Gp130-loaded engineered extracellular vesicle and application thereof

Abstract

The invention discloses a gp130-loaded engineered extracellular vesicle and application thereof, belongs to the technical field of biological medicines, and relates to a medicine, which comprises a scaffold protein EN144 membrane externally hung gp130 protein-loaded engineered extracellular vesicle, the scaffold protein EN144 membrane externally hung gp130 protein-loaded engineered extracellular vesicle, and the gp130 protein-loaded engineered extracellular vesicle. The sequence of the scaffold protein EN144 is SEQ ID NO: 1; according to the present invention, the EN144-EVhgp130 provides the anti-inflammatory effect in a variety of diseases; the consistency of in-vivo and in-vitro research results shows that the EN144-EVhgp130 and the EN144-EVmgp130 can be used for effectively inhibiting the generation of sepsis inflammatory factors on RNA (Ribonucleic Acid) and protein levels; compared with a clinically approved drug TCZ, the EN144-EVhgp130 shows a higher survival rate and more remarkable inflammation improvement in a sepsis mouse model, and the IC50 of the EN144-EVhgp130 is lower than that of the TCZ.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an engineered extracellular vesicle loaded with gp130 and an application thereof. Background Art

[0002] Although activated protein C, a drug approved by the U.S. Food and Drug Administration for the treatment of sepsis, has shown positive effects in promoting thrombolysis, inhibiting thrombosis and reducing inflammatory response, its associated higher risk of bleeding limits its use in patients with impaired immune function or requiring renal dialysis. In addition, biological agents are expensive.

[0003] At present, the treatments for OA include medication, physical therapy, surgery and other methods, but the efficacy of these methods usually gradually weakens within 3 months after the start of treatment. For patients with advanced OA, total knee replacement needs to be considered to relieve severe symptoms, but this requires patients to bear the dual pressure of surgical costs and medical risks. Therefore, it is particularly necessary to find new drugs and treatments for inflammatory diseases. In the previous patent CN202310429909.8, the sorting effect of EN144 was used to sort MS2 protein into exosomes. In addition, the MS2 recognition stem-loop structure loop was placed in the non-coding region of the target mRNA, and the specific binding of RNA to the binding protein was used to achieve efficient loading of mRNA in exosomes; therefore, exploring more applications of EN144 is a very valuable study. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an engineered extracellular vesicle loaded with gp130 and its application;

[0005] A drug comprises an engineered extracellular vesicle with a scaffold protein EN144 carrying a gp130 protein outside the membrane; the sequence of the scaffold protein EN144 is SEQ ID NO: 1.

[0006] Preferably, the engineered extracellular vesicles are derived from Expi293F cells.

[0007] Preferably, the engineered extracellular vesicles further comprise an affinity peptide CAP that specifically targets chondrocytes.

[0008] A method for preparing a drug, comprising the following steps:

[0009] The plasmid is transfected into Expi293F cells, and the cell supernatant is collected and centrifuged to collect the engineered extracellular vesicles; the plasmid includes EN144-mgp130, EN144-hgp130 or EN144-hgp130-CAP.

[0010] Preferably, the engineered extracellular vesicles further comprise an affinity peptide CAP that specifically targets chondrocytes.

[0011] Preferably, the cell supernatant is collected 48 h or 72 h after plasmid transfection.

[0012] The application of engineered extracellular vesicles containing scaffold protein EN144, which are loaded with affinity peptide CAP and gp130 protein specifically targeting chondrocytes, in the preparation of drugs for treating cartilage inflammation.

[0013] Application of engineered extracellular vesicles with gp130 protein mounted on the extracellular membrane of scaffold protein EN144 in the preparation of drugs for the treatment of sepsis.

[0014] The beneficial effects of the present invention include:

[0015] 1. EN144-EV of the present invention hgp130 It has anti-inflammatory effects in a variety of diseases.

[0016] 2. The results of in vitro and in vivo studies have consistently shown that EN144-EV hgp130 and EN144-EV mgp130 It can effectively inhibit the production of sepsis inflammatory factors at both RNA and protein levels.

[0017] 3. Compared with the clinically approved drug TCZ, EN144-EV hgp130 EN144-EV showed higher survival rate and more significant improvement in inflammation in a mouse model of sepsis. hgp130 The IC50 of TCZ is lower than that of TCZ.

[0018] 4. The presence of CAP peptide promoted the uptake of bait EVs by chondrocytes and prolonged the survival of EN144-EV hgp130-CAP Retention time in cartilage area.

