Preparation method and application of enzyme-responsive nanovesicles taking pillararene as carrier

Abstract

The invention relates to the technical field of nano-drug carriers, in particular to a preparation method and application of enzyme-responsive nano-vesicles with pillararene as a carrier, a compound 30 is used as a starting raw material and condensed with a compound 31 under the action of EEDQ to obtain a compound 32, the compound 32 is subjected to Fmoc protection removal through piperidine and diethyl ether precipitation to obtain a compound 33, and the compound 33 is subjected to enzyme-responsive reaction to obtain the enzyme-responsive nano-vesicles with pillararene as the carrier. A compound 33 and a compound 34 react in DMF to obtain a compound 35, the compound 35 is subjected to trifluoroacetic acid deprotection to obtain a compound 36, the compound 36 and a compound 37 react and are subjected to column chromatography separation to obtain a compound 38, the compound 38 and terminal alkyne pillar arene 11 are subjected to 1, 3-dipole addition under the catalysis of Cu I / TBTA to obtain a compound 39, acetyl protecting groups are removed through sodium methoxide to obtain MP5, MP5 is dissolved in deionized water containing 1% of DMSO, dialysis is performed after ultrasonic treatment, and the compound 38 is obtained. The MP5 nano vesicle solves the problem that common antibiotics cannot play a role in intracellular parasitic salmonella, so that infection is difficult to radically treat.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomedicine carriers, and particularly relates to a preparation method and application of an enzyme-responsive nanovesicle with a pillar arene as a carrier. BACKGROUND

[0002] When bacteria invade the body, the immune system such as macrophages can quickly start the response mechanism and show extremely complex chemotactic response behavior. Through this chemotactic response behavior, macrophages can gather to the infection site through blood circulation and kill bacteria, thereby protecting the body from bacterial invasion. For this reason, macrophages have become the primary target of Salmonella and many other bacteria when they enter the body. When Salmonella and other bacteria enter macrophages, part of them are killed, and a small part of them survive in macrophages, which can avoid the bactericidal effect of gentamicin and other antibiotics, causing persistent infection. The protective effect of macrophages makes it difficult for many commonly used antibiotics to enter cells or accumulate enough intracellular antibacterial concentration. Although traditional antibiotic delivery systems can enter cells through passive transport and improve the intracellular concentration of antibiotics, they lack targeting and are prone to off-target effects and toxic side effects.

[0003] To solve this problem, targeted drug delivery systems have become a research hotspot. Macrophages, as the main host cells of intracellular parasitic bacteria, have high expression of mannose receptors on their surface, which can specifically recognize sugar ligands such as mannose, providing an ideal target for targeted delivery. Studies have shown that mannose-modified carriers can enhance the uptake of drugs by macrophages through receptor-mediated endocytosis and improve the intracellular drug concentration. However, existing delivery systems have the problem of poor controllability of drug release, and drugs are prone to premature release at non-target sites, leading to increased toxic side effects and poor antibacterial effect.

[0004] Enzyme-responsive carriers can trigger drug release in a specific enzyme environment, providing a new idea for the precise delivery of intracellular drugs. Cathepsin B is a cysteine protease highly expressed in macrophages, which can specifically recognize and break dipeptide sequences such as Val-Cit, making it suitable as a trigger signal for intracellular drug release. Pillar arenes are a new type of supramolecular host compound with rigid structure and easy modification characteristics. Their amphiphilic derivatives can self-assemble into nanovesicles in aqueous solution, making them good drug carriers with good stability and biocompatibility.

[0005] Currently, there is no report on the combination of mannose-targeted modification, cathepsin B-responsive release, and pillar arene nanovesicle carriers to construct a drug delivery system that targets macrophages and has intracellular enzyme-controlled release function, for improving the clearance effect of antibiotics on intracellular parasitic bacteria.

[0006] Therefore, the application develops an enzyme-responsive nanovesicle based on a pillararene, solves the problem that common antibiotics have good antibacterial effect on extracellular bacteria, but are difficult to penetrate into host cells such as macrophages, cannot play a role on intracellular parasitic salmonella, and lead to difficult-to-cure infection. SUMMARY

[0007] The application aims to provide a preparation method and application of an enzyme-responsive nanovesicle with a pillararene as a carrier, solve the problem that common antibiotics have good antibacterial effect on extracellular bacteria, but are difficult to penetrate into host cells such as macrophages, cannot play a role on intracellular parasitic salmonella, and lead to difficult-to-cure infection.

[0008] To achieve the above-mentioned purpose, the application provides a preparation method of an enzyme-responsive nanovesicle with a pillararene as a carrier, comprising the following steps:

[0009] The L-citrulline compound 30 protected by chloroformic acid-9-fluorenylmethyl ester is used as a starting material, reacts with aniline derivative 31 containing an azide group under the action of condensation reagent EEDQ, and compound 32 is obtained in a yield of 75%;

[0010] The compound 32 is subjected to removal of the Fmoc protecting group under the action of organic base piperidine, and is precipitated by using ether to obtain compound 33;

[0011] Then, the compound 33 is reacted with Boc-protected L-valine activated ester 34 in DMF as a solvent, and compound 35 is separated in a yield of 57%;

[0012] The compound 35 is subjected to removal of the protecting group under the action of trifluoroacetic acid to obtain compound 36;

[0013] The compound 36 is reacted with mannose derivative 37, and compound 38 is separated by column chromatography in a yield of 45.9%;

[0014] The compound 38 is reacted with terminal alkyne pillararene 11 under the action of a catalytic equivalent of CuI and with TBTA as a ligand, 1,3-dipolar addition is carried out, compound 39 is obtained in a yield of 76%, and the acetyl protecting group is removed in a methanol solution containing sodium methoxide to obtain amphiphilic pillararene MP5 in a yield of 99%;

[0015] The amphiphilic pillararene compound MP5 is dissolved in deionized water containing 1% DMSO, is ultrasonically dissolved, is loaded into a regenerated cellulose dialysis bag with a molecular weight cut-off of 8000 Da, is dialyzed in ultrapure water until no DMSO is left, and MP5 nanovesicles are obtained.

[0016] The ultrasonic dissolution condition is that the power is 200-300W, and the time is 10-15 minutes.

[0017] The MP5 nanovesicle is self-assembled to form a spherical hollow structure, the hydrodynamic radius is 80-134nm, the surface potential is 29.1mV, and the critical micelle concentration is 41muM.

[0018] The 1,3-dipolar addition reaction of the compound 38 and the terminal alkyne columnar arene 11 is carried out under N2 protection, the reaction temperature is room temperature, and the reaction time is 24 hours.

