A highly efficient tumor-targeting bacterial outer membrane vesicle and a preparation method thereof

Abstract

The application provides a kind of high-efficiency tumor-targeting bacterial outer membrane vesicle and its preparation method, it is related to biological medicine technical field.The target protein PDL1 binding peptide is successfully modified on the surface of bacterial outer membrane vesicle to prepare bacterial outer membrane vesicle PDL1-OMVs, its particle size range is between 20-250nm, with obvious tea tray like double membrane structure, meet the structural characteristics of bacterial outer membrane vesicle.The PDL1-OMVs provided by the application improves the weak targeting efficiency of PD-L1 binding peptide on the one hand, significantly improves the targeting ability of PD-L1 binding peptide, on the other hand, realizes the targeted functional modification of bacterial outer membrane vesicle, and PDL1-OMVs can realize specific targeting effect on tumor cells, which provides conditions for it as a targeted delivery tumor drug carrier.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a highly efficient bacterial outer membrane vesicle for targeting tumors and its preparation method. Background Technology

[0002] Breast cancer is the most common malignant tumor worldwide and a leading cause of cancer death. It is estimated that one in four women will develop breast cancer, and one in eight will die from it. Breast cancer is a hereditary disease caused by alterations in its genome structure. Genetic changes in tumor suppressor and oncogenes transform breast epithelial cells into a malignant phenotype. These genetic alterations also affect the behavior of breast cancer, including its response to treatment and clinical outcomes. Currently, there are various treatments for breast cancer, including surgery, radiotherapy, chemotherapy, targeted drug therapy, endoscopic therapy, and immunotherapy. While local surgical procedures such as surgery and radiotherapy are becoming less invasive and more precise, the accompanying systemic changes in patients are becoming increasingly complex. Currently, most researchers are focused on developing targeted therapies that have less impact on normal cells. Therefore, developing novel drug delivery systems that are highly efficient, low in toxicity, highly targeted, and biocompatible is a key direction in cancer treatment research.

[0003] Bacterial outer membrane vesicles (OMVs) are membranous vesicles primarily secreted by Gram-negative bacteria, composed of lipids, LPS, proteins, and RNA. OMVs serve as long-distance delivery vehicles, involved in a range of physiological processes, including intracellular and extracellular communication, toxin delivery, and polysaccharide hydrolysis. Due to their similar composition and properties to cell membranes, OMVs are adept at crossing cell barriers. Based on their membrane stability, immunogenicity, safety, and permeability, OMVs are currently widely used in the development of vaccines and drug delivery systems. Compared to traditional drug delivery carriers, OMVs offer several advantages, including larger drug loading space and stability, higher biocompatibility, appropriate immunogenicity, and lower cytotoxicity. However, OMVs themselves do not possess good targeting capabilities, preventing them from achieving maximum drug efficacy when used as drug delivery carriers. Based on genetic engineering research and the ease of modification of OMVs, tumor-specific antibodies or functionalized peptides can be conjugated to OMVs to directionally alter their properties, thereby improving their yield, safety, and targeting capabilities during drug delivery and providing a new reference for the clinical translation and application of OMVs.

[0004] Programmed death-ligand 1 (PD-L1) on cancer cells is an essential immune checkpoint protein. Its binding to programmed cell death-1 (PD-1) on T cells provides negative feedback regulation of activated T cells, preventing the immune system from attacking cancer cells and facilitating cancer immune escape. Since many cancer cells express PD-L1, blocking the PD-1 / PD-L1 axis is a crucial step in the anti-cancer immune response. Numerous studies have shown that PD-L1 protein can be degraded in proteases or lysosomes. Immune checkpoint inhibitors such as PD-L1 monoclonal antibodies can promote PD-L1 degradation, thereby enhancing cancer immunotherapy. However, only a small percentage of patients achieve treatment success, and severe treatment toxicity and side effects occur during and after treatment.

