Brain-targeted extracellular vesicle as well as preparation method and application thereof

Abstract

The embodiment of the invention discloses a brain-targeted extracellular vesicle as well as a preparation method and application thereof. The method comprises the following steps: centrifugally separating blood to obtain red blood cell precipitate, and then carrying out gradient centrifugation to obtain RBCEVs; the RBCEVs and DBCO-sulfo-NHS are subjected to a coupling reaction, and DBCO-RBCEVs is obtained; the DBCO-RBCEVs and RVG29-N3 are subjected to a click chemical reaction without copper azide, and the brain-targeted extracellular vesicles are obtained. The RVG-RBCEVs is successfully prepared, is used as a drug carrier (such as loading siRNA, miRNA, mRNA, viruses, Cas9 gene editing tools and the like), and has the capability of entering the brain in a targeted manner, so that the targeted delivery of central nervous system disease drugs is realized. The invention provides a simple, efficient and reliable brain targeting realization method, can be used for rapidly preparing available biological agents, and has a better application prospect.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedical technology, and particularly to a brain-targeted extracellular vesicle and its preparation method and application. Background Art

[0002] Subarachnoid hemorrhage (SAH) is a highly harmful hemorrhagic cerebrovascular disease mainly caused by the rupture of intracranial aneurysms. It has a high fatality rate among various cerebrovascular diseases, accounting for about 5% of all stroke cases and ranking second among the diseases that cause stroke patients to die. Approximately 30% of the survivors will have permanent disabilities, seriously affecting their quality of life. From a pathological mechanism perspective, the brain injury after SAH mainly includes early brain injury (EBI) and cerebral vasospasm, among which EBI is the key pathogenic factor, involving various physiological dysfunction such as inflammatory injury, aggravated cerebral edema, and neuronal apoptosis.

[0003] Small extracellular vesicles (sEVs) are derived from intraluminal vesicles with a diameter less than 200 nm; they can freely penetrate the blood-brain barrier (BBB). sEVs are important mediators of intercellular communication. They carry various biomolecules from donor cells, such as RNA, proteins, metabolites, and lipids, for transfer to recipient cells. The BBB is a highly selective biological (physiological) barrier; more than 98% of drugs cannot cross the BBB. Due to the nano effect, biocompatibility, long-distance targeting, circulatory stability, low immunogenicity, and the ability to cross the BBB of sEVs, they have been used as carriers for brain-targeted drugs to treat various CNS diseases. However, one major dilemma currently faced is that natural sEVs lack targeting specificity for specific organs and are difficult to accurately deliver the carried therapeutic substances to specific lesion areas in the brain, which limits their efficacy and application scope in the treatment of brain diseases. Summary of the Invention

[0004] Therefore, the embodiments of the present invention provide a brain-targeted extracellular vesicle and its preparation method and application.

[0005] To achieve the above object, the embodiments of the present invention provide the following technical solutions:

[0006] According to the first aspect of the embodiments of the present invention, the present invention provides a preparation method of a brain-targeted extracellular vesicle, and the method includes the following steps:

[0007] (1) Blood is centrifuged to obtain a red blood cell precipitate, and then gradient centrifugation is performed to obtain extracellular vesicles derived from red blood cells, namely RBCEVs;

[0008] (2) RBCEVs are subjected to a coupling reaction with DBCO-sulfo-NHS to obtain DBCO-bound RBCEVs, namely DBCO-RBCEVs;

[0009] (3) The DBCO-RBCEVs and RVG29-N3 undergo a copper-free azide click chemical reaction to obtain RVG-RBCEVs, which are the brain-targeting extracellular vesicles.

[0010] Furthermore, in step (1), the gradient centrifugation includes: centrifuging at 3000×g for 15 min at 4°C, passing the supernatant through a 0.22 μm filter membrane, and then successively centrifuging at 10,000×g for 30 min and at 100,000×g for 4 h at 4°C, discarding the supernatant, and collecting the bottom precipitate RBCEVs.

