Bone-targeted hybrid extracellular vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a bone-targeted hybrid extracellular vesicle as well as a preparation method and application thereof. Probiotics are modified through a synthetic biological technology, so that the exovesicles secreted by the probiotics carry hCXCR4 and ClyA genes to realize bone targeting, meanwhile, the Yam exovesicles are rich in diosgenin and can promote differentiation and mineralization of osteoblasts, and the two types of vesicles are hybridized to prepare the bone targeting'plant-bacterium 'hybridized extracellular vesicles. Animal experiments prove that the bone-targeted extracellular vesicles can effectively regulate bone metabolism and remarkably improve osteoporosis symptoms represented by ovarian-removed female mice. The invention provides a novel, safe and effective biomedical material for treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and specifically provides a bone-targeted hybrid extracellular vesicle and a preparation method and application thereof. Background Art

[0002] Osteoporosis (OP) is a common bone metabolic disease characterized by decreased bone density and destruction of bone microstructure, which leads to increased bone brittleness and increased risk of fractures. Traditional treatments mainly rely on medications, such as bisphosphonates and estrogen, but these drugs have certain side effects and may cause patient dependence. Therefore, there is an urgent need to develop a new type of targeted therapy that can effectively promote bone formation while reducing side effects.

[0003] Extracellular vesicles (EVs) have excellent properties such as good biocompatibility, unique nanostructure and stable drug delivery capacity, and are therefore regarded as a promising nano-drug delivery carrier. In previous studies, extracellular vesicles (MEVs) derived from natural and engineered mammals have been developed for targeted delivery of miRNA to treat OP. However, the low extraction efficiency and complex engineering process of MEVs limit their further clinical application. In recent years, bacterial-derived extracellular vesicles (BEVs) have attracted widespread attention due to their rapid reproduction ability, mature high-density culture technology and rich synthetic biology tools. It is widely recognized that intestinal microorganisms play an indispensable role in the pathogenesis of OP. In general, intestinal microorganisms can be divided into probiotics, harmful bacteria and neutral bacteria. Among them, a variety of probiotics such as Akkermansia muciniphila (AKK), Escherichia coli (E. coli) Nissle 1917 and Lactobacillus rhamnosus GG (LGG) are particularly important in this process. Proteus mirabilis (PM)-derived EVs can inhibit osteoclasts, thereby reducing bone loss in ovariectomized (OVX) mice. By modifying the surface of probiotics using synthetic biology techniques, they can overexpress bone tissue receptor genes during the fusion process, thereby giving BEVs bone-targeting properties. In addition, BEVs were further modified to enhance their targeting and ability to promote osteogenesis.

[0004] Plant extracellular vesicles (PEVs) contain a variety of components, including DNA, RNA, cytoplasmic proteins, peptidoglycan, and various enzymes. These PEVs act as extracellular messengers, stimulating intercellular communication and biological defense against pathological diseases. Recently, PEVs have been widely explored as drug delivery systems in various therapies by isolating a variety of plant sources (including starchy roots and tubers, nuts and seeds, and fresh and dried plants). Because they are not only safe, biocompatible, and biodegradable, without any negative impact on intestinal barrier function or other organ toxicity, but also can be prepared in large quantities. PEVs are also rich in a variety of active ingredients, such as growth factors, antioxidants, and polyphenols, which help promote the proliferation and differentiation of osteocytes. PEVs can not only promote osteogenesis, but also may have multiple functions such as anti-inflammatory and antioxidant, and have a comprehensive therapeutic effect on the treatment of osteoporosis. PEVs are relatively easy to obtain and have low production costs, making them suitable for large-scale application. Studies have shown that 8-shogaol in ginger targets TAK1 to relieve rheumatoid arthritis. The miRNA and mRNA of Pueraria lobata extracellular vesicles also significantly promoted the osteogenic differentiation of hBMSCs. Yam extracellular vesicles (YEVs) activated the differentiation and proliferation of osteoblasts through the BMP-2 / p-p38-dependent Runx2 pathway. Therefore, YEVs are a potentially useful therapeutic agent for the treatment of OP with excellent biocompatibility and safety.