[0005] PD-L1 binding peptide (amino acid sequence CNYSKPTDRQYHF) is a small molecule inhibitory peptide that targets and binds to the PD-L1 receptor. It prevents PD-L1 from binding to the PD-1 receptor on the surface of T cells, thereby relieving immunosuppression of T cells and helping to restore their ability to recognize and kill tumor cells, thus enhancing the anti-tumor immune response. In addition, PD-L1 binding peptide can also be used as a tool for tumor-targeted imaging or precise drug delivery. However, PD-L1 binding peptide suffers from a short half-life and weak targeting ability, limiting its application in tumor immunotherapy or molecular imaging tools. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a highly efficient bacterial outer membrane vesicle for targeting tumors and its preparation method.

[0007] The present invention first constructs a fusion protein ClyA-PDL1 composed of a PD-L1 binding peptide and a membrane protein ClyA on the surface of bacterial outer membrane vesicles. The amino acid sequence of the fusion protein ClyA-PDL1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0008] Furthermore, the present invention also constructed a recombinant plasmid and recombinant bacteria capable of stably expressing the above-mentioned fusion protein ClyA-PDL1.

[0009] Finally, the present invention successfully prepared bacterial outer membrane vesicles that stably express the above-mentioned fusion protein ClyA-PDL1 using the above-mentioned recombinant bacteria, referred to as PDL1-OMVs.

[0010] In addition, this invention also provides a method for preparing PDL1-OMVs, specifically including the following steps:

[0011] (1) Design of the fusion protein ClyA-PDL1 sequence

[0012] Based on the PD-L1 binding peptide and the membrane protein cytolysin A (ClyA), a fusion protein ClyA-PDL1 with the structure Ncol-ClyA-Linker-3×MYC-Linker-PDL1-Xhol was designed, and the nucleotide sequence of the fusion protein is shown in SEQ ID NO.2.

[0013] (2) Construction of recombinant plasmids

[0014] The gene shown in SEQ ID NO.2 was cloned into the multiple cloning site of the pGEX-6P-1 vector, which is suitable for fusion protein expression, and the recombinant plasmid ClyA-PDL1-Pgex-6P-1 was successfully constructed. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.3.

[0015] (3) Construction of recombinant bacteria

[0016] The above recombinant plasmid was introduced into Escherichia coli strain W3110, and single colonies were selected. Based on the primers, bacterial PCR reaction, positive bands of the product agarose gel electrophoresis, and Western Blot, single colony C-PDL1 strains that stably express ClyA-PDL1 fusion protein were screened.

[0017] (4) Expression of the fusion protein ClyA-PDL1

[0018] C-PDL1 bacterial culture was inoculated into ampicillin-resistant LB medium and shaken at 37°C and 220 rpm for 4 h. Then, 1 M IPTG was added to bring the final IPTG concentration in the medium to 1 mM. After adding IPTG, the culture was incubated overnight at 21°C and 200 rpm to induce the expression of the exogenous protein, thus obtaining recombinant C-PDL1 strain that stably expresses the fusion protein ClyA-PDL1.

[0019] (5) Preparation of PDL1-OMVs

[0020] The C-PDL1 bacterial culture that stably expressed the fusion protein ClyA-PDL1 was centrifuged at 5000×g for 15 min at 4 °C. The supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. It was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 hours. The supernatant was discarded, and the precipitate was PDL1-OMVs.

[0021] This invention utilizes the targeting effect of PD-L1 binding peptides and the ease of modification of bacterial outer membrane vesicles to organically combine the two, resulting in bacterial outer membrane vesicles PDL1-OMVs with highly efficient tumor targeting. The PDL1-OMVs prepared by this invention have a particle size range of 20-250 nm, exhibit a distinct saucer-like bilayer membrane structure, consistent with the structural characteristics of bacterial outer membrane vesicles. Furthermore, the target protein PDL1 binding peptide is successfully modified onto the surface of the bacterial outer membrane vesicles, demonstrating excellent tumor-targeting activity. The PDL1-OMVs provided by this invention, on the one hand, improve the weak targeting efficacy of PD-L1 binding peptides, significantly enhancing their targeting ability; on the other hand, they achieve targeted functional modification of bacterial outer membrane vesicles, enabling PDL1-OMVs to achieve specific targeting of tumor cells, thus providing conditions for their use as carriers for targeted delivery of tumor drugs. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid ClyA-PDL1-Pgex-6P-1.