[0011] Furthermore, in step (2), the concentration of the RBCEVs is 0.5 mg / mL, and the concentration of DBCO-PEG4-NHS is 2 μM;

[0012] The conditions for the conjugation reaction are: pH 7 - 8, 20 - 30°C, 2 - 5 h.

[0013] Furthermore, in step (3), the concentration of the DBCO-RBCEVs is 0.4 mg / mL, and the concentration of RVG29-N3 is 0.6 mg / mL;

[0014] The conditions for the copper-free azide click chemical reaction are: pH 7 - 8, 1 - 5°C, 10 - 15 h.

[0015] Furthermore, the method further includes: placing the reaction solution of the copper-free azide click chemical reaction in PBS, centrifuging at 7500×g, and then concentrating using an ultrafiltration column with a molecular weight cut-off of 100KDa.

[0016] According to the second aspect of the embodiments of the present invention, the present invention provides a brain-targeting extracellular vesicle prepared by the preparation method described in any one of the above.

[0017] According to the third aspect of the embodiments of the present invention, the present invention provides the application of the above-described brain-targeting extracellular vesicle as a delivery system in the preparation of a drug for treating central nervous system diseases.

[0018] Furthermore, the drug includes siRNA, miRNA, mRNA, virus, Cas9 gene editing tool.

[0019] Furthermore, the central nervous system disease includes subarachnoid hemorrhage.

[0020] The embodiments of the present invention have the following advantages:

[0021] Through a coupling reaction, the present invention obtains DBCO-conjugated RBCEVs. Subsequently, using the bioorthogonal copper-free azide-alkyne cycloaddition method (click chemistry), the rabies virus glycoprotein peptide (RVG) with the ability of brain targeting is conjugated to the surface of red blood cell extracellular vesicles (RBCEVs), and RVG-RBCEVs are successfully prepared. Experimental verification shows that the prepared RVG-RBCEVs, as drug carriers (such as loading siRNA, miRNA, mRNA, viruses, Cas9 gene editing tools, etc.), have the ability to target the brain, thereby realizing the targeted delivery of drugs for central nervous system diseases. The present invention provides a simple, efficient and reliable method for achieving brain targeting, which can quickly prepare available biological agents and has good application prospects. Brief Description of the Drawings

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0023] Figure 1 Showing the modification and characterization of RBCEVs. A. Schematic diagram of conjugating RVG and Cy5.5 fluorescent group to the RBCEVs membrane through a two-step reaction. B. Transmission electron micrographs of unmodified RBCEVs and RVG-RBCEVs. Scale bar is 100 nm. C. Western blot analysis results of CD63, TSG101, Alix, and HBA relative to GAPDH in red blood cells and RBCEVs. D. Particle size distribution of unmodified RBCEVs and RVG-RBCEVs measured by nanoparticle tracking analysis (NTA). E. Fluorescent images of unmodified RBCEVs and RVG-RBCEVs. Green represents the dio-stained lipid membrane, and red is Cy5.5. Scale bar is 5 mm.

[0024] Figure 2Show the characteristics of RVG-RBCEVs / siRNA and their ability to target the SAH lesion area. A. Schematic diagram of loading siRNA onto vesicles by electroporation. B. TEM of unmodified RBCEVs / siRNA and RVG-RBCEVs / siRNA. Scale bar is 100 nm. C. Particle size distribution of unmodified RBCEvs / siRNA and RVG-RBCEvs / siRNA measured by NTA. D. Relative concentration of siRNA in the electroporation supernatant. E. Quantitative analysis of fluorescence intensity in the brain tissues of SAH mice 24 hours after intravenous injection of PBS, RBCEvs / Cy5.5, RBCEvs / siRNA / Cy5.5, or RVG-RBCEvs / siRNA / Cy5.5. G. Representative fluorescence images of dissected organs of SAH mice treated with intravenous injection of RBCEVs / Cy5.5, RBCEVs / siRNA / Cy5.5, or RVG-RBCEVs / siRNA / Cy5.5 for 24 hours. The right block diagram shows the positions of the six analyzed organs. H. Quantitative analysis of fluorescence intensity in different organs of mice after SAH. I. Immunofluorescence images of brain tissues of SAH mice treated with RVG-RBCEvs / siRNA / Cy5.5. Scale bar is 50 μm. All quantitative data are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001). Detailed implementation mode