[0019] The MP5 nanovesicle can load hydrophilic antibiotics, when the mass concentration ratio of gentamicin and the amphiphilic columnar arene MP5 is 7:10, the encapsulation rate is 41.6%, and the drug loading rate is 29.4%.

[0020] The application also provides application of the enzyme-responsive nanovesicle with columnar arene as a carrier, and the preparation method of the enzyme-responsive nanovesicle with columnar arene as a carrier is based on the preparation method of the enzyme-responsive nanovesicle with columnar arene as a carrier;

[0021] The MP5 nanovesicle is applied to the medicine for treating salmonella infection.

[0022] The medicine for treating salmonella infection is the MP5 nanovesicle loaded with gentamicin.

[0023] The MP5 nanovesicle plays a role through the following mode:

[0024] The surface mannose group is combined with the mannose receptor on the surface of macrophages, and is endocytosed into cells through receptor-mediated endocytosis;

[0025] Under the action of cathepsin B in macrophages, the Val-Cit dipeptide sequence is broken, the loaded gentamicin is released, and intracellular salmonella is removed.

[0026] The application discloses a preparation method and application of a pillar arene enzyme-responsive nanovesicle taking pillar arene as a carrier, and specifically discloses the following steps: taking a chloroformic acid-9-fluorenylmethyl ester protected L-citrulline compound 30 as a starting material, reacting with aniline derivative 31 containing an azide group under the action of a condensation reagent EEDQ to obtain compound 32 at a yield of 75%; removing a Fmoc protecting group from the compound 32 under the action of an organic base piperidine and precipitating the compound 32 with ether to obtain compound 33; then, reacting the compound 33 with a Boc protected L-valine activated ester 34 in DMF to separate compound 35 at a yield of 57%; removing a protecting group from the compound 35 under the action of trifluoroacetic acid to obtain compound 36; reacting the compound 36 with a mannose derivative 37, and separating the compound 38 at a yield of 45.9% through column chromatography; reacting the compound 38 with terminal alkyne pillar arene 11 under the action of a catalytic equivalent of CuI and taking TBTA as a ligand to obtain compound 39 at a yield of 76% through 1,3-dipolar addition, and removing an acetyl protecting group from the compound 39 in a methanol solution containing sodium methoxide to obtain an amphiphilic pillar arene compound MP5 at a yield of 99%; dissolving the amphiphilic pillar arene compound MP5 in deionized water containing 1% DMSO, ultrasonically dissolving the compound, loading the compound into a regenerated cellulose dialysis bag with a molecular weight cut-off of 8000 Da, dialyzing the compound in ultrapure water until no DMSO is left, and obtaining MP5 nanovesicles, so that the problem that common antibiotics have good antibacterial effect on extracellular bacteria, but are difficult to penetrate cell membranes to enter host cells such as macrophages, and cannot play a role on intracellular salmonella, leading to difficulty in curing infection, is solved. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced.

[0028] Figure 1 It is a flow chart of the preparation method of the pillar arene enzyme-responsive nanovesicle taking pillar arene as a carrier of the present application.

[0029] Figure 2 It is a preparation schematic diagram of MP5 of the present application.

[0030] Figure 3 It is a schematic diagram of the structure of MP5, self-assembly drug loading process and intracellular bacteria resistance process of the present application.

[0031] Figure 4 It is a schematic diagram of the critical micelle concentration of MP5 of the present application.

[0032] Figure 5 It is a schematic diagram of DSL and TEM of the MP5 vesicle of the present application.

[0033] Figure 6is a schematic diagram showing the length of the MP5 molecule of the present application.

[0034] Figure 7 is a schematic diagram showing the standard curve of gentamicin and PI of the present application.

[0035] Figure 8 is a schematic diagram showing the DLS and TEM of the MP5-Gen vesicle of the present application.

[0036] Figure 9 is a schematic diagram showing the activity of MP5-Gen against intracellular Salmonella of the present application.

[0037] Figure 10 is a schematic diagram showing the cytotoxicity of MP5 and MP5-Gen of the present application. DETAILED DESCRIPTION

[0038] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, the embodiments described below by reference to the drawings are exemplary, and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0039] Referring to Figures 1-10 , Figure 1 is a flow chart of the preparation method of the pillar arene-based enzyme-responsive nanovesicle of the present application, Figure 2 is a schematic diagram showing the preparation of MP5 of the present application, Figure 3 is a schematic diagram showing the structure of MP5, self-assembly drug loading process and anti-intracellular bacteria process of the present application, Figure 4 is a schematic diagram showing the critical micelle concentration of MP5 of the present application, Figure 5 is a schematic diagram showing the DSL and TEM of the MP5 vesicle of the present application, Figure 6 is a schematic diagram showing the length of the MP5 molecule of the present application, Figure 7 is a schematic diagram showing the standard curve of gentamicin and PI of the present application, Figure 8 is a schematic diagram showing the DLS and TEM of the MP5-Gen vesicle of the present application, Figure 9 is a schematic diagram showing the activity of MP5-Gen against intracellular Salmonella of the present application, Figure 10 is a schematic diagram showing the cytotoxicity of MP5 and MP5-Gen of the present application.

[0040] The present application provides a preparation method of cross-linked polyolefin lithium ion battery diaphragm, comprising the following steps:

[0041] S1; with chloroformic acid-9-fluorenylmethyl ester protected L-citrulline compound 30 as starting material, under the action of condensation reagent EEDQ, react with aniline derivative 31 containing azido group, to obtain compound 32 with a yield of 75%;

[0042] S2; the compound 32 is precipitated by removing the Fmoc protective group under the action of an organic base piperidine and using ether to precipitate to obtain compound 33;

[0043] S3; then the compound 33 is reacted with a Boc-protected L-valine activated ester 34 in DMF to obtain compound 35 in a yield of 57%;

[0044] S4; the compound 35 is removed from the protective group under the action of trifluoroacetic acid to obtain compound 36;

[0045] S5; the compound 36 is reacted with a mannose derivative 37, and compound 38 is separated by column chromatography in a yield of 45.9%;

[0046] S6; the compound 38 is reacted with the terminal alkyne columnar arene 11 under the action of a catalytic equivalent of CuI and with TBTA as a ligand to obtain compound 39 by 1,3-dipolar addition in a yield of 76%, and the acetyl protective group is removed in a methanol solution containing sodium methoxide to obtain the amphiphilic columnar arene MP5 in a yield of 99%;

[0047] S7; the amphiphilic columnar arene compound MP5 is dissolved in deionized water containing 1% DMSO, ultrasonically dissolved, loaded into a regenerated cellulose dialysis bag with a molecular weight cut-off of 8000 Da, and dialyzed in ultrapure water until no DMSO residue is left to obtain MP5 nanovesicles.