[0023] Figure 2 Results of Sanger sequencing identification of the prokaryotic expression plasmid ClyA-PDL1-pGEX-6P-1 (A), agarose gel electrophoresis image of recombinant plasmid ClyA-PDL1-Pgex-6P-1 (B), and results of identification of the expression of fusion protein ClyA-PDL1 in Escherichia coli (C).

[0024] Figure 3 Transmission electron microscope images of OMVs(A) and PDL1-OMVs(B).

[0025] Figure 4 Particle size distribution (A) and polydispersity index (PDI) (B) of OMVs and PDL1-OMVs.

[0026] Figure 5 The expression of the PDL1-OMVs surface fusion protein ClyA-PDL1 was detected by Western blotting.

[0027] Figure 6 The results of flow cytometry analysis were used to detect the targeting binding ability of OMVs and PDL1-OMVs after co-incubation with MDA-MB-231 and GES-1 for 2 h; *P<0.05, ****P<0.0001.

[0028] Figure 7 The results of flow cytometry analysis were used to detect the targeting binding ability of OMVs and PDL1-OMVs after co-incubation with MDA-MB-231 and GES-1 for 4 h; *P<0.05, ****P<0.0001.

[0029] Figure 8 Results of cell fluorescence microscopy detection of the binding ability of OMVs and PDL1-OMVs to MDA-MB-231 and GES-1 (A); and results of PDL1-OMVs binding ability to MDA-MB-231 and GES-1 (B); *P<0.05, ****P<0.0001.

[0030] Figure 9 The effects of co-incubating PDL1-OMVs and PDL1 single peptides with cells for 24 h on the signaling pathway of MDA-MB-231 breast cancer cells were investigated; compared with PDL1-OMVs, ***P<0.001. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] Construction of recombinant bacteria stably expressing the fusion protein ClyA-PDL1

[0034] (1) Design of the fusion protein ClyA-PDL1 sequence

[0035] Based on the PD-L1 binding peptide (sequence shown in SEQ ID NO.4) and the membrane protein cytolysin A (ClyA, sequence shown in SEQ ID NO.5), a fusion protein ClyA-PDL1 with the structure Ncol-ClyA-Linker-3×MYC-Linker-PDL1-Xhol was designed, and the sequence is shown in SEQ ID NO.2.

[0036] (2) Construction of recombinant plasmids

[0037] The gene shown in SEQ ID NO.2 was cloned into the multiple cloning site of the pGEX-6P-1 vector, which is suitable for fusion protein expression, and the recombinant plasmid ClyA-PDL1-Pgex-6P-1 was successfully constructed. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.3, and the structure of the recombinant plasmid is as follows. Figure 1 As shown.

[0038] (3) Construction of recombinant bacteria

[0039] The above recombinant plasmid was introduced into Escherichia coli strain W3110, and single colonies were selected. Based on the primers, bacterial PCR reaction, positive bands of the product agarose gel electrophoresis, and Western Blot, single colony C-PDL1 strains that stably express ClyA-PDL1 fusion protein were screened.

[0040] (4) Expression of the fusion protein ClyA-PDL1

[0041] C-PDL1 bacterial culture was inoculated into ampicillin-resistant LB medium and shaken at 37°C and 220 rpm for 4 h. Then, 1 M IPTG was added to bring the final IPTG concentration in the medium to 1 mM. After adding IPTG, the culture was incubated overnight at 21°C and 200 rpm to induce the expression of the exogenous protein, thus obtaining recombinant C-PDL1 strain that stably expresses the fusion protein ClyA-PDL1.