[0019] 5.EN144-EV hgp130 and EN144-EV hgp130-CAP They showed significant anti-inflammatory, cartilage protection and repair functions. They effectively inhibited the expression of inflammatory factors in in vitro and in vivo OA models, promoted the expression of COL2A1, and effectively delayed the progression of OA pathology. Moreover, EN144-EV with chondrocyte targeting ability hgp130-CAP , its therapeutic effect is more significant.

[0020] 6. In terms of safety assessment, EN144-EV hgp130-CAP It showed good biocompatibility, and no obvious pathological changes were observed in the organs of the treated rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 .Expression of mgp130 in extracellular vesicles;

[0023] Expression of mgp130 protein in EVs derived from Expi293F after transfection with Lamp2b-mgp130, PTGFRN-mgp130, and EN144-mgp130, respectively.

[0024] Figure 2 .Expression of gp130 in extracellular vesicles;

[0025] Expression of Flag protein in Expi293F cells (upper panel) and derived EVs (lower panel) after transfection with EN144-mgp130 and EN144-hgp130 plasmids.

[0026] Figure 3 .EN144-EV mgp130 and EN144-EV hgp130 In vitro anti-inflammatory effect;

[0027] AB.EN144-EV hgp130 or EN144-EV mgp130 After co-culture with LPS-induced Raw 264.7 inflammatory cells, the expression levels of IL-6 mRNA (A) and protein (B) in cells; C. EN144-EV hgp130 D.EN144-EV hgp130 Compared with the IC50 of TCZ. **P<0.01.

[0028] Figure 4 .EN144-EV mgp130 、EN144-EV hgp130 and the effect of TCZ in treating sepsis;

[0029] AB. Tail vein injection of EN144-EV in septic mice mgp130 、EN144-EV hgp130 Changes in mouse survival rate (A) and body weight (B) after TCZ or TCZ. 5 animals in each group. CE. Tail vein injection of EN144-EV in septic mice mgp130 、EN144-EV hgp130The mRNA expression levels of IL-6 in the liver (C), spleen (D), and lung (E) after treatment with TCZ or TCZ.

[0030] Figure 5 .EN144-EV hgp130-CAP Protein expression and particle size distribution;

[0031] AB. Expression levels of Flag protein in cells (A) and in EVs (B) produced by EN144, EN144-hgp130 or EN144-hgp130-CAP plasmid transfection; C. hgp130-CAP and EN144-EV hgp130 Particle size distribution diagram.

[0032] Figure 6 .EN144-EV hgp130-CAP In vitro targeting effect analysis;

[0033] AB.EN144-EV hgp130 or EN144-EV hgp130-CAP Comparison (A) and quantitative analysis (B) of the proportion of DiI-positive cells after treatment of primary chondrocytes; C. EN144-EV hgp130 or EN144-EV hgp130-CAP Representative images of DiI-positive cells after treatment of primary chondrocytes. Scale bar: 75 μm. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

[0034] Figure 7 .EN144-EV hgp130-CAP In vivo targeting effect analysis;

[0035] A. In situ injection of EN144-EVs and EN144-EV into the joint cavity of OA model hgp130 or EN144-EV hgp130-CAP 24 hours later, the in vivo distribution image of DiR-labeled EVs (upper figure) and the representative fluorescence image of DiI-labeled EVs in cartilage tissue (lower figure); B. In situ injection of EN144-EVs and EN144-EVs into the joint cavity of arthritis rat model hgp130 or EN144-EV hgp130-CAP After 7 days, the in vivo distribution image of DiR-labeled EVs. Color scale, Min: 2.00×10 7 ,Max:6.00×10 7 .

[0036] Figure 8 .EN144-EV hgp130-CAP In vitro anti-inflammatory effect;

[0037] PBS, EN144-EVs, TCZ, EN144-EV hgp130 or EN144-EV hgp130-CAP Protein expression levels of COL2A1, MMP13 and IL-6 in cells after co-culture with OA-like cell model for 24 h.