[0048] The ultrasonic dissolution conditions are: power 200-300 W, time 10-15 minutes; the dialysis solution is replaced every 2 hours during the dialysis process, and the total dialysis time is not less than 24 hours.

[0049] The MP5 nanovesicles self-assemble into a spherical hollow structure, with a hydrodynamic radius of 80-134 nm, a surface potential of 29.1 mV, and a critical micelle concentration of 41 μM.

[0050] The 1,3-dipolar addition reaction of the compound 38 and the terminal alkyne columnar arene 11 is carried out under N2 protection, the reaction temperature is room temperature, and the reaction time is 24 hours.

[0051] The MP5 nanovesicles can load hydrophilic antibiotics, and when gentamicin is loaded, the mass concentration ratio of gentamicin to the amphiphilic columnar arene MP5 is 7:10, the encapsulation efficiency is 41.6%, and the drug loading rate is 29.4%.

[0052] Preparation of the MP5 nanovesicles;

[0053] MP5 (2.0 mg) was dissolved in 2 mL of deionized water containing 1% DMSO, ultrasonic dissolution, and left overnight. Then the formed supramolecular nanovesicle solution was loaded into a regenerated cellulose dialysis bag (MW: 8000 Da), dialyzed in ultrapure water, and the dialysate was changed every 2 hours until no DMSO was detected. After dialysis, the sample solution was loaded into a 5 mL centrifuge tube for standby. The particle size and morphology of the nanovesicles were characterized by dynamic light scattering analysis (DLS) and transmission electron microscopy (TEM).

[0054] Standard curve determination of gentamicin and PI;

[0055] We used ultraviolet absorption spectrum to analyze the content of gentamicin. 16.6 mg of gentamicin sulfate (Gen) was precisely weighed, in which gentamicin was 10 mg, and was dissolved in 10 mL of borate buffer solution to prepare a 1 mg / mL stock solution. The gentamicin standard solution was accurately diluted to 800, 500, 300, 200, 150, 100, 80, 60, 50, 40, 30, 20, 10 μg / mL, respectively. 0.5 mL of the above prepared gentamicin standard solution was taken into a sample bottle wrapped with tin foil, 0.5 mL of o-phenylenediamine solution (0.04% OPA, 0.1% v / v ether, 0.2% v / v β-mercaptoethanol in boric acid solution) was added, and the borate buffer solution was quantitatively added to 3 mL, and reacted for 1 h. Then the UV absorption at 380 nm was measured by ultraviolet spectrophotometer, and the concentration-absorption peak relationship was converted.

[0056] The content of iodinated acridine (PI) was determined by HPLC. An appropriate amount of PI was precisely weighed, dissolved with phosphate buffer solution, and prepared into a 10 mg / mL standard solution, which was stored at -30°C. The PI standard solution was diluted with the mobile phase to prepare 0.5, 1, 2, 5, 10, 20, 30, 40, 60, 80 and 100 μg / mL PI standard solutions, respectively. 0.8 mL of the above prepared PI standard solution was taken into an HPLC sample bottle for standby. Chromatographic conditions: column: C18 column (Extend-C18, 250 x 4.6 mm, 5 μm, Agilent Technologies); mobile phase: MeCN-0.05M KH2PO4 buffer (9.5:90.5); flow rate: 1 mL / min; ultraviolet detection wavelength: 493 nm; room temperature; injection volume: 20 μL.

[0057] Preparation of nanovesicles loaded with gentamicin and iodinated acridine;

[0058] ​Accurately weigh 10 mg MP5 into 10 mL ultrapure water and sonicate for 10 min to form nanovesicles. Then add a certain amount of gentamicin and sonicate for another 30 min. After standing overnight, load the mixture into a regenerated cellulose dialysis bag (MW: 5000 Da) and dialyze against ultrapure water. Change the dialysis solution every 2 h and detect the residual amount of gentamicin in the dialysate by the method described in 3.4.2 until no gentamicin is detected. Then collect the MP5-Gen aqueous solution and store at 4°C for later use. The content of gentamicin is determined by UV absorption spectrum. MP5-PI is prepared in a similar manner and the residual amount is determined by HPLC.

[0059] Determination of drug loading and encapsulation efficiency of MP5-Gen by UV method;

[0060] Take 4 mL of the prepared MP5-Gen aqueous solution and freeze-dry to obtain MP5-Gen samples, which are dissolved in 2 mL of DMSO / H2O (volume ratio: 2:1). Then filter the solution through an ultrafiltration membrane (MW: 3000 Da), collect the filtrate and freeze-dry. Then determine the encapsulation efficiency (DLE%) and drug loading (DLC%) of gentamicin by UV absorption spectrum. MP5-PI is treated in a similar manner and the DLE% and DLC% are determined by HPLC.

[0061] Drug encapsulation efficiency (%) = (mass of encapsulated drug) ÷ (total drug mass) x 100%

[0062] Drug loading efficiency (%) = (mass of encapsulated drug) ÷ (mass of MP5) x 100%

[0063] Anti-S. enterica activity experiment of MP5-Gen vesicles;

[0064] Construction of S. enterica infection model in RAW264.7 cells: Culture RAW264.7 cells in 24-well culture dishes containing 10% fetal bovine serum DMEM medium at 106 cells per well. Then infect with 1 x 107 CFU / mL of S. enterica for 1 h, remove the bacterial-containing culture medium, replace it with culture medium containing 100 μg / mL gentamicin, and continue to culture for 0.5 h to remove extracellular S. enterica. Then remove the culture medium and wash with PBS 5 times, and continue to culture in DMEM medium for 4 h to complete the construction of the S. enterica infection model in RAW264.7 cells.

[0065] Plate count method to observe MP5-Gen treatment of RAW264.7 cells infected with Salmonella: After RAW264.7 cells were infected with Salmonella for 24 h, test drugs were added, and the final drug concentration was 5, 10, 15, 20, 25, and 30 μg / mL. Drug treatment groups were blank control group (PBS, Control), carrier control group (MP5, Control), gentamicin group (Gen), and mannoseylated pillararene-loaded gentamicin group (MP5-Gen).

