Click chemistry mediated macrophage bearing chromosome vesicle drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a click chemistry mediated macrophage bearing chromosome vesicle drug delivery system as well as a preparation method and application thereof. According to the invention, chromosome vesicles extracted from thermophilic bacteria are used as drug carriers, drug-loaded chromosome vesicles are prepared by adopting an ultrasonication method, and then diphenyl cyclooctyne modified lipid chains and azide modified lipid chains are anchored on the surfaces of the chromosome vesicles and macrophages respectively by adopting a post-insertion method. Finally, coupling the color carrier vesicles and the macrophages through a click chemical reaction to construct the color carrier vesicle delivery system carried by the macrophages, and the prepared color carrier vesicle delivery system carried by the macrophages has good loading capacity; targeted delivery can be realized by utilizing the homing characteristic of the macrophages under the condition that the adhesion capacity of the macrophages is not influenced, and the macrophages have a wide application prospect in the aspect of treating diseases such as atherosclerosis, tumors and brain inflammations.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and pharmaceutical preparation, and particularly relates to a click chemistry mediated macrophage back-loaded chromosomal vesicle drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Macrophages are a kind of natural immune cells widely distributed in tissues, which play a key role in immune regulation and inflammatory response of the body, and can actively migrate and accumulate in the lesion sites such as inflammatory, damaged or tumor microenvironment. Therefore, using the natural chemotaxis and targeting ability of macrophages to construct a drug delivery system based on them has become an important direction of biomedical research in recent years. The existing macrophage delivery methods mainly include drug endocytosis, surface modification and coupling with drug carriers, but there are still problems such as limited drug loading capacity, unstable delivery efficiency and impaired cell function.

[0003] On the other hand, most therapeutic drugs have the disadvantages of poor water solubility and low bioavailability, and need to be loaded by suitable carriers to enhance the therapeutic effect. As a new type of drug delivery carrier, vesicles have the characteristics of controllable structure, strong loading capacity and good biocompatibility. Studies have shown that the chromosomal vesicles extracted from thermophilic bacteria can realize self-driven movement to acidic environment, and have the potential to enhance tissue penetration and improve drug delivery efficiency. However, the application of chromosomal vesicles alone still has the disadvantage of insufficient targeting.

[0004] Therefore, coupling macrophages with chromosomal vesicles can combine the targeting homing characteristics of macrophages with the efficient delivery function of vesicles, and has application prospects in the fields of treating atherosclerosis, tumors and brain inflammation. SUMMARY

[0005] The purpose of the present application is to improve the problems of limited drug loading capacity, unstable delivery efficiency, insufficient vesicle retention in vivo and insufficient targeting of the existing macrophage drug delivery, and provide a click chemistry mediated macrophage back-loaded chromosomal vesicle drug delivery system and a preparation method and application thereof.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions: A click chemistry mediated macrophage back-loaded chromosomal vesicle drug delivery system comprises macrophages and bacterial chromosomal vesicles, the chromosomal vesicles load drugs, and the chromosomal vesicles are covalently coupled to the surface of the macrophages through click chemistry reaction, so as to realize the targeted drug delivery of the macrophage back-loaded chromosomal vesicles.

[0007] Further, the chromosomal vesicles are derived from thermophilic bacteria.

[0008] Further, the click chemistry reaction is realized by azide-alkyne cycloaddition reaction, and specifically includes: anchoring the azide-modified lipid chain on the surface of the macrophage, and anchoring the diphenyl cyclooctyne-modified lipid chain on the surface of the exosome vesicle, and completing the coupling through the specific reaction of the lipid chains.

[0009] Further, the azide-modified lipid chain is DSPE-PEG-N3, and the diphenyl cyclooctyne-modified lipid chain is DSPE-PEG-DBCO or CHOL-PEG-DBCO.

[0010] Further, the drug loaded in the exosome vesicle is selected from an anti-inflammatory drug, an anti-tumor drug, an anti-atherosclerosis drug or an anti-infection drug.

[0011] A preparation method of a click chemistry-mediated macrophage back-loaded exosome vesicle drug delivery system, comprising the following steps: (1) extracting the exosome vesicle from thermophilic bacteria, and loading the drug through an ultrasonic crushing method to obtain a drug-loaded exosome vesicle; (2) inserting the diphenyl cyclooctyne-modified lipid chain into the surface of the drug-loaded exosome vesicle to obtain a DBCO-modified drug-loaded exosome vesicle; (3) inserting the azide-modified lipid chain into the surface of the macrophage to obtain an N3-modified macrophage; (4) co-incubating the DBCO-modified drug-loaded exosome vesicle and the N3-modified macrophage through a click chemistry reaction to realize coupling, and obtaining a macrophage back-loaded exosome vesicle drug delivery system.

