The E. coli strains used in this study included Top 10F’ as a host for plasmid propagations and cloning procedures, ∆msbB/∆pagP W3110, and BL21 (DE3), which were used to produce the engineered OMVs and recombinant protein, respectively. The mutant host strain was kindly provided by Prof. Sang Hyun Kim (KRIBB, Republic of Korea) and the remaining two along with the protein expression vectors pET-32a (+) and pET-28a (+) were purchased from Invitrogen (Carlsbad, CA, United States). E. coli strains were grown in Luria–Bertani (LB) medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl, pH 7.0]. The growth medium was supplemented with ampicillin (100 µg/ml) and kanamycin (50 µg/ml) when required. Restriction endonucleases and T4 DNA ligase were supplied by Fermentas (Waltham, United States) and Roche companies (Penzberg, Germany).

MDA-MB-468 (human breast carcinoma), HEK293T (human embryonic kidney), and THP-1 (human monocytic) cell lines were obtained from the cell bank of Pasture Institute of Iran (NCBI). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) or RPMI1640 medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ml streptomycin (GibcoBRL, Rockville, IN, United States) at 37°C with 5% CO₂ in a humidified atmosphere inside a CO₂ incubator. All reagents used were of analytical grade and obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (MO, United States). EGF antigen was a kind gift from Dr. Majid Golkar (Pasteur Institute of Iran, Tehran, Iran).

A codon-optimized 1,356 bp DNA fragment encoding His tag (Gly₄Ser)₃, myc tag, Z_EGFR:1907 affibody (Gly₄Ser)₃, and GALA fused to the C-terminus of Cytolysin-A gene was synthesized by GeneRay Biotech (Shanghai, China) and delivered in an intermediate pMD18-T plasmid. To facilitate the expression procedure in E. coli W3110, we first generated a modified version of pET32a (mpET32a) in which the T7 promoter was replaced by Tac promoter. This step was performed to bypass the T7 RNA polymerase-dependent expression of our construct in E. coli W3110. Next, the ClyA-affibody-GALA construct was cloned in the HindIII/NcoI site of mpET32a. Finally, the recombinant plasmid, mpET32a/ClyA-affibody-GALA, was transformed into the ∆msbB/∆pagP W3110 E. coli strain and the transformed strain was maintained on the solid medium supplemented with ampicillin (100 μg/ml).

OMVs were isolated according to the procedure described previously by Chen et al. (2010) with some modifications. Briefly, the overnight saturated cultures of E. coli were inoculated into 1 L of LB medium supplemented with ampicillin (1:100 v:v). Subculturing was carried out in the baffled flask using a 1:5 (v:v) ratio of liquid to air to enhance the yield of vesicle production. The culture was shaken at 30°C for 5 h until cells reached the mid-log phase (OD600 ∼ 0.6–0.8). Then, the required amount of isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the cultivation was continued for a further 20 h at 20°C to induce expression of the recombinant protein. Afterward, the bacterial pellet was precipitated by centrifugation at 5,000 × g (20 min) and cell-free culture supernatant was collected and filtered through a 0.45 μm filter and concentrated to 80 ml by ultrafiltration through an Amicon filter with 100 kDa cutoff (Millipore, Billerica, MA, United States). Lastly, OMVs were isolated by ultracentrifugation (Ti98 rotor, Beckman-Coulter, California, United States) at 170,000 × g for 2 h at 4°C. The purified OMVs were washed with phosphate-buffered saline (PBS) at 170,000× g for an additional 2 h, followed by resuspending the pellet in 1 ml of PBS and passing through Detoxi-Gel endotoxin-removing columns (Pierce, Thermo Scientific, United States) and 0.20 μm cellulose acetate filters. The purified OMVs were stored at −20°C until use. The total protein concentration of OMVs was quantified using Bradford assay, and the bovine serum albumin was used as a standard.

