Bacterial outer membrane vesicle modified by anti-tumor peptide and preparation method of bacterial outer membrane vesicle

Abstract

The invention provides an anti-tumor peptide modified bacterial outer membrane vesicle and a preparation method thereof, and relates to the technical field of engineering bacterial outer membrane vesicles. According to the invention, a recombinant plasmid capable of expressing a ClyA-A25 fusion protein in a prokaryote is designed and constructed, and the engineering bacterium outer membrane vesicle W-A25OMV modified by the anti-tumor peptide A25 is successfully prepared by using the recombinant plasmid. The W-A25OMV retains the basic structure of bacterial outer membrane vesicles, enhances the anti-tumor biological activity of A25 peptide, has good stability, shows a remarkable inhibition effect on invasion and migration of tumor cells, can effectively promote apoptosis of the tumor cells, and has development and application potential as an anti-tumor drug.

Description

Technical Field

[0001] This invention relates to the field of engineered bacterial outer membrane vesicle technology, specifically to a bacterial outer membrane vesicle modified with an anti-tumor peptide and its preparation method. Background Technology

[0002] Since the beginning of the 21st century, the incidence and mortality rates of cancer have been rising continuously, gradually becoming one of the major threats to human health. The development of safe and effective anti-tumor drugs has become an urgent problem to be solved in medicine. While traditional chemotherapy has certain efficacy, the resulting cytotoxicity and drug resistance problems greatly restrict its clinical application. In recent years, rapidly developing new cancer therapies such as targeted therapy and immunotherapy have achieved some breakthroughs, but they also have many limitations, such as long drug development cycles, high prices, lack of universal applicability, and serious drug resistance, making it difficult to widely apply and promote them in precision cancer treatment, posing a huge challenge to cancer treatment.

[0003] A25 is a 25-amino acid polypeptide whose sequence includes the integrin-targeting ligand RGD sequence and the antitumor polypeptide ES-2 sequence. Studies have found that A25 polypeptide has the effect of killing various tumor cells, including gastric cancer, breast cancer, colorectal cancer, and ovarian cancer. Mechanistic studies show that it can interact with integrin αVβ3 and its co-receptor heparin sulfate glycoprotein on the surface of vascular endothelial cells, thereby inhibiting tumor angiogenesis by inhibiting the migration of tumor vascular endothelial cells. A25 has good antitumor effects and mild toxic side effects, showing potential for the development and application of antitumor drugs. However, A25's insufficient stability and short half-life in vivo make it difficult to effectively accumulate in tumors, thus limiting its drug development and clinical application.

[0004] In recent years, the rapid development of nanotechnology and materials science has brought new opportunities to cancer treatment. Nanomaterials, with their nanoscale structure and size, can be used to load small molecule compounds or biomacromolecules to form nanomedicine systems. Compared with traditional small molecule drugs or biomacromolecules, nanomedicines can improve cellular recognition and uptake efficiency or enhance bioavailability through the regulation of surface properties or size. Furthermore, compared with free drugs, targeted delivery based on nanoparticles helps reduce cytotoxicity, protect drugs from degradation, and enhance drug solubility. Currently, there are many types of nanomedicine carriers, with polymer micelles, liposomes, and biological proteins being among the most widely studied. These materials have met the requirements for in vivo drug delivery via blood circulation and drug release, but their therapeutic effects remain limited, with a lack of tumor penetration being one of the main bottlenecks restricting their efficacy.

[0005] Bacterial outer membrane vesicles (OMVs) are nanoscale lipid bilayer vesicles secreted by Gram-negative bacteria during growth via budding. They have a diameter of approximately 20-300 nm. They are primarily composed of the bacterial outer membrane and periplasmic components. The outer membrane includes glycerophospholipids, phosphatidylethanolamine, and phosphatidylglycerol. The vesicle lumen contains various compounds from the periplasm or cytoplasm, such as proteins, DNA, RNA, and peptidoglycans. Compared to traditional nanomaterials used for drug delivery, the unique membrane structure and surface proteins of OMVs enable them to protect drugs from clearance by the phagocytic system. Furthermore, OMVs exhibit excellent tumor permeability, allowing them to cross tumor barriers and achieve precise drug delivery. These superior properties make bacterial outer membrane vesicles a promising new anti-tumor drug carrier. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention utilizes OMV as a carrier and engineers it by modifying the surface of OMV with A25 peptide to obtain bacterial outer membrane vesicles W-A25 OMV with anti-tumor effects.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] First, the present invention provides a bacterial outer membrane vesicle modified with an antitumor peptide, wherein the surface of the bacterial outer membrane vesicle is modified with an A25 peptide with an amino acid sequence as shown in SEQ ID NO.1, or an A25 peptide with a nucleotide sequence as shown in SEQ ID NO.8.