[0042] We first performed gene sequencing on the constructed recombinant plasmid ClyA-PDL1-Pgex-6P-1, and the results showed that the gene sequence was consistent with the expectation. Figure 2 As shown in Figure A. Subsequent agarose gel electrophoresis confirmed that the band of the recombinant plasmid ClyA-PDL1-Pgex-6P-1 was located approximately 1000 bp from the DNA DL 5000 Marker, consistent with the theoretical value of 1107 bp. The results are as follows. Figure 2 As shown in B. Western blot analysis then confirmed that the ClyA-PDL1 fusion protein was expressed in the C-PDL1 strain, but not in the wild-type W3110 strain, consistent with expectations. Figure 2 As shown in Figure C. The above results demonstrate that the recombinant plasmid ClyA-PDL1-Pgex-6P-1 was successfully constructed in this invention, and a single clone of Escherichia coli stably expressing the ClyA-PDL1 fusion protein was successfully screened.

[0043] Example 2

[0044] 1. Preparation of PDL1-OMVs

[0045] The C-PDL1 bacterial culture that stably expressed the fusion protein ClyA-PDL1 prepared in Example 1 was centrifuged at 5000×g for 15 min at 4 °C. The supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was concentrated 20 times using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD. It was then filtered again through a 0.22 μm filter membrane and centrifuged at 150,000×g for 3 hours. The supernatant was discarded, and the precipitate was PDL1-OMVs.

[0046] Wild-type bacterial outer membrane vesicles (OMVs) were prepared using the same method as described above, except that the bacterial solution used was wild-type W3110 bacterial solution.

[0047] 2. Identification of PDL1-OMVs

[0048] (1) Morphological identification

[0049] Transmission electron microscopy results show ( Figure 3 The diameters of OMVs and PDL1-OMVs ranged from 20 to 250 nm, both exhibiting a distinct saucer-like bilayer membrane structure, appearing singly, with a relatively calm background and few contaminants. These results indicate that the expression of heterologous proteins did not affect the basic structure of bacterial outer membrane vesicles, providing structural support for their widespread application.

[0050] (2) Particle size determination

[0051] The particle size of OMVs and PDL1-OMVs was further determined using dynamic light scattering (DLS), and the results are as follows: Figure 4 As shown, the particle sizes of OMVs and PDL1-OMVs are concentrated at approximately 133.1 nm and 144.2 nm, respectively, and the particle size distribution exhibits a unimodal normal distribution. The polydispersity index (PDI) of OMVs and PDL1-OMVs are 0.269 and 0.311, respectively, indicating good particle dispersibility. The defined diameter of bacterial outer membrane vesicles is 20-250 nm. The above results demonstrate that the PDL1-OMVs prepared in this invention conforms to the diameter range of bacterial outer membrane vesicles while maintaining a good nanostructure.

[0052] (3) Identification of the expression of the target protein PDL1 binding peptide on the surface of PDL1-OMVs membrane

[0053] We used Western blotting to detect the marker membrane protein ompA and the fusion protein tag MYC in bacterial OMVs to identify the expression location of the target protein in PDL1-OMVs. The results are as follows: Figure 5 As shown, OMVs, PDL1-OMVs, and OMVs and PDL1-OMVs treated with proteinase K all expressed the membrane protein ompA. When OMVs and PDL1-OMVs had their lipid membranes disrupted by SDS and were simultaneously digested with proteinase K, the membrane protein ompA was destroyed and no longer expressed. However, the MYC-tagged protein in PDL1-OMVs with intact lipid membranes could be digested by proteinase K, and the expression of the MYC-tagged protein was detectable in untreated PDL1-OMVs. These results indicate that the target protein PDL1-binding peptide was successfully modified onto the surface of bacterial outer membrane vesicles, providing conditions for it to exert its targeting effect.

[0054] Example 3

[0055] Validation of the targeting effect of PDL1-OMVs on tumor cells

[0056] 1. Staining and Marking Treatment

[0057] PDL1-OMVs and OMVs obtained from 1,000 mL of bacterial culture were resuspended separately in 900 μL of PBS buffer. DiR staining working solution was added at a 1:1 volume ratio, and the mixture was thoroughly mixed by pipetting. The mixture was then incubated at 37°C in the dark for 30 min. After incubation, the mixture was centrifuged at 150,000 × g for 3 hours. The supernatant was discarded, and the mixture was allowed to dry completely. The stained PDL1-OMVs and OMVs were then resuspended in 500 μL of PBS.