[0025] The following specific embodiments illustrate the implementation mode of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] Construction of animal model:

[0027] Animal model: All mice (male, C57BL / 6J, 8 - 10 weeks old, 21 - 26 g) were purchased from Collective Pharmachem Biotechnology Co., Ltd. All mouse experiments were conducted in accordance with the guidelines of the National Institutes of Health and were approved by the Animal Welfare and Ethics Committee of the First Affiliated Hospital of Wannan Medical College (approval number: LLSC - 2023 - 112). The mice were anesthetized with 2% isoflurane, and a sharp nylon monofilament (6 - 0) was gently inserted from the external carotid artery into the end of the internal carotid artery until there was a slight sense of breakthrough, and then the nylon monofilament was removed to obtain the SAH model. The Sham group underwent the same surgical procedure without puncturing the artery. After sacrificing the SAH mice at the designated time points, the brain tissue around the temporal base hemorrhage was sampled and analyzed.

[0028] Detection methods:

[0029] Near - infrared fluorescence (NIRF) imaging: To determine the biodistribution of RVG - RBCEVs / siRNA, 100 μg of RBCEVs / Cy5.5, RBCEVs / siRNA / Cy5.5, RVG - RBCEVs / siRNA / Cy5.5 or PBS was administered 2 hours after SAH. At 24 hours after administration, each organ (lung, heart, spleen, kidney, brain, and liver) was dissected and imaged using an in - vivo imaging system spectral system and real - time imaging software (PerkinElmer, Waltham, MA, USA) to examine the biodistribution of each group.

[0030] Western blot: Erythrocytes or brain tissues were lysed with RIPA lysis buffer containing protease inhibitors, and then the protein supernatant was extracted by centrifugation. Subsequently, the protein sample concentration was measured using a BCA protein concentration assay kit (Beyotime, Shanghai, China). The protein supernatant was mixed with 5x Protein Loading Buffer and denatured at 95 °C for 10 minutes. Dual-color pre-stained protein markers (Epizyme Biotech, Shanghai, China) and protein samples were electrophoretically separated on 10% SDS-PAGE (Epizyme Biotech, Shanghai, China). After electrophoresis, the electrophoresis was transferred to a PVDF membrane (Millipore, USA). The PVDF membrane was blocked in 5% skim milk at room temperature for 2 h. The blocked PVDF membrane was incubated with specific primary antibodies overnight at 4 °C. The primary antibodies included: rabbit anti-TSG101 (1:50000, ab133586, Abcam, USA), rabbit anti-CD63 (1:50000, ab134045, Abcam, USA), rabbit anti-ALIX (1:1000, ab275377, Abcam, USA), rabbit anti-hemoglobin α (HBA, 1:1000, ab92492, Abcam, USA), rabbit anti-GAPDH (1:10000, Abcam, USA). The PVDF membrane was washed three times with TBST and then incubated with an enzyme-labeled anti-rabbit secondary antibody (1:50000, HA1001 HUABIO, China) at room temperature for 1 h. Finally, the bands were visualized using a super-sensitive ECL chemiluminescence kit (Beyotime, Shanghai), and the bands were quantitatively analyzed using Image J software (USANIH).

