Engineering bacterium driven micro-rod robot drug delivery system and preparation method thereof

Abstract

The invention provides an engineered bacterium-driven micro-rod robot drug delivery system and a preparation method thereof, and the preparation method comprises the following steps: grafting a thrombolytic drug to the surface of a carbon nanotube through amidation to obtain a drug-loaded carbon nanotube; and modifying the drug-loaded carbon nanotube on the surface of escherichia coli through a biotin-streptavidin binding principle, and modifying drug-loaded platelet membrane vesicles on the surface of the obtained drug-loaded escherichia coli through the biotin-streptavidin binding principle to obtain the engineering bacterium driven micro-rod robot drug delivery system. According to the drug delivery system, the rod-shaped structure of escherichia coli is reserved, the penetrability and interaction time of the drug delivery system to thrombus tissue are increased, and the adopted modification materials are low-toxicity nanoscale carbon nanotubes and platelet membranes derived from organisms, so that the toxicity is low, and the biocompatibility is high. The drug delivery system can be applied to treatment of thrombotic diseases, has a thrombus active targeting function, can quickly target thrombus tissues, and realizes penetrability treatment of the thrombus tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of thrombotic tissue targeted drug delivery, and in particular to an engineered bacteria-driven microrod robot drug delivery system and a preparation method thereof. Background Art

[0002] At present, the methods for engineering E. coli into drug delivery systems are mainly manifested in the following two aspects: 1. Drug delivery system that destroys the structure of E. coli: The method of extracting the bacterial membrane and reintegrating it into a nanovesicle drug delivery system destroys the rod-shaped structure of the bacteria (cutting and punching the bacteria). This method reduces the time of interaction with thrombotic tissue, and the penetration of thrombotic tissue is poor, resulting in a longer thrombolysis time and a lower thrombolysis rate.

[0003] 2. Drug delivery system for modified Escherichia coli membrane surface: Most of the existing materials for modifying the surface of Escherichia coli are exogenous substances such as chemically synthesized macromolecular materials, polymer materials, and metal materials. They have high immunogenicity and are easily cleared by the endothelial reticular system, resulting in low drug bioavailability. In addition, the introduced exogenous substances have high biological toxicity and poor biocompatibility.

[0004] In addition, most of the current drug delivery systems engineered with Escherichia coli are used in the diagnosis and treatment of tumors, and are rarely used in the treatment of thrombotic diseases. Summary of the invention

[0005] The purpose of the present invention is to provide an engineered bacteria-driven microrod robot drug delivery system and a preparation method thereof. Drug-loaded carbon nanotubes are first modified on the surface of Escherichia coli, and the obtained drug-loaded Escherichia coli is then modified with drug-loaded platelet membrane vesicles through the biotin-streptavidin binding principle. An engineered bacteria-driven microrod robot drug delivery system with good penetrability and strong interaction with thrombus tissue can be obtained. The drug delivery system can target the delivery of thrombolytic drugs to the inside of the thrombus, thereby achieving a rapid thrombolytic effect.

[0006] The present invention solves the technical problem by adopting the following technical solutions.

[0007] The present invention provides a method for preparing an engineered bacteria-driven microrod robot drug delivery system, comprising the following steps: S1. Obtain Escherichia coli; S2, grafting the thrombolytic drug onto the surface of the carbon nanotubes through amidation to obtain drug-loaded carbon nanotubes; S3, modifying the drug-loaded carbon nanotubes on the surface of the Escherichia coli by the principle of biotin-streptavidin binding to obtain drug-loaded Escherichia coli; S4. The drug-loaded platelet membrane vesicles are modified on the surface of the drug-loaded Escherichia coli by the biotin-streptavidin binding principle to obtain an engineered bacteria-driven microrod robot drug delivery system.

[0008] The present invention provides an engineered bacteria-driven microrod robot drug delivery system, which is prepared according to the preparation method.

