5-Fu released was detected by HPLC (chromatographic column: XBridge C18, column temperature 35°C, mobile phase: water / methanol gradient, detection wavelength 210 nm); the results showed that, as shown in Figure 6 , the amount of 5Fu released in the intermittent ultrasound group reached 78% after 5 times of ultrasound, similar to the amount released in the continuous ultrasound group, and only a small amount of 5Fu was released during the no-ultrasound period; while the amount released in the no-ultrasound group was <5%, and it was proved that the composite drug delivery system had the characteristics of ultrasound-responsive drug release.

[0051] Example 5: In vitro anti-tumor experiment of the ultrasound-responsive bacterial composite drug delivery system

[0052] KPC-LUC cells were inoculated in 96-well plates (5x10 3 cells / well) and cultured for 24 h; they were divided into 6 groups: ① PBS (control group); ② 5-fluorouracil (simple chemotherapy drug) group (780 pg / mL); ③ simple bacteria group (10 6 CFU / mL); ④ manganese carbonate+ultrasound (50 pg / mL, ultrasound treatment); ⑤ ultrasound-responsive bacterial composite drug delivery group (10 6 CFU / mL); ⑥ ultrasound-responsive bacterial composite drug delivery+ultrasound group (10 6 CFU / mL, ultrasound treatment); after 24 h of culture, CCK8 solution (10 pL / well) was added, incubated for 4 h, and the absorbance at 450 nm was measured; the results showed that, as shown in Figure 7 , the cell survival rate of the ultrasound-responsive bacterial composite drug delivery+ultrasound group was the lowest (19.01%±2.45%), which was significantly lower than that of the other groups.

[0053] Example 6: Tumor targeting experiment of the ultrasound-responsive bacterial composite drug delivery system

[0054] Female C57BL / 6 mice (16-18 g) were anesthetized by intraperitoneal injection of 10% chloral hydrate, the abdomen was incised to expose the pancreas, and 1x10 6 KPC-LUC cells (containing matrigel) were injected, and the incision was sutured; they were fed for 7 days, and when the tumor volume reached 100 mm 3 , they were used for experiments. The Cy5.5-labeled bacterial drug (100 pL, 10 6CFU / mL) into tumor-bearing mice, and live fluorescence imaging was performed at 1, 4, 8, 12, 24 h, respectively; the results showed that the fluorescence intensity at the tumor site reached a peak at 8 h, and was significantly higher than that in other organs, such as Figure 8 , 9; it is proved that the ultrasound-responsive bacterial composite drug delivery system has good tumor targeting.

[0055] Example 7: In vivo anti-tumor experiment of ultrasound-responsive bacterial composite drug delivery system

[0056] The tumor-bearing mice were randomly divided into 5 groups (n=5): ① control group; ② 5-fluorouracil group; ③ manganese carbonate+ultrasound group; ④ ultrasound-responsive bacterial composite drug delivery group; ⑤ ultrasound-responsive bacterial composite drug delivery group+ultrasound group; the administration mode was tail vein injection, once every 3 days, for a total of 5 times; the ultrasound treatment was performed at 8 h after administration, and the tumor site was irradiated with ultrasound (1.0 MHz, 1.5 W / cm 2 , 5 min); after 18 days of treatment, the mice were sacrificed, and the tumors were peeled off and weighed; the results showed that the tumor weight of the VNP@PTK5-Fu@MnCO3+US group was the smallest, as shown in Figure 10 .

[0057] The embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above embodiments, and here, all the embodiments are not required to be exhausted or cannot be exhausted. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. An ultrasound-responsive bacterial composite drug delivery system, characterized in that, The system uses engineered bacteria as a carrier, grafts chemotherapeutic precursor molecules onto its surface via atom transfer radical polymerization, and then loads a sonosensitive agent.

2. The ultrasound-responsive bacterial composite drug delivery system according to claim 1, characterized in that, The chemotherapy prodrug molecule comprises one or more of the following: TK5-Fu, gemcitabine, and doxorubicin. The molecular structure of the prodrug molecule TK5-Fu is as follows: 。 3. The ultrasound-responsive bacterial composite drug delivery system according to claim 1, characterized in that, The engineered bacteria are one or more of attenuated Salmonella VNP20009 and Escherichia coli.

4. The ultrasound-responsive bacterial composite drug delivery system according to claim 1, characterized in that, The sound-sensing agent is an inorganic sound-sensing agent, including manganese carbonate nanoparticles and titanium dioxide nanoparticles.

5. A method for preparing an ultrasound-responsive bacterial composite drug delivery system as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Synthesize and prepare chemotherapy prodrug molecules; Step 2: Preparation of bacterial ATRP polymerization modified prodrug; Step 3: Preparation of synthetic sound-sensing agent; Step 4: The bacterial ATRP-modified precursor drug was resuspended in PBS, incubated with an equal volume of acoustic sensitizer solution, centrifuged, and washed several times to obtain an ultrasonic-responsive bacterial composite drug delivery system.

6. The method for preparing the ultrasound-responsive bacterial composite drug delivery system according to claim 5, characterized in that, In step one, the steps for synthesizing and preparing the prodrug molecule TK5-Fu include: Starting with TK-1, TK-2 was generated by acyl chloride substitution reaction, and then reacted with p-nitrophenyl chloroformate (NPC) to generate TK-3. Finally, TK-3 was reacted with 5-fluorouracil under the catalysis of triethylamine (TEA) and 4-dimethylaminopyridine (DMAP). The mixture was purified by silica gel column chromatography (n-hexane / ethyl acetate = 2:1) to obtain TK5-Fu.

7. The method for preparing the ultrasound-responsive bacterial composite drug delivery system according to claim 5, characterized in that, In step two, the steps for preparing the attenuated Salmonella ATRP polymerized modified prodrug include: Attenuated Salmonella VNP20009 was cultured to OD600=0.8, centrifuged, and resuspended in PBS to a final concentration of OD600=3. Initiator was added to a final concentration of 1mM, and the mixture was cultured on a shaker. After centrifugation and washing, the mixture was transferred to ATRP reaction solution containing 12mM TK5-Fu, 21mM CuBr, 2mM TPMA, and ascorbic acid reducing agent. The mixture was incubated in the dark, centrifuged, and washed to obtain VNP@PTK5-Fu.

8. The method for preparing the ultrasound-responsive bacterial composite drug delivery system according to claim 7, characterized in that, The monomer conversion rate of the ATRP reaction is not less than 70%.

9. The use of an ultrasound-responsive bacterial composite drug delivery system as described in any one of claims 1 to 3 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The antitumor drug is administered via intravenous injection and activated and monitored using ultrasound.

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