Bacterial outer membrane vesicle drug carrier with targeting effect and preparation

Abstract

The invention provides a bacterial outer membrane vesicle drug carrier with a targeting effect and preparation, and relates to the technical field of biological medicines.The bacterial outer membrane vesicle protein ClyA derived from an escherichia coli W3110 strain is combined with iRGD protein capable of being combined with an integrin receptor alpha v beta 3, a ClyA-iRGD-pGEX-6P-1 recombinant plasmid for expressing ClyA-iRGD fusion protein is constructed, and the ClyA-iRGD-pGEX-6P-1 recombinant plasmid is used for preparing the bacterial outer membrane vesicle drug carrier with the targeting effect. By endogenous expression of the plasmid in W3110 escherichia coli, the C-iRGD-OMVs which has a tumor targeting effect and shows ClyA-iRGD fusion protein on the surface is prepared. The C-iRGD-OMVs shows efficient targeting binding capacity to MCF-7 breast cancer cells, overcomes the defect that iRGD single peptide is prone to aggregation and crystallization, has the potential of loading drugs, lays a foundation for the C-iRGD-OMVs serving as a targeting carrier for delivering breast cancer treatment drugs, and is expected to be further developed into a targeting treatment preparation for breast cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a bacterial outer membrane vesicle drug carrier with targeting effect and preparation. BACKGROUND

[0002] Breast cancer accounts for about one-third of all malignant tumors in women, and its mortality rate accounts for about 15% of the total number of diagnosed cases. Genetic, environmental and lifestyle factors can all lead to the occurrence of breast cancer, so these complex pathogenesis mechanisms pose a major obstacle to the effective treatment and prevention of breast cancer. At present, there are various therapies for breast cancer, including surgery, radiotherapy, chemotherapy, endoscopic treatment and immunotherapy. Despite these therapies, the incidence and mortality of breast cancer are still high, and traditional chemotherapeutic drugs and targeted drugs have many shortcomings, including causing treatment recurrence or drug resistance and the need to enter tumor tissue to destroy tumor cells and adverse secondary effects such as nausea, vomiting and strong systemic secondary effects. Therefore, the development of new drug delivery systems with high efficiency, low toxicity, targeting properties, good biocompatibility and penetration has become an important direction of tumor treatment research.

[0003] Bacterial outer membrane vesicles (OMVs) are a kind of membrane vesicles secreted by gram-negative bacteria, and the main components include bacterial lipopolysaccharide, outer membrane protein and lipid. The types and yields of OMVs are regulated by bacterial growth conditions and external environmental factors. Studies have shown that OMVs from various bacteria can stimulate different degrees of immune response, thereby providing corresponding immune protection for the host. Escherichia coli is a classic model organism with mature genetic manipulation technology, which can regulate the composition of OMVs (such as knocking out LPS to reduce toxicity, overexpressing tumor targeting peptides to enhance specificity) or loading exogenous therapeutic molecules (such as antigens, antibody fragments) through genetic modification. In addition, E. coli has low culture cost and mature fermentation technology, which is conducive to the large-scale production of OMVs to meet the needs of clinical transformation. Therefore, E. coli-derived OMVs have the following advantages in application: (1) formed spontaneously from the outer membrane of bacteria, with good biocompatibility; (2) with nanoscale size, it is easy to escape the capture of the reticuloendothelial system in vivo; (3) can load nucleic acids, small molecule drugs or proteins, and enter target cells through membrane fusion, thereby efficiently delivering drugs; (4) OMVs can be modified by genetic modification to have different functional properties; (5) large-scale production, relatively low cost.