[0009] The beneficial effects of the engineered bacteria-driven microrod robot drug delivery system and the preparation method thereof according to the embodiment of the present invention are: 1. Compared with the streamline motion of spherical particles, rod-shaped particles undergo twisting, rotation and tumbling during blood circulation, thereby enhancing the interaction with clots on the blood vessel wall and having a high ability to penetrate thrombotic tissue. The engineered E. coli-driven microrod robot drug delivery system of the present invention retains the rod-shaped structure of E. coli, increases the penetration and interaction time of the drug delivery system with thrombotic tissue, shortens the thrombolysis time, and is more meaningful for the treatment of thrombotic diseases with a short treatment time window.

[0010] 2. The modified materials used in the engineered E. coli driven microrod robot drug delivery system of the present invention are low-toxic nanoscale carbon nanotubes and platelet membranes derived from organisms, which have low toxicity and high biocompatibility.

[0011] 3. Based on the natural rod-shaped structure and large specific surface area of ​​E. coli, it is very suitable for the treatment and diagnosis of thrombotic diseases after modification. The application field of the engineered E. coli driven microrod robot drug delivery system of the present invention is the treatment of thrombotic diseases. The natural rod-shaped structure of E. coli is utilized to achieve deep penetration of thrombotic tissue. And based on the large specific surface area of ​​carbon nanotubes, the drug loading capacity of E. coli is increased. The drug delivery system can deliver thrombolytic drugs to the inside of the thrombus in a targeted manner, so that the thrombus tissue disintegrates from the inside, thereby achieving a rapid thrombolytic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 This is a flow chart for preparing the engineered bacteria-driven microrod robot drug delivery system of the present invention; Figure 2 TEM images, SEM images and atomic force microscope (AFM) images of SWCNT-rt-PA and SWCNT-COOH of Example 2 of the present invention; Figure 3 TEM images and SEM images of platelets, platelet membranes, and drug-loaded platelet membrane vesicles of Example 4 of the present invention; Figure 4 TEM images and SEM images of EcN of Example 1, EcN-(SWCNT-rt-PA) of Example 3, and (SWCNT-rt-PA)-EcN-PMV(L-Arg) of Example 4 of the present invention; Figure 5 This is the laser confocal fluorescence imaging of EcN; Figure 6 This is the laser confocal fluorescence imaging of EcN-(SWCNT-rt-PA); Figure 7 This is the laser confocal fluorescence imaging of (SWCNT-rt-PA)-EcN-PMV(L-Arg). DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0015] The engineered bacteria-driven microrod robot drug delivery system and its preparation method according to an embodiment of the present invention are described in detail below.

[0016] Reference Figure 1 As shown, a method for preparing an engineered bacteria-driven microrod robot drug delivery system provided by an embodiment of the present invention comprises the following steps: S1. Obtain Escherichia coli.

[0017] Furthermore, in a preferred embodiment of the present invention, the Escherichia coli is cultured using Luria-Bertani medium. Specifically, the present invention uses a liquid culture method to culture a large number of non-pathogenic Escherichia coli E. coli Nissle1917 (EcN) to obtain a large number of colonies with good activity.

[0018] S2. The thrombolytic drug is grafted onto the surface of the carbon nanotubes through amidation to obtain drug-loaded carbon nanotubes (SWCNT-rt-PA).

[0019] Furthermore, in a preferred embodiment of the present invention, the thrombolytic drug is alteplase (rt-PA), the carbon nanotubes are carboxylated single-walled carbon nanotubes (SWCNT-COOH), and the step of grafting the thrombolytic drug onto the surface of the carbon nanotubes by amidation is: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the protein solution of alteplase, stirred at room temperature for 30 minutes, and then the carboxylated single-walled carbon nanotubes are added and stirred overnight. After centrifugation and washing, drug-loaded carbon nanotubes are obtained.