[0066] In the competitive inhibition experiment, electron microscope slides were added to the bottom of the culture dish, then RAW 264.7 cells were cultured and infected with Salmonella for 1 h, followed by 100 μg / mL gentamicin in the culture medium to remove extracellular bacteria. After treatment, the Salmonella-infected cells were incubated with D-mannosamine hydrochloride (50 mM) for 1 h before being treated with MP5-Gen for 24 h. After incubation, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 30 min, washed with sterile PBS 4 times, then perforated with 0.1% Triton-X (v / v) for 5 min, followed by 4 washes with sterile PBS, incubation with LIVE / DEAD diluent at room temperature for 20 min, 4 washes with PBS, and finally observation of intracellular Salmonella survival by laser confocal microscopy (Leica TCS SP8, Solms, Germany).

[0067] Characterization of MP5 vesicles;

[0068] ①Critical micelle concentration (CMC) of pillararene MP5

[0069] To confirm whether the amphiphilic pillararene MP5 has the conditions to form nanovesicles, we need to verify the critical micelle concentration (CMC) of MP5 in aqueous solution. In this study, the relationship between surface tension and MP5 concentration was used to determine the CMC. As shown in the accompanying drawings Figure 4 , a nonlinear fitting curve was used to determine that the CMC was about 41 μM. This result indicates that MP5 is an amphiphilic compound with certain water solubility and has the prerequisite conditions for self-assembly into nanoparticles.

[0070] ②Particle size and morphology characterization of MP5 vesicles

[0071] When MP5 and deionized water were mixed and ultrasonically dissolved, it was found that a Tyndall phenomenon was produced, indicating that a large number of gel particles were formed in the system. Subsequently, we characterized the particle size and morphology of MP5 nanoparticles by DLS and TEM. As shown in the accompanying drawings Figure 5As shown, the hydrodynamic radius of the prepared MP5 nanoparticles was about 80-134 nm and the monodispersity coefficient was 0.39 by DLS analysis. The zeta potential of MP5 nanoparticles was 29.1 mV due to the presence of a large number of mannose derivatives on the outer surface of the nanoparticles. TEM characterization further showed that the average particle size of MP5 supramolecular nanoparticles was about 128 nm, which was consistent with the DSL results. At the same time, it was found by TEM observation that MP5 nanoparticles were spherical hollow structures with uneven size, and the outer layer was about 7 nm thick, which was similar to the length of the MP5 molecule calculated by ChemDraw 3D simulation (see attached drawings Figure 6 ). The above results showed that MP5 could self-assemble into a monolayer nanovesicle structure in aqueous solution through hydrophobic interaction, π-π interaction and hydrogen bonding.

[0072] Characterization of MP5-Gen and MP5-PI vesicles

[0073] Gentamicin has good extracellular antibacterial activity, but it is difficult to enter cells and shows negligible antibacterial activity against intracellular bacteria. Therefore, we used gentamicin as a model drug and loaded it into MP5 nanovesicles (MP5-Gen) to verify whether it could increase the intracellular accumulation concentration and antibacterial activity of gentamicin.

[0074] ① Standard curve of gentamicin and iodinated acridine

[0075] First, we analyzed gentamicin and iodinated acridine (PI) by liquid chromatography to obtain the standard curve of gentamicin and iodinated acridine (see attached drawings Figure 7 ).

[0076] ② Drug encapsulation efficiency and drug loading efficiency of MP5-Gen nanovesicles

[0077] Subsequently, we determined the drug loading efficiency and encapsulation efficiency of MP5 loaded with gentamicin. By fixing the concentration of MP5 (1 mg / mL) and adjusting the concentration of gentamicin, we sought the optimal encapsulation efficiency and drug loading efficiency. As shown in the following table;

[0078] Table 3.3 DLE(%)andDLC(%)

[0079]

[0080] a Weight ratio of M Gen / M MP5 ; b Weight ratio of M PI / M MP5

[0081] When the concentration ratio of gentamicin and MP5 was 1:10, the DLE was the highest, 67.3%, but the DLC was only 6.3%. With the increase of the concentration of gentamicin, the corresponding DLE gradually decreased, and the DLC increased. When the concentration ratio of gentamicin and MP5 was 7:10, the DLE was 41.6%, and the DLC was 29.4%. With the further increase of the concentration of gentamicin, the DLC gradually decreased. Therefore, considering the size of DLE and DLC, we finally used the mass concentration ratio of gentamicin and MP5 as 7:10. When the concentration ratio of PI and MP5 was 7:10, the final DLE was 39.2%, and the DLC was 25.4%.

[0082] ③Particle size and morphology of MP5-Gen nanovesicles

[0083] After determining the optimal drug loading rate and encapsulation efficiency, we analyzed the particle size and morphology of MP5-Gen vesicles by DLS and TEM. As shown in the description Figure 7 , the particle size of MP5-Gen nanovesicles loaded with gentamicin increased to about 184 nm, and the monodispersity coefficient was 0.14. Compared with MP5 nanovesicles, MP5-Gen nanovesicles were more uniform. At the same time, it was found by TEM observation that the particle size of MP5-Gen vesicles was basically consistent with the particle size obtained by DLS test, about 175 nm, and the center of the nanovesicle had a very heavy black shadow, the edge of the vesicle became irregular, and presented a relatively uniform spherical particle. From the results of DLS and TEM, it can be seen that MP5 vesicles loaded with gentamicin form solid MP5-Gen spherical vesicles.

[0084] Intracellular anti-salmonella activity of MP5-Gen vesicles

[0085] After determining that MP5-Gen vesicles could improve the intracellular concentration of gentamicin, we constructed a model of salmonella-infected mouse RAW264.7 macrophages, and studied the inhibitory effect of free gentamicin and MP5-Gen on intracellular salmonella. As shown in the description Figure 9 (a), in the model of salmonella-infected mouse macrophages, the inhibitory effect of MP5-Gen on intracellular salmonella was significantly better than that of free gentamicin, and this trend became more and more significant with the increase of the concentration of gentamicin. In the competition test, we treated the infected macrophages with mannose amine, then incubated with MP5-Gen, and observed the survival of intracellular salmonella by laser confocal microscope, and found that the survival rate of salmonella in the mannose amine group was higher than that in the MP5-Gen group (description Figure 9 (b)). It is proved that MP5-Gen can enter the cell through mannose receptor-mediated endocytosis, and enhance the anti-intracellular salmonella activity of gentamicin.

[0086] Cytotoxicity of MP5 and MP5-Gen vesicles

[0087] After confirming that MP5-Gen vesicles could significantly reduce the number of intracellular Salmonella, we further detected the cytotoxicity of MP5 and MP5-Gen nanovesicles to RAW 264.7 cell line by MTT method. The concentration range of MP5 and MP5-Gen nanovesicles was 1-30 μg / mL, and the survival rate of cells within 72 h was more than 90% (see the attached drawings of the specification Figure 9 ). The experimental results showed that MP5 and MP5-Gen nanovesicles had good cell biocompatibility, which provided a safety guarantee for subsequent animal experiments.