[0004] iRGD is a novel cyclic peptide consisting of 9 amino acids, which contains the Arg-Gly-Asp (RGD) characteristic sequence in its structure, can specifically recognize and bind to the αvβ3 integrin highly expressed on the surface of tumor cells, and has a significant selective binding capacity. Compared with traditional RGD peptide segments, iRGD not only can bind to integrin, but also can recognize the neuropilin-1 receptor overexpressed in tumor tissue at the same time, thereby showing stronger tumor targeting. In addition, thanks to its disulfide bond-mediated cyclic structure, iRGD has more excellent tissue penetration capacity, which can effectively promote the enrichment and penetration of drugs coupled or combined with it in tumor tissue, thereby significantly improving the anti-tumor effect. iRGD peptide can deliver different types of functional molecules into cells for treatment and diagnosis, and has good application prospect in clinic, but there are still some deficiencies that need to be optimized, for example: studies have shown that when the iRGD peptide is added to the cell culture medium with a pH value of 6.5 to 7.5 and the concentration is higher than 50 μM, molecular aggregation often occurs. In view of the fact that the physiological pH environment (7.2-7.4) of human blood circulation is in this range, this aggregation effect can significantly reduce the bioavailability of iRGD peptide when administered intravenously. In addition, although the disulfide bond cyclic structure of iRGD peptide gives it higher conformational stability than linear peptides, it still has the risk of being degraded by plasma proteases, resulting in a shorter half-life in the body and limiting the exertion of long-acting therapeutic effect. In order to improve the clinical application value of iRGD peptide, the current research mostly adopts the strategy of covalently coupling it with carrier molecules, which not only can effectively enhance its structural stability, but also can maintain its biological activity, providing important technical support for the clinical transformation of iRGD peptide. SUMMARY

[0005] (One) technical problems solved

[0006] In view of the deficiencies of the prior art, the present application provides a bacterial outer membrane vesicle drug carrier with targeting effect and preparation.

[0007] (Two) technical solutions

[0008] To achieve the above purpose, the present application is implemented by the following technical solutions:

[0009] On the one hand, the present application provides an Escherichia coli outer membrane vesicle with targeting effect, the surface of the Escherichia coli outer membrane vesicle comprises a fusion protein ClyA-iRGD, the amino acid sequence of the fusion protein ClyA-iRGD is shown as SEQ ID NO. 1, and the fusion protein ClyA-iRGD is sequentially connected by the following parts:

[0010] (1) the ClyA protein with the amino acid sequence shown as SEQ ID NO. 2;

[0011] (2) Linker1 protein with an amino acid sequence as shown in SEQ ID NO. 3;

[0012] (3) MYC tag protein with an amino acid sequence as shown in SEQ ID NO. 4;

[0013] (4) Linker2 protein with an amino acid sequence as shown in SEQ ID NO. 5;

[0014] (5) Targeting peptide iRGD with an amino acid sequence as shown in SEQ ID NO. 6.

[0015] Further, the fusion protein is connected in a manner that the N terminus of the 3×MYC tag protein is connected to the C terminus of the ClyA protein through Linker1, and then the N terminus of the targeting peptide iRGD is connected to the C terminus of the 3×MYC protein through Linker2.

[0016] In a second aspect, the present application provides a recombinant plasmid for expressing the ClyA-iRGD fusion protein, characterized in that the structure of the recombinant plasmid is ClyA-iRGD-pGEX-6P-1, and the nucleotide sequence is shown in SEQ ID NO. 7.

[0017] In a third aspect, the present application provides a preparation method of E. coli outer membrane vesicles with targeting effect, comprising the following steps:

[0018] (1) Constructing the ClyA-iRGD fusion protein

[0019] Linker1, MYC tag protein sequence, Linker2 and iRGD protein sequence are sequentially inserted into the C terminus of the ClyA protein of the natural E. coli W3110 strain, and an Ncol site is introduced at the 5' end of the fusion protein, and an Xhol site is introduced at the 3' end, thereby constructing a fusion protein gene with the structure of Ncol-ClyA-Linker1-3×MYC-Linker2-iRGD-Xhol, and the sequence of the fusion protein gene is shown in SEQ ID NO. 7;