[0020] Further, in a preferred embodiment of the present invention, the molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the NHS and the alteplase is 10:10:1; the protein dissolving solution is a mixed solution of solution A and solution B in a volume ratio of 9:1, wherein the solution A comprises a sodium carbonate solution, a sodium bicarbonate solution and a sodium chloride solution, and in the solution A, the concentration of the sodium carbonate solution is 0.0159 g / mL, and the sodium bicarbonate solution is 0.016 g / mL. The concentration of A is 0.0126 g / mL, the concentration of the sodium chloride solution is 0.0008 g / mL, and the mass ratio of sodium carbonate, sodium bicarbonate and sodium chloride is 19.875:15.75:1; the B liquid is a mixed solution of sodium carbonate solution and sodium bicarbonate solution in a volume ratio of 1:9, and its pH is 9.33. In the B liquid, the concentration of the sodium carbonate solution is 0.0159 g / mL, and the concentration of the sodium bicarbonate solution is 0.0126 g / mL.

[0021] S3. Modifying the drug-loaded carbon nanotubes on the surface of the Escherichia coli by the principle of biotin-streptavidin binding to obtain drug-loaded Escherichia coli (EcN-(SWCNT-rt-PA)).

[0022] Furthermore, in a preferred embodiment of the present invention, the step of modifying the drug-loaded carbon nanotubes on the surface of the Escherichia coli through the principle of biotin-streptavidin binding is: reacting the drug-loaded carbon nanotubes with sulfonyl NHS-biotin to synthesize SWCNT-rt-PA-Biotin, and then incubating the Escherichia coli suspension with sulfonyl NHS-biotin, streptavidin and the SWCNT-rt-PA-Biotin in sequence, and obtaining the drug-loaded Escherichia coli after centrifugation and washing.

[0023] Furthermore, in a preferred embodiment of the present invention, the incubation temperature is 37°C, the incubation time is 1 h, and the concentration of the E. coli suspension is 1×10 9 CFU / mL.

[0024] S4. The drug-loaded platelet membrane vesicles (PMV(L-Arg)) are modified on the surface of the drug-loaded Escherichia coli by the biotin-streptavidin binding principle to obtain an engineered bacteria-driven microrod robot drug delivery system ((SWCNT-rt-PA)-EcN-PMV(L-Arg)).

[0025] Furthermore, in a preferred embodiment of the present invention, the step of modifying the drug-loaded platelet membrane vesicles on the surface of the drug-loaded Escherichia coli by the biotin-streptavidin binding principle is: Obtaining platelet membranes, and subjecting the platelet membranes to repeated freeze-thaw treatments and centrifugation to obtain platelet membrane vesicles; The platelet membrane vesicles are resuspended in PBS buffer and washed with PBS buffer, then resuspended in L-arginine solution and ultrasonically treated for 15 minutes, extruded through a polycarbonate porous membrane 10 times using an Avestin micro-extruder, and then the excess L-arginine is removed by dialysis to obtain the drug-loaded platelet membrane vesicles; The drug-loaded platelet membrane vesicles were reacted with sulfonyl NHS-biotin to synthesize PMV(L-Arg)-Biotin, and then EcN(SWCNT-rt-PA) was incubated with sulfonyl NHS-biotin, streptavidin and the PMV(L-Arg)-Biotin at 37°C for 1 h to obtain the engineered bacteria-driven microrod robot drug delivery system.

[0026] Furthermore, in a preferred embodiment of the present invention, the step of obtaining the platelet membrane is: Whole blood was extracted from the orbits of C57BL / 6J mice, allowed to stand and then centrifuged, the platelet-rich plasma obtained was centrifuged at 800 rpm for 10 min, and the supernatant was discarded to obtain platelets; After the ammonium oxalate solution is added to the platelets, the platelets are centrifuged at 3500 rpm for 10 minutes and the supernatant is discarded. Then, a cold protein extract is added, mixed thoroughly and oscillated at 4°C for 20-30 minutes, and then centrifuged at 4°C and 12000 g for 10 minutes. The obtained supernatant is water bathed at 37°C for 10 minutes, and then centrifuged at 37°C and 1000 g for 3 minutes. The upper layer solution is removed, and the obtained lower layer solution is dissolved with a membrane protein dissolving solution to obtain the platelet membrane, wherein the mass concentration of the ammonium oxalate solution is 1%.