[0088] In this study, we first designed and synthesized mannose-modified cathepsin B-sensitive pillararene monomer MP5. We proved by DLS and TEM that MP5 could self-assemble into spherical hollow vesicles with a particle size of 80-134 nm. We also prepared drug-loaded nanovesicles of MP5 loaded with gentamicin, and determined the drug loading rate of gentamicin by spectrophotometer. It was then proved that MP5-Gen nanovesicles could release gentamicin rapidly under the action of cathepsin B, and the release rate was proportional to the concentration of cathepsin B. We also proved by cell uptake experiment that MP5 nanovesicles could be recognized by the mannose receptor on the surface of macrophages, and improve the uptake ability of macrophages to MP5 vesicles. It was then found that MP5-Gen loaded with gentamicin had no significant extracellular antibacterial activity, but in the presence of cathepsin B, it had extracellular anti-Salmonella activity comparable to free gentamicin. On the RAW 264.7 cell model infected with Salmonella, it was found that MP5-Gen enhanced the anti-intracellular Salmonella activity of gentamicin and had good biocompatibility.

[0089] Synthesis of compound 30: L-citrulline (8.8 g, 50 mmol) was dissolved in DME (200 mL) and H2O (100 mL), Na2CO3(10.6 g, 100 mmol) was added, chloroformic acid-9-fluorenylmethyl ester (25.9 g, 100 mmol) was added under ice bath condition, the reaction was allowed to warm to room temperature for 12 h. After completion of the reaction, the solvent was removed under reduced pressure, pH was adjusted to neutral with 1 N HC1, filtered, washed with water, acetone to obtain Fmoc protected product 30 (14 g, 70.4 %).1H NMR (400 MHz, DMSO-d6): δ 7.85 (d, J = 8.0 Hz, 2H), 6.79 (t, J = 8.0 Hz, 2H), 7.38 (t, J = 8.0 Hz, 2H), 7.30 (t, J = 8.0 Hz, 2H), 4.25 (d, J = 8.0 Hz, 2H), 4.21 - 4.18 (m, 1H), 3.95 - 3.89 (m, 1H), 3.78 - 3.72 (m, 1H), 3.42 - 3.38 (m, 1H), 3.94 (t, J = 8.0 Hz, 2H), 1.95 - 1.88 (m, 1H), 1.69 - 1.67 (m, 1H), 1.57 - 1.55 (m, 1H), 1.42 - 1.40 (m, 2H), 1.02 - 1.00 (m, 4H);13C NMR (101 MHz, DMSO-d6): δ 166.9, 159.4, 156.6, 144.3, 144.2, 141.1, 128.1, 127.5, 125.8, 120.6, 66.1, 54.1, 47.1, 28.7, 27.2, 25.9.

[0090] 174.4, 159.4, 156.6, 144.3, 144.2, 141.1, 128.1, 127.5, 125.8, 120.6, 66.1, 54.1, 47.1, 28.7, 27.2, 25.9.

[0091]

[0092] Synthesis of compound 31 : To a solution of aminophenol (10.9 g, 100 mmol) and Et3N (10.1 g, 100 mmol) in CH2Cl2(300 mL) was added Boc20 (21.8 g, 100 mmol) dropwise under ice bath condition, then the reaction was allowed to warm to room temperature for 1 h. After completion of the reaction, it was washed with saturated Na2CO3, extracted with CH2Cl2, and dried over Na2SO4. The CH2Cl2was removed under reduced pressure to give the crude product, which was recrystallized from petroleum ether to give tert-butyl (4-hydroxyphenyl)carbamate (20.9 g, ~100%) without further purification, which was used directly in the next step.1H NMR (400 MHz, CDC13): δ 7.12 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 1.49 (s, 9H);13C NMR (101 MHz, CDC13): δ 152.3, 130.6, 121.7, 115.8, 28.4. Then the above compound (10.5 g, 50 mmol) and K2CO3(27.6 g, 200 mmol) were dissolved in anhydrous acetone (200 mL), and 1,4-dibromobutane (43 g, 200 mmol) was added with stirring, then heated to reflux for 18 h. After completion of the reaction, it was filtered, the acetone was removed under reduced pressure, washed with water, extracted with CH2Cl2, dried over Na2SO4, and separated by column chromatography (CH2Cl2 / EA = 100: 1) to give the product tert-butyl (4-(4-bromobutoxy)phenyl)carbamate (12.3 g, 71.5%).1H NMR (400 MHz, CDC13): δ 7.24 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 8.0 Hz, 2H), 6.38 (s, 1H), 3.94 (t, J = 8.0 Hz, 2H), 3.47 (t, J = 8.0 Hz, 2H), 2.03 (q, J = 8.0 Hz, 2H), 1.91 (q, J = 8.0 Hz, 2H), 1.50 (s, 9H);13C NMR (101 MHz, CDC13): δ 154.9, 131.5, 120.5, 114.8, 110.0, 67.1, 33.5, 29.5, 28.4, 27.9.tert-Butyl (4-(4-bromobutoxy)phenyl)carbamate (12.3 g, 35.8 mmol) was dissolved in DMF (50 mL) and NaN3(4.6 g, 70 mmol) was added. The reaction was stirred at 70 °C for 24 h. After the reaction was completed, N,N-dimethylformamide was removed by distillation under reduced pressure to give the crude product, which was then washed with saturated NaCl solution, extracted with CH2Cl2, dried over Na2SO4, and CH2Cl2was removed by distillation under reduced pressure to give the crude tert-butyl (4-(4-azidobutoxy)phenyl)carbamate (10.9 g, ~100%) which was used directly in the next step.1H NMR (400 MHz, CDCl3): δ 7.23 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.0 Hz, 2H), 6.54 (s, 1H), 3.92 (t, J = 8.0 Hz, 2H), 3.32 (t, J = 8.0 Hz, 2H), 1.83-1.73 (m, 4H), 1.48 (s, 9H);13C NMR (101 MHz, CDCl3): δ 162.5, 154.8, 131.6, 120.5, 114.8, 67.5, 51.2, 36.4, 28.3, 26.5, 25.7. The above compound (10.9 g, 35.8 mmol) was dissolved in anhydrous CH2Cl2 / TFA (5:1, 200 mL) and the reaction was stirred at room temperature for 4 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure to give the crude product, 200 mL of CH2Cl2was added and the pH was adjusted to weakly basic with saturated Na2CO3solution. After drying over anhydrous sodium sulfate, the CH2Cl2was removed to give the product 31 (7.4 g, ~100%).1H NMR (400 MHz, CDCl3): δ 9.43 (s, 1H), 7.13 (d, J = 8.0 Hz, 2H), 6.73 (d, J = 8.0 Hz, 2H), 3.88 (t, J = 8.0 Hz, 2H), 3.32 (t, J = 8.0 Hz, 2H), 1.83-1.71 (m, 4H);13C NMR (101 MHz, CDCl3): δ 158.8, 124.0, 122.8, 115.3, 67.4, 51.1, 26.3, 25.6.