[0031] Immunofluorescence staining: Mouse brain tissue paraffin sections were dewaxed, rehydrated, and then antigen retrieval was performed in a modified sodium citrate antigen retrieval solution (Beyotime, Shanghai, China). Subsequently, the sections were incubated with hydrogen peroxide at room temperature for 15 minutes to block endogenous peroxidase activity, then incubated with Triton X-100 for 15 minutes to permeabilize the membrane, and then blocked with 5% BSA for 1 h. Next, the sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies were: rabbit anti-LIAS (1:1000, ab246917, Abcam, USA), goat anti-IBA-1 (1:1000, ab289874, Abcam, USA), mouse anti-NEUN (1:50, HA601111, HUABIO, China), and mouse anti-GFAP (1:50, EM140707, HUABIO, China). After that, the slides were rinsed 3 times with PBS and then incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 h. The secondary antibodies were: anti-goat iFluor TM488 (1:1000, HA1131, HUABIO, China), anti-mouse iFluor TM 488 (1:5000, HA1125, HUABIO, China), and anti-rabbit iFluor TM 594 (1:5000, HA1122, HUABIO, China). Finally, an anti-fluorescence quenching blocking solution containing DAPI (Beyotime, Shanghai, China) was added dropwise to block the glass slides. Images were obtained by an independent observer under a fluorescence microscope (AXIO observer 3, Zeiss, Germany).

[0032] Statistical analysis: All statistical analyses were performed using Prism 6.0 (GraphPad Software, USA) and MedCalc version 19.0.4 (Broekstraat 529030, Mariakerke, Belgium). All data were expressed as mean ± standard deviation. Before analysis, the Kolmogorov-Smirnov test was used to test the normal distribution of each dataset. The Mann-Whitney U and / or Student's t-tests were used to evaluate the differences between two groups. The Kruskal-Wallis test or one-way analysis of variance was used for multiple comparisons of data with more than two groups. The Spearman correlation coefficient analysis was used to evaluate the correlation between the LIAS level and the WFNS score. A P < 0.05 was considered statistically significant.

[0033] Example 1

[0034] Preparation and characterization of RVG-RBCEVs

[0035] (1) Isolation and identification of sEVs

[0036] Blood samples of type O were obtained from Wuhu Central Blood Station. The isolated red blood cells were diluted with physiological saline. To isolate sEVs, the red blood cells and cell debris were centrifuged at 3000×g for 15 min at 4°C; the supernatant was passed through a 0.22-μm filter membrane; then, the supernatant was centrifuged at 10,000×g for 30 min at 4°C; then centrifuged at 100,000×g for 4 h at 4°C; the supernatant was discarded, and the bottom precipitate of RBCEVs (red blood cell-derived extracellular vesicles) was collected. Subsequently, the RBCEV precipitate was resuspended with PBS and RIPA lysis buffer.

[0037] The Hitachi HT7700 transmission electron microscope (TEM; Hitachi, Japan) was used. The RBCEVs samples were fixed on copper grid plates with 2% formaldehyde. Subsequently, they were washed three times in deionized water, uranyl acetate, and lead citrate, and then dropped onto copper plates for chemical staining. Finally, the grids were examined using TEM. The Zetaview-PMX120 instrument (Particle Metrix GmbH, Ammersee, Bavaria, Germany) was used to quantify the particle size distribution of RBCEVs. Western blot was used to detect the RBCEVs markers.

[0038] (2) Coupling of RVG29-azide with RBCEVs

[0039] RVG29-azide (RVG29-N3) was synthesized by GL Biochem (Shanghai) Ltd. After reacting 2 μM DBCO-sulfo-NHS (No. 762040, Sigma, St. Louis, MO, USA) with 0.5 mg / mL RBCEVs at room temperature (25 °C) and pH 7.4 for 4 h, an ultrafiltration column (100 KDa, Millipore) was used to wash and centrifuge at 7500×g to purify the unreacted DBCO-sulfo-NHS, obtaining DBCO-conjugated RBCEVs (DBCO-RBCEVs). Subsequently, DBCO-RBCEVs and RVG29-azide underwent a copper-free azide click chemical reaction. DBCO-RBCEVs (0.4 mg / mL), RVG29-azide (0.6 mg / mL), and sulfo-cy5.5-azide (0.4 μM, B7330, Lumiprobe Co., Hallandale Beach, FL, USA) were reacted at 4 °C and pH 7.4 for 12 h to generate RVG-RBCEVs.