[0027] The present invention extracts and separates platelet cells from mouse bone marrow, separates the platelet membrane and retains the activity of its surface adhesion protein, and the obtained drug-loaded platelet membrane vesicles are modified on the surface of drug-loaded Escherichia coli through the biotin-streptavidin binding principle, thereby obtaining an engineered bacteria-driven microrod robot drug delivery system. The engineered bacteria-driven microrod robot drug delivery system retains the rod-shaped structure of Escherichia coli, increases the penetration and interaction time of the drug delivery system to thrombotic tissue, and the modified materials used are low-toxic nanoscale carbon nanotubes and platelet membranes derived from organisms, which have low toxicity and high biocompatibility. In addition, the application field of the bacteria-driven microrod robot drug delivery system is the treatment of thrombotic diseases, and it has an active thrombus targeting function, can quickly target thrombus tissue, and achieve penetrating treatment of thrombus tissue.

[0028] The present invention also provides an engineered bacteria-driven microrod robot drug delivery system, which is prepared according to the above-mentioned preparation method.

[0029] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0030] Example 1 In this embodiment, a liquid culture method is used to culture a large number of non-pathogenic Escherichia coli E. coli Nissle1917 (EcN) to obtain a large number of colonies with good activity, wherein the culture medium is Luria-Bertani medium, and the specific steps are as follows: Weigh 49.4 g of the purchased LB1 medium powder (Guangdong Huankai Microbiological Technology Co., Ltd., catalog number: 028320), add 1 L of deionized water, stir and heat to boil until completely dissolved, dispense into Erlenmeyer bottles, and sterilize at 121°C for 15 min. Cool the sterilized medium to about 45°C, and add 1 tube of LB1 supporting reagent (SR0100) per 225 mL of base to prepare LB1 medium.

[0031] Pre-culture of E. coli: Pick the preserved E. coli strain (Guangdong Huankai Microbiological Technology Co., Ltd., item number: BNCC361741) and paste it on a high-clean culture dish, and repeatedly apply the colonies by cross-coating method. Use sterile porcelain beads to gently press the colonies so that they are evenly dispersed on the culture dish. After pasting the bacteria, turn the culture dish upside down to avoid condensation on the lid. Then put the culture dish in the incubator, keep it warm at 37℃, and pre-culture it after 24 hours. After pre-culture, take an appropriate amount of E. coli colony liquid, add it to the culture medium, and culture it on a shaker. The culture conditions are generally 37℃, the shaker speed is 200~250 rpm, and after 20 hours of culture, a well-active E. coli colony is obtained for standby use.

[0032] Example 2 In this example, purchased surface carboxylated single-walled carbon nanotubes (SWCNT-COOH, Jiangsu Xianfeng Nanomaterial Technology Co., Ltd., catalog number: 101369) and alteplase (rt-PA, alteplase, MedChemExpress (Monmouth Junction, NJ, USA), catalog number: HY-108865) were used to synthesize drug-loaded carbon nanotubes (SWCNT-rt-PA) by amidation reaction. The specific steps are as follows: First, prepare a protein solution for use as a reaction solution, that is, prepare solution A and solution B. The preparation steps of solution A are as follows: dissolve 0.159 g sodium carbonate, 0.126 g sodium bicarbonate and 0.008 g sodium chloride in 10 mL deionized water respectively. The preparation steps of solution B are as follows: take 1 mL of the prepared sodium carbonate solution and mix it with 9 mL sodium bicarbonate solution, and adjust the pH to 9.33 with sodium hydroxide. The protein solution is obtained by mixing 9 mL of solution A and 1 mL of solution B. Then, EDC and NHS are added to 1 mg of rt-PA protein solution (the molar ratio of EDC / NHS / rt-PA is 10 / 10 / 1), stirred at room temperature for 30 minutes, and then 10 mg of SWCNT-COOH is added, and stirring is continued overnight. After the reaction solution is centrifuged at 1200 g for 5 minutes, SWCNT-rt-PA is obtained by repeated washing 3 times with PBS buffer.

