CN116162597A - Engineered extracellular vesicles loaded with Wnt proteins and uses thereof - Google Patents

Abstract

An engineered extracellular vesicle loaded with Wnt protein and its application. The extracellular vesicle loaded with Wnt protein is isolated from cells, and the extracellular vesicle can be applied to the preparation of medicines for tissue repair and regeneration.

Description

Engineered extracellular vesicle loaded with Wnt protein and its application

technical field

The invention relates to the technical field of biomedicine, in particular to engineered extracellular vesicles loaded with Wnt proteins and applications thereof.

technical background

Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by persistent airflow limitation, with high morbidity and mortality. According to the statistics of the World Health Organization, COPD is the third leading cause of death in the world and the fifth in the world's economic burden of disease. Long-term inflammatory damage from chronic bronchitis and emphysema is the most common cause of COPD. Normal lungs have endogenous regeneration potential. When lung tissue is damaged, resting alveolar stem cells can rapidly proliferate and differentiate into type I alveolar epithelial cells, replace and replenish damaged and dead cells, and maintain the integrity of the alveolar barrier structure, thus Improvement in lung function after injury is achieved. However, the endogenous regeneration mechanisms of the lungs of COPD patients are severely impaired, making it difficult to initiate repair and regeneration processes spontaneously.

Further studies have found that the repair and regeneration mechanisms mediated by the Wnt signaling pathway in alveolar epithelial cells play a key role in the occurrence and development of COPD. In recent years, treating COPD by activating the Wnt signaling pathway has become a research hotspot in this field. However, CHIR99021, LiCl and other small molecule drugs that activate Wnt signaling pathway have strong toxic and side effects, and the hydrophobicity of Wnt protein makes it difficult to make drugs, which limits the clinical transformation. Although some researchers are currently trying to develop Wnt receptor agonists, their clinical effects have yet to be further verified. Therefore, it is still necessary to develop a feasible new method that can promote the repair and regeneration of tissues, especially damaged lungs, so as to provide more possibilities for clinical transformation.

Contents of the invention

In view of this, one of the main purposes of the present invention is to propose an engineered extracellular vesicle loaded with Wnt protein and its application, so as to transport biologically active Wnt protein to tissue cells, and promote tissue regeneration by activating the Wnt signaling pathway. Cellular repair and regeneration.

In order to achieve the above purpose, as one aspect of the present invention, an engineered extracellular vesicle loaded with Wnt protein is provided.

As another aspect of the present invention, the application of V kinds of engineered extracellular vesicles as described above in the preparation of drugs for tissue repair and regeneration is provided.

As yet another aspect of the present invention, a use of the above-mentioned engineered extracellular vesicles in the preparation of a medicament for treating chronic obstructive pulmonary disease is provided.

Based on the above technical scheme, it can be seen that the engineered extracellular vesicle loaded with Wnt protein and its application of the present invention have one or part of the following beneficial effects:

The engineered extracellular vesicles loaded with Wnt protein provided by the present invention have biological activity, and can exert a therapeutic effect on damaged tissues such as damaged lungs by activating the endogenous regeneration mechanism mediated by the Wnt signaling pathway, and The engineered extracellular vesicle has good biocompatibility, which is of great significance for clinical transformation and application research.

Description of drawings

Fig. 1 is a transmission electron micrograph of engineered extracellular vesicles loaded with Wnt3a protein in Example 1 of the present invention.

Fig. 2 is the nanoparticle analysis result of the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Figure 3 is the Western blot identification results of marker proteins in engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Fig. 4 is the Western blot identification result of Wnt3a in the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Fig. 5 is the result of the TOPFLASH reporter test of HEK293T cells in Example 1 of the present invention.