[0040] After the reaction was completed, the solution containing RVG-RBCEVs was centrifuged at 7500×g in PBS, and continuously concentrated three times using a 100K ultrafiltration column. The cy5.5-conjugated RVG-RBCEVs (1×10 8 particles / mL) were stained with DIO lipophilic dye and incubated at room temperature for 10 min. They were continuously centrifuged three times at 7500×g through a 100K ultrafiltration column to remove the unreacted dye. Since RVG-RBCEVs were stained red by cy5.5 and green by DIO, fluorescence microscopy imaging could be used to evaluate whether the coupling reaction of RVG29 with RBCEVs was successful.

[0041] The preparation of the engineered vesicles RVG-RBCEVs was divided into two parts (Figure 1 A). First, water-soluble DBCO-sulfo-NHS forms a covalent bond with the surface proteins or phosphatidylethanolamine amino groups of RBCEVs. Next, DBCO-conjugated RBCEVs and RVG9-azide form a stable triazole bond through a copper-free click chemical reaction. Meanwhile, Cy5.5 azide is also conjugated to the DBCO group to achieve the tracking of RBCEVs.

[0042] The morphologies of unmodified RBCEVs and RVG-RBCEVs were observed using transmission electron microscopy (TEM). Figure 1 B). Then, nanoparticle tracking analysis (NTA) was used to evaluate the size distributions of unmodified RBCEVs and RVG-RBCEVs. The results showed that the average diameter of unmodified RBCEVs was about 86 nm, and the average diameter of RVG-RBCEVs increased to 134 nm. Figure 1 C). Western blot analysis showed that CD63, TSG101, ALIX, and the erythrocyte marker protein hemoglobin A (HBA) were all expressed in purified RBCEVs. Figure 1 D). To verify the successful synthesis of RVG-RBCEVs, under a fluorescence microscope, DIO-labeled RBCEVs were green, and cy5.5 conjugated to DBCO-RBCEVs was red. The overlap of the two colors confirmed the successful conjugation of RVG to the surface of RBCEVs. Figure 1 E).

[0043] Example 2

[0044] Loading siRNA of LIAS into rabies virus glycoprotein-modified red blood cell extracellular vesicles (RVG / RBCEVs).

[0045] (1) Synthesis of siRNA

[0046] According to the LIAS gene, siRNA that inhibits the expression of the LIAS gene was screened out, and its sequence is as follows:

[0047] Sense strand: 5’-CCGGAUAUUUGGAAGAUAUTT-3’;

[0048] Antisense strand: 5’-AUAUCUUCCAAAUAUCCGGTT-3’.

[0049] At the same time, a negative control siRNA was set up. It was synthesized by Shanghai GenePharma Co., Ltd.

[0050] (2) Preparation of RVG-RBCEVs / siRNA

[0051] RVG-RBCEVs with a total protein concentration of 20 μg (using BCAAssay kit, Beyotime, Shanghai, China) were mixed with 20 μl of Lias siRNA or negative control siRNA in 180 μl of nucleofection buffer (Cell Line nucleoectorkitV, Amaxa) and transferred to the nucleoector TM IIs / 2b device, and electroporation was performed under the conditions of 350 V and 150 μF. To remove unbound siRNA, RVG-RBCEVs were sequentially ultracentrifuged and washed in PBS (4 °C).

[0052] (3) Characterization of RVG-RBCEVs / siRNA and its targeting ability

[0053] Lias siRNA was loaded into unmodified RBCEVs and RVG-RBCEVs by electroporation ( Figure 2 A). Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology and size distribution of unmodified RBCEVs / siRNA and RVG-RBCEVs / siRNA ( Figure 2 B, C). To evaluate the encapsulation effect of RVG-RBCEVs on siRNA, the concentrations of Lias siRNA or NC control that unloaded siRNA oligomers after electroporation were detected using a NanoDrop ND-2000 spectrophotometer. Compared with the total siRNA solution, the concentrations of LiassiRNA or NC control in the supernatant of unbound siRNA were significantly reduced ( Figure 2 D).