[0012] Further, in step (1), the drug loading adopts the ultrasonic crushing method, the mass ratio of the loaded drug to the exosome vesicle is 1:1-1:50, and the solvent of the loaded drug is any one of methanol, ethanol, acetone and dimethyl sulfoxide.

[0013] Further, in step (2), the final concentration of the diphenyl cyclooctyne-modified lipid chain is 0.1-20 μM, and the final concentration of the azide-modified lipid chain is 0.1-0.5 mg / mL. The application of the click chemistry-mediated macrophage back-loaded exosome vesicle drug delivery system in the preparation of a drug for treating atherosclerosis, tumors or brain inflammation.

[0014] Compared with the prior art, the application has the following beneficial effects: 1) The application constructs a macrophage back-loaded exosome vesicle drug delivery system, which takes the exosome vesicle as a drug carrier, modifies the vesicle through lipid insertion and anchors the vesicle on the surface of the macrophage, and a large number of vesicles are observed to be adsorbed on the surface of the macrophage under an electron microscope, thereby solving the problem of low loading amount of traditional drugs on the macrophage.

[0015] 2) The macrophage back-loaded chromophore vesicle drug delivery system prepared by the application improves the poor water solubility of most lipid-soluble drugs, the drug loading of the chromophore vesicle is significantly improved, and the ability to cross the biological barrier is improved, the composite system has the biological activity of macrophages and the self-driving characteristics of vesicles, and has good biocompatibility and safety as a whole, and has been proved to still have endothelial cell adhesion ability in vitro, and drug enrichment can be observed at the treatment site 4h after tail vein administration in mice, so as to achieve more superior efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a transmission electron micrograph of the chromophore vesicle of embodiment 1 of the application; Figure 2 It is a release curve diagram of the curcumin-loaded Chroma at pH 7.4 in vitro of embodiment 3 of the application; Figure 3 It is an MTT result diagram of the chromophore vesicle of embodiment 4 of the application; Figure 4 It is a SEM image of the macrophage back-loaded chromophore vesicle of embodiment 7 of the application; Figure 5 It is the adhesion effect of inflammatory macrophages on non-inflammatory and inflammatory endothelial cells of embodiment 8 of the application; Figure 6 It is a fluorescence distribution diagram of the aorta of the macrophage back-loaded chromophore vesicle of embodiment 9 of the application and the chromophore vesicle injected into the tail vein of the atherosclerotic mouse in vivo, which is determined by using small animal live imaging. DETAILED DESCRIPTION

[0017] The application will be further described below in combination with the drawings and specific embodiments. The embodiments described in the application are only used to illustrate the application and not to limit the scope of the application. Any non-substantial changes made by those skilled in the art on the basis of the embodiments of the application will result in all other embodiments within the protection scope of the application.

[0018] Embodiment 1

[0019] This embodiment extracts the chromophore vesicle, and the specific method is as follows: (1) Take 500 mL LB culture based on a 1 L conical flask, add 5 mL of thermophilic bacteria original bacteria solution, gently shake the conical flask, seal with a sealing film, and culture at 55°C.

[0020] (2) The thermophilic bacteria solution is broken by 400 W ultrasonic for 1 h, centrifuged at 3500 g for 30 min to remove the broken bacteria, centrifuged at 10000 g for 1 h to remove impurities, and ultracentrifuged at 140000 g for 1 h. After resuspension with PBS buffer, it is stored at -20°C to obtain the chromophore vesicle.

[0021] Figure 1 For the electron micrograph of the carrier vesicle in this embodiment, the vesicles exhibit typical spherical structures, the lipid bilayer is clearly visible, the size is relatively uniform, and the distribution is uniform, with a particle size distribution of 50 nm to 150 nm.

[0022] Example 2

[0023] Preparation of curcumin-loaded vesicles (Cur@Chroma) Taking curcumin as an example, 40 mg of curcumin was dissolved in 1 mL of DMSO to prepare a 40 mg / mL curcumin solution. 20 μL of the solution was added to 1 mL of a PBS solution containing 3.4 mg of carrier vesicles, vortexed for 1 minute, ultrasonicated at 200 W for 30 minutes under ice bath, incubated at 37°C for 1 hour, and centrifuged at 6000 rpm for 10 minutes to remove free drugs, thereby obtaining drug-loaded carrier vesicles Cur@Chroma, with an average particle size of 184.3 ± 9.3 nm and a zeta potential of -24.4 ± 3.3.

[0024] Example 3

[0025] The in vitro release kinetics of Cur@Chroma was studied by dialysis method, and the Cur content was determined by UV spectrophotometry. A certain amount of Cur@Chroma was diluted in 0.01M pH=7.4 PBS, then loaded into a dialysis bag with a molecular weight cut-off MD=100kD, and placed in 30 mL of 0.01M pH=7.4 PBS containing 0.5% Tween 80 and 0.5% SDS, and released at 37°C with 100 rpm shaking. Samples were taken at 0.5-24 h and replenished with an equal volume of release medium. Each time point sample was taken in triplicate, the absorbance value was determined, the cumulative release rate was calculated according to the cumulative release formula, and the release curve was plotted.