Fig. 6 is the analysis result of Annexin-V in the in vitro repair function test of the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Fig. 7 is the result of immunostaining analysis in the in vitro repair function test of the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Fig. 8 is the mouse model test results of the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Figure 9 shows the in vivo safety results of the engineered extracellular vesicles loaded with Wnt3a in Example 1 of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

The invention provides an engineered extracellular vesicle loaded with Wnt protein and application thereof. In the process of realizing the present invention, it was found that by co-transfecting the two constructed plasmids in model cells, extracellular vesicles loaded with Wnt3a protein could be successfully isolated, and the extracellular vesicles were tested by in vitro models and mouse models The functional verification test verified that it can achieve the effect of proliferating lung tissue cells by activating the Wnt signaling pathway, indicating that extracellular vesicles loaded with Wnt protein can promote tissue repair and regeneration based on the activation of the Wnt signaling pathway. It has application potential in related diseases, especially in the treatment of chronic obstructive pulmonary disease.

definition:

As used herein, the term "extracellular vesicles" (Extracellular Vesicles, EVs) is a membrane vesicle structure secreted by cells with a diameter of 30-200nm (exosomes) or <1000nm (microvesicles, microvesicles), containing Abundant inclusions (including proteins, lipids, nucleic acids, etc.), participate in the signal transmission between cells. In recent years, as an important carrier of cell communication, extracellular vesicles have shown great prospects in the fields of diagnosis and detection, immunotherapy, nucleic acid drugs, protein, and small molecule delivery therapy by participating in normal physiological and pathological processes. Due to the natural generation properties of cells, extracellular vesicles have good biocompatibility and extremely low immunogenicity and toxicity. At the same time, extracellular vesicles are also natural nanocarriers, which can be loaded with functional proteins, RNA, chemical drugs, etc. The surface of extracellular vesicles is rich in membrane proteins, which determines its excellent characteristics of recognizing target cells. It can also be endowed with cell and tissue targeting specificity by modifying the surface molecules of extracellular vesicles, and further make extracellular vesicles The loaded effective molecules are delivered to specific pathological tissues and organs. Combined with the advantages of the above extracellular vesicles, they have attracted extensive attention as potential therapeutic agents in tissue repair and regeneration. At present, the technology based on extracellular vesicles is still in the early stage of clinical translation and application research, facing many challenges but also accompanied by the possibility of numerous new discoveries and new technologies.

The term "Wnt signaling pathway" as used herein is a large family of secreted proteins that mediate developmental and postdevelopmental physiology by regulating cellular processes. There are two types of Wnt secretory proteins discovered so far, one is the canonical Wnt signaling pathway that relies on β-catenin (β-catenin), including Wnt1, Wnt2, Wnt3 and Wnt3a; the other is not dependent on β-catenin Non-canonical Wnt signaling pathway, including Wnt4a, etc.

Specifically, according to some embodiments of the present invention, there is provided an engineered extracellular vesicle loaded with Wnt protein.

According to an embodiment of the present invention, the extracellular vesicle loaded with Wnt protein can promote the repair and regeneration of tissue under the condition of activating the Wnt signaling pathway by exerting its biological activity.

According to an embodiment of the present invention, the above-mentioned Wnt protein is Wnt3a protein, which provides extracellular vesicles loaded with Wnt3a protein, but is not limited thereto, for example, it may also be other Wnt proteins such as Wnt4a protein.

According to the embodiments of the present invention, taking Wnt3a protein as an example, Wnt3a is the main ligand of the canonical Wnt signaling pathway, which can promote cell proliferation and inhibit cell apoptosis. Can exert its biological activity, so as to achieve the effect of promoting tissue repair and regeneration.

According to an embodiment of the present invention, the above-mentioned engineered extracellular vesicles are derived from HEK293T cells, that is, HEK293T is used as a model cell and an extracellular vesicle supply platform to obtain extracellular vesicles that efficiently carry Wnt3a and have biological activity .

According to an embodiment of the present invention, the plasmids required for the preparation of the above-mentioned engineered extracellular vesicles are derived from the design and construction of our laboratory, which are respectively Wnt3a-T2A-WLS plasmid and GPC6 ^ΔGPI -C1C2 plasmid having the sequence shown in SEQ ID NO.2. By designing and constructing plasmids corresponding to Wnt proteins, extracellular vesicles loaded with different Wnt proteins can be realized to target specific tissue cells to deliver Wnt proteins.

According to an embodiment of the present invention, the above-mentioned engineered extracellular vesicles are specifically prepared by the following preparation method: co-transfecting the Wnt3a-T2A-WLS plasmid and the GPC6 ^ΔGPI -C1C2 plasmid into HEK293T cells; after transfection, The above engineered extracellular vesicles were isolated from the culture supernatant of HEK293T cells.

According to an embodiment of the present invention, the above preparation method further includes: before transfection, seeding HEK293T cells in a 10 cm culture dish, and washing the cells with PBS when the cell confluence reaches 70%.