[0009] Specifically, the A25 peptide is modified on the surface of bacterial outer membrane vesicles by binding to the bacterial outer membrane vesicle surface protein ClyA.

[0010] Specifically, the A25 peptide binds to the bacterial outer membrane vesicle surface protein ClyA to obtain the fusion protein ClyA-A25 with an amino acid sequence as shown in SEQ ID NO.2 or a nucleotide sequence as shown in SEQ ID NO.3.

[0011] Furthermore, this invention also provides a method for preparing bacterial outer membrane vesicles modified with an anti-tumor peptide, wherein the bacterial outer membrane vesicles are abbreviated as W-A25 OMV, and the W-A25 OMV is prepared according to the following steps:

[0012] (I) ClyA-A25 fusion protein gene synthesis

[0013] The gene sequence of the fusion protein ClyA-A25 was obtained using overlap extension PCR technology. The amino acid sequence of the fusion protein ClyA-A25 is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.3. The structure of the fusion protein ClyA-A25 is ClyA-Linker-3×Myc-Linker2-A25.

[0014] (II) Construction of recombinant plasmid expressing fusion protein ClyA-A25

[0015] The gene encoding the nucleotide sequence shown in SEQ ID NO.3 was cloned into the multiple cloning site on the pGEX-6P-1 vector to construct the recombinant plasmid ClyA-A25-pGEX-6P-1; the recombinant plasmid sequence is shown in SEQ ID NO.9.

[0016] (III) Construction of recombinant bacteria expressing the fusion protein ClyA-A25

[0017] W3110 E. coli competent cells were thawed on ice, and the recombinant plasmid ClyA-A25-pGEX-6P-1 was added to the competent cells. The cells were then incubated on ice for 30 min, followed by heat shock at 42°C for 60 s and incubation on ice for 2 min. 800 μL of antibiotic-free LB medium was then added, and the cells were cultured at 200 rpm and 37°C for 1 h. The bacterial culture was then spread onto LB agar plates containing ampicillin and cultured overnight at 37°C. The next day, single clones were picked, cultured in a shake culture, and sequenced to identify the recombinant bacteria expressing the fusion protein ClyA-A25, which is referred to as strain W-A25.

[0018] (iv) Preparation of W-A25 OMV

[0019] (1) Add 500 mL of ampicillin-resistant LB medium to a 1 L Erlenmeyer flask, inoculate 10 μL of W-A25 bacterial culture into the medium, and shake the culture at 37 °C and 220 rpm for 4 h. Measure the OD value of the bacterial culture every 20 minutes. When the OD value reaches 0.5-0.6, add 500 μL of 1 M IPTG to make the final IPTG concentration in the medium 1 mM. Then, induce the expression of exogenous protein overnight at 21 °C and 200 rpm to obtain 500 mL of W-A25 bacterial culture.

[0020] (2) After centrifuging the W-A25 bacterial culture at 4℃ and 5,000×g for 15 min, collect the supernatant and filter it with a 0.45μm filter membrane. Concentrate the filtrate 20 times with an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kDa. Filter it again with a 0.22μm filter membrane, and then centrifuge it at 150,000×g for 3 hours. Discard the supernatant and the precipitate is W-A25 OMV.

[0021] The bacterial outer membrane vesicles W-A25 OMV modified with the antitumor peptide A25 provided by this invention can be used to prepare drugs for treating tumors, including gastric cancer, breast cancer, colorectal cancer, cervical cancer, etc.

[0022] This invention designed and constructed a recombinant plasmid capable of expressing the ClyA-A25 fusion protein in prokaryotes. Using this recombinant plasmid, engineered bacterial outer membrane vesicles (W-A25 OMVs) modified with the antitumor peptide A25 were successfully prepared. Characterization results showed that the A25 peptide was successfully modified on the outer membrane surface of the OMVs without affecting their particle size, structure, or other characteristics, providing a foundation for the use of W-A25 OMVs in the preparation of antitumor drugs.