[0020] (2) Constructing the ClyA-iRGD-pGEX-6P-1 recombinant plasmid

[0021] The Ncol-ClyA-Linker1-3×MYC-Linker2-iRGD-Xhol fusion protein gene is cloned into the multiple cloning site on the pGEX-6P-1 vector suitable for fusion protein expression, thereby successfully constructing the recombinant plasmid ClyA-iRGD-pGEX-6P-1, and the nucleotide sequence is shown in SEQ ID NO. 8;

[0022] (3) Establishing an E. coli monoclonal strain stably expressing ClyA-iRGD fusion protein

[0023] The above recombinant plasmid ClyA-iRGD-pGEX-6P-1 is transformed into E. coli W3110 competent cells, and a strain C-iRGD stably expressing ClyA-iRGD fusion protein is obtained through screening;

[0024] (4) Extracting E. coli outer membrane vesicles

[0025] The C-iRGD strain is inoculated into LB medium containing ampicillin, and IPTG is added to induce expression of the exogenous protein, so that the final concentration of IPTG in the medium is 1 mM, and C-iRGD bacterial solution is obtained; the bacterial solution is concentrated by ultrafiltration and extracted by ultracentrifugation to obtain outer membrane vesicles, which are named C-iRGD-OMVs.

[0026] In a fourth aspect, the application provides a use of the extracellular membrane vesicle with targeting effect, and the bacterial outer membrane vesicle is used for in vitro targeted binding of MCF-7 breast cancer cells.

[0027] In a fifth aspect, the application provides a use of the bacterial outer membrane vesicle as a drug carrier for targeted tumor treatment.

[0028] (III) Beneficial effects

[0029] The application provides a bacterial outer membrane vesicle co-delivery carrier for tumor treatment and a preparation method. The bacterial outer membrane vesicle protein ClyA from E. coli W3110 strain is combined with iRGD protein having a binding with integrin receptor αvβ3, a ClyA-iRGD-pGEX-6P-1 recombinant plasmid expressing ClyA-iRGD fusion protein is constructed, the plasmid is endogenously expressed in W3110 E. coli, and C-iRGD-OMVs with surface display of ClyA-iRGD fusion protein and having a targeting tumor effect are prepared. The vesicle retains the original morphology of the bacterial outer membrane vesicle and also retains the targeting tumor activity of iRGD, and shows high efficient targeted binding capacity for MCF-7 breast cancer cells. In addition, compared with the technical defect that free iRGD monopeptide is prone to aggregation and crystallization, the C-iRGD-OMVs significantly improve the physical stability of iRGD by fusing and displaying iRGD on the surface of the vesicle, and avoid aggregation and inactivation of iRGD. Meanwhile, the natural hollow capsule structure of the bacterial outer membrane vesicle endows the C-iRGD-OMVs with the potential of loading various therapeutic drugs such as small molecule chemotherapeutic drugs and nucleic acid drugs, lays a foundation for the C-iRGD-OMVs as a targeted carrier for delivery of breast cancer therapeutic drugs, and is expected to be further developed as a targeted therapeutic preparation for breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1The ClyA-iRGD-pGEX-6P-1 prokaryotic expression plasmid was identified using Sanger sequencing.

[0031] Figure 2 Agarose gel electrophoresis image of recombinant plasmid ClyA-iRGD-pGEX-6P-1.

[0032] Figure 3 The expression status of ClyA-iRGD fusion protein in C-iRGD E. coli marker protein.

[0033] Figure 4 For W3110-OMVs ( Figure 4 A), C-iRGD-OMVs ( Figure 4 Transmission electron microscope image B).

[0034] Figure 5 For W3110-OMVs ( Figure 5 A), C-iRGD-OMVs ( Figure 5 B) Particle size distribution diagram.

[0035] Figure 6 The expression of the C-iRGD-OMVs surface fusion protein was detected by Western blotting.