[0058] 2. Cell flow cytometry assay to detect the effects of PDL1-OMVs and OMVs uptake on breast cancer cells MDA-MB-231 and human gastric mucosal epithelial cells GES-1.

[0059] MDA-MB-231 and GES-1 were used at 2×10 5 Cells were evenly seeded into 12-well plates at a uniform density and cultured in the corresponding complete medium for 2 days. Two wells were then incubated with 20 μL of DIR-labeled LOMVs and 20 μL of PDL1-OMVs for 2 h each. Similarly, two more wells were incubated with 20 μL of DIR-labeled W3110-OMVs and 20 μL of C-PDL1-OMVs for 4 h each. Subsequently, 100 μL of trypsin was added to each well for digestion, and the cells were transferred to 1.5 mL sterile centrifuge tubes. The tubes were centrifuged at 800 rpm for 3 min, and the supernatant was discarded. 400 μL of sterile PBS was added to each tube, and the mixture was centrifuged at 1,000 rpm for 5 min. The supernatant was discarded, and 400 μL of sterile PBS buffer was added again for remixing. Flow cytometry was used to detect the number and proportion of cells bound to C-PDL1-OMVs.

[0060] The results are as follows Figure 6 , Figure 7 The results showed that, under different incubation times with cells, both GES-1 cells with low expression of the receptor gene PDL1 and cells with high expression of the receptor gene PDL1 exhibited significantly higher binding rates to PDL1-OMVs than to wild-type OMVs. This indicates that the PDL1-OMVs provided by this invention have a significant targeted binding effect on cells expressing the receptor gene PDL1 (results are shown in Figure 1). Figure 6 C Figure 7 (As shown in C). Furthermore, we calculated the differences in binding rates between PDL1-OMVs, OMVs, and GES-1 cells, and the differences in binding rates between PDL1-OMVs, OMVs, and MDA-MB-231 cells, and performed statistical analysis on the two differences. The results are shown in [Figure C]. Figure 6 D、 Figure 7As shown in Figure D, the results indicate that the PDL1-OMVs provided by this invention have a significantly higher targeting binding effect on MDA-MB-231 cells that highly express the receptor gene PDL1 than on GES-1 cells that low express the receptor gene PDL1 (P<0.05, P<0.0001). PDL1-OMVs can achieve specific targeting of cancer cells.

[0061] 3. Immunofluorescence microscopy was used to observe the uptake of PDL1-OMVs and OMVs by MDA-MB-231 breast cancer cells and GES-1 human gastric mucosal epithelial cells.

[0062] OMVs and PDL1-OMVs labeled with the fluorescent dye DIR were co-incubated with breast cancer cells MDA-MB-231 and human gastric mucosal epithelial cells GES-1 for 4 hours, respectively. The uptake was observed using immunofluorescence microscopy, and the fluorescence intensity was quantitatively analyzed.

[0063] like Figure 8 As shown in Figure A, after different incubation methods with cells, the fluorescence intensity of GES-1 cells with low expression of the receptor gene PDL1 and cells with high expression of the receptor gene PDL1 after incubation with PDL1-OMVs was significantly higher than that after incubation with wild-type OMVs (P<0.05, P<0.0001), indicating that the PDL1-OMVs provided by this invention have a significant targeted binding effect on cells expressing the receptor gene PDL1. Furthermore, as... Figure 8 As shown in Figure B, the fluorescence intensity of A549 cells after incubation with PDL1-OMVs was significantly higher than that of GES-1 cells after incubation with PDL1-OMVs (P<0.001). This indicates that PDL1-OMVs have a stronger targeting and binding ability to MDA-MB-231 in breast cancer cells and have a specific targeting effect on tumor cells. This result is consistent with the results of cell flow cytometry experiments.