[0038] Fig. 9 .OARSI scores of cartilage damage in different groups;

[0039] Fig.10 .Representative images of cartilage tissue staining in different groups;

[0040] Fig.11 .EN144-EV hgp130-CAP Effects on gene expression in articular cartilage;

[0041] A. OA rats were injected intra-articularly with PBS, EN144-EVs, TCZ, EN144-EVs for 4 consecutive weeks (once a week). hgp13 、EN144-EV hgp13-CAP The expression levels of COL2A1, IL-6 and MMP13 proteins in cartilage tissues of different groups after HA or PBS (n=3). Scale bar: 50 μm. BD. OA rats were injected with PBS, EN144-EVs, TCZ, EN144-EVs, and HA intra-articularly for 4 consecutive weeks (once a week). hgp13 、EN144-EV hgp13-CAP Semi-quantitative statistical analysis of the expression levels of COL2A1 (B), IL-6 (C) and MMP13 (D) proteins in cartilage tissues of different groups after HA or HA (n=3). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

[0042] Fig.12 .HE staining was used to evaluate the pathological changes of important organs in each treatment group;

[0043] Fig.13 .Evaluation of the toxic effects of each treatment group on important organs.

[0044] Changes in serum ALT (A), ALP (B), AST (C), LDH (D), BUN (E) and CREA (F) levels in each treatment group. *P<0.05. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The amino acid sequence of EN144 is SEQ ID NO: 1:

[0047] MERDGCAGGGSRGGEGGRAPREGPAGNGRDRGRSHAAEAPGDPQAAA SLLAPMDVGEEPLEKAARARTAKDPNTYKVLSLVLSVCVLTTILGCIFGLKPS CAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPE.

[0048] The sequence of hgp130 protein is SEQ ID NO: 2:

[0049] .

[0050] Example 1: Extracting extracellular vesicles displaying mouse mgp130 on the surface and comparing the content of mgp130 protein loaded by various scaffold proteins.

[0051] 20 μg of plasmids (EN144-mgp130, Lamp2b-mgp130, and PTGFRN-mgp130) and 60 μL of 1 mg / mL PEI transfection reagent were added to 500 μL of DMEM medium without dual antibodies. Then, the two were gently mixed and incubated at room temperature in the dark for 15 minutes to form a DNA-PEI cationic nucleic acid transfection reagent complex. The complex was then evenly added to a 1×10 7 The supernatant was collected after 48 h and 72 h of culture in a shaker. The cells were removed by centrifugation at 300 g for 5 min. Subsequently, the cells were centrifuged at 10,000 g for 30 min at 4 °C to remove dead cells and cell debris. Next, the supernatant was passed through a 0.22 μm filter to remove large vesicles. The filtrate was added to an ultracentrifuge tube and balanced to a value not exceeding 0.0001 g. The EVs were precipitated by centrifugation at 130,000 g for 90 min at 4 °C. This step was repeated to wash the EVs. Finally, the EVs were resuspended in 1 mL of PBS.

[0052] For proteomic analysis, extracellular vesicle (EVs) lysates (3 × 10 9 ) were chromatographically separated on an nLC 1200 system equipped with a C18 column (2.2 μm, Michrom Bioresources). Chromatographic separation was performed at a flow rate of 300 nL / min using 0.1% formic acid in water (solvent A) and 80% acetonitrile solution containing 0.1% formic acid (solvent B) as mobile phases.

[0053] The column was first equilibrated with 100% liquid A, followed by a gradient elution: 2-8% B from 0-3 min, 8-40% B from 3-81 min, 40-95% B from 81-83 min, and held at 95% B from 83-90 min. Data acquisition was performed in data-dependent acquisition (DDA) mode, and MS1 settings included a resolution of 60,000, an AGC target of 3e6, a maximum injection time of 30 ms, and a scan range of 400-1200 m / z. For MS2, settings included a resolution of 30,000, an AGC target of 1e5, a maximum injection time of 50 ms, a cycle number of 20, a collision energy of 28, and a dynamic exclusion time of 40 s. Raw data were searched using Proteome Discoverer 2.3 software, and false discovery rates (FDRs) for both proteins and peptides were set to 1% to ensure high data quality.

[0054] EN144-EV mgp130 、Lamp2b-EV mgp130and PTGFRN-EV mgp130 The expression of mgp130 protein was detected in EN144-EV mgp130 The protein content of mgp130 was the highest in the EV, significantly higher than that of Lamp2b-EV. mgp130 and PTGFRN-EV mgp130 The results showed that EN144 had a higher loading level and higher protein loading efficiency than Lamp2b and PTGFRN. Figure 1 , Ps<0.001).

[0055] Example 2 Extraction of EN144-EV surface displaying mouse mgp130 (EN144-EV mgp130 ) or human hgp130 (EN144-EV hgp130 ) of extracellular vesicles.