[0005] EVs have unique advantages in treating OP. We hybridized YEVs with engineered E. coli EVs. Through synthetic biology methods, we endowed E. coli EVs with targeting and combined the characteristics of PEVs to enhance their ability to promote osteogenesis, thereby customizing a bone-targeted "plant-bacteria" hybrid extracellular vesicle with anti-osteoporosis function to alleviate OP in OVX mice. This customized hybrid vesicle can be delivered to the bone microenvironment, thereby enhancing osteogenic activity and ultimately alleviating the OVX-induced OP phenotype. In summary, this engineered hybrid vesicle provides a promising, safe and effective therapeutic strategy for the treatment of refractory OP. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides a hybrid extracellular vesicle with good biocompatibility and bone targeting. Specifically, bacteria, Chinese yam and its outer membrane vesicles (OMVs) are used to prepare bone-targeted "plant-bacteria" hybrid extracellular vesicles that can be used for the treatment of osteoporosis. Experiments show that the hybrid extracellular vesicles provided by the present invention show obvious advantages in cell internalization and targeted treatment of orthopedic diseases. In addition, the preparation method of the hybrid extracellular vesicle is economical and environmentally friendly, can be industrialized and mass-produced, and is easy to obtain. It provides a new way to prevent and treat osteoporosis.

[0007] The purpose of the present invention can be specifically achieved through the following technical solutions: In a first aspect, the present invention provides a bone-targeted bacterial extracellular vesicle, wherein the structure of the bone-targeted bacterial extracellular vesicle is a double-layer phospholipid layer vesicle.

[0008] Furthermore, the particle size of the bone-targeted bacterial extracellular vesicles is 20-2000 nm, and further, preferably 60-100 nm.

[0009] Furthermore, the bacteria of the bacterial extracellular vesicles are selected from Escherichia coli Nissle 1917, LGG, AKK edible probiotics; preferably Escherichia coli Nissle 1917.

[0010] In a second aspect, the present invention provides a bone-targeted "plant-bacteria" hybrid extracellular vesicle, wherein the hybrid extracellular vesicle comprises bacterial extracellular vesicles and yam extracellular vesicles.

[0011] Furthermore, the hybrid extracellular vesicles are prepared by the following process: after NTA detection of bacterial extracellular vesicles and yam extracellular vesicles, the two vesicles are diluted to a suitable concentration (usually 10 6 ~10 13 ), and then the two vesicles are mixed in equal volumes and subjected to ultrasonic treatment, preferably for 10 to 30 minutes.

[0012] Furthermore, in the preparation process, the particle number ratio of bacterial extracellular vesicles to yam extracellular vesicles is 1:1, the preferred concentration is 10*10 / ml, and the ultrasound is protected from light, and the time is preferably 20 minutes.

[0013] The third aspect of the present invention provides a method for preparing the bone-targeted bacterial extracellular vesicles, wherein the hCXCR4 and ClyA genes are fused to the surface of the probiotic membrane by biosynthesis technology to complete the strain construction, and then the bacteria are cultured by bacterial fermentation and the bacterial extracellular vesicles are collected.

[0014] Furthermore, the bacterial culture process includes the following operations: first, the bacteria are cultured at 37°C, 5% CO2 and 220 rpm for 12 to 72 hours, and then transferred to secondary culture, and continue to grow at 37°C for 6 to 24 hours, while low-temperature protein expression is performed at 16°C to complete the bacterial culture. Next, the culture fluid is collected by low-speed centrifugation at 100 to 200 g to obtain a bacterial fermentation fluid that efficiently expresses exogenous CXCR4.

[0015] Furthermore, 50 mg / L kanamycin may be added during the above-mentioned bone-targeted bacterial culture.

[0016] Furthermore, the BEVs collection process includes the following operations: the fermentation broth is first centrifuged at a low speed of 1000~13000g, and the centrifugation time can be selected to be 5~50min. To further precipitate the bacteria, the above process can be repeated 2~3 times; the supernatant after centrifugation is filtered with a 0.22μm sterile filter to obtain a bacterial solution to remove residual bacteria, and the bacterial solution is collected by ultracentrifugation. An ultrafiltration membrane (usually 50 KDa~100 KDa) is required to concentrate bacterial extracellular vesicles and remove non-bacterial extracellular vesicle-related proteins. The speed of high-speed centrifugation is 50000~200000g, and the time is 60~120min. To obtain a high-purity extracellular vesicle suspension, centrifugation can be repeated 2~4 times; the precipitate is collected and used immediately or stored at -80℃.