[0036] Figure 7 The targeting binding ability of C-iRGD-OMVs to MCF-7 breast cancer cells after co-incubation for 2 hours was detected by cell flow cytometry.

[0037] Figure 8 The targeting binding ability of C-iRGD-OMVs to MCF-7 breast cancer cells after co-incubation for 4 h was detected by cell flow cytometry.

[0038] Figure 9 To detect the targeting binding ability of C-iRGD-OMVs to MCF-7 breast cancer cells using cell fluorescence microscopy.

[0039] Figure 10 The effects of C-iRGD-OMVs and iRGD single peptides co-incubated with serum for 1 h and 6 h on the signaling pathways of MCF-7 breast cancer cells.

[0040] Figure 11 This represents the degree of aggregation of iRGD monopeptides and C-iRGD-OMVs at the same concentration. Detailed Implementation

[0041] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0042] Embodiment 1

[0043] Construction method of C-iRGD-OMVs

[0044] 1 Construction of ClyA-iRGD fusion protein

[0045] Linker1, MYC tag protein sequence, Linker2 and iRGD protein sequence were sequentially inserted into the C-terminal end of the ClyA protein of the natural E. coli W3110 strain, and a Ncol site was introduced at the 5' end of the fusion protein and a Xhol site was introduced at the 3' end, to construct a fusion protein gene with the structure of Ncol-ClyA-Linker1-3xMYC-Linker2-iRGD-Xhol, and the sequence of the fusion protein gene is shown as SEQ ID NO. 7.

[0046] 2 Construction of ClyA-iRGD-pGEX-6P-1 recombinant plasmid

[0047] The Ncol-ClyA-Linker1-3xMYC-Linker2-iRGD-Xhol fusion protein gene was cloned into the multiple cloning site on the pGEX-6P-1 vector suitable for fusion protein expression, and a recombinant plasmid ClyA-iRGD-pGEX-6P-1 was successfully constructed, and the nucleotide sequence is shown as SEQ ID NO. 8.

[0048] 3 Establishment of E. coli monoclonal strain stably expressing ClyA-iRGD fusion protein

[0049] The above-mentioned recombinant plasmid ClyA-iRGD-pGEX-6P-1 was transformed into E. coli W3110 competent cells, and then the competent cells were incubated on ice for 30 min, and then quickly placed in a 42℃ water bath, heat shocked for 90 s, and then placed back on ice for incubation for 2 min. Then 700 μL of antibiotic-free LB medium was added, and the culture was incubated at 37℃, 200 rpm for 1 h. The bacterial solution was centrifuged at 3000xg for 2 min, the supernatant was discarded, and 50 μL of the bacterial solution was used to resuspend the bacterial pellet. The resuspended bacterial solution was spread on LB plate medium supplemented with ampicillin and incubated at 37℃ overnight. The next day, single colony strains were picked and the strain was named C-iRGD strain.

[0050] The selected monoclonal colonies were subjected to colony PCR primary screening, and the PCR products were analyzed by 1% agarose gel electrophoresis. The positive colonies were sent for sequencing to verify the correctness of the recombinant gene sequence, and the results are shown in Figure 1 The sequencing results of the fusion gene are consistent with the theoretical sequence, indicating that the plasmid ClyA-iRGD-pGEX-6P-1 is successfully constructed. Subsequently, the positive colonies with correct sequencing were selected for expansion culture, and the recombinant plasmid was extracted and identified by agarose gel electrophoresis, and the results are shown in Figure 2 The band of the recombinant plasmid ClyA-iRGD-pGEX-6P-1 is located near 500 bp, which is consistent with the theoretical value of 509 bp. To verify the expression of the fusion protein, the target strain (C-iRGD) and the wild type control (W3110) were induced with IPTG (final concentration of 1 mM) at 21°C when the OD600 reached 0.5-0.6. The bacterial cells were collected and lysed, and SDS-PAGE protein electrophoresis was performed, and the membrane was transferred to the PVDF membrane. Western blot analysis was performed using anti-MYC tag antibody, and the results are shown in Figure 3 The ClyA-iRGD fusion protein is expressed in the C-iRGD strain, and the wild type W3110 strain does not express, which is consistent with the expectation. The above results show that the recombinant plasmid ClyA-iRGD-pGEX-6P-1 is successfully constructed, and the E. coli monoclonal strain stably expressing ClyA-iRGD fusion protein is successfully screened.