[0033] Example 3 This example uses the principle of biotin-streptavidin binding to prepare EcN-(SWCNT-rt-PA), and the specific steps are as follows: The drug-loaded carbon nanotubes were reacted with sulfo-NHS-biotin to synthesize SWCNT-rt-PA-Biotin. Then, sulfo-NHS-biotin (1 mM, 500 µL), streptavidin (1 mM, 500 µL) and synthesized SWCNT-rt-PA-Biotin (1 mM, 500 µL) were incubated in 1 mL of EcN suspension (1×10 9 CFU / mL) for 1 hour. Finally, the reaction solution was centrifuged at 2000 rpm for 10 min and then washed three times with PBS to obtain EcN-(SWCNT-rt-PA). It was placed in a 4°C refrigerator for later use.

[0034] Example 4 This example is the preparation of (SWCNT-rt-PA)-EcN-PMV(L-Arg), and the specific steps are: (1) Extraction of platelet membrane: Whole blood was extracted from the orbits of C57BL / 6J mice (Guangdong Yaokang Biotechnology Co., Ltd., 6-8 weeks old, SPF grade, female mice), placed in an anticoagulant centrifuge tube, and centrifuged at 800 rpm for 10 minutes after standing for 30 minutes. The supernatant was platelet-rich plasma. The supernatant was aspirated and placed in a clean test tube, centrifuged at 3500 rpm for 10 minutes, and the supernatant was discarded. The sediment at the bottom of the tube was platelets.

[0035] Add 50~100 µL of 1% ammonium oxalate solution to the test tube, stir gently with a glass rod, then add 2~3 mL of 1% ammonium oxalate solution, and let it stand for 5 minutes to dissolve the red blood cells. Then centrifuge at 3500 rpm for 10 minutes, discard the supernatant, add 300~500 µL of cold protein extract, mix thoroughly and shake at 4℃ for 20~30 minutes. Then centrifuge at 4℃, 12000 g for 10 minutes, take the supernatant and place it in a 37℃ water bath for 10 minutes, and centrifuge at 37℃, 1000 g for 3 minutes. At this time, the liquid is divided into 2 layers, carefully remove the upper layer solution, leave about 30~50 µL of the lower layer at the bottom of the tube, and dissolve it with 1~2 times the volume of membrane protein dissolution solution to obtain platelet membrane.

[0036] (2) The platelet membrane obtained in step (1) was repeatedly frozen and thawed three times in a liquid nitrogen-37°C water bath. After the last thawing, the frozen platelet suspension was further centrifuged (2000g, 5min) to prepare platelet membrane vesicles. The obtained frozen platelet membrane vesicles (PMVs) were resuspended in PBS and washed three times with PBS (containing protease inhibitors) (2000g, 5min) to separate the platelet membrane and its organelles. The purified PMVs were then resuspended in L-arginine (10 mM) solution and sonicated for 15 minutes. Finally, an Avestin microextruder was used to extrude 10 times through a 200 nm polycarbonate porous membrane. Excess L-arginine in the emulsion was removed by dialysis, and the obtained drug-loaded platelet membrane vesicles were stored in PBS at 4°C.

[0037] (3) The drug-loaded platelet membrane vesicles were modified onto the surface of EcN(SWCNT-rt-PA) by using the principle of biotin-streptavidin binding to prepare (SWCNT-rt-PA)-EcN-PMV(L-Arg): First, the drug-loaded platelet membrane vesicles were reacted with sulfo-NHS-biotin to synthesize PMV(L-Arg)-Biotin. Then, sulfo-NHS-biotin (1 mM, 500 µL), streptavidin (1 mM, 500 µL) and synthesized PMV(L-Arg)-Biotin (1 mM, 500 µL) were incubated with EcN(SWCNT-rt-PA) at 37°C for 1 h to obtain (SWCNT-rt-PA)-EcN-PMV(L-Arg), which was placed in a 4°C refrigerator for use.