[0017] The fourth aspect of the present invention provides a method for preparing the yam extracellular vesicles, the specific process comprising: peeling and squeezing the fresh hairy yam, filtering the juice with gauze, and then subjecting the filtrate to differential centrifugation to obtain the yam extracellular vesicles.

[0018] Furthermore, since the yam filtrate is too viscous, the filtrate can be diluted with PBS to control the volume ratio of fresh yam filtrate to PBS to 2-10:1 before subsequent centrifugation operation; preferably 3:1.

[0019] Furthermore, the differential centrifugation step includes: centrifuging the filtrate at 500-1000g for 10-20 minutes at 4°C to collect a first supernatant; then, centrifuging the first supernatant at 2000-6000g for 20-40 minutes at 4°C to collect a second supernatant; then, centrifuging the second supernatant at 8000-12000g for 30-60 minutes at 4°C to collect a third supernatant, and the third centrifugation process can be repeated once to obtain a fourth supernatant; finally, centrifuging the fourth supernatant at 120000-150000g for 90-120 minutes at 4°C to collect the bottom precipitate, and the fourth centrifugation process can be repeated 2-4 times, and the collected precipitate can be used immediately or stored at low temperature to complete the differential centrifugation operation.

[0020] The fifth aspect of the present invention provides a use of the bone-targeted "plant-bacteria" hybrid extracellular vesicles or the bone-targeted bacterial extracellular vesicles in the preparation of biomedical materials for treating osteoporosis.

[0021] Compared with the prior art, the present invention has the following effects: First, the present invention provides a bone-targeted bacterial extracellular vesicle, and then uses the engineered bacterial extracellular vesicles and yam extracellular vesicles to fuse to obtain bone-targeted "plant-bacteria" hybrid extracellular vesicles, which can not only give the hybrid extracellular vesicles bone targeting, but also make the hybrid extracellular vesicles have the characteristics of promoting osteoblast differentiation.

[0022] Secondly, the preparation method provided by the present invention is simple in process, easy to operate, and easy to carry out industrial production. The hybrid extracellular vesicles obtained by the construction method disclosed in the present invention, as an innovative biomedical material, can be widely used in the fields of drug delivery, bioimaging, disease prevention and diagnosis, etc. With the continuous maturity and optimization of related technologies, it is expected to bring more possibilities and breakthroughs to clinical treatment.

[0023] Finally, the hybrid extracellular vesicles provided by the present invention have good targeting in mice, increase the amount of extracellular vesicles in bone tissue, efficiently promote osteogenic differentiation and mineralized nodule production, and promote the expression of osteogenic differentiation genes such as ALP, OCN, RUNX2 and COL1, which is a strategy for targeted treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 . Transmission electron microscopy image and nanoparticle tracking analysis image of bacterial extracellular vesicles modified with CXCR4 in an embodiment of the present invention.

[0025] Figure 2 . Transmission electron microscopy image of extracellular vesicles of yam and nanoparticle tracking analysis image in an embodiment of the present invention.

[0026] Figure 3 . Fluorescence analysis diagram of hybridization of bacterial exovesicles and yam exovesicles in an embodiment of the present invention.

[0027] Figure 4 . Statistical graph of cell proliferation after co-culture of hybrid extracellular vesicles and bone marrow mesenchymal stem cells in an embodiment of the present invention.

[0028] Figure 5 . ALP activity diagram of hybrid extracellular vesicles in an embodiment of the present invention.

[0029] Figure 6 . Fluorescence distribution diagram of major organs and femur of mice 4h, 8h, and 16h after tail vein injection of Cy5-labeled hybrid extracellular vesicles in the example of the present invention. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0031] Example 1 Preparation of bone-targeted bacterial extracellular vesicles Strain construction: pCla-CXCR4 was constructed using a one-step cloning kit and pCla-CXCR4 was transferred and replicated to the membrane surface of E. coli Nissle 1917 to obtain engineered ECN-CXCR4 for the production of BEV-CXCR4.