[0023] Furthermore, we investigated and verified the antitumor effect of W-A25 OMV provided by this invention. The experimental results showed that W-A25 OMV significantly inhibited the invasion and migration of tumor cells and effectively promoted tumor cell apoptosis, while wild-type OMV did not show obvious antitumor effect.

[0024] Although A25 monopeptide possesses broad-spectrum antitumor activity, its poor stability and short half-life in blood severely hinder its antitumor effect. Therefore, we further investigated the effects of W-A25 OMV and A25 monopeptide incubated with serum for different times on tumor cell invasion, migration, and apoptosis. Experimental results showed that W-A25 OMV significantly inhibited tumor cell migration and invasion compared to A25 monopeptide, and also significantly promoted tumor cell apoptosis. The W-A25 OMV prepared in this invention retains the basic structure of bacterial outer membrane vesicles while enhancing the antitumor biological activity of A25 peptide.

[0025] The key point is that W-A25 OMV still showed an inhibitory effect on the invasion and migration of tumor cells after co-incubation with serum for 1 h and 6 h, and effectively promoted the apoptosis of tumor cells. However, after co-incubation with serum for 1 h or 6 h, A25 monopeptide did not show a significant inhibitory effect on the invasion and migration of tumor cells, and the promoting effect on the apoptosis of tumor cells was also significantly reduced. This indicates that W-A25 OMV prepared in this invention has better stability in serum, effectively solving the problems of poor stability and short half-life of A25 monopeptide, and has the potential for development and application as an anti-tumor drug. Attached Figure Description

[0026] Figure 1Characterization results of ClyA-A25-pGEX-6P-1, where (A) is the identification of the ClyA-A25-pGEX-6P-1 prokaryotic expression plasmid by agarose gel electrophoresis; (B) is the identification of the ClyA-A25-pGEX-6P-1 prokaryotic expression plasmid by sequencing.

[0027] Figure 2 Western blot was used to identify the expression of W-A25 OMV and W3110 OMV marker proteins and exogenous proteins, using OmpA and Myc antibodies.

[0028] Figure 3 Particle size analysis (A), PDI distribution (B), and transmission electron microscopy identification of structure and morphology of W-A25 OMV and W3110 OMV (C).

[0029] Figure 4 Inhibitory effect of W-A25 OMV and W3110 OMV on breast cancer cell migration; **P<0.01.

[0030] Figure 5 Inhibitory effects of W-A25 OMV and W3110 OMV on the invasion of gastric cancer cells (Figure A) and breast cancer cells (B); *P<0.05, ***P<0.001.

[0031] Figure 6 The effects of W-A25 OMV and W3110 OMV on promoting apoptosis in gastric cancer cells (Figures A and C) and breast cancer cells (Figures B and D); **P<0.01, ***P<0.001.

[0032] Figure 7 Comparison of the inhibitory effects of W-A25 OMV and A25 peptide on tumor cell migration after incubation with serum for different times; *P<0.05, **P<0.01.

[0033] Figure 8 Comparison of the inhibitory effects of W-A25 OMV and A25 peptide on tumor cell invasion after incubation with serum for different times; *P<0.05, **P<0.01.

[0034] Figure 9 Comparison of the effects of W-A25 OMV and A25 peptide on promoting tumor cell apoptosis after incubation with serum for different times; **P<0.01. Detailed Implementation

[0035] 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.

[0036] Example 1

[0037] Preparation and identification of bacterial outer membrane vesicles W-A25 OMV modified with antitumor peptide A25

[0038] 1. Design and synthesis of the ClyA-A25 fusion protein gene sequence

[0039] We designed a method to sequentially insert a Linker gene, a 3×Myc tag protein gene, a Linker2 protein gene, and an A25 peptide gene into the 3' end of the E. coli ClyA gene to obtain the fusion protein ClyA-A25 gene. The specific structure of the fusion protein encoded by this gene is: ClyA-Linker-3×Myc-Linker2-A25. The amino acid sequence of this fusion protein is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.3.

[0040] The gene sequences of the fusion protein ClyA-A25 were obtained using overlap extension PCR technology, wherein the ClyA gene sequence is shown in SEQ ID NO.4; the Linker protein gene sequence is shown in SEQ ID NO.5; the 3×Myc tag protein gene sequence is shown in SEQ ID NO.6; the Linker2 protein gene sequence is shown in SEQ ID NO.7; and the A25 peptide gene sequence is shown in SEQ ID NO.8.