[0038] Test Example 1 In this test example, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy were used to analyze the SWCNT-rt-PA synthesized in Example 2. The specific steps are: The SWCNT-rt-PA synthesized in Example 2 was prepared into a concentration of 1 mg / mL with ultrapure water. SWCNT-COOH (1 mg / mL) and SWCNT-rt-PA (1 mg / mL) were observed under three microscopes.

[0039] like Figure 2 The TEM, SEM and atomic force microscopy (AFM) images of SWCNT-rt-PA and SWCNT-COOH are shown. Figure 2 It can be seen that rt-PA protein is modified on the surface of carboxylated single-walled carbon nanotube SWCNT-COOH in large quantities.

[0040] Test Example 2 In this test example, transmission electron microscopy and scanning electron microscopy were used to characterize the platelet cell membrane extracted in Example 4. The specific steps were: PBS was used to prepare a platelet cell membrane concentration of 1 mg / mL, and the platelet cell membrane was observed under two microscopes.

[0041] like Figure 3 Shown are TEM images and SEM images of platelets, platelet membranes, and drug-loaded platelet membrane vesicles of Example 4. Figure 3 (a) and Figure 3 (b) TEM and SEM images of platelets, respectively; Figure 3 (c) and Figure 3 (d) TEM and SEM images of platelet membrane, respectively; Figure 3 (e) and Figure 3 (f) TEM and SEM images of drug-loaded platelet membrane vesicles. Figure 3 It can be seen that the extracted platelet cell membrane has intact morphology and a size of approximately 400 nm.

[0042] Test Example 3 In this test example, transmission electron microscopy and scanning electron microscopy were used to analyze the EcN of Example 1, the EcN-(SWCNT-rt-PA) of Example 3, and the (SWCNT-rt-PA)-EcN-PMV(L-Arg) of Example 4.

[0043] like Figure 4 The TEM and SEM images of EcN of Example 1, EcN-(SWCNT-rt-PA) of Example 3, and (SWCNT-rt-PA)-EcN-PMV(L-Arg) of Example 4 are shown. Figure 4 (a) TEM images of EcN, EcN-(SWCNT-rt-PA) and (SWCNT-rt-PA)-EcN-PMV(L-Arg); Figure 4(b) SEM images of EcN, EcN-(SWCNT-rt-PA) and (SWCNT-rt-PA)-EcN-PMV(L-Arg); red arrows indicate SWCNT-rt-PA; blue arrows indicate SWCNT-rt-PA and PMV(L-Arg). Figure 4 It can be seen that the cultured EcN has a good morphology and a complete structure. SWCNT-rt-PA and PMV (L-Arg) are successfully modified on the surface of EcN.

[0044] Test Example 4 In order to further verify whether SWCNT-rt-PA and PMV (L-Arg) were successfully modified on the surface of EcN, confocal fluorescence microscopy was used to analyze EcN, EcN-(SWCNT-rt-PA), and (SWCNT-rt-PA)-EcN-PMV (L-Arg). EcN was stained with SYTO9 (5µM); rt-PA protein was stained with Cy5.5-NHS (10µM) to prepare SW-rt-PA-Cy5.5; platelet membrane was stained with cell membrane staining probe DiR (5µM) to prepare DiR-PMV (L-Arg); and (SWCNT-rt-PA)-EcN-PMV (L-Arg) stained with SYTO9, Cy5.5-NHS and DiR were prepared. The specific steps are as follows: SYTO9 (5 µM) staining method for EcN: 1 mL of SYTO9 (5 µM) solution was mixed with EcN (1×10 9 The 100 μL (400 μL) culture medium solution was mixed evenly and placed at room temperature for 30 min, and then centrifuged at 3000 rpm for 10 min to remove unmodified SYTO9 and obtain SYTO9-stained EcN.

