An antibody-conjugated exosome and a preparation method and application thereof

Abstract

The application relates to an antibody-coupled exosome and a preparation method and application thereof, and relates to the technical field of biotechnology. The antibody-coupled exosome comprises an anti-TIMP-1 antibody and an exosome, and the anti-TIMP-1 antibody is coupled to the surface of the exosome. The exosome is coupled to the surface of the exosome in a chemical coupling mode, the anti-TIMP-1 antibody is coupled to the surface of the exosome, the antibody-coupled exosome has the treatment functions of degrading collagen and repairing tissues, and the treatment effect of diseases is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antibody-conjugated exosome, its preparation method, and its application. Background Technology

[0002] Pulmonary fibrosis, as a major disease threatening human health, has received widespread attention. Early intervention with antifibrotic drugs or lung transplantation in later stages are currently common treatment options for pulmonary fibrosis. However, the mechanisms of antifibrotic drugs are mainly focused on inhibiting the TGF-β pathway and fibroblasts; they can only slow the disease progression, not cure it, and their efficacy is poor for late-stage patients. Furthermore, lung transplantation is expensive, resources are scarce, and complications are frequent. Therefore, further research is needed to discover new treatment strategies. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides an antibody-conjugated exosome. This exosome is chemically conjugated with an anti-TIMP-1 antibody, enabling the antibody-conjugated exosome to possess both collagen degradation and tissue repair therapeutic functions, thereby improving the therapeutic effect of the disease.

[0004] To achieve the above objectives, the present invention provides an antibody-conjugated exosome comprising an anti-TIMP-1 antibody and an exosome, wherein the anti-TIMP-1 antibody is conjugated to the surface of the exosome.

[0005] Currently, the main drugs used clinically to treat pulmonary fibrosis are pirfenidone and nintedanib. Their mechanism of action primarily involves blocking the fibrosis-related TGF-β pathway and inhibiting fibroblast activation, but they have no direct effect on existing collagen fibers. Therefore, these drugs can only be used to delay disease progression in the early and middle stages of pulmonary fibrosis, and their efficacy is poor in patients with advanced stages. With a deeper understanding of extracellular matrix metabolism and protease homeostasis, therapeutic strategies targeting extracellular matrix regulation have gained attention. In our research, we discovered that matrix metalloproteinases (MMPs) are a group of peptidases involved in extracellular matrix degradation. Tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) is an endogenous inhibitor of MMPs, binding 1:1 to MMPs and thus inhibiting their activity in degrading the extracellular matrix. Extracellular matrix metabolism is dynamically regulated by the MMP / TIMP-1 protease system. TIMP-1, as an endogenous inhibitor of MMPs, binds 1:1 to MMPs and inhibits their activity, reducing extracellular matrix degradation and thus promoting collagen deposition. Based on this, during their research on extracellular matrix metabolic balance, the inventors discovered TIMP-1, a potential target for the treatment of pulmonary fibrosis. Therefore, by neutralizing excess TIMP-1 with anti-TIMP-1 antibodies and blocking TIMP-1, their inhibitory effect on MMPs can be relieved, helping MMPs regain activity and thus promoting the degradation of the extracellular matrix to treat pulmonary fibrosis. This restores protease system homeostasis and promotes collagen degradation, providing a new strategy for the treatment of pulmonary fibrosis. Therefore, the inventors proposed combining exosomes with anti-TIMP-1 antibodies for the treatment of pulmonary fibrosis, preparing the aforementioned antibody-conjugated exosomes. This nanomedicine (i.e., antibody-conjugated exosomes) combines the characteristics of exosomes and anti-TIMP-1 antibodies, possessing both the function of inhibiting fibroblast activation and degrading the extracellular matrix. Simultaneously, this nanomedicine also has reactive oxygen species (ROS) response capabilities, releasing anti-TIMP-1 antibodies on demand and prolonging the residence time of anti-TIMP-1 antibodies in the lungs. This provides a new strategy for the treatment of pulmonary fibrosis. Furthermore, the anti-TIMP-1 antibody is loaded on the surface of exosomes, allowing it to detach before entering cells and treat the target.

[0006] In one embodiment, the exosomes are mesenchymal stem cell exosomes.

[0007] Mesenchymal stem cell exosomes have the functions of tissue repair and fibrosis inhibition, and can reduce fibroblast activation at the source.

[0008] In one embodiment, the exosomes are human umbilical cord mesenchymal stem cell exosomes.

[0009] Human umbilical cord mesenchymal stem cell exosomes can promote lung tissue repair, inhibit fibroblast activation, reduce the source of extracellular matrix production, and work synergistically with anti-TIMP-1 antibodies, showing promise for treating mid-to-late stage pulmonary fibrosis.