[0054] To identify the targeting ability of RVG-RBCEVs / siRNA to the SAH brain, cy5.5-labeled RBCEVs, RBCEVs / siRNA, or RVG-RBCEVs / siRNA were intravenously injected into mice with SAH for 2 hours. After 24 hours, the brain tissues were dissected for NIRF imaging analysis ( Figure 2 E). After administration of RVG-RBCEVs / siRNA, the fluorescence intensity at the bottom surface of the temporal lobe was significantly higher than that of unmodified RBCEVs and RBCEV / siRNAs. These findings indicate that RVG-RBCEVs / siRNA increases the brain targeting of RBCEVs and can stay in the SAH bleeding area for a longer time ( Figure 2 F).

[0055] For the organ distribution of RBCEVs, the main organs (lung, heart, spleen, kidney, brain, liver) were dissected and subjected to NIRF imaging analysis ( Figure 2G, H). Compared with the injection of unmodified RBCEVs and RBCEVs / siRNA, the RVG-RBCEVs / siRNA injection group had stronger fluorescence signals in the brain. These results are consistent with the previous research results on RVG-modified exosomes. In addition, the targeting ability of RVG-RBCEVs / siRNA to neurons was studied by immunofluorescence staining of brain tissue, and the signal of RVG-RBCEVs / siRNA was significantly increased at the lesion site ( Figure 2 I).

[0056] In summary, the present invention successfully prepared RVG / RBCEVs and verified their ability to deliver nucleic acid drugs with brain targeting, showing good application prospects in central nervous system diseases.

[0057] Although the present invention has been described in detail with general descriptions and specific examples above, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for preparing brain-targeted extracellular vesicles, characterized in that: The method comprises the following steps: (1) Blood is centrifuged to obtain red blood cell sediment, which is then subjected to gradient centrifugation to obtain red blood cell-derived extracellular vesicles, i.e., RBCEVs; (2) RBCEVs undergo coupling reaction with DBCO-sulfo-NHS to obtain DBCO-conjugated RBCEVs, namely DBCO-RBCEVs; (3) DBCO-RBCEVs and RVG29-N3 undergo copper-free azide click chemistry reaction to obtain RVG-RBCEVs, i.e., the brain-targeted extracellular vesicles.

2. The method for preparing brain-targeted extracellular vesicles according to claim 1, characterized in that: In step (1), the gradient centrifugation includes: centrifugation at 3000×g for 15 min at 4°C, passing the supernatant through a 0.22 μm filter membrane, and then centrifuging at 10,000×g for 30 min and 100,000×g for 4 h at 4°C, discarding the supernatant, and collecting the RBCEVs precipitated at the bottom.

3. The method for preparing brain-targeted extracellular vesicles according to claim 1, characterized in that: In step (2), The concentration of the RBCEVs was 0.5 mg / mL, and the concentration of DBCO-sulfo-NHS was 2 μM; The coupling reaction conditions are: pH 7-8, 20-30°C, 2-5h.

4. The method for preparing brain-targeted extracellular vesicles according to claim 1, characterized in that: In step (3), The concentration of DBCO-RBCEVs is 0.4 mg / mL, and the concentration of RVG29-N3 is 0.6 mg / mL; The conditions of the copper-free azide click chemistry reaction are: pH 7-8, 1-5° C., 10-15 h.

5. The method for preparing brain-targeted extracellular vesicles according to claim 1, characterized in that: The method further comprises: The reaction solution of the copper-azide-free click chemistry reaction was placed in PBS, centrifuged at 7500×g, and then concentrated using an ultrafiltration column with a molecular weight cutoff of 100 KDa.

6. A brain-targeted extracellular vesicle, characterized in that: The invention is prepared by the preparation method described in any one of claims 1 to 5.

7. Use of the brain-targeted extracellular vesicles according to claim 6 as a delivery system in the preparation of drugs for treating central nervous system diseases.

8. The use according to claim 7, characterized in that: The drugs include siRNA, miRNA, mRNA, viruses, and Cas9 gene editing tools.

9. The use according to claim 7, characterized in that: The central nervous system disease includes subarachnoid hemorrhage.

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