[0045] The steps of staining rt-PA protein with Cy5.5-NHS (10µM) are as follows: the cryopreserved Cy5.5-NHS is placed at room temperature to warm up for at least 20 min. 1000μl of high-quality anhydrous DMSO is added to 1mg of powder to fully dissolve. 10µM Cy5.5-NHS is added to the 10µM rt-PA protein solution. (The dye and protein are usually in a mass ratio of 1:1). Then 15μl of triethylamine is added, and the reaction mixture is stirred at 4°C in the dark overnight. The reaction solution is dialyzed with PBS to obtain Cy5.5-NHS-labeled rt-PA. Cy5.5-NHS-labeled SWCNT-rt-PA is prepared according to the steps of Example 2 using fluorescently labeled rt-PA. The fluorescently labeled SWCNT-rt-PA is prepared into EcN-(SWCNT-rt-PA) according to the steps of Example 3.

[0046] Steps for staining platelet membrane with cell membrane staining probe DiR (5µM): The cryopreserved DiR was placed at room temperature for at least 20 min. 1000μl of high-quality anhydrous DMSO was added to 1mg of powder to fully dissolve it. 5µM Cy5.5-NHS was added to 5µM platelet membrane protein solution. (The dye and protein are usually in a mass ratio of 1:1). Then 15μl of triethylamine was added, and the reaction mixture was stirred at 4°C in the dark overnight. The reaction solution was dialyzed with PBS to obtain DiR-labeled platelet membrane. Thus, fluorescently labeled platelet membrane drug-loaded nanoparticles PMV (L-Arg) were prepared. According to the steps of Example 4, the fluorescently labeled PMV (L-Arg) was modified on the surface of fluorescent EcN-(SWCNT-rt-PA), and EcN was stained with SYTO9 (5 µM) to obtain Cy5.5, DiR and SYTO9-labeled (SWCNT-rt-PA)-EcN-PMV (L-Arg).

[0047] like Figure 5 Shown is the laser confocal fluorescence imaging of EcN. Figure 6 Shown is the laser confocal fluorescence imaging of EcN-(SWCNT-rt-PA). Figure 7 The figure shows the laser confocal fluorescence imaging of (SWCNT-rt-PA)-EcN-PMV(L-Arg). Figure 5~Figure 7 In the figure, green fluorescence refers to EcN labeled with the probe SYTO9; blue fluorescence refers to SWCNT-rt-PA labeled with the probe Cy5.5; red fluorescence refers to PMV (L-Arg) labeled with the probe DiR; white arrows refer to SWCNT-rt-PA; and yellow arrows refer to PMV (L-Arg). Figure 5 It can be seen that EcN was successfully stained with green fluorescence by SYTO9 and its morphology was intact. Figure 6 It can be seen that EcN was successfully modified on the surface by SWCNT-rt-PA. Figure 7 It can be seen that SWCNT-rt-PA and PMV (L-Arg) were successfully modified on the EcN surface, and the EcN morphology was intact and the bacterial survival rate was high.

[0048] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing an engineered bacteria-driven microrod robot drug delivery system, characterized in that: The following steps are involved: S1. Obtain Escherichia coli; S2, grafting the thrombolytic drug onto the surface of the carbon nanotubes through amidation to obtain drug-loaded carbon nanotubes; S3, modifying the drug-loaded carbon nanotubes on the surface of the Escherichia coli by the principle of biotin-streptavidin binding to obtain drug-loaded Escherichia coli; S4. The drug-loaded platelet membrane vesicles are modified on the surface of the drug-loaded Escherichia coli by the biotin-streptavidin binding principle to obtain an engineered bacteria-driven microrod robot drug delivery system.

2. The preparation method according to claim 1, characterized in that: In step S1, the Escherichia coli is cultured using Luria-Bertani medium.