The gene fragment encoding His tag, G4S, myc tag, Z_EGFR:1907 affibody, G4S, and GALA peptide was PCR-amplified using affibody forward (5-GAG​GTA​CCG​GAA​GTC​GGG​GGC​GG -3′) and affibody reverse (5′-CTC​TAG​ATT​ATT​TAG​GAG​CCT​GTG​CAT​C-3′) primers using pMD18-T vector as a template. The amplified sequence was then cloned between KpnI and XbaI sites of the pET28a expression vector and the final construct was transformed and expressed in E. coli BL21 (DE3) strain. To express the recombinant protein, a 500 ml of bacterial culture supplemented with kanamycin was set and the protein expression was induced by adding IPTG (1 mM) at 37°C for 4 h. The bacterial pellet was then collected and lysed using lysis buffer (300 mM NaCl, 50 mM NaH₂PO₄, and 10 mM imidazole; pH8.0) and sonication (12 cycles of 20 s ON/OFF). Following centrifugation at 5,000 × g for 20 min, the supernatant was loaded on the Ni-NTA agarose column (QIAGEN; Hilden, Germany) equilibrated with the same lysis buffer. After washing, the recombinant protein was eluted using the elution buffer (300 mM NaCl, 50 mM NaH₂PO₄, and 250 mM imidazole; pH8.0) and finally, imidazole was removed from the protein solution and replaced with PBS using an Amicon ultrafiltration system (3 kDa cutoff filter) (Millipore, Billerica, MA, United States). The concentration of the purified fusion protein was determined by the Bradford assay.

Expression of the exogenous protein was detected by electrophoresis. Briefly, bacterial cells and OMV samples were suspended in lysis buffer (4% CHAPS, 7M Urea, 2M Thiourea, and 40 mM Tris-HCl) and boiled for 15 min in a loading buffer containing β-mercaptoethanol. After cooling to room temperature, samples were loaded onto a 10% polyacrylamide gel and separated electrophoretically. To visualize protein bands, SDS-PAGE gels were stained with Coomassie Brilliant Blue R-250 (BioRad, United States).

For Western blot analysis, bacterial cell lysates and OMV samples were separated by SDS-PAGE as described above and the protein bands were subsequently transferred to a PVDF membrane via semidry transfer. Membranes were blocked using skim milk in PBS (3% w/v) and the fusion protein band was detected by a mouse horseradish peroxidase (HRP)-labeled monoclonal antibody against the 6XHis-tag (1:1,000 v/v, Sigma-Aldrich, United States, A7058). For further verification, specific antibodies, including goat polyclonal anti-affibody primary antibody (1/4,000 v/v, Abcam, United Kingdom, ab50345) and HRP-conjugated anti-goat secondary antibody (Abcam, United Kingdom), were used. Bands were visualized using HRP substrate Clarity ECL kit (GE Health Care Life Sciences, Buckinghamshire, United Kingdom).

To confirm the surface distribution of ClyA-affibody-GALA in bacteria and OMVs, immune staining of the samples was performed and the samples were analyzed using flow cytometry. In brief, bacterial cells were centrifuged at 8,000 × g for 5 min and then washed with PBS at 8,000 ×g for another 5 min. Afterward, bacteria were blocked in PBS containing 3% (w/v) BSA for 15 min and incubated with anti-affibody antibody in 1% (w/v) BSA (1:500) for 1 h. Following washing, samples were incubated with FITC -conjugated secondary antibody (1:2000) (Abcam, United Kingdom) for 30 min. Flow cytometry analysis was performed using the CyFlow® SL machine (Partec, Munster, Germany). For OMVs, at the end of each step, OMVs were collected using a 50 kDa cutoff ultrafiltration tube (Millipore, Billerica, MA, United States) and centrifugation at 5,000 × g for 30 min (Huang et al., 2016).

Transmission electron microscope (TEM) analysis was performed for structural analysis of vesicles. For this purpose, fresh non-targeted and EGFR-targeted OMVs (modified OMVs, mOMVs) (250 µg/ml) were resuspended in 3% glutaraldehyde with the ratio of 1:1 to fix them. Next, one drop of this solution was mounted on a Formvar coated grid. Samples were then negatively stained with 2% uranyl acetate for 2 min and air-dried for 1 hour. Upon washing with distilled water, the completely dried copper grids were examined under a transmission electron microscope Zeiss EM900, 80 kV (Germany), to assess the size and morphology of OMVs.