According to an embodiment of the present invention, the above-mentioned transfection adopts liposome method. For example, the above transfection operation includes: using Lipo2000 transfection reagent to co-transfect the above two plasmids into HEK293T cells.

According to an embodiment of the present invention, the above-mentioned separation operation includes: continuing to culture HEK293T cells after transfection, and collecting the culture supernatant of HEK293T cells; performing multiple centrifugations on the collected culture supernatant to obtain the above-mentioned engineered extracellular vesicles Bubble.

According to some embodiments of the present invention, there is also provided an application of the above-mentioned engineered extracellular vesicles in the preparation of drugs for tissue repair and regeneration, especially the application in the preparation of drugs for lung tissue repair and regeneration .

According to an embodiment of the present invention, a use of the above-mentioned engineered extracellular vesicles in the preparation of a drug for treating chronic obstructive pulmonary disease is also provided.

According to an embodiment of the present invention, the above-mentioned engineered extracellular vesicles can activate the endogenous regeneration mechanism mediated by the Wnt signaling pathway to promote tissue regeneration such as lung tissue repair and regeneration.

The technical solution of the present invention will be further elaborated below through specific embodiments in conjunction with the accompanying drawings. It should be noted that the following specific embodiments are only for illustration, and the protection scope of the present invention is not limited thereto. The medicines or reagents used in the following examples are all commercially available or self-made through known preparation methods. The methods used in the following examples, such as H&E staining and Western blot, are all methods well known in the art, and can be performed through descriptions in textbooks or related literature, and will not be repeated here.

Example 1

Isolation and characterization of Wnt3a-loaded extracellular vesicles.

1. Cell transfection and collection of culture supernatant.

The culture system of HEK293T cells is: DMEM basal medium + 10% FBS + 1X penicillin-streptomycin mixed solution. The cells were digested with 0.25% trypsin, and then the cells were seeded into 10em culture dishes and adhered overnight. Using Lipo2000 transfection reagent, transfect Wnt3a-T2A-WLS and GPC6 ^ΔGPI -C1C2 plasmids into HEK293T cells according to the operating instructions, and collect the culture supernatant at 24h, 48h and 72h after transfection, and store at -80°C Long-term preservation.

2. Isolation of extracellular vesicles.

Wnt3a ^WG EVs were isolated from the collected culture supernatant by ultracentrifugation. The detailed rotational speed and time of the ultracentrifugation method are as follows: Centrifuge the culture supernatant at 300g for 10min, collect the supernatant into a new centrifuge tube, and continue to use 2000g Centrifuge for 10 min, collect the supernatant into a new centrifuge tube, and continue centrifuging at 100,000 g for 70 min, discard the supernatant, add sufficient sterile PBS to the pellet, continue centrifuging at 100,000 g for 70 min to rinse extracellular vesicles, and finally The extracellular vesicles were resuspended in PBS and stored at -80°C for long-term storage.

Comparative example 1

Using a similar operation to Example 1, the difference is that the transfection operation of the two plasmids is not performed to obtain native extracellular vesicles (Native EVs), or only Wnt3a-T2A-WLS is transfected to obtain extracellular vesicles in the control group ( Control EVs).

Performance tests and results:

1. Identification of extracellular vesicles.

1) Use a transmission electron microscope to detect the isolated extracellular vesicles. As shown in Figure 1, B, the extracellular vesicles Wnt3a ^WG EVs isolated in this example all present a biconcave disc shape, and the size is 100nm Left and right size; similar to the shape of unmodified native extracellular vesicles (Native EVs) also derived from HEK293T cells in A, indicating that EVs loaded with Wnt3a protein will not significantly change their morphological characteristics.

2) Use a nanoparticle analyzer to detect the particle size distribution and particle concentration of extracellular vesicles. Figure 2 shows the particle concentration of Wnt3a ^WG EVs and Naive EVs under different particle size distributions. It can be seen that the collected extracellular vesicles The size distribution of Wnt3a ^WG EVs was in the range of 150-200nm.

3) The expressions of positive marker proteins (including TSG101 and CD63) and negative marker proteins (Calnexin) of donor cells (TCL) and extracellular vesicles were detected by Western blot (Western blot test, referred to as WB for short). As shown in Figure 3, the collected extracellular vesicles all expressed TSG101 and CD63, but not Calnexin, indicating that the extracellular vesicles were successfully prepared with high purity.