3. The preparation method according to claim 1, characterized in that: In step S2, the thrombolytic drug is alteplase, the carbon nanotubes are carboxylated single-walled carbon nanotubes, and the step of grafting the thrombolytic drug onto the surface of the carbon nanotubes by amidation is: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the protein solution of alteplase, and after stirring at room temperature for 30 minutes, the carboxylated single-walled carbon nanotubes were added and stirring was continued overnight. After centrifugation and washing, drug-loaded carbon nanotubes were obtained.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the alteplase is 10:10:1; the protein dissolving solution is a mixed solution of solution A and solution B in a volume ratio of 9:1, wherein the solution A includes sodium carbonate solution, sodium bicarbonate solution and sodium chloride solution, in the solution A, the concentration of the sodium carbonate solution is 0.0159 g / mL, the concentration of the sodium bicarbonate solution is 0.0126 g / mL, the concentration of the sodium chloride solution is 0.0008 g / mL, and the mass ratio of sodium carbonate, sodium bicarbonate and sodium chloride is 19.875:15.75:1; the solution B is a mixed solution of sodium carbonate solution and sodium bicarbonate solution in a volume ratio of 1:9, and its pH is 9.33, in the solution B, the concentration of the sodium carbonate solution is 0.0159 g / mL, and the concentration of the sodium bicarbonate solution is 0.0126 g / mL.

5. The preparation method according to claim 1, characterized in that In step S3, the step of modifying the drug-loaded carbon nanotubes on the surface of the Escherichia coli through the principle of biotin-streptavidin binding is: reacting the drug-loaded carbon nanotubes with sulfonyl NHS-biotin to synthesize SWCNT-rt-PA-Biotin, and then incubating the Escherichia coli suspension with sulfonyl NHS-biotin, streptavidin and the SWCNT-rt-PA-Biotin in sequence, and obtaining the drug-loaded Escherichia coli after centrifugation and washing.

6. The preparation method according to claim 5, characterized in that In step S3, the incubation temperature is 37°C, the incubation time is 1 h, and the concentration of the E. coli suspension is 1×10 9 CFU / mL.

7. The preparation method according to claim 1, characterized in that: In step S4, the step of modifying the drug-loaded platelet membrane vesicles on the surface of the drug-loaded Escherichia coli by the biotin-streptavidin binding principle is as follows: Obtaining platelet membranes, and subjecting the platelet membranes to repeated freeze-thaw treatments and centrifugation to obtain platelet membrane vesicles; The platelet membrane vesicles are resuspended in PBS buffer and washed with PBS buffer, then resuspended in L-arginine solution and ultrasonically treated for 15 minutes, extruded through a polycarbonate porous membrane 10 times using an Avestin micro-extruder, and then the excess L-arginine is removed by dialysis to obtain the drug-loaded platelet membrane vesicles; The drug-loaded platelet membrane vesicles were reacted with sulfonyl NHS-biotin to synthesize PMV(L-Arg)-Biotin, and then EcN(SWCNT-rt-PA) was incubated with sulfonyl NHS-biotin, streptavidin and the PMV(L-Arg)-Biotin at 37°C for 1 hour to obtain the engineered bacteria-driven microrod robot drug delivery system.

8. The preparation method according to claim 7, characterized in that: The steps of obtaining the platelet membrane are: Whole blood was extracted from the orbits of C57BL / 6J mice, allowed to stand and then centrifuged, the platelet-rich plasma obtained was centrifuged at 800 rpm for 10 min, and the supernatant was discarded to obtain platelets; After the ammonium oxalate solution is added to the platelets, the platelets are centrifuged at 3500 rpm for 10 minutes and the supernatant is discarded. Then, a cold protein extract is added, mixed thoroughly and oscillated at 4°C for 20-30 minutes, and then centrifuged at 4°C and 12000 g for 10 minutes. The obtained supernatant is water bathed at 37°C for 10 minutes, and then centrifuged at 37°C and 1000 g for 3 minutes. The upper layer solution is removed, and the obtained lower layer solution is dissolved with a membrane protein dissolving solution to obtain the platelet membrane, wherein the mass concentration of the ammonium oxalate solution is 1%.

9. An engineered bacteria-driven microrod robot drug delivery system, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

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