Size distribution of non-targeted OMVs and the affi_EGFR-OMVs was measured by dynamic light scattering (DLS) using a ZetaSizer Analyzer (Malvern Instruments Ltd. Nano-ZS ZEN3600, United Kingdom) equipped with a 5 mW HeNe laser and operating at an angle of 173°. To detect Z-average size and polydispersity index (PDI) of the OMVs, the OMV frozen samples were thawed at room temperature and diluted to 50 μg/ml (total protein concentration) in PBS. Z-average defines the mean diameter of the vesicles in nm (d.nm), while PDI describes the particle size distribution. Tracing analysis was performed in three independent runs for each sample in single-use polystyrene zeta cell DTS1060 with a path length of 10 mm by measuring the backscattering intensity at 25°C. Scattering light detected at 173° was automatically adjusted by laser attenuation filters. The software used to collect and analyze the data was the Zetasizer software version 7.01. Size distribution by intensity was preferred to measurements by number or by volume to have more reproducible results.

Furthermore, the alteration in size and PDI of affi_EGFR-OMVs reflecting vesicles stability was tested during storage for a period of 3 days at 4°C.

The Tyndall effect was applied to verify the existence of OMVs as nanoparticles in the solution using a red laser pointer as the light source. A visible light beam path can be observed in the nanoparticle solution due to Tyndall scattering.

Enzyme-linked immunosorbent assay (ELISA) was carried out to explore the specificity of the engineered OMVs for EGFR receptors according to a previously described protocol with some modifications (Kim et al., 2012). Briefly, 100 μL of EGFR protein (5 µg/ml) (Abcam, United Kingdom) and bovine serum albumin (BSA) (5 μg/ml in PBS) were coated onto 96-well plates (SPL, Korea) and incubated at 4°C overnight. Following removal of the coating solution, 200 μL of blocking buffer containing 2% of BSA was added and further incubated for 2 h at 25°. Afterward, the plates were washed four times with PBS containing 0.1% of Tween-20 and exposed to 100 μL of the OMVs or affi_EGFR-OMVs at different concentrations (5–150 μg/ml) and incubated for 2 h at 25°C. The affi_EGFR-OMVs were then detected using 100 μL of goat polyclonal anti-affibody primary antibody (1/4,000 in PBST), followed by four times washing and incubating with horseradish peroxidase (HRP)-conjugated secondary antibody. The immunoreactivity was observed using the chromogenic HRP substrate 3,3′,5,5′-tetramethybenzidine (TMB) (Abcam, United Kingdom) and the absorbance was measured at 450 nm. Finally, the function of the engineered OMVs was compared with the affi_EGFR protein (0.1–10 μg/ml).

ELISA was also performed with cancer cell lines. MDA-MB-468 (EGFR-positive) and HEK293T (EGFR-negative) cell lines were grown in a DMEM medium containing 10% FBS. Cells (10⁶/ml) were lysed by sonication (15 cycles: 10 s on/10 s off) at 80 A and then 100 μL of the cell lysate was coated onto each well (Banisadr et al., 2018). Upon blocking and washing, coated wells were incubated with 100 μL of the affi_EGFR-OMVs and OMVs at 55 μg/ml as well as affi_EGFR (3.5 μg/ml) and Cetuximab (Erbitox®, Merck) (1.5 μg/ml) as the positive control. HRP-conjugated anti-human secondary antibody (Cytomatin gene, Isfahan, Iran) was applied to detect Cetuximab binding.

Cell specificity and binding capacity of the affi_EGFR-OMVs were examined by flow cytometry as described before (Zarei et al., 2014). In brief, 5×10⁵ cells of each of MDA-MB-468 and HEK293T were spun down at 800×g for 5 min. Cells were resuspended in 100 μL of FACS buffer (PBS, 5% FBS) and incubated with 25 μg of OMVs and affi_EGFR-OMVs or 1.5 μg of the affi_EGFR for 2 h at 4°C. Following washing two times with FACS buffer and blocking, cells were incubated with 100 μL of anti-affibody antibody (1/500) for a further 2 h at 4°C, then stained with FITC -conjugated secondary antibody (1/2000) in the dark for 30 min at 4°C, and finally resuspended in PBS. The fluorescent intensity of the cells was measured by flow cytometry. All samples were compared to negative control. For the negative control, the cells were processed and stained in the same manner with anti-affibody and FITC-conjugated secondary antibody, except for incubating with OMVs or affi_EGFR. To quantify the difference in cell binding, the mean fluorescence intensity was calculated for all the treatments.