[0064] Example 4

[0065] Comparison of the targeting effects of PDL1-OMVs and PDL1-binding peptides

[0066] MDA-MB-231 breast cancer cells were injected at a rate of 4 × 10⁻⁶. 5 The OMVs, PDL1-OMVs, and PDL1 binding peptide were added evenly to each well in a 6-well plate and cultured for 2 days. After incubating 20 μL each of OMVs, PDL1-OMVs, and PDL1 binding peptide with MDA-MB-231 for 24 h, RNA was extracted and reverse transcribed into cDNA. PCR was then performed to detect the activation of the receptor gene PDL1.

[0067] The PDL1 gene is the receptor gene targeted by the PDL1-binding peptide. The expression level of PDL1 mRNA can reflect the activation effect of the targeted substance, and further assess the integrity of the targeted substance's function. Results are as follows... Figure 9 The results showed that, compared with OMVs and PDL1 monopeptides, PDL1-OMVs significantly enhanced the activation ability of receptor genes (P<0.001). These results indicate that the PDL1-OMVs provided by this invention can significantly enhance the targeting effect of PDL1-binding peptides.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fusion protein ClyA-PDL1 consisting of a PD-L1 binding peptide and a membrane protein colysozyme A (ClyA) on the surface of a bacterial outer membrane vesicle, wherein the amino acid sequence of the fusion protein ClyA-PDL1 is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO.

2. 2.A recombinant plasmid for expressing the fusion protein ClyA-PDL1 according to claim 1. 3.A recombinant bacteria for expressing the fusion protein ClyA-PDL1 according to claim 1. 4.A bacterial outer membrane vesicle (PDL1-OMVs) for expressing the fusion protein ClyA-PDL1 according to claim 1.

5. The method of producing bacterial outer membrane vesicles PDL1-OMVs according to claim 4, characterized in that, Specifically comprising the following steps: (1) Designing the sequence of the fusion protein ClyA-PDL1 Based on the PD-L1 binding peptide and the membrane protein colysozyme A (ClyA), a fusion protein ClyA-PDL1 sequence with the structure of Ncol-ClyA-Linker-3×MYC-Linker-PDL1-Xhol is designed, wherein the nucleotide sequence of the fusion protein is shown as SEQ ID NO.2; (2) Construction of the recombinant plasmid The gene shown as SEQ ID NO.2 is cloned into the multiple cloning site on the pGEX-6P-1 vector suitable for fusion protein expression, and a recombinant plasmid ClyA-PDL1-Pgex-6P-1 is successfully constructed, wherein the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.3; (3) Construction of the recombinant bacteria The above-mentioned recombinant plasmid is introduced into the W3110 Escherichia coli strain, and a single colony strain C-PDL1 stably expressing the ClyA-PDL1 fusion protein is selected by picking a single colony, performing a bacterial liquid PCR reaction according to the primer, and screening a positive band by agarose gel electrophoresis and Western Blot; (4) Expression of the fusion protein ClyA-PDL1 The C-PDL1 bacterial liquid is inoculated into an ampicillin-resistant LB medium, and after shaking the bacteria at 37 ℃ and 220 rpm for 4 h, 1 M IPTG is added to make the final concentration of IPTG in the medium 1 mM; after adding, the expression of the exogenous protein is induced overnight at 21 ℃ and 200 rpm, and the recombinant bacteria C-PDL1 stably expressing the fusion protein ClyA-PDL1 are obtained; (5) Preparation of PDL1-OMVs After the C-PDL1 bacterial liquid stably expressing the fusion protein ClyA-PDL1 is centrifuged at 4 ℃ and 5000×g for 15 min, the supernatant is collected, filtered with a 0.45 μm filter membrane, and concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass of 100 kD; the filtrate is filtered again with a 0.22 μm filter membrane, and then centrifuged at 150,000×g for 3 hours, and the supernatant is discarded, and the precipitate is PDL1-OMVs.

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