[0010] In one embodiment, the method for preparing the antibody-conjugated exosomes includes the following steps: modifying the surface of the exosomes with an azide group, purifying them to obtain modified exosomes, modifying dibenzocyclooctylene with anti-TIMP-1 antibody, purifying it to obtain a modified antibody, mixing the modified exosomes with the modified antibody, incubating, centrifuging, taking the precipitate, and obtaining antibody-conjugated exosomes.

[0011] The antibody-conjugated exosomes prepared by the above method were analyzed and verified by Western blotting, and the proteins on them were found to contain exosome marker proteins and antibodies, respectively.

[0012] The present invention also provides a method for preparing the antibody-conjugated exosomes, comprising the following steps:

[0013] Preparation of modified exosomes: Azide groups were modified on the surface of exosomes, and the exosomes were purified to obtain modified exosomes;

[0014] Preparation of modified antibodies: Anti-TIMP-1 antibody was modified with dibenzocyclooctylene, purified, and the modified antibody was obtained;

[0015] Preparation of antibody-conjugated exosomes: Mix modified exosomes with modified antibodies, incubate, centrifuge, collect the precipitate, and obtain antibody-conjugated exosomes.

[0016] The above preparation method is simple, efficient, and easy to operate. It requires simple sources of exosomes, has low acquisition costs, and a short extraction time.

[0017] In one embodiment, modifying the exosome surface with azide groups includes the following steps: mixing the exosomes with azide-polyethylene glycol-phenylboronic acid and incubating.

[0018] In one embodiment, the ratio of the amount of the azide-polyethylene glycol-phenylboronic acid to the exosome is (100-120) μM: 1 mg / mL; in the step of modifying the surface of the exosome with azide groups, the incubation temperature is 3-5°C and the time is 2-4 h.

[0019] In one embodiment, the anti-TIMP-1 antibody-modified dibenzocyclooctyne includes the following steps: mixing diphenylcyclooctyne-tetraethylene glycol-active ester with the anti-TIMP-1 antibody and incubating.

[0020] In one embodiment, the molar ratio of the diphenylcyclooctyn-tetraethylene glycol-active ester to the anti-TIMP-1 antibody is 50:1; in the step of modifying dibenzocyclooctyn with the anti-TIMP-1 antibody, the incubation temperature is 22-28°C and the time is 1 hour.

[0021] In one embodiment, the mass ratio of the modified exosomes to the modified antibody is 5:1.

[0022] In one embodiment, in the step of preparing antibody-conjugated exosomes, the incubation temperature is 3-5°C, the incubation time is 2-4 hours, the centrifugation speed is 100,000-120,000 g, and the centrifugation time is 1-1.5 hours.

[0023] The present invention also provides the application of the antibody-conjugated exosomes in the preparation of anti-pulmonary fibrosis drugs.

[0024] Anti-TIMP-1 antibody can effectively degrade collagen.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention discloses an antibody-conjugated exosome, its preparation method and application. The antibody-conjugated exosome has a uniform structure and is obtained by chemically conjugating an antibody to the surface of the exosome. It combines an anti-TIMP-1 antibody and exosomes, and has both collagen degradation and tissue repair therapeutic functions, thereby improving the therapeutic effect of pulmonary fibrosis. Attached Figure Description

[0027] Figure 1 Electron microscopy images (scarbar: 100 nm) of Mexo prepared in Example 1 and Mexo-cl-aT prepared in Example 2 are shown.

[0028] Figure 2 The particle size and potential statistics of Mexo prepared in Example 1 and Mexo-cl-aT prepared in Example 2 are shown in the following figures.

[0029] Figure 3 This is a graph showing the colocalization efficiency of Mexo and aT observed by cofocusing fluorescence microscopy in Example 2.

[0030] Figure 4 WB images of Mexo prepared in Example 1 and Mexo-cl-aT prepared in Example 2;

[0031] Figure 5 This is a standard curve of the aT drug loading of Mexo-cl-aT prepared in Example 2;

[0032] Figure 6The graph shows the aT release curve under the reactive oxygen species response of the Mexo-cl-aT prepared in Example 2.

[0033] Figure 7 The image shows the confocal fluorescence results of mouse lung fibroblasts treated with Mexo prepared in Example 1 and Mexo-cl-aT prepared in Example 2.

[0034] Figure 8 Organ imaging images of the Mexo-cl-aT group prepared in Example 2 after in vivo drug administration. Detailed Implementation

[0035] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0037] source:

[0038] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0039] Example 1

[0040] Extraction of exosomes from human umbilical cord mesenchymal stem cells.

[0041] HUMSC cells were cultured in serum-free HMSC-specific medium. When the cells reached 80%-90% confluence, the culture medium was collected. The cells were then centrifuged at 1000g for 10 min, and the supernatant was collected. This process was repeated with centrifugation at 2000g for 20 min, followed by another centrifugation at 10000g for 45 min, and finally, centrifugation at 100000g for 1 h. The precipitate was collected and resuspended in PBS to obtain a human umbilical cord mesenchymal stem cell exosome (Mexo) solution. The resuspended cells were then stored at -80°C.