The specific binding of the modified OMVs was more investigated in the presence of the EGFR competitors. For this goal, 5×10⁵ cells of EGFR-positive MDA-MB-468 cells were pretreated with an excess amount of EGF (7.5 μg) or Cetuximab (186.5 μg) in 200 μL of FACS buffer (equal to 12.5 μM) for 90 min at 4°C. Having washed the cells, they were then treated with 25 μg of OMVs or affi_EGFR-OMVs for a further 90 min at 4°C. Anti-affibody antibody and FITC-conjugated secondary antibody were applied as previously described. The fluorescent intensity of the cells was measured by flow cytometry.

The binding specificity of the affi_EGFR-OMVs was also examined with the increasing concentrations of EGF as an EGFR competitor. To do it, MDA-MB-468 cells (5×10⁵ cells/200 μL per tube) were incubated with 25 μg of affi_EGFR-OMVs in the presence of 15 ng to 7.5 μg (3.25 nM–12.5 μM final concentration) of EGF for 2 h at 4°C. Anti-affibody and FITC were used for staining cell-bound affi_EGFR-OMVs. MFI was recorded and % of cell binding of affi_EGFR-OMVs + EGF tests was calculated relative to affi_EGFR-OMVs alone.

To detect expression of EGFR in MDA-MB-468 and HEK293T cells, the cells were seeded at 2.5×10⁵ in 6 well plates and incubated at the standard growth culture for 24 h. For the EGFR stimulation test, MDA-MB-468 cells were plated and treated with affi_EGFR-OMVs (250 μg/ml) or EGF (100 nM) on the next day following overnight serum starvation. The cells were then lysed using RIPA lysis buffer (Sigma-Aldrich) containing protease inhibitors. The protein concentration was determined using the Bradford protein assay and protein samples were separated by 10% SDS-PAGE electrophoresis. Western blotting analysis was then performed as described before. Images were quantified using the ImageJ 1.4.3.67 (NIH software). The protein content was normalized to the level of β-actin. The antibodies used were as follows: EGFR (1/1,000, orb225296, Biorbyt, United Kingdom), p-EGFR (1/200, SC-81488, Santa Cruz Biotechnology, United States) and β-actin (1/1,000, 4967S, Cell Signaling, United States).

To visualize the cell binding of OMVs, confocal microscopy was applied (Gujrati et al., 2014). In a few words, 1.5 ×10⁵ of MDA-MB-468 and HEK293T cells were seeded on 8-well glass slides (SPL Life Sciences, Korea) in a DMEM medium containing 10% FBS and antibiotics for 24 h to reach 60% of confluency. Cells were then fixed with paraformaldehyde (PFA, 3.7% in PBS) for 15 min, blocked by 2% of BSA for 1 h at room temperature, and then further incubated with the affi_EGFR-OMVs (25 μg) for 2 h at 4°C. Samples were sequentially exposed to anti-affibody primary antibody (1:500) and FITC-labeled secondary antibody (1:2,000). Cells were finally evaluated under a confocal microscope (Olympus Fluoview TM FV 1000, Olympus, Japan).

For tracing the cellular uptake of OMVs, MDA-MB-468 cells at 60% of confluency were exposed to the OMVs and affi_EGFR OMVs (25 μg) for 2 h at the standard growth culture. After being washed with PBS, cells were fixed in 3.7% of PFA at room temperature for 10 min. Fixed cells were then washed and permeabilized by 0.1% Triton X-100 for 10 min. Avoiding nonspecific interaction of antibodies, cells were treated with 3% (w/v) of bovine albumin serum (BSA) in PBS for 1 h at room temperature. Cells were finally incubated with anti-affibody primary antibody and FITC-labeled anti-goat secondary antibody. The nuclei were stained with propidium iodide (PI, 1 μg/ml) (Sigma-Aldrich, MO, United States) and cells images were taken by the confocal microscope.