[0051] 4. Extraction of E. coli outer membrane vesicles

[0052] The C-iRGD strain was inoculated into 500 mL of LB medium containing ampicillin, and the bacterial liquid was shaken at 37°C and 220 rpm for 4 h. The OD value of the bacterial liquid was measured every 20 min. When the OD value reached 0.5-0.6, IPTG was added to make the final concentration of IPTG in the culture medium 1 mM. Subsequently, the expression of foreign proteins was induced at 21°C and 200 rpm overnight to obtain C-iRGD bacterial liquid. At the same time, the W3110 strain was inoculated in the non-resistant medium and cultured overnight under the same conditions as the control to obtain W3110 bacterial liquid. The bacterial liquid was centrifuged at 5000 x g at 4°C for 15 min, and the supernatant was collected. The supernatant was filtered with a 0.45 μm filter membrane, and the filtrate was concentrated 20 times with an ultrafiltration centrifuge tube with a relative molecular mass of 100 kD. The concentrated supernatant was filtered again with a 0.22 μm filter membrane, and then centrifuged at 150000 x g for 3 h. The supernatant was discarded and resuspended with 200 μL PBS buffer to obtain C-iRGD-OMVs and W3110-OMVs, respectively.

[0053] Example 2

[0054] Identification of C-iRGD-OMVs

[0055] 1. Morphological identification

[0056] The morphology and structure of the C-iRGD-OMVs and W3110-OMVs prepared in Example 1 were observed using transmission electron microscopy. The results are as follows: Figure 4 As shown, W3110-OMVs ( Figure 4 A), C-iRGD-OMVs( Figure 4 B) has a diameter range of 20-250 nm, a distinct saucer-like double-layer film structure, is individually distributed, has a relatively clear background, and few contaminants.

[0057] 2. Dynamic light scattering (DLS) measurement of particle size

[0058] The particle size of W3110-OMVs and C-iRGD-OMVs prepared in Example 1 was determined using dynamic light scattering (DLS). The results are as follows: Figure 5 As shown, W3110-OMVs ( Figure 5 A), C-iRGD-OMVs( Figure 5 B) has a concentrated particle size distribution of around 133.1 nm and 144.2 nm, and the particle size distribution is a unimodal normal distribution. The polydispersity index (PDI) is around 0.269 and 0.311, indicating that the particles are well dispersed.

[0059] 3. Western Blot detection of OMV marker proteins

[0060] Western blotting was used 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 OMVs. Results are as follows: Figure 6 As shown, OmpA protein was detected in both wild-type W3110-OMVs and C-iRGD-OMVs, regardless of whether they were treated with proteinase K, confirming the integrity of the OMVs. The OmpA signal disappeared after the lipid membrane of the OMVs was disrupted by SDS and then digested with proteinase K. MYC tag protein expression was detected only in the untreated C-iRGD-OMVs group; however, when the lipid membrane was intact, the MYC signal disappeared in C-iRGD-OMVs treated with proteinase K. These experimental results confirm that the ClyA-iRGD fusion protein was successfully anchored to the outer surface of the E. coli OMV membrane, providing crucial experimental evidence for surface display strategies in targeted delivery systems.