[0042] Example 2

[0043] Prepare antibody-conjugated exosomes.

[0044] I. Preparation of modified exosomes.

[0045] BCA was used to quantify Mexo protein concentration. PBA-PEG4-N3 was added to the Mexo solution (final concentration PBA-PEG4-N3:Mexo = 100 μM: 1 mg / mL), mixed, and incubated at 4 °C for 2 h; the solution was purified by 100 kDa ultrafiltration, the filtrate was discarded, and the upper layer of the filter membrane (Mexo-N3) was collected after resuspending in PBS.

[0046] II. Preparation of modified antibodies.

[0047] NHS-PEG4-DBCO was added to an anti-TIMP-1 antibody (aT) solution (DBCO:aT = 50:1 molar ratio). The anti-TIMP-1 antibody can be a commercially available product; in this example, the brand Abclonal, catalog number A1389, was selected. The mixture was stirred and incubated on a shaker at room temperature for 1 hour. The solution was purified by 10 kDa ultrafiltration, the filtrate was discarded, and the upper layer of the filter membrane (aT-DBCO) was collected after resuspending in PBS. The concentrations of Mexo-N3 and aT-DBCO proteins were quantified using the BCA method.

[0048] III. Preparation of antibody-conjugated exosomes.

[0049] Mexo-N3 solution and aT-DBCO solution were mixed, with the mass ratio of Mexo-N3 (modified exosomes) to aT-DBCO (modified antibody) in the Mexo-N3 solution and aT-DBCO solution being 5:1. The mixture was incubated at 4°C for 2 hours. After centrifugation at 100,000g for 1 hour, the precipitate was collected and resuspended in PBS to obtain an antibody-conjugated exosome (Mexo-cl-aT) solution.

[0050] Example 3

[0051] The Mexo prepared in Example 1 and the Mexo-cl-aT prepared in Example 2 were characterized respectively, and the experimental results are as follows.

[0052] I. Electron microscopy observation.

[0053] Take 2 μL each of Mexo and Mexo-cl-aT solutions and drop them onto an amorphous carbon film copper mesh carrier. After 1 min, dry the copper mesh, then drop 2 μL of 3% uranium acetate dye onto it, let it stand for 30 s, and then dry it rapidly. Finally, observe the copper mesh using a HITACHI transmission electron microscope. The results are as follows: Figure 1 As shown.

[0054] II. Particle size.

[0055] The particle size and potential of the two types of particles were measured using a Malvern particle size analyzer, and the results are as follows: Figure 2 As shown.

[0056] III. Confocal observation.

[0057] Mexo and aT were labeled with DiI and FITC secondary antibodies, respectively. The co-localization efficiency of Mexo and aT was observed by laser confocal microscopy. The results are as follows: Figure 3 As shown, Mexo and aT are co-located, indicating that aT is successfully coupled to the Mexo surface.

[0058] IV. Protein characterization by Western blot gel electrophoresis.

[0059] First, protein quantification of the two types of particles was performed using the BCA method to determine the final loading amount as 20 μg and the required loading volume. A 20 mL 10% separating gel was prepared (7.9 mL H₂O, 6.7 mL 30% Acrylamide, 5.0 mL 1.5 M Tris-HCl (pH 8.80), 0.2 mL 10% SDS, 0.2 mL 10% ammonium persulfate, 0.008 mL TEMED), quickly mixed, and poured into a mold. After 30 seconds, water was added for liquid sealing, and the mixture solidified after 30 minutes. A 4 mL stacking gel was prepared (2.7 mL H₂O, 0.67 mL 30% Acrylamide, 0.5 mL 1.0 M Tris (pH 6.8), 0.04 mL 10% SDS, 0.04 mL...). Add 10% ammonium persulfate (0.004 mL TEMED), discard the sealing water, add the stacking gel, and immediately add the comb to solidify for 30 min; add 500 mL of 1× electrophoresis buffer to the electrophoresis tank, and remove the comb after 15 min. Add samples aT, Mexo, and Mexo-cl-aT sequentially (protein concentrations of 4 μg, 20 μg, and 20 μg, respectively); electrophoresis is first performed at 80 V and 200 mA for 15 min, then at 120 V until the buffer reaches the bottom of the gel; remove the gel, place it in an electrophoresis transfer apparatus, and electrophores at 120 V for 2 h. After that, remove the PVDF membrane, place it in an antibody cassette, prepare 5% BSA blocking buffer with PBST, and block at room temperature for 2 h. The results are as follows. Figure 4 As shown, Mexo-cl-aT contains Mexo and aT components.