To investigate the uptake of affi_EGFR-OMVs by MDA-MB-468 cells, EGFR-overexpressing cells were initially incubated with EGF (7.5 μg) or Cetuximab (186.5 μg) for 2 h in a standard culture medium. Following washing, cells were exposed to 25 μg of OMVs or affi_EGFR-OMVs and incubated for a further 2 h at 37°C. The procedure was similar to that described for tracing cell uptake. Finally, the treated cells were evaluated using a confocal microscope.

Endotoxicity of affi_EGFR-OMVs in both the intact and lysed (with 0.5% of sodium deoxycholate) forms was evaluated using the commercial gel clot LAL kit (PYROTELL, Associates of Cape Cod Inc. United States). Serial dilutions of affi_EGFR-OMVs and its lysate (250 down to 7.81 μg/ml) were applied in this test, and endotoxin activity was assayed as per the manufacturer’s instructions in duplicate. Control standard endotoxin (CSE) (E. coli strain O111:B4, 0.5 EU/mL) and LAL reagent water (LRW), provided by the Associates of Cape Cod Inc., were used as positive and negative controls, respectively.

To assess sterility of the affi_EGFR-OMVs, OMV preparations were plated on LB-agar plates and grown overnight at 37°C. Cytolytic or hemolytic activity assay was performed using horse blood agar plates. To do this, the affi_EGFR-OMVs-derived (500 μg/ml) and its parent strains (transformed ∆msbB/∆pagP W3110) were plated on the horse blood agar plates and incubated at 37°C overnight. β-Streptococcus was also plated as a positive control. The next day, plates were analyzed for any clear zone as a result of red blood cells lysis.

A liquid hemolysis assay was also performed to show the hemolysis activity quantitatively. To this end, human erythrocytes were washed and diluted with a ratio of 2:100 in PBS. Aliquots of the washed erythrocytes were then transferred to a v-bottom 96-well plate and subjected to affi_EGFR-OMVs (6.25–400 μg/ml) for 1 h at 37°C. Upon centrifugal sedimentation of the cells and debris at 2000 rpm for 5 min, the supernatant hemoglobin was quantified by spectrophotometric detection at 540 nm. PBS and deionized water were used as negative and positive controls, respectively. Hemolysis activity was reported relative to the erythrocytes lysed in deionized water.

Cell viability was measured following OMVs exposure via MTT assay in the MDA-MB-468 and HEK293T cell lines. In short, a total of 1 × 10⁵ cells were seeded in 96-well plates overnight and treated with both OMVs and the affi_EGFR-OMVs (25–400 μg/ml) for 4 h. The medium was then refreshed and the cells were further incubated for 72 h in a standard growth condition. To confirm the nontoxic effect of OMVs, the percentage of cell viability was calculated. Cisplatin (Sigma-Aldrich, MO, United States) was also applied as a positive control.

The procedure for this assay was as described previously with certain modifications (Widdrington et al., 2018). Briefly, THP-1 cells were grown as a suspension culture in RPMI-1640 medium containing 10% FBS. Cells were then exposed to 200 nM of phorbol myristate acetate (PMA) for 48–72 h to induce differentiation. Afterward, the obtained adherent cells were treated with affi_EGFR-OMVs (25–400 μg/ml) along with PBS and LPS (100 ng/ml) (Sigma-Aldrich, MO, United States) as negative and positive controls, respectively. Following a 24 h incubation, human TNF-α was quantified using ELISA (Quantikine HS, United States) as per the manufacturer’s instructions.

All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, United States). Differences were considered significant if the p value was <0.05. All graphed values represent the mean, and the error bars show standard error. One-way analysis of variance (ANOVA) followed by post hoc Tukey’s test was used for intergroup comparisons.

The ∆msbB/∆pagP W3110 E. coli K12 strain is a nonpathogenic strain harboring two mutations in msbB and pagP genes to highly reduce endotoxin activity (Lee et al., 2011). ClyA-affi_EGFR-GALA construct (Figure 1A) was transformed into this strain to prepare the affi_EGFR-OMVs with the least cytotoxic effect.

As illustrated by SDS-PAGE, a clear protein band of ∼50 kDa, corresponding to the full-length ClyA-affi_EGFR-