[0061] Example 3

[0062] Validating the targeting of ClyA-iRGD fusion protein to E. coli outer membrane vesicles

[0063] 1. Staining markers C-iRGD-OMVs

[0064] C-iRGD-OMVs extracted from 1000 mL of bacterial culture were resuspended 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 and incubated at 37 °C in the dark for 30 min. After incubation, the mixture was centrifuged at 150,000 × g for 3 h, the supernatant was discarded, and the supernatant was allowed to dry completely. The precipitate was resuspended in 500 μL of PBS to obtain DiR-labeled C-iRGD-OMVs. Wild-type W3110-OMVs were labeled using the same method as a control.

[0065] 2. Cell flow cytometry assay to detect the uptake of C-iRGD-OMVs by MCF-7 and GES-1 cells

[0066] MCF-7 breast cancer cells and GES-1 human gastric mucosal epithelial cells were respectively used at 2×10 5 Cells were evenly seeded at a density of cells / well in 12-well plates and incubated at 37°C with 5% CO2 for 48 h. After cell attachment, 20 μL of DiR-labeled W3110-OMVs or C-iRGD-OMVs were added to each group, and incubated for 4 h. A time-gradient group was also set up, with 20 μL of DiR-labeled W3110-OMVs or C-iRGD-OMVs added to each group, and incubated for 2 h. After incubation, cells were collected by trypsin digestion and transferred to sterile centrifuge tubes. The cells were centrifuged at 800 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in PBS and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 400 μL of sterile PBS buffer. The percentage of fluorescently labeled cells was detected by flow cytometry.

[0067] Flow cytometry analysis results as follows Figure 7 , Figure 8 As shown, at different time points, the binding rates of MCF-7 and GES-1 cells with C-iRGD-OMVs were significantly higher than those with W3110-OMVs (P<0.001), which is closely related to the expression of α5β3 receptor in both cell types. Comparing the differences in binding rates between MCF-7 and GES-1 cells with the C-iRGD-OMVs and W3110-OMVs groups, the results showed that in the 2h and 4h incubation groups, the binding efficiency of C-iRGD-OMVs with MCF-7 cells was significantly higher than that with GES-1 cells (P<0.001). These results indicate that compared to GES-1 cells with low α5β3 receptor expression, C-iRGD-OMVs exhibit a stronger targeting binding ability to MCF-7 cells with high α5β3 receptor expression.

[0068] 3. Immunofluorescence microscopy was used to observe the uptake of C-iRGD-OMVs by MCF-7 and GES-1 cells.

[0069] To verify the targeting ability of C-iRGD-OMVs expressing ClyA-iRGD fusion protein to tumor cells with high expression of receptor genes, C-iRGD-OMVs labeled with the fluorescent dye DiR were co-incubated with MCF-7 breast cancer cells with high expression of αvβ3 receptor and GES-1 human gastric mucosal epithelial cells with low expression of αvβ3 receptor for 4 h. The uptake of vesicles by cells was observed by immunofluorescence microscopy.

[0070] The results are as follows Figure 9 As shown, in GES-1 cells, there was no significant difference in fluorescence intensity / area between the W3110-OMVs group and the C-iRGD-OMVs group (P>0.05), indicating that ClyA-iRGD fusion protein modification did not significantly promote vesicle uptake by GES-1 cells. In MCF-7 cells, the fluorescence intensity / area of ​​the C-iRGD-OMVs group was significantly higher than that of the W3110-OMVs group (P<0.0001), indicating that ClyA-iRGD fusion protein modification could significantly enhance vesicle uptake by MCF-7 cells. This suggests that ClyA-iRGD may promote vesicle internalization and entry into cells by binding to specific receptors on the surface of MCF-7 cells, giving C-iRGD-OMVs higher targeting in MCF-7 cells. This targeting provides experimental evidence for subsequent vesicle-based tumor-targeted delivery applications.

[0071] Example 4

[0072] Verify the alteration of iRGD single peptide properties by C-iRGD-OMVs.