[0032] Bacterial culture: E. coli Nissle 1917 and recombinant strains were cultured in LB medium containing 5 g / L yeast extract, 10 g / L tryptophan and 10 g / L NaCl. Primary culture and secondary culture were carried out at 220 rpm, 5% CO2 and 37 °C for 12 h. After 8 h, 1 mL of the secondary culture was inoculated into 50 mL LB for batch culture. The two-step temperature method was used for efficient expression of exogenous CXCR4 under the conditions of growth at 37 °C and protein expression at 16 °C, and 50 mg / L kanamycin was added.

[0033] Extraction and characterization of bacterial extracellular vesicles: First, E. coli Nissle 1917 was centrifuged from the fermentation broth at low speed (12000g, 30min, twice), and then the supernatant after centrifugation was filtered with a 0.22μm sterile filter and a 100KDa ultrafiltration membrane. The filtrate was subjected to ultracentrifugation (150000g, 90min) to collect the bacterial extracellular vesicles. The obtained extracellular vesicles can be purified again by ultracentrifugation (150000g, 90min), and the final collected extracellular vesicles can be stored at -80°C. The morphology of bacterial extracellular vesicles was observed by transmission electron microscopy (TEM). The morphology of BEV-CXCR4 and the nanoparticle tracking analysis are shown in Figure 2. Figure 1 As shown in the figure, the particle size of the exosomes modified with CXCR4 is about 100nm, showing a bilayer membrane structure. The particle size is mainly concentrated between 50 and 300nm, and the exosomes of about 100nm are the most.

[0034] Extraction and characterization of yam extracellular vesicles: Peel and squeeze the fresh hairy yam, filter it with gauze, and obtain the filtrate. Since the yam filtrate is too viscous, the filtrate can be diluted 3 times with PBS and then subjected to differential centrifugation. The diluted filtrate was centrifuged at 500g for 10 minutes at 4°C to collect the first supernatant; then, the first supernatant was centrifuged at 2000g for 20 minutes at 4°C to collect the second supernatant; then, the second supernatant was centrifuged at 10000g for 30 minutes at 4°C to collect the third supernatant, and the fourth supernatant was obtained by repeating the process; finally, the fourth supernatant was centrifuged at 150000g for 90 minutes at 4°C to collect the bottom precipitate, and the obtained extracellular vesicles can be purified by centrifugation at 150000g for 90 minutes at 4°C again, and the precipitate was collected and used immediately or stored at -80°C. The morphology of yam extracellular vesicles was observed by transmission electron microscopy (TEM). The morphology of yam extracellular vesicles and nanoparticle tracking analysis are shown in the figure. Figure 2 As shown in the figure, the particle size of the extracellular vesicles of yam cells is about 100nm, showing a bilayer membrane structure. The particle size is mainly concentrated between 50 and 300nm, and the extracellular vesicles around 100nm are the most.

[0035] Preparation of hybrid extracellular vesicles: After the particle size analysis of the bacterial extracellular vesicles and yam extracellular vesicles, the concentration of 10*10 / ml was selected and mixed in equal volumes, wrapped in tin foil and ultrasonically treated for 20 minutes, the yam extracellular vesicles were stained with Dio, and the bacterial extracellular vesicles were stained with Dil dye, and then the hybridization effect was observed under a fluorescence microscope. Figure 3 As shown, there is a good hybridization effect under the microscope.

[0036] Example 2 Cytotoxicity Test Cytotoxicity test: In a 96-well plate, bone marrow mesenchymal stem cells were plated at a density of 2000 cells per well, and the cells adhered to the wall after one day of culture. On the second day, bacterial extracellular vesicles, yam extracellular vesicles and hybrid extracellular vesicles were added to the 96-well plate at a concentration of 10*10 / ml; on the third day, 10μL CCK-8 solution was added to each well, mixed thoroughly without bubbles, and incubated in a 37℃ incubator for 1h. The OD value at 450nm was detected with an enzyme labeling instrument to evaluate cell viability. The statistical graph of cell proliferation is shown in the figure below. Figure 4 As shown, in different types of extracellular vesicle suspensions, the proliferation rate of co-cultured cells was greater than 85%, indicating that the hybrid extracellular vesicles were not toxic to cells.