[0060] V. Detect the drug loading of aT.

[0061] The aT drug loading of Mexo-cl-aT prepared in Example 2 was detected.

[0062] Detection Method: To determine the aT drug loading, the inventors prepared aT standard solutions of known concentrations and established a standard curve using ELISA. The inventors fixed the Mexo concentration at 1 mg / mL and measured the concentration of uncoupled aT. The drug loading was calculated using the following formula, and the results are as follows: Figure 5 As shown.

[0063] The formula for calculating drug loading is: Drug loading = 0.7322 × total aT + 0.1858.

[0064] VI. aT release rate under reactive oxygen species response.

[0065] Detection method: To determine the aT release rate, the inventors labeled aT with FITC secondary antibody. The inventors added the labeled Mexo-cl-aT at the same concentration to a 1000 kDa dialysis membrane. The dialysate was 0 mM, 0.1 mM, and 1 mM hydrogen peroxide PBS solution. The dialysate was collected at a series of time points, and the fluorescence intensity was measured. The results are as follows: Figure 6 As shown.

[0066] VII. In vitro antifibrotic ability of Mexo-cl-aT.

[0067] Detection method: To evaluate the anti-fibrotic ability of Mexo-cl-aT at in vitro levels, the inventors performed immunofluorescence staining on mouse lung fibroblast fibrosis markers α-SMA and COL1A1. The fluorescence intensity of the markers was observed by confocal fluorescence microscopy. The results are as follows: Figure 7 As shown.

[0068] 8. The sustained-release capacity of Mexo-cl-aT.

[0069] Detection method: To evaluate the sustained-release capacity of Mexo-cl-aT at in vivo levels, the inventors labeled aT with Alexa Fluor647 secondary antibody and administered Mexo-cl-aT to mice via intratracheal injection. Mice were dissected 72 hours later, and fluorescence signals in major organs were observed using a small animal in vivo imaging system. The results are as follows: Figure 8 As shown.

[0070] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An antibody-conjugated exosome, characterized in that, The antibody-conjugated exosomes include an anti-TIMP-1 antibody and exosomes, wherein the anti-TIMP-1 antibody is conjugated to the surface of the exosomes, and the exosomes are mesenchymal stem cell exosomes; The method for preparing the antibody-conjugated exosomes includes the following steps: modifying the surface of exosomes with azide groups, purifying them to obtain modified exosomes; modifying the anti-TIMP-1 antibody with dibenzocyclooctylene, purifying it to obtain modified antibodies; mixing the modified exosomes with the modified antibodies, incubating, centrifuging, and collecting the precipitate to obtain antibody-conjugated exosomes; the modification of the exosome surface with azide groups includes the following steps: mixing the exosomes with azide-polyethylene glycol-phenylboronic acid and incubating.

2. The method for preparing antibody-conjugated exosomes according to claim 1, characterized in that, Includes the following steps: Preparation of modified exosomes: The surface of exosomes is modified with azide groups, purified, and modified exosomes are obtained; the modification of the exosome surface with azide groups includes the following steps: mixing exosomes with azide-polyethylene glycol-phenylboronic acid and incubating; Preparation of modified antibodies: Anti-TIMP-1 antibody was modified with dibenzocyclooctylene, purified, and the modified antibody was obtained; Preparation of antibody-conjugated exosomes: Mix modified exosomes with modified antibodies, incubate, centrifuge, collect the precipitate, and obtain antibody-conjugated exosomes.

3. The preparation method according to claim 2, characterized in that, The ratio of the amount of azide-polyethylene glycol-phenylboronic acid to the exosomes is (100-120) μM: 1 mg / mL; in the step of modifying the surface of the exosomes with azide groups, the incubation temperature is 3-5℃ and the time is 2-4 h.

4. The preparation method according to claim 2, characterized in that, The modification of the anti-TIMP-1 antibody with dibenzocyclooctyn includes the following steps: mixing dibenzocyclooctyn-tetraethylene glycol-active ester with the anti-TIMP-1 antibody and incubating.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the dibenzocyclooctyn-tetraethylene glycol-active ester to the anti-TIMP-1 antibody is 50:1; in the step of modifying the anti-TIMP-1 antibody with dibenzocyclooctyn, the incubation temperature is 22-28℃ and the time is 1h.

6. The preparation method according to claim 2, characterized in that, The mass ratio of the modified exosomes to the modified antibody is 5:

1.

7. The preparation method according to claim 2, characterized in that, In the step of preparing antibody-conjugated exosomes, the incubation temperature is 3-5℃, the incubation time is 2-4h, the centrifugation force is 100000-120000g, and the centrifugation time is 1-1.5h.

8. The use of the antibody-conjugated exosomes of claim 1 in the preparation of anti-pulmonary fibrosis drugs.

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