[0041] 2. Construction of recombinant plasmid expressing fusion protein ClyA-A25

[0042] The gene fragment and pGEX-6P-1 plasmid were digested with EcoNI and XhoI as shown in SEQ ID NO.3. The digestion products were then separated by 1% agarose gel electrophoresis, and the digested fragments were recovered using a gel recovery kit. EcoNI-ClyA-Linker-3×Myc-Linker2-A25-XhoI and pGEX-6P-1 were ligated using T4 ligase to obtain the recombinant plasmid ClyA-A25-pGEX-6P-1 expressing the fusion protein ClyA-A25. The sequence of the recombinant plasmid is shown in SEQ ID NO.9.

[0043] The ligated plasmids were separated by 1% agarose gel electrophoresis, and the electrophoretic bands were obtained as follows: Figure 1 As shown in Figure A, the observed band of the ClyA-A25-pGEX-6P-1 plasmid is consistent with the theoretical value of 5410 bp. Subsequently, the plasmid was sequenced using Sanger sequencing, and the results are as follows... Figure 1 As shown in B, the 5' end sequence, 3' end sequence of the fusion gene, and the upstream and downstream vector sequences of the gene are consistent with the expected sequences, indicating that the vector was successfully constructed.

[0044] 3. Transform W3110 Escherichia coli with ClyA-A25-pGEX-6P-1 plasmid.

[0045] W3110 *E. coli* competent cells were thawed on ice, and the recombinant plasmid ClyA-A25-pGEX-6P-1 was added to the competent cells. The cells were then incubated on ice for 30 min, followed by heat shock at 42°C for 60 s and incubation on ice for 2 min. 800 μL of antibiotic-free LB medium was then added, and the cells were cultured at 200 rpm and 37°C for 1 h. The bacterial culture was then plated onto LB agar plates supplemented with ampicillin and incubated overnight at 37°C. The next day, single clones were picked, cultured in a shake culture, and sequenced for identification. The strain stably transformed with the ClyA-A25-pGEX-6P-1 plasmid was identified as the single clone expressing the ClyA-A25 fusion protein, and this strain is abbreviated as W-A25 strain.

[0046] 4. Induction of ClyA-A25 protein expression in strain W-A25

[0047] Add 500 mL of ampicillin-resistant LB medium to a 1 L Erlenmeyer flask, and inoculate 10 μL of W-A25 bacterial culture into the medium, labeling it W-A25. Add 500 mL of non-antibiotic LB medium to a 1 L Erlenmeyer flask, and inoculate 10 μL of W3110 bacterial culture into the medium, labeling it W3110. After shaking the W-A15 and W3110 cultures at 37℃ and 220 rpm for 4 h, measure the OD value of the bacterial cultures every 20 minutes. When the OD value reaches 0.5-0.6, add 500 μL of 1 M IPTG to bring the final IPTG concentration in the medium to 1 mM. Then, induce exogenous protein expression overnight at 21℃ and 200 rpm to obtain 500 mL of each of W-A25 and W3110 bacterial cultures.

[0048] 5. Extraction of W-A25 OMV

[0049] After centrifuging W-A25 and W3110 bacterial cultures at 4°C and 5,000×g for 15 min, 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 kDa. 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 precipitates were W-A25 OMV and W3110 OMV.

[0050] 6. Identification of W-A25 OMV bacterial exovesicles

[0051] OMV precipitate was dissolved in PBS to obtain W-A25 OMV solution. W3110 wild-type E. coli OMV was prepared using the same method. Three parallel groups were set up for the extracted W3110 OMV and W-A25 OMV: control group without treatment, proteinase K digestion group, and group that underwent both SDS lipid membrane disruption and proteinase K digestion. The expression of the tag protein Myc and the membrane protein OmpA was detected by Western blot using anti-Myc antibody and anti-OmpA antibody.