[0093]

[0094] Synthesis of compound 32: Compound 31 (7.4 g, 35.8 mmol) and compound 30 (14 g, 35.2 mmol) were weighed into CH2Cl2 / MeOH (v:v, 3:1, 150 mL) and protected with N2, then EEDQ (25.9 g, 105 mmol) was added and the reaction was allowed to proceed at room temperature for 14 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain the crude product 32. The product 32 (15.1 g, 75%) was then recrystallized with diethyl ether.1H NMR (400 MHz, DMSO-d6): δ 7.86 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.71 (dd, J = 8.0 Hz, 4.0 Hz, 2H), 7.64-7.57 (m, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 2H), 7.31 (t, J = 8.0 Hz, 2H), 6.00-5.92 (m, 1H), 5.42-5.38 (m, 2H), 4.29-4.13 (m, 3H), 4.02-3.91 (m, 1H), 3.59 (t, J = 8.0 Hz, 1H), 3.38-3.34 (m, 3H), 3.04-2.92 (m, 2H), 1.74-1.53 (m, 4H), 1.46-1.39 (m, 2H);13C NMR (101 MHz, DMSO-d6): δ 173.3, 171, 159.2, 156.5, 154.9, 144.2, 141.2, 132.6, 129.3, 128.1, 127.7, 127.5, 125.8, 125.7, 121.8, 121.1, 120.5, 120.4, 114.9, 67.5, 66.1, 54.2, 52.3, 50.9, 47.1, 28.6, 27.2, 26.4, 25.6. HRMS (ESI-TOF) m / z calcd for C31H35N7NaO5+(M+Na)+608.2592, found 608.2593.

[0095]

[0096] Synthesis of compound 33: Compound 32 (15.1 g, 25.8 mmol) was dissolved in DMF (80 mL) and piperidine (4.4 g, 51.6 mmol) was added at room temperature and the reaction was allowed to proceed at room temperature for 4 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure and the crude product was recrystallized with diethyl ether and ethyl acetate to obtain compound 33 (8.9 g, 94.7%) which was used directly in the next step without further purification.

[0097]

[0098] Synthesis of compound 35: L-valine (11.7 g, 100 mmol) and Na2CO3(15.9 g, 150 mmol) were dissolved in 1,4-dioxane / H2O (v:v, 1:1, 300 mL), Boc2O (21.8 g, 100 mmol) was added dropwise under ice bath condition, the reaction was allowed to warm to room temperature for 16 h. After the reaction was completed, 1 N NaHSO4solution was added to adjust the pH to neutral, extracted with EA, dried over sodium sulfate, and EA was removed by distillation under reduced pressure to obtain Boc-protected L-valine. Without further purification, EDCI (38.3 g, 200 mmol) and NHS (17.3 g, 150 mmol) were added and dissolved in CH2Cl2(200 mL), and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain crude product 34.1H NMR (400 MHz, CDCl3): δ 2.82 (s, 4H), 1.44 (s, 9H), 1.05-1.02 (m, 6H). Subsequently, an excess of activated ester 34 and compound 33 (8.9 g, 24.5 mmol) were dissolved in DMF (100 mL), and DIPEA (6.3 g, 49 mmol) was added dropwise, and the reaction was allowed to proceed at room temperature for 8 h. After the reaction was completed, DMF was removed by distillation under reduced pressure, recrystallized with diethyl ether, and EA to obtain product 35 (7.9 g, 57%).1H NMR (400 MHz, CD3OD): δ 7.06 (d, J = 8.0 Hz, 2H), 6.47 (d, J = 8.0 Hz, 2H), 4.13 (t, J = 8.0 Hz, 1H), 4.04-4.01 (m, 2H), 3.60-3.53 (m, 4H), 2.99-2.92 (m, 2H), 2.84-2.77 (m, 1H), 2.73-2.70 (m, 4H), 1.66-1.61 (m, 2H), 1.53-1.42 (m, 2H), 1.40-1.31 (m, 2H), 1.21-1.12 (m, 2H), 1.05 (s, 9H), 1.66-1.61 (m, 2H), 0.89-0.82 (m, 1H), 0.58 (d, J = 8.0 Hz, 3H), 0.54 (d, J = 8.0 Hz, 3H);13C NMR (101 MHz, CD3OD): δ 173.2, 172.4, 160.7, 156.5, 155.8, 131.0, 121.7, 114.2, 79.1, 67.2, 59.9, 53.5, 52.2, 51.3, 50.9, 30.7, 28.4, 27.4, 26.3, 25.4, 18.4, 17.2. HRMS (ESI-TOF) m / z calcd for C26H43N8O6+(M+H)+563.3300, found 563.3300.

[0099]

[0100] Synthesis of compound 36: Compound 35 (7.9 g, 14.1 mmol) was dissolved in anhydrous CH2Cl2 / TFA (v:v, 1:1, 40 mL) and reacted at room temperature for 4 h. After the reaction was completed, CH2Cl2and TFA were removed by distillation under reduced pressure, the crude product was dissolved in CH2Cl2again, Et3N was added, TFA was removed, followed by removal of CH2Cl2, recrystallization with ether and EA crystallization to obtain product 36 (6.0 g, 92%).1H NMR (400 MHz, DMSO-d6): δ 9.99 (s, 1H), 8.65 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.76 (t, J = 12.0 Hz, 2H), 6.84 (t, J = 12.0 Hz, 2H), 4.45 (q, J = 8.0 Hz, 4.0 Hz, 1H), 3.92 (t, J = 8.0 Hz, 2H), 3.65-3.61 (s, 1H), 3.65 (t, J = 8.0 Hz, 2H), 3.04-2.95 (m, 2H), 2.05 (q, J = 8.0 Hz, 4.0 Hz, 1H), 1.74-1.59 (m, 6H), 1.50-1.40 (m, 2H), 0.92 (d, J = 8.0 Hz, 6H);13C NMR (101 MHz, DMSO-d6): δ 167.0, 168.2, 159.7, 155.0, 132.4, 121.1, 114.9, 67.5, 57.7, 53.6, 50.9, 30.3, 29.8, 27.1, 26.4, 25.6, 25.5, 18.7, 18.1. HRMS (ESI-TOF) m / z calcd for C21H35N8O4+(M+H)+463.2776, found 463.2779.