[0037] Example 3 Evaluation of in vitro osteogenic function In vitro osteogenic function evaluation: 8×10 3Bone marrow mesenchymal stem cells at a concentration of 100 μg / mL were inoculated into the culture medium and cultured in 24-well plates with α-MEM (10% fetal bovine serum + 1% penicillin and streptomycin). After the cells adhered to the wall, the culture medium was changed to osteogenic induction medium (DMEM-HG + 10% fetal bovine serum + 1% penicillin and streptomycin + 10mmol / L sodium B-glycerophosphate + 50ug / mL ascorbic acid + 10nmol / L dexamethasone). The culture medium was changed every 2 days. After 5 days of culture, the ALP content in the cells was detected using an alkaline phosphatase (ALP) detection kit, and the active area statistics were calculated as shown in the figure below. Figure 5 As shown, the hybrid extracellular vesicles had the highest initial osteogenic activity, while the cells in the blank group showed poor osteogenic activity.

[0038] Example 4 In vivo targeting tracking Eight-week-old C57BL / 6 female mice weighing 20-22 g were purchased from Slack Laboratory Animal Company. They were allowed free access to food and water to maintain a circadian rhythm.

[0039] 100 μl of bone-targeted hybrid extracellular vesicles were mixed with 100 μl of 1 μg / ml cy5, kept in a dark place at 4°C for 12 hours, and then ultracentrifuged at 100,000 g for 60 minutes to remove excess dye. The mixture was injected into mice using the tail vein injection method. The heart, liver, spleen, lung, kidney and femur were removed 4h, 8h and 16h after the injection, and the distribution of vesicles in various organs was photographed under imaging equipment. The results are shown in Figure 2. Figure 6 As shown, both BEVs and YBEVs groups had a good bone targeting.

Claims

1. A bone-targeted bacterial extracellular vesicle, characterized in that: The structure of the bone-targeting bacterial extracellular vesicle is a double-layer phospholipid layer vesicle.

2. The bacterial extracellular vesicle according to claim 1, characterized in that The particle size of the bone-targeted bacterial extracellular vesicles is 20-2000 nm.

3. The bacterial extracellular vesicle according to claim 1, characterized in that The bacteria of the bacterial extracellular vesicles are selected from Escherichia coli Nissle 1917, LGG, and AKK edible probiotics.

4. A bone-targeted "plant-bacteria" hybrid extracellular vesicle, characterized in that: The hybrid extracellular vesicles include bacterial extracellular vesicles and yam extracellular vesicles.

5. The hybrid extracellular vesicle according to claim 4, characterized in that The hybrid extracellular vesicles are prepared by the following process: after NTA detection of bacterial extracellular vesicles and yam extracellular vesicles, the two vesicles are diluted to a suitable concentration, usually 10 6 ~10 13 Then, equal volumes of the two vesicles were mixed and sonicated for 10 to 30 minutes.

6. The hybrid extracellular vesicle according to claim 5, characterized in that In the preparation process, the particle number ratio of bacterial extracellular vesicles to yam extracellular vesicles is 1:1, the concentration is 10*10 / ml, and the ultrasound is performed in a dark environment for 20 minutes.

7. A method for preparing the bone-targeted bacterial extracellular vesicles according to claim 1 or claim 4, characterized in that: The hCXCR4 and ClyA genes were fused to the surface of the probiotic membrane through biosynthesis technology to complete the strain construction, and then the bacteria were cultured and the bacterial extracellular vesicles were collected by bacterial fermentation.

8. A method for preparing the yam extracellular vesicles according to claim 4, characterized in that: The specific process includes: peeling and squeezing the fresh hairy yam, filtering the juice with gauze, and then subjecting the filtrate to differential centrifugation to obtain the yam extracellular vesicles.

9. Use of the bone-targeted bacterial extracellular vesicles according to claim 1 in preparing biomedical materials for treating osteoporosis.

10. Use of the bone-targeted "plant-bacteria" hybrid extracellular vesicles according to claim 4 in the preparation of biomedical materials for treating osteoporosis.

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