[0052] The results are as follows Figure 2 As shown, the OMV marker protein OmpA was detected in the untreated W-A25 OMV and W3110 OMV groups. Additionally, the Myc tag of the ClyA-A25 fusion protein was detected in the W-A25 OMV group, but not in the W3110 OMV group. In the W-A25 OMV and W3110 OMV groups digested with proteinase K, OmpA expression was detected, but Myc-tagged protein expression was not detected in the ClyA-A25 OMV. In the W-A25 OMV and W3110 OMV groups treated with both SDS and proteinase K, neither Myc nor OmpA proteins were detected. OmpA is an internal OMV membrane protein and cannot be directly digested by proteinase K. However, SDS has a cell membrane disrupting effect, and co-incubation with SDS and proteinase K can digest the internal OMV protein. Therefore, the above results show that the W-A25OMV and W3110 OMV groups express the OMV marker protein, indicating that the OMV prepared in this invention is indeed OMV, and that the A25 protein was successfully modified on the outer membrane surface of bacterial outer membrane vesicles.

[0053] The particle size distribution and PDI of W-A25 OMV and W3110 OMV were then analyzed using a particle size analyzer. The particle size distribution is shown below. Figure 3As shown in Figure A, the average particle sizes of W-A25 OMV and W3110 OMV are 64.8 nm and 62.7 nm, respectively. There is no significant difference in particle size between the two groups, and the particle sizes are consistent with the theoretical OMV particle size. The PDI measurement results are as follows... Figure 3 As shown in Figure B, the PDI values ​​for the W-A25 OMV group and the W3110 OMV group are 0.50 and 0.44, respectively, indicating that the OMV particles are well dispersed.

[0054] Finally, the appearance and morphology of OMV were identified using transmission electron microscopy, and the results are as follows: Figure 3 As shown in Figure C, both W-A25 OMV and W3110 OMV exhibit typical saucer-like bilayer membrane structures with intact structures and clear backgrounds, indicating that high-purity OMV has been prepared. There is no morphological difference between W-A25 OMV and W3110 OMV, suggesting that surface modification of the A25 protein has no effect on the morphological structure of the OMV.

[0055] The above results indicate that the A25 peptide was successfully modified on the outer surface of the OMV without affecting the particle size, structure, or other characteristics of the OMV, providing a basis for the use of W-A25 OMV in the preparation of antitumor drugs.

[0056] Example 2

[0057] Antitumor effects of W-A25 OMV

[0058] 1. W-A25 OMV inhibits tumor cell migration

[0059] Tumor cell migration is a key ability of tumors to metastasize to distant sites. Triple-negative breast cancer, for example, has a high metastatic potential, which can shorten patient survival. We first conducted a cell scratch assay to investigate the inhibitory effect of W-A25 OMV on the migration of triple-negative breast cancer tumor cells.

[0060] The specific method is as follows: Inoculate 5×10 5 MDA-MB-231 breast cancer cells were cultured overnight in 6-well plates until 80% confluence was achieved. The wells were then scored using a pipette tip. Three control groups were established: a W-A25 OMV group, a W3110 OMV control group, and a negative control group (NC group). The final concentration of A25 peptide in the W-A25 OMV group was 0.2 μmol·L⁻¹. -1 The W3110 OMV control group received an equal volume of W3110 OMV, while the NC group received an equal volume of PBS. Cell scratch region images were recorded under a microscope at 0h, 12h, and 24h, and cell migration rate was calculated.

[0061] The results are as follows Figure 4As shown in the figure, compared with the NC group, the cell migration rate of the W-A25 OMV group decreased significantly by 42.6% and 34.6% after 12 h and 24 h of drug administration, respectively, with significant differences (P<0.01); while the W3110 OMV group had no significant inhibitory effect on the migration of breast cancer cells (P>0.05). These results indicate that the W-A25OMV prepared in this invention has a significant inhibitory effect on the migration of MDA-MB-231 breast cancer cells.

[0062] 2. W-A25 OMV inhibits tumor cell invasion.

[0063] Before metastasizing, tumor cells must breach the basement membrane and surrounding matrix to enter the bloodstream or lymphatic system; therefore, cell invasion is a key capability for early tumor metastasis. We investigated the inhibitory effect of W-A25 OMV on tumor cell invasion using a Transwell assay.

[0064] The specific method is as follows: Place the Transwell chamber in a 24-well plate to construct a two-layer culture chamber, and then place 2×10⁻⁶ cells into the well. 5 HGC-27 cells and MDA-MB-231 cells were seeded into the upper wells, respectively. A W-A25 OMV group, a W3110 OMV control group, and a negative control group (NC group) were set up. The W-A25 OMV group received A25 peptide at a final concentration of 0.2 μmol·L⁻¹. -1 The control group of W-A25 OMV and W3110 OMV was given an equal volume of W3110 OMV, while the control group of NC group was given an equal volume of PBS. After culturing for 24 h and 48 h, crystal violet staining was performed, and the cells were observed and counted under a microscope. Each group was set up with 3 parallel replicates.