[0026] Figure 2 The cumulative release curve of Cur@Chroma is shown in the figure. The drug is basically not released within 1 h, which is sufficient to complete the subsequent coupling process.

[0027] Example 4

[0028] In vitro cell biocompatibility experiment of carrier vesicles RAW264.7 cells and MAEC cells in the logarithmic growth phase were inoculated in a 96-well plate (100 μL per well, 5 × 10 3The blank control group (only add culture medium) and the negative control group (only add culture medium containing cells) were set up, and the cells were incubated in the carbon dioxide incubator for 24 h. After incubation, the chroma purification liquid was taken and diluted 10 times, 20 times, 40 times, 100 times and 200 times with complete culture medium, respectively, and each group had 6 holes in parallel. After incubation for 24 h, the original culture medium was removed, 100 μL of fresh serum-free culture medium and 10 μL of MTT (concentration of 5 mg / mL, prepared with sterile PBS buffer and filtered through a microporous filter) were added to each hole, and the culture was continued for 4 h. After 4 h, the old culture medium was discarded, 150 μL of DMSO was added to each hole, and the sample was placed on a constant temperature shaker in the dark for 10 min. The OD value at 562 nm was detected using a fluorescence microplate reader.

[0029] Figure 3 MTT results of the chroma-loaded vesicles, Figure 3 A in which is a cytotoxicity graph of the chroma-loaded vesicles of different concentrations on MAEC cells, Figure 3 B in which is a cytotoxicity graph of the chroma-loaded vesicles of different concentrations on RAW cells, indicating that the chroma-loaded vesicles have good biocompatibility.

[0030] Example 5

[0031] Preparation of DBCO-modified chroma-loaded vesicles DSPE-PEG2000-DBCO was dissolved in anhydrous ethanol and mixed with drug-loaded Chroma at 20 μM:10 11 μM vesicles in PBS at 37°C for 1 h, then loaded into a 100 kD dialysis tube, centrifuged at 3000g for 10 min to remove unreacted DBCO, resuspended in PBS, and ultrafiltrated again and repeated twice, finally resuspended in PBS to 1 mL, to prepare DBCO-modified chroma-loaded vesicles.

[0032] Example 6

[0033] On the basis of DBCO-modified drug-loaded chroma-loaded vesicles, this embodiment further prepared a macrophage drug delivery system with drug-loaded chroma-loaded vesicles on the surface, and the specific steps were as follows: (1) The macrophages were inoculated into cell slides, and the cell culture box was cultured to 80% confluence; (2) DSPE-PEG2000-N3 was dissolved in serum-free culture medium to a final concentration of 200 μg / mL; (3) Incubation insertion: the original culture medium was discarded, 3 mL of DSPE-PEG2000-N3 solution was added, and the culture was incubated at 37°C in the dark for 2 h, then washed twice with pre-cooled PBS to remove the un-inserted DSPE-PEG2000-N3; (4) Click reaction coupling: Without affecting the macrophage activity, N3-modified macrophages and DBCO-modified chromosome vesicles were mixed in serum-free culture medium and incubated at 37°C in the dark for 1 h to complete N3-DBCO coupling and obtain macrophage back-borne chromosome vesicle drug delivery system (MP-Chroma).

[0034] Example 7

[0035] After conjugation of the macrophage-borne chromosome vesicle drug delivery system (MP-Chroma), the cells were fixed with paraformaldehyde for 1 h, then dehydrated twice each with 50% ethanol, 75% ethanol, and 95% ethanol, and sputter-coated with gold. The cells were then observed using SEM. Figure 4 As shown, Figure 4 In the image, A and B are SEM images of macrophages and their magnified portions, respectively. Figure 4 C and D in the image are SEM images of macrophages carrying chromosomal vesicles and their magnified local images, respectively, proving that the chromosomal vesicles and macrophages were successfully coupled.

[0036] Example 8

[0037] To verify the adhesion ability of MP-Chroma to inflammatory endothelial cells in Example 6, this example uses DIO-labeled macrophages to determine their adhesion to endothelial cells. The specific experimental method is as follows: (1) MAEC cells 10 5 Cells were seeded per well in 6-well plates and cultured for 48 hours until adherent and tightly connected. Then, 20 ng / mL TNF-α and 1 μg / mL LPS were added and incubated for 24 hours to induce inflammatory cells. (2) RAW with 5×10 5 Cells were seeded at 1000 cells / mL in a culture dish, treated with 0.2 mg / mL DSPE-PEG-N3 for 2 h, washed with PBS to remove free DSPE-PEG-N3, stained with 10 μM DIO for 20 min, free DIO was removed, washed with PBS, and chromatographs with inserted DBCO were added and incubated for 1 h. Cells were digested, and free chromatographs were removed by centrifugation to obtain MP-Chroma. (3) MP-Chroma (approximately 25 × 10⁻⁶) 4 MAECs were treated with (number per mL) for 30 min, followed by 3 washes with PBS. (4) Observe and quantify the fluorescence count using an inverted microscope.