[0056] 20 μg of plasmids (EN144-mgp130 and EN144-hgp130) were transfected into Expi293F cells, and the cell supernatants were collected at 48 h and 72 h, respectively. Extracellular vesicles were collected by ultracentrifugation, and 10 μL (total volume of 3 × 10 9 10 μg of EVs and 10 μg of protein lysate were added and mixed with 10 μL of SDS-PAGE protein loading buffer. The samples were then heated at 95°C for 10 min to promote protein denaturation and dissociation. The electrophoresis parameters were set to 190 V for 70 min. The transfer conditions were set to 275 mA for 90 min. Protein expression was detected after incubation with primary and secondary antibodies.

[0057] Subsequently, in transfected cells, EN144-EV mgp130 、EN144-EV hgp130 The expression of gp130 was detected in Figure 2 The results showed that transfected cells, EN144-EV mgp130 、EN144-EV hgp130 Can promote cartilage repair.

[0058] Example 3EN144-EV mgp130 and EN144-EV hgp130 Effectively reduces cellular inflammation.

[0059] Raw 264.7 cells were digested and counted, and 4×10 5Cells / well were placed in a 6-well plate and cultured in a cell culture incubator for 24 hours. The next day, 1 mL of basal medium containing lipopolysaccharide (LPS) was replaced with a final concentration of 1 μg / mL of LPS. After 3 hours of co-culture, the induced Raw 264.7 cell inflammation model was obtained. Subsequently, 1 mL of basal medium containing LPS was replaced, and different therapeutic drugs were added and cultured for 24 hours. EN144-EV mgp130 and EN144-EV hgp130 The final concentration was 1×10 10 / mL. After 24h or 48h of co-culture, cells were collected for subsequent RNA and protein detection.

[0060] With EN144-EV mgp130 Similar, EN144-EV hgp130 It can effectively reduce the expression of IL-6 protein and RNA in inflammatory cells, thus exerting a powerful anti-inflammatory effect ( Figure 3 A-3B). To quantify gp130 on EVs, we performed protein grayscale analysis. Using this method, we estimated that each EV displayed an average of 400 hgp130 ( Figure 3 C) Next, we tried to test EN144-EV hgp130 The efficacy of EN144-EV was compared with that of TCZ (tocilizumab, a clinically approved IL-6R monoclonal antibody). hgp130 The half maximal inhibitory concentration (IC50) value of Figure 3 D).

[0061] Example 4EN144-EV mgp130 、EN144-EV hgp130 and evaluation of the anti-inflammatory effect of TCZ in vivo.

[0062] Three hours after intraperitoneal injection of LPS (10 mg / kg), different drugs were injected through the tail vein. The liver, lungs, and spleen were harvested 6 hours after the tail vein injection for the detection of inflammatory factors. The survival time was recorded from the time of tail vein administration. The final dose of TCZ group was 10 μg / mouse, and EN144-EV mgp130 Group is 5×10 9 / pcs,EN144-EV hgp130 Group is 1×10 10 / Only.

[0063] EN144-EV hgp130 The survival rate of the treatment was 80%, significantly higher than that of EN144-EV mgp130 40% and 20% of TCZ ( Figure 4A). In addition, the bait EV-treated mice regained body weight earlier (about 50 h) compared with the TCZ-treated mice (about 60 h) ( Figure 4 B) Bait EV, especially EN144-EV hgp130 , showed excellent protection against LPS-induced inflammation, as evidenced by the reduction of IL-6 levels in the liver, lung, and spleen ( Figure 4 C).

[0064] Example 5 Extraction of EVs for targeted chondrocyte delivery of hgp130.

[0065] In order to improve the affinity between bait EVs and chondrocytes, a cartilage targeting peptide (CAP peptide) sequence was linked to the C-terminus of EN144-hgp130 to form EN144-hgp130-CAP. Subsequently, the plasmid was transiently transfected into Expi293F cells, and the target EVs were isolated by ultracentrifugation.

[0066] (EN144-EV hgp130-CAP Flag protein expression was detected in all transfected cells and EVs ( Figure 5 A-5B).

[0067] When using NTA for analysis, you first need to install a chip with a range of 60nm-200nm on the test card. Next, use a pipette to draw 200μL PBS buffer, aim the pipette tip at the groove of the sample slot chip of the test card, quickly inject the liquid at one time, and then add 200μL PBS buffer in the same way to the groove on the other side. After the liquid is added, be sure that there are no bubbles in the rubber ring hole, and there should be no liquid leakage from the test card. Before testing the sample, it is necessary to record that the number of particles in the PBS buffer within 60s is less than 5. After diluting the test sample 20-100 times, record the size of 100 EVs particles.