[0073] 1. PCR detection of downstream gene activation

[0074] MCF-7 breast cancer cells were used at a rate of 4 × 10 5 C-iRGD-OMVs and iRGD peptides were seeded at a density of 1 cell / well in 6-well plates and cultured for 2 days. C-iRGD-OMVs and iRGD peptides were co-incubated with serum for 1 h and 6 h, respectively. After co-incubation, C-iRGD-OMVs and iRGD peptides were co-incubated with MCF-7 cells for 24 h. Total RNA was extracted and reverse transcribed into cDNA. The downstream gene ITGA5 was detected by PCR. Figure 10 A), ITGB3 ( Figure 10 The activation status of B).

[0075] The results are as follows Figure 10As shown, there was no significant difference in the activation ability of downstream target genes ITGA5 and ITGB3 after C-iRGD-OMVs co-incubated with serum for 1 h and 6 h, respectively, and then co-cultured with MCF-7 cells for the same time (P>0.05); while after iRGD monopeptide co-incubated with serum for 1 h and 6 h, respectively, and then co-cultured with MCF-7 cells under the same conditions, the activation ability of ITGA5 and ITGB3 genes of iRGD monopeptide co-incubated with serum for 6 h was significantly lower than that of iRGD monopeptide co-incubated with serum for 1 h (P<0.01). The above results show that iRGD monopeptide is prone to degradation in serum environment, and its activation ability to downstream genes decreases significantly with the extension of incubation time; while C-iRGD-OMVs have stronger stability in the same serum conditions and can maintain stable receptor activation ability.

[0076] 2DLS determination of particle size

[0077] The particle size of C-iRGD-OMVs and iRGD monopeptide at 20 μM and 200 μM was detected by dynamic light scattering (DLS), and the results are shown in Figure 11 As shown, the particles of C-iRGD-OMVs and iRGD monopeptide at 20 μM were concentrated at about 127.6 nm and 68.23 nm, respectively, and the particle size distribution was single-peak normal distribution, and the polydispersity coefficient PDI was about 0.278 and 0.345, respectively, indicating good particle dispersion; while the particles of C-iRGD-OMVs and iRGD monopeptide at 200 μM were concentrated at about 113.1 nm and 2147.78 nm, respectively, and the particle size distribution was single-peak normal distribution, and the polydispersity coefficient PDI was about 0.488 and 0.314, respectively, indicating that compared with iRGD monopeptide, C-iRGD-OMVs were not prone to molecular aggregation and had better solubility.

[0078] iRGD is a novel cyclic peptide consisting of 9 amino acids, which contains the Arg-Gly-Asp (RGD) characteristic sequence in its structure, and can specifically recognize and bind to the αvβ3 integrin highly expressed on the surface of tumor cells, and has a significant selective binding capacity. Compared with the traditional RGD peptide segment, iRGD can not only bind to the integrin, but also recognize the neuropilin-1 receptor overexpressed in tumor tissues at the same time, thereby showing stronger tumor targeting. In addition, thanks to its disulfide bond-mediated cyclic structure, iRGD has more excellent tissue penetration capacity, which can effectively promote the enrichment and penetration of drugs coupled or co-administered with it in tumor tissues, thereby significantly improving the anti-tumor effect. However, although the disulfide bond cyclic structure of the iRGD peptide gives it higher conformational stability than linear peptides, it still has the risk of being degraded by plasma proteases, resulting in a shorter half-life in the body, which limits the exertion of long-acting therapeutic effect. The present application combines the bacterial outer membrane vesicle protein ClyA derived from the E. coli W3110 strain with the iRGD protein that binds to the integrin receptor αvβ3 to construct a ClyA-iRGD-pGEX-6P-1 recombinant plasmid expressing a ClyA-iRGD fusion protein. By endogenously expressing the plasmid in W3110 E. coli, C-iRGD-OMVs with tumor-targeting effect are prepared. The vesicle retains the original morphology of the bacterial outer membrane vesicle and also retains the tumor-targeting binding activity of iRGD, showing high-efficiency targeting binding capacity to MCF-7 breast cancer cells. In addition, compared with the technical defect that free iRGD monopeptide is prone to aggregation and crystallization, C-iRGD-OMVs significantly improve the physical stability of iRGD by fusing and displaying iRGD on the surface of the vesicle, avoiding its aggregation and inactivation; at the same time, the natural hollow capsule structure of the bacterial outer membrane vesicle endows C-iRGD-OMVs with the potential to load various therapeutic drugs such as small molecule chemotherapeutic drugs and nucleic acid drugs, laying a foundation for its use as a targeted carrier for delivering breast cancer treatment drugs, and is expected to be further developed as a targeted therapeutic preparation for breast cancer.