[0101]

[0102] Synthesis of compound 37: Diethylene glycolamine (10.5 g, 100 mmol) was dissolved in anhydrous CH2Cl2 (200 mL), and CS2 (15.2 g, 200 mmol) was added at -78 °C. The reaction was continued for 3 h, followed by the dropwise addition of 30% H2O2 (30 mL), and the reaction was stirred for another 1 h. The mixture was then brought to room temperature, extracted with CH2Cl2, dried over Na2SO4, and separated by column chromatography (PE / EA = 1:1) to obtain the oily product 2-(2-isothiocyanatoethoxy)ethan-1-ol (10.1 g, 71.8%). ¹H NMR (400 MHz, CDCl3): δ 3.77–3.75 (m, 2H), 3.69 (dd, J = 8.0, 4.0 Hz, 4H), 3.64–3.62 (m, 2H). Subsequently, compound 2-(2-isothiocyanatoethoxy)ethan-1-ol (10.1 g, 68.5 mmol), compound 4 (34.5 g, 70 mmol), and activated... Molecular sieve (5 g) was dissolved in dry CH2Cl2 (200 mL), and BF3·Et2O (19.4 g, 137 mmol) was added dropwise under ice bath conditions. The reaction was carried out under ice bath conditions for 2 h, and then the temperature was raised to room temperature and the reaction was continued for 1 h. The reaction progress was monitored by TLC. After the reaction was completed, CH2Cl2 was removed by vacuum distillation, the crude product was redissolved in CH2Cl2, washed with saturated Na2CO3 solution, the organic phase was collected, dried over Na2SO4, and separated by column chromatography (PE / EA = 1:1) to obtain an oily product 37 (21.8 g, 66%). 1HNMR (400MHz, CDCl3): δ5.31-5.26(m,1H),5.22-5.20(m,1H),4.83(s,1H),4.24(dd,J=8.0Hz,4.0H 13C NMR (101MHz, CDCl3): δ170.6,170.0,169.9,169.7,97.6,70.1,69.4,69.3,69.0,68.4,67.3,66.0,62.4,45.3,20.9,20.7,20.7,20.7.

[0103]

[0104] Synthesis of compound 38: Compound 36 (6 g, 13 mmol) and compound 37 (9.5 g, 20 mmol) were dissolved in DMF / pyridine (v:v, 4:1, 80 mL) and reacted at room temperature for 8 h. After the reaction was completed, DMF and pyridine were removed by distillation under reduced pressure, and the product 38 (5.6 g, 45.9%) was isolated by column chromatography (CH2Cl2 / MeOH = 30:1).1H NMR (400 MHz, CDCl3): δ 9.13 (s, 1H), 7.82-7.68 (s, 1H), 7.39 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 8.0 Hz, 2H), 5.30-5.21 (m, 4H), 4.85 (s, 1H), 4.57 (s, 1H), 4.24 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.11-4.08 (m, 1H), 3.97 (s, 1H), 3.85-3.75 (m, 4H), 3.64-3.57 (m, 6H), 3.34-3.31 (m, 2H), 3.19-3.07 (m, 2H), 2.21 (s, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.77-1.52 (m, 8H), 0.95 (s, 6H);13C NMR (101 MHz, CDCl3): δ 170.8, 170.6, 170.5, 170.3, 170.2, 170.1, 169.7, 160.3, 155.5, 130.9, 121.8, 114.4, 97.4, 69.7, 69.5, 69.2, 68.5, 67.3, 67.0, 65.8, 62.4, 51.1, 45.9, 44.5, 39.4, 30.8, 28.8, 26.5, 25.7, 21.0, 20.8, 20.7, 19.4, 18.4, 15.3, 8.6. HRMS (ESI-TOF) m / z calcd for C40H62N9O15S+(M+H)+940.4008, found 940.4007.

[0105]

[0106] Synthesis of compound 39: Compound 38 (5.6 g, 6.0 mmol) and columnarene 11 (0.5 g, 0.5 mmol) were dissolved in DMF (10 mL), CuI (95 mg, 0.5 mmol) was added, N2 protection, followed by the addition of TBTA (0.27 g, 0.5 mmol), and the reaction was allowed to proceed at room temperature for 24 h. After the reaction was completed, the DMF was removed by distillation under reduced pressure, and the product 39 (3.6 g, 76%) was obtained by recrystallization from diethyl ether, EA, and methanol.1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 10H), 8.31 (s, 10H), 8.17 (s, 10H), 7.79 (s, 10H), 7.60 (s, 10H), 7.45 (d, J = 8.0 Hz, 20H), 7.35-7.26 (m, 20H), 7.25 (d, J = 8.0 Hz, 10H), 6.79 (d, J = 8.0 Hz, 20H), 5.97 (s, 10H), 5.57 (s, 10H), 5.41 (s, 20H), 5.09 (s, 30H), 4.90 (s, 10H), 4.81 (t, J = 12.0 Hz, 10H), 4.74-4.65 (m, 10H), 4.37-4.29 (m, 30H), 4.81 (dd, J = 8.0 Hz, 4.0 Hz, 10H), 4.81 (d, J = 12.0 Hz, 10H), 3.96 (s, 10H), 3.81 (s, 20H), 3.74-3.71 (m, 20H), 3.62-3.50 (m, 80H), 2.07 (s, 1H), 1.95 (s, 1H), 1.94 (s, 1H), 1.88 (s, 1H);13C NMR (101 MHz, DMSO-d6): δ 170.5, 170.4, 170.4, 170.1, 169.9, 162.8, 162.7, 159.3, 154.8, 136.4, 132.5, 129.2, 128.5, 128.2, 121.0, 121.0, 114.8, 97.0, 69.5, 69.4, 69.4, 69.4, 69.4, 69.1, 69.1, 69.1, 69.0, 68.2, 67.0, 65.8, 62.4, 53.5, 49.5, 36.2, 31.7, 31.2, 29.8, 27.3, 27.1, 26.2, 21.1, 21.0, 21.0, 20.9, 20.9, 20.9, 19.4. MALDI-TOF-MS. Calcd. for C465H664N91O160S10+(M+NH4)+, 10402.382, found 10402.385.