[0068] NTA results show that EN144-EV hgp130-CAP With EN144-EV hgp130 There is no significant change in size ( Figure 5 C).

[0069] Example 6EN144-EV hgp130-CAP Targeted evaluation.

[0070] First, 2.5×10 10 / mL EVs were mixed with 1mM DiI dye at a volume ratio of 400:1 and incubated at 37°C in the dark for 30min. After incubation, free DiI dye was removed through a 0.22μm filter to obtain DiI-labeled EVs. Subsequently, rat chondrocytes were plated in a 6-well plate and 1×1010 The cells were collected after overnight culture and their targeting properties were evaluated by confocal microscopy and flow cytometry.

[0071] After 24 h of co-culture, EN144-EV hgp130-CAP The treated cells showed a large area of ​​bright red fluorescence, which was similar to that of EN144-EV. hgp130 Compared with the control group, the number of fluorescent cells increased by about 30% (P<0.001, Figure 6 A). This verifies EN144-EV hgp130-CAP Stronger chondrocyte targeting ability in vitro ( Figure 6 B).

[0072] Take 1×10 11 5 μL of 10 mM DiR dye was added to EVs / mL and incubated at 37°C in the dark for 30 min. After incubation, free DiR dye was removed through a 0.22 μm filter to obtain DiR-labeled EVs. 10 The labeled EVs were injected orthotopically into rat joints.

[0073] In vivo, with EN144-EV hgp130 Compared with EN144-EV hgp130-CAP The intra-articular injection of the group showed higher fluorescence intensity after 24 and 168 hours, and no systemic spread ( Figure 7 A-7B). This indicates that EN144-EV hgp130-CAP The retention and chondrocyte targeting of the endothelial cells were improved.

[0074] Example 7EN144-EV hgp130 With EN144-EV hgp130-CAP In vitro evaluation of therapeutic effects

[0075] P3 chondrocytes were obtained and digested to prepare a single cell suspension. 6 Cells / well were plated in a six-well plate and incubated overnight at 37°C and 5% CO2. On the second day, the cell culture medium containing 50 ng / mL IL-1β was replaced and cultured for 24 h to establish an OA-like chondrocyte model in vitro. For the EVs co-culture group, 100 μL of 1×10 11 / mL EVs, 10 μg TCZ was added to each well. After 24 h of co-culture, the expression of MMP13, IL-6 and COL2A1 was detected.

[0076] The study found that EN144-EV hgp130 and EN144-EV hgp130-CAPThe treatments reduced the levels of MMP13 and IL-6 proteins and upregulated the expression of COL2A1 ( Figure 8 ), and EN144-EV hgp130-CAP The effect of EN144-EV was better, while TCZ treatment had no significant effect on gene expression. hgp130-CAP It can effectively improve the expression of inflammatory proteins induced by IL-1β and upregulate the content of collagen.

[0077] Example 8 EN144-EV hgp130 With EN144-EV hgp130-CAP In vivo evaluation of therapeutic efficacy

[0078] After the rats were fully anesthetized, a 1-cm longitudinal medial parapatellar incision was made on both sides of the knee joints to expose the knee joints, the anterior cruciate ligament was cut, and the wounds were sutured layer by layer. The sham group (Sham group) only underwent skin incision on the rats. After surgery, the incisions were sutured and the OA model rats were placed in cages to recover slowly on their own. Four weeks after ACLT surgery, the rats were randomly divided into 6 groups (n=5 in each group), namely: PBS group, hyaluronic acid (HA, 10 mg / mL) group, TCZ group (100 μg / mL), EN144-EV group (10×10 11 / mL)、EN144-EV hgp130 Group (10×10 11 / mL) and EN144-EV hgp130-CAP Group (10×10 11 OA model rats were injected with 100 μL of the drug into each leg once a week through the joint cavity for 4 consecutive weeks. The rats were killed in the 5th week, and cartilage, heart, liver, spleen, lung, and kidney were collected for histological analysis, and blood was collected from the abdominal aorta for blood biochemical analysis.