[0065] The results are as follows Figure 5 As shown in the figure. Compared with the NC group, after 24 h of drug administration, W-A25 OMV significantly inhibited the invasion of HGC-27 gastric cancer cells, reducing the number of invasive cells by 31.8% (P<0.05), and W-A25 OMV significantly inhibited the invasion of MDA-MB-231 breast cancer cells, reducing the number of invasive cells by 57.1% (P<0.001). After 48 h of drug administration, W-A25 OMV showed highly significant inhibitory effects on the invasion of both HGC-27 and MDA-MB-231 cells, reducing the number of invasive cells by 42.8% and 71.0%, respectively (P<0.001). Furthermore, W3110 OMV showed no significant inhibitory effect on the invasion of HGC-27 and MDA-MB-231 cells at any time point (P>0.05). The above results indicate that the W-A25 OMV prepared in this invention has a significant inhibitory effect on the invasive ability of HGC-27 gastric cancer cells and MDA-MB-231 breast cancer cells.

[0066] 3. W-A25 OMV promotes tumor cell apoptosis.

[0067] Apoptosis rate is a core tool for evaluating the efficacy of tumor drug treatment. Therefore, we used flow cytometry to investigate the promoting effect of W-A25 OMV on tumor cell apoptosis.

[0068] The specific method is as follows: 5×10 5 HGC-27 cells and MDA-MB-231 cells were seeded into 6-well plates. When cell confluence reached 80%, a W-A25 OMV group and a W3110 OMV control group were established. The W-A25 OMV group received A25 peptide at a final concentration of 0.2 μmol·L⁻¹. -1 W-A25 OMV and W3110 OMV control groups were supplemented with an equal amount of W3110 OMV. After culturing for 6 h and 24 h, the cells were digested, and the apoptosis rate was detected by flow cytometry.

[0069] The results are as follows Figure 6 As shown in the figure, under 6h and 24h conditions, the apoptosis rate of MDA-MB-231 cells in the W-A25 OMV group was significantly higher than that in the W3110 OMV group (P<0.01); under 24h conditions, the apoptosis rate of HGC-27 cells in the W-A25 OMV group was significantly higher than that in the W3110 OMV group (P<0.001). These results indicate that the W-A25 OMV prepared in this invention has a significant promoting effect on apoptosis of HGC-27 gastric cancer cells and MDA-MB-231 breast cancer cells.

[0070] The above results indicate that, compared with wild-type OMV, the A25 peptide modified on the surface of bacterial outer membrane vesicles still retains its anti-tumor biological activity. W-A25 OMV shows a significant inhibitory effect on the invasion and migration of tumor cells and can effectively promote tumor cell apoptosis, thus exhibiting significant anti-tumor effects.

[0071] Example 3

[0072] Although A25 monopeptide possesses broad-spectrum antitumor activity, its poor stability and short half-life in blood severely hinder its antitumor effect. Therefore, we further investigated the effects of W-A25OMV and A25 monopeptide incubated with serum for different durations on tumor cell invasion, migration, and apoptosis.

[0073] 1. Effect of serum incubation time on the inhibitory effect of W-A25 OMV and A25 monopeptide on cell migration

[0074] W-A25 OMV and A25 monopeptide were incubated in serum for 0 h, 1 h, and 6 h, respectively, and then added to 6-well plates seeded with MDA-MB-231 breast cancer cells to achieve a theoretical final concentration of 0.2 μmol·L⁻¹. -1 A negative control group (NC group) with added PBS was also set up. The cell culture wells were scratched using pipette tips, and the cell scratched areas were recorded under a microscope at 0h and 24h, and the cell migration rate was calculated.

[0075] Experimental results are as follows Figure 7 As shown in the figure. After 0 h of incubation in serum, both W-A25 OMV and A25 monopeptide showed significant inhibitory effects on cell migration compared to the NC group (P<0.01), with the cell migration rate of the W-A25 OMV group being significantly lower than that of the A25 monopeptide group (P<0.05). After 1 h of incubation in serum, compared to the NC group, the W-A25 OMV group showed a significant inhibitory effect on cell migration (P<0.05), but the A25 monopeptide group had no inhibitory effect on tumor cell migration (P>0.05).