[0107]

[0108] Synthesis of Pillararene MP5: Compound 39 (3.6 g, 0.4 mmol) was dissolved in MeOH (20 mL) under N2protection, 30% sodium methoxide in MeOH was used to adjust pH to 9. The reaction was carried out at room temperature for 5 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, the pH was adjusted to neutral with 0.5 N HC1 solution, and dialyzed with regenerated cellulose dialysis bag (MW: 5000 Da), and freeze-dried to obtain Pillararene MP5 (3.3 g, ~99%).1H NMR (400 MHz, DMSO-d6): δ 9.78 (s, 10H), 8.30 (s, 10H), 8.17 (s, 10H), 7.43 (s, 10H), 7.59 (s, 10H), 7.44 (d, J = 8.0 Hz, 20H), 7.33-7.26 (m, 10H), 6.79-6.78 (m, 20H), 5.9 (s, 10H), 5.56-5.42 (m, 20H), 4.84-4.79 (m, 10H), 4.73-7.72 (m, 20H), 4.61-4.58 (m, 20H), 4.48-4.46 (m, 10H), 4.35-4.29 (m, 30H), 3.82-3.79 (m, 10H), 3.62-3.42 (m, 150H), 3.98-3.92 (m, 20H), 2.03-1.98 (m, 10H), 1.84 (s, 20H), 1.65-1.56 (m, 30H), 1.42-1.34 (m, 20H), 0.82 (s, 60H);13C NMR (101 MHz, DMSO-d6): δ 170.4, 159.4, 154.9, 149.2, 143.6, 135.9, 132.5, 129.2, 129.2, 128.5, 128.2, 128.2, 121.0, 114.8, 100.4, 100.4, 74.4, 71.3, 70.7, 70.6, 69.8, 69.4, 67.4, 67.2, 66.1, 61.7, 53.6, 49.5, 44.2, 40.5, 40.3, 40.1, 39.9, 39.7, 39.5, 39.3, 31.729.8, 29.8, 27.3, 27.1, 27.0, 26.2, 26.2, 19.4, 18.5, 18.4. MALDI-TOF-MS. Calcd. for C385H580KN90O120S10+(M+K)+8742.889, found 8742.885.

[0109]

[0110] The application further provides an application of the pillararene-based enzyme-responsive nanovesicle, and a preparation method of the pillararene-based enzyme-responsive nanovesicle.

[0111] The MP5 nanovesicle is applied to a medicine for treating salmonella infection.

[0112] The medicine for treating salmonella infection is the MP5 nanovesicle loaded with gentamicin.

[0113] The MP5 nanovesicle plays a role in the following manner:

[0114] The surface mannose group is combined with the mannose receptor on the surface of macrophages, and enters the cells through receptor-mediated endocytosis;

[0115] Under the action of cathepsin B in the macrophages, the Val-Cit dipeptide sequence is broken, the loaded gentamicin is released, and the intracellular salmonella is cleared.

[0116] The above only discloses one or more preferred embodiments of the application, and cannot limit the scope of the application, and those skilled in the art can understand that all or part of the above-mentioned embodiments can be implemented, and equivalent changes made according to the claims of the application still belong to the scope of the application.

Claims

1. A method for preparing enzyme-responsive nanovesicles using columnar aromatic hydrocarbons as carriers, characterized in that, Includes the following steps; Starting from L-citrulline compound 30 protected by 9-fluorenyl chloroformate, it was reacted with aniline derivative 31 containing an azide group in the presence of condensing reagent EEDQ to give compound 32 in 75% yield. Compound 32 was deprotected by the Fmoc protecting group under the action of the organic base piperidine, and then precipitated with diethyl ether to obtain compound 33; Subsequently, using DMF as a solvent, compound 33 was reacted with Boc-protected L-valine activated ester 34 to separate compound 35 in 57% yield. Compound 35 was deprotected by trifluoroacetic acid to obtain compound 36; The compound 36 was then reacted with a mannose derivative 37, and the compound 38 was obtained by column chromatography in a yield of 45.9%. Compound 38 and terminal alkyne columnar aromatic hydrocarbon 11 were reacted with 1,3-dipolar addition in the presence of catalytic equivalent CuI and with TBTA as a ligand to give compound 39 in 76% yield. The acetyl protecting group was then removed in a methanol solution containing sodium methoxide to give amphiphilic columnar aromatic hydrocarbon MP5 in 99% yield. The amphiphilic columnar aromatic compound MP5 was dissolved in deionized water containing 1% DMSO. After ultrasonic dissolution, it was placed into a regenerated cellulose dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed in ultrapure water until no DMSO residue was found, thus obtaining MP5 nanovesicles.

2. The method for preparing enzyme-responsive nanovesicles with columnar aromatic hydrocarbons as a carrier as described in claim 1, characterized in that, The conditions for ultrasonic dissolution are: power 200-300W, time 10-15 minutes; the dialysate is changed every 2 hours during the dialysis process, and the total dialysis time is not less than 24 hours.

3. The method for preparing enzyme-responsive nanovesicles with columnar aromatic hydrocarbons as a carrier as described in claim 1, characterized in that, The MP5 nanovesicles are self-assembled into a spherical hollow structure with a hydrodynamic radius of 80-134 nm, a surface potential of 29.1 mV, and a critical micelle concentration of 41 μM.

4. The method for preparing enzyme-responsive nanovesicles with columnar aromatic hydrocarbons as a carrier as described in claim 1, characterized in that, The 1,3-dipolar addition reaction of compound 38 with terminal alkyne columnar aromatic hydrocarbon 11 was carried out under N2 protection at room temperature for 24 hours.

5. The method for preparing enzyme-responsive nanovesicles with columnar aromatic hydrocarbons as a carrier as described in claim 1, characterized in that, The MP5 nanovesicles can be loaded with hydrophilic antibiotics. When loaded with gentamicin, the encapsulation efficiency is 41.6% and the drug loading efficiency is 29.4% when the mass concentration ratio of gentamicin to the amphiphilic columnar aromatic hydrocarbon MP5 is 7:

10.

6. An application of an enzyme-responsive nanovesicle using columnar aromatic hydrocarbons as a carrier, based on the preparation method of the enzyme-responsive nanovesicle using columnar aromatic hydrocarbons as a carrier as described in any one of claims 1-5, characterized in that, The MP5 nanovesicles are used in drugs for treating Salmonella infections; The drug for treating Salmonella infection is the MP5 nanovesicle loaded with gentamicin.

7. The application of the enzyme-responsive nanovesicles with columnar aromatic hydrocarbons as a carrier as described in claim 6, characterized in that, The MP5 nanovesicles function in the following ways: The surface mannose group binds to the mannose receptor on the surface of macrophages and enters the cell via receptor-mediated endocytosis; Under the action of cathepsin B in macrophages, the Val-Cit dipeptide sequence is cleaved, releasing the loaded gentamicin and clearing intracellular Salmonella.

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