2. Biological activity verification

1) Wnt3a, Alix and β-actin contents in donor cells and extracellular vesicles were detected by WB assay. As shown in Figure 4, the unmodified donor cells (Native TCL) and Native EVs themselves do not contain Wnt3a, while the Wnt3a protein is expressed in the modified donor cells and extracellular vesicles, and the two plasmids are transfected at the same time. Compared with the transfection of Wnt3a-T2A-WLS plasmid alone, the amount of Wnt3a contained in the cell vesicles was significantly more than that of the control group, indicating that the simultaneous transfection of the two plasmids can effectively promote the efficient loading of Wnt3a, which is beneficial to biological Increased activity.

2) The cells transfected with TOPFlash luciferase and pRL-TK reporter plasmid were treated with EVs and corresponding conditioned medium (CM) obtained without transfection plasmid or co-transfection of the two plasmids for 48 hours, and then the fluorescence was detected Sulfase activity. As shown in Figure 5, it is not the ability to express Wnt3a protein to exert biological activity to activate the Wnt signaling pathway, while Wnt3a ^wG EVs transfected with two plasmids can significantly activate the Wnt signaling pathway.

3) In vitro repair function test: A549 cells were treated with LPS (50 μg/mL) for 2 days to construct a human alveolar epithelial cell injury model, and then LiCl (5 mM), rhWnt3a (200 ng/mL), Native EVs (2×10 ⁹ grains/mL) and Wnt3a ^WG EVs (2×109 grains/mL) for 2 days. Cells were collected for Annexin-V and immunostaining analysis. As shown in Figures 6 and 7, Wnt3a ^WG EVs can significantly increase cell proliferation and effectively inhibit cell apoptosis.

4) Verification of repair function in vivo: Elastase (100 U/kg) was infused into the mouse trachea to construct a mouse model of emphysema. On day 7, Native EVs (2×10 ⁹ grains/dose) and Wnt3a ^WG EVs (2×10 ⁹ grains/dose) were intravenously injected into mice, once every 2 days; LiCl (200 mg/dose) was injected intraperitoneally every day. kg) was considered as positive control treatment. Mice were sacrificed after 7 days of treatment for further analysis. As shown in Figure 8, the H&E stained lung tissue sections in Figure A and the mean linear intercept (MLI) measurements of the alveoli in Figure B showed that the lung tissue of mice injected with Wnt3a ^WG EVs was repaired and regenerated; as shown in Figure 9 The body weight of the mice shows that the mice injected with Wnt3a ^WG EVs are robust, grow fast, and do not show toxic side effects.

From the foregoing, it can be known that Wnt3a ^WG EVs, compared with other agents, can promote cell proliferation by activating the Wnt signaling pathway, thereby achieving the effect of tissue regeneration and repair.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (8)

1. An engineered extracellular vesicle loaded with Wnt protein. 2. The engineered extracellular vesicle according to claim 1, wherein the Wnt protein is Wnt3a protein. 3. The engineered extracellular vesicle according to claim 2, wherein the engineered extracellular vesicle is derived from HEK293T cells. 4. The engineered extracellular vesicle according to claim 3, wherein the engineered extracellular vesicle is prepared by the following method: Co-transfecting the Wnt3a-T2A-WLS plasmid with the sequence shown in SEQ ID NO.1 and the GPC6 ^ΔGPI -C1C2 plasmid with the sequence shown in SEQ ID NO.2 into the HEK293T cells; After transfection, the engineered extracellular vesicles were isolated from the culture supernatant of the HEK293T cells. 5. The engineered extracellular vesicle according to claim 4, characterized in that, the transfection adopts liposome method. 6. A use of the engineered extracellular vesicle according to any one of claims 1 to 5 in the preparation of a drug for tissue repair and regeneration. 7. A use of the engineered extracellular vesicle according to any one of claims 1 to 5 in the preparation of a medicament for treating chronic obstructive pulmonary disease. 8. The application according to claim 6 or 7, wherein the engineered extracellular vesicles can activate the endogenous regeneration mechanism mediated by the Wnt signaling pathway to promote tissue repair and regeneration.

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