[0038] Figure 5 The fluorescence results are from the adhesion experiment. Figure 5 From top to bottom, A represents the fluorescence microscopy images of macrophages adhering to endothelial cells in the non-inflammatory, inflammatory, and inflammatory condition coupled groups. Figure 5B in the figure is a column chart of cell adhesion amount, indicating that macrophages have stronger adhesion ability to inflammatory endothelial cells, and the adhesion ability of the macrophages will not be affected after being coupled with the color carrier vesicles.

[0039] Example 9

[0040] Evaluation of in vivo targeting of the drug delivery system: The color carrier vesicles were labeled with DID as a fluorescent probe to detect the aortic targeting, and the macrophages were given the color carrier vesicles (DID@MP-Chroma) and the color carrier vesicles (DID@Chroma) at 0.02 mg / mL (calculated based on the concentration of DID). Four hours after the administration, the aorta was dissected and sampled, and the fluorescence of the aorta was detected using a small animal live imaging instrument. As shown in FIG. 8, the fluorescence of the DID@MP-Chroma group was stronger than that of the DID@Chroma group, indicating that the macrophages have better targeting after being carried. Figure 6 ​

Claims

1. A click chemistry-mediated macrophage dorsal chromosomal vesicle drug delivery system, characterized in that, It includes macrophages and bacterial chromosome vesicles, with drugs loaded within the chromosome vesicles. The chromosome vesicles are covalently coupled to the surface of macrophages via click chemistry, thereby achieving targeted drug delivery to macrophages carrying chromosome vesicles.

2. The click chemistry-mediated macrophage dorsal chromosomal vesicle drug delivery system as described in claim 1, characterized in that, The chromosome vesicles are derived from thermophilic bacteria.

3. The click chemistry-mediated macrophage dorsal chromosomal vesicle drug delivery system as described in claim 1, characterized in that, The click chemistry reaction is achieved through an azide-alkyne cycloaddition reaction, specifically including: anchoring azide-modified lipid chains to the surface of macrophages and anchoring diphenylcyclooctyne-modified lipid chains to the surface of chromosome vesicles, and completing the coupling through the specific reaction of the lipid chains.

4. The click chemistry-mediated macrophage dorsal chromosomal vesicle drug delivery system as described in claim 3, characterized in that, The lipid chain modified with azide is DSPE-PEG-N3, and the lipid chain modified with diphenylcyclooctyne is DSPE-PEG-DBCO or CHOL-PEG-DBCO.

5. The click chemistry-mediated macrophage dorsal chromosomal vesicle drug delivery system as described in claim 1, characterized in that, The drugs loaded in the chromosome vesicles are selected from anti-inflammatory drugs, anti-tumor drugs, anti-atherosclerotic drugs, or anti-infective drugs.

6. A method for preparing a click chemistry-mediated macrophage-borne chromosome vesicle drug delivery system as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Chromosome vesicles were extracted from thermophilic bacteria and loaded with drugs by ultrasonic disruption to obtain drug-loaded chromosome vesicles; (2) The lipid chain modified by diphenylcyclooctylene was inserted into the surface of the drug-loaded chromosome vesicle to obtain the drug-loaded chromosome vesicle modified by DBCO. (3) Insert the azide-modified lipid chain into the surface of macrophages to obtain N3-modified macrophages; (4) The drug-loaded chromosome vesicles modified with DBCO were co-incubated with N3-modified macrophages through a click chemical reaction to achieve coupling, thereby obtaining a macrophage-borne chromosome vesicle drug delivery system.

7. The preparation method according to claim 6, characterized in that, In step (1), the drug loading is performed by ultrasonic disruption. The mass ratio of the loaded drug to the chromogenic vesicle is 1:1 to 1:

50. The solvent for loading the drug is any one of methanol, ethanol, acetone, or dimethyl sulfoxide.

8. The preparation method according to claim 6, characterized in that, In step (2), the final concentration of the lipid chain modified with diphenylcyclooctyn is 0.1-20 μM, and the final concentration of the lipid chain modified with azide is 0.1-0.5 mg / mL.

9. The use of the click chemistry-mediated macrophage dorsal chromosomal vesicle drug delivery system as described in claim 1 in the preparation of medicaments for treating atherosclerosis, tumors, or brain inflammation.

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