[0076] 2. Effect of serum incubation time on the inhibitory effect of W-A25 OMV and A25 peptide on cell invasion

[0077] W-A25 OMV and A25 peptide were incubated in serum for 0 h, 1 h, and 6 h, respectively, and then added to chambers seeded with MDA-MB-231 cells to achieve a theoretical final concentration of 0.2 μmol·L⁻¹ of A25 peptide. -1 A negative control group (NC group) with added PBS was also set up. After 24 h and 48 h of culture, crystal violet staining was performed, and the cells were observed and counted under a microscope. Each group was set up with 3 parallel replicates.

[0078] Experimental results are as follows Figure 8 As shown, after incubation in serum for 0 h, both W-A25 OMV and A25 peptide significantly inhibited cell invasion compared to the NC group. After 24 h of culture following drug administration, the number of invasive cells in the W-A25 OMV group decreased by 57.6%, and the number of invasive cells in the A25 peptide group decreased by 42.1%, with significant differences (P<0.01). After 48 h of culture following drug administration, the number of invasive cells in the W-A25 OMV group decreased by 56.6%, and the number of invasive cells in the A25 peptide group decreased by 42.2% (P<0.05, P<0.01). Among them, the inhibitory effect of W-A25 OMV on tumor cell invasion was significantly better than that of A25 peptide (P<0.05).

[0079] After incubation in serum for 1 hour, compared with the NC group, W-A25 OMV and A25 peptide still showed significant inhibitory effects on cell invasion. After 24 hours of culture following drug administration, the number of invasive cells in the W-A25 OMV group decreased by 49.4%, and the number of invasive cells in the A25 peptide group decreased by 23.9% (P<0.01). After 48 hours of culture following drug administration, the number of invasive cells in the W-A25 OMV group decreased by 44.7%, and the number of invasive cells in the A25 peptide group decreased by 21.6% (P<0.05, P<0.01). Among them, the inhibitory effect of W-A25 OMV on tumor cell invasion was significantly better than that of A25 peptide (P<0.01).

[0080] After 6 hours of incubation in serum, W-A25 OMV still significantly inhibited cell invasion compared to the NC group. After 24 hours of culture following drug administration, the number of invasive cells in the W-A25 OMV group decreased by 32.6%, and after 24 hours of culture following drug administration, the number of invasive cells in the W-A25 OMV group decreased by 39.4% (P<0.01). After 6 hours of incubation in serum, A25 monopeptide did not inhibit tumor cell invasion.

[0081] (3) Effect of serum incubation time on the apoptosis-inducing ability of W-A25 OMV and A25 peptide

[0082] W-A25 OMV and A25 peptide were incubated in serum for 0 h, 1 h, and 6 h, respectively, and then added to six-well plates seeded with MDA-MB-231 cells to achieve a theoretical final concentration of 0.2 μmol·L⁻¹. -1 A negative control group with added PBS was also set up. After 24 hours of culture, the cells were digested, stained, and analyzed by flow cytometry.

[0083] Experimental results are as follows Figure 9 As shown in the figure, in the W-A25 OMV group, the apoptosis rate was 22.7% after 0 h of co-incubation with serum. After 1 h and 6 h of co-incubation with serum, the apoptosis rates were 21.6% and 18.5%, respectively. The apoptosis rate of the W-A25 OMV group did not decrease significantly with increasing incubation time with serum, and the difference was not statistically significant (P>0.05). In the A25 peptide group, the apoptosis rate was 16.8% after 0 h of co-incubation with serum. After 1 h and 6 h of co-incubation with serum, the apoptosis rates were 10.7% and 4.8%, respectively, which were significantly lower than the apoptosis rate after 0 h of co-incubation with serum (P<0.01). These results indicate that the W-A25 OMV group still has good anti-tumor activity after 6 h of incubation in serum and can effectively promote cell apoptosis.

[0084] Based on the above results, W-A25 OMV exhibits significantly better inhibitory effects on tumor cell migration and invasion than A25 monopeptide, and its effect on promoting tumor cell apoptosis is also significantly better than that of A25 monopeptide. The W-A25 OMV prepared in this invention retains the basic structure of bacterial outer membrane vesicles while enhancing the antitumor biological activity of A25 peptide. Importantly, W-A25 OMV, after co-incubation with serum for 1 h and 6 h, still showed inhibitory effects on tumor cell invasion and migration, and effectively promoted tumor cell apoptosis; while A25 monopeptide, after co-incubation with serum for 1 h or 6 h, did not show significant inhibitory effects on tumor cell invasion and migration, and its promoting effect on tumor cell apoptosis significantly decreased. This indicates that the W-A25 OMV prepared in this invention has better stability in serum, effectively solving the problems of poor stability and short half-life of A25 monopeptide, and has the potential for development and application as an antitumor drug.

[0085] 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. An antitumor peptide-modified bacterial outer membrane vesicle, characterized in that, The bacterial outer membrane vesicle is modified on the surface with an A25 peptide with an amino acid sequence as shown in SEQ ID NO.

1.

2. A bacterial outer membrane vesicle modified with an anti-tumor peptide according to claim 1, characterized in that, The A25 peptide is modified on the surface of the bacterial outer membrane vesicle by binding with a surface protein ClyA of the bacterial outer membrane vesicle.

3. A bacterial outer membrane vesicle modified with an antitumor peptide according to claim 1, characterized in that, The A25 peptide binds with the surface protein ClyA of the bacterial outer membrane vesicle to obtain a fusion protein ClyA-A25 with an amino acid sequence as shown in SEQ ID NO. 2 or a nucleotide sequence as shown in SEQ ID NO.

3.

4. The method for preparing bacterial outer membrane vesicles modified with an antitumor peptide as described in any one of claims 1 to 3, characterized in that, The bacterial outer membrane vesicle is referred to as W-A25 OMV, and the W-A25 OMV is prepared according to the following steps: (1) Synthesis of ClyA-A25 fusion protein gene The fusion protein ClyA-A25 gene sequence is obtained by using overlap extension PCR technology, the amino acid sequence of the fusion protein ClyA-A25 is as shown in SEQ ID NO. 2, and the nucleotide sequence is as shown in SEQ ID NO. 3; the structure of the fusion protein ClyA-A25 is ClyA-Linker-3xMyc-Linker2-A25; (2) Construction of expression fusion protein ClyA-A25 recombinant plasmid The coding gene with the nucleotide sequence as shown in SEQ ID NO. 3 is cloned into a multiple cloning site on a pGEX-6P-1 vector to construct a recombinant plasmid ClyA-A25-pGEX-6P-1; the sequence of the recombinant plasmid is as shown in SEQ ID NO. 9; (3) Construction of expression fusion protein ClyA-A25 recombinant bacteria W3110 E. coli competent cells are thawed on ice, the recombinant plasmid ClyA-A25-pGEX-6P-1 is added to the competent cells, ice bath for 30 min, then 42℃ heat shock for 60 s, and incubate on ice for 2 min; then 800 μL of antibiotic-free LB medium is added, and cultured at 200 rpm and 37℃ for 1 h, then the bacterial solution is spread on LB plate medium added with ampicillin, and cultured at 37℃ overnight; the next day, a single colony strain is picked, and after shake culture, sequencing identification is performed, to obtain the recombinant bacteria expressing the fusion protein ClyA-A25, which is referred to as W-A25 strain; (4) Preparation of W-A25 OMV (1) A 1L conical flask is added with 500mL of ampicillin-resistant LB medium, 10μL of W-A25 bacterial solution is inoculated into the medium, and the bacterial solution is cultured at 37℃ and 220rpm for 4h, then the OD value of the bacterial solution is measured every 20min, when the OD value reaches 0.5-0.6, 500μL of IPTG with a concentration of 1M is added to make the final concentration of IPTG in the medium 1mM; then the expression of the exogenous protein is induced at 21℃ and 200rpm overnight to obtain 500mL of W-A25 bacterial solution; (2) The W-A25 bacterial liquid was centrifuged at 4°C, 5,000xg for 15 min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was concentrated 20 times by using an ultrafiltration centrifuge tube with a relative molecular mass cut-off of 100 kDa. The filtrate was filtered again through a 0.22 μm filter membrane, and then centrifuged at 150,000xg for 3 hours. The supernatant was discarded, and the precipitate was the W-A25 OMV.

5. Use of the anti-tumor peptide-modified bacterial outer membrane vesicle W-A25 OMV according to any one of claims 1 to 3 in the preparation of a drug for treating a tumor.

Images