[0079] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A targeted Escherichia coli outer membrane vesicle, characterized in that, The outer membrane vesicles of the *E. coli* contain a fusion protein ClyA-iRGD, the amino acid sequence of which is shown in SEQ ID NO.1 and is composed of the following parts linked together in sequence: (1) The amino acid sequence of the ClyA protein as shown in SEQ ID NO.2; (2) The Linker1 protein with the amino acid sequence shown in SEQ ID NO.3; (3) The amino acid sequence of the MYC-tagged protein is shown in SEQ ID NO.4; (4) The Linker2 protein with the amino acid sequence shown in SEQ ID NO.5; (5) The amino acid sequence of the target peptide iRGD is shown in SEQ ID NO.

6.

2. A recombinant plasmid expressing the ClyA-iRGD fusion protein, characterized in that, The recombinant plasmid structure is ClyA-iRGD-pGEX-6P-1, and its nucleotide sequence is shown in SEQ ID NO.

7.

3. A method for preparing Escherichia coli outer membrane vesicles with targeting effect, characterized in that, Includes the following steps: (1) Construction of ClyA-iRGD fusion protein Linker1, MYC tag protein sequence, Linker2, and iRGD protein sequence were sequentially inserted into the C-terminus of the ClyA protein of natural Escherichia coli strain W3110. An Ncol site was introduced at the 5' end of the fusion protein and an Xhol site was introduced at the 3' end to construct a fusion protein gene with the structure Ncol-ClyA-Linker1-3×MYC-Linker2-iRGD-Xhol. The sequence of the fusion protein gene is shown in SEQ ID NO.

7. (2) Construction of ClyA-iRGD-pGEX-6P-1 recombinant plasmid The Ncol-ClyA-Linker1-3×MYC-Linker2-iRGD-Xhol fusion protein gene 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-iRGD-pGEX-6P-1 was successfully constructed. Its nucleotide sequence is shown in SEQ ID NO.

8. (3) Establish a stable Escherichia coli monoclonal strain expressing the ClyA-iRGD fusion protein The recombinant plasmid ClyA-iRGD-pGEX-6P-1 was transformed into Escherichia coli W3110 competent cells, and strains that stably express the ClyA-iRGD fusion protein were screened. (4) Extraction of Escherichia coli outer membrane vesicles The C-iRGD strain was inoculated into LB medium containing ampicillin, and IPTG was added to induce the expression of exogenous proteins. The final concentration of IPTG in the medium was 1 mM to obtain C-iRGD bacterial solution. The bacterial solution was concentrated by ultrafiltration and extracted by ultracentrifugation to obtain outer membrane vesicles, which were named C-iRGD-OMVs.

4. The application of an extracellular membrane vesicle with targeting effect as described in claim 1, characterized in that, The bacterial outer membrane vesicles are used to target and bind to MCF-7 breast cancer cells in vitro.

5. A bacterial outer membrane vesicle with targeting function as described in claim 1, characterized in that, The bacterial outer membrane vesicles are used as drug carriers in targeted tumor therapy.

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