[0079] According to the score of Osteoarthritis Research Society International (OARSI), EN144-EV hgp130-CAP The recovery of the group was the best and the score reduction was the most significant. hgp130 In contrast, targeted modification made EN144-EV hgp130-CAP Has a stronger cartilage protection effect ( Fig. 9 ). After treatment, staining was performed to evaluate cartilage degeneration in each group. The PBS group showed cartilage thinning and decreased proteoglycan levels, while HA, TCZ, and EN144-EV hgp130 These characteristics can be partially improved ( Fig.10 ). However, EN144-EV hgp130-CAP It has a better effect on alleviating cartilage degradation and wear in OA rats ( Fig.10). IHC showed that compared with the PBS group, the expression of COL2A1 in the treatment group was increased, and the expressions of IL-6 and MMP13 were decreased. hgp130-CAP The strongest effect on enhancing COL2A1 and inhibiting IL-6 / MMP13 was better than EN144-EV hgp130 and TCZ group ( Fig.11 ). These results showed that TCZ group, EN144-EV hgp130 Group and EN144-EV hgp130-CAP Both groups can promote cartilage repair, among which the latter has the best effect.

[0080] Example 9EN144-EV hgp130-CAP Analysis of toxic effects.

[0081] The hearts, livers, spleens, lungs, and kidneys of rats treated for 4 weeks were collected, and the damage to tissues and organs in vivo by different treatments was evaluated by HE. Blood was collected from the abdominal aorta and gently added to a 15 mL centrifuge tube along the tube wall. Too fast speed or dripping can easily cause hemolysis. Let it stand overnight at 4°C and take the upper serum. Centrifuge at 3,000g for 10 minutes, and the supernatant was used to detect alkaline phosphatase (ALP) and alanine aminotransferase (ALT) indicators of liver damage, blood urea nitrogen (BUN) and blood creatinine (CERA) indicators of renal damage, and aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) commonly used markers of heart damage.

[0082] Compared with the Sham group, the PBS group, HA group, TCZ group, EN144-EVs group, and EN144-EVs group showed hgp130 Group and EN144-EV hgp130-CAP There were no obvious pathological changes in the organs of the rats in the group, indicating that EN144-EV hgp130-CAP Better safety ( Fig.12 ). Consistent with the HE results, compared with the Sham group, the blood biochemical indexes in each treatment group did not change significantly by more than 1 times ( Fig.13 These results indicate that the use of EN144-EV to deliver hgp130-CAP does not cause pathological changes in the main organs of rats. hgp130-CAP It is a safe and feasible treatment for cartilage damage.

[0083] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0084] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A drug, characterized in that The invention relates to an engineered extracellular vesicle comprising a scaffold protein EN144 and a gp130 protein mounted outside the membrane; the sequence of the scaffold protein EN144 is SEQ ID NO:

1.

2. The drug according to claim 1, characterized in that The engineered extracellular vesicles are derived from Expi293F cells.

3. The drug according to claim 1, characterized in that The engineered extracellular vesicles further include an affinity peptide CAP that specifically targets chondrocytes.

4. A method for preparing a drug, characterized in that: The method comprises the following steps: The plasmid is transfected into Expi293F cells, and the cell supernatant is collected and centrifuged to collect the engineered extracellular vesicles; the plasmid includes EN144-mgp130, EN144-hgp130 or EN144-hgp130-CAP.

5. The method for preparing the drug according to claim 4, characterized in that: The engineered extracellular vesicles further include an affinity peptide CAP that specifically targets chondrocytes.

6. The method for preparing the drug according to claim 4, characterized in that: The time for collecting the cell supernatant is 48h or 72h after plasmid transfection.

7. Application of engineered extracellular vesicles containing the scaffold protein EN144 and the affinity peptide CAP and gp130 protein specifically targeting chondrocytes mounted outside the membrane in the preparation of drugs for the treatment of cartilage inflammation.

8. Application of engineered extracellular vesicles with gp130 protein mounted on the extracellular membrane of scaffold protein EN144 in the preparation of drugs for the treatment of sepsis.

9. The use of engineered extracellular vesicles of the scaffold protein EN144 with affinity peptide CAP and gp130 protein specifically targeting chondrocytes mounted outside the membrane according to claim 7 in the preparation of drugs for treating cartilage inflammation, characterized in that: The engineered extracellular vesicles are derived from Expi293F cells.

10. Use of the engineered extracellular vesicles of the scaffold protein EN144 with gp130 protein mounted outside the membrane according to claim 8 in the preparation of a drug for treating sepsis, characterized in that: The engineered extracellular vesicles are derived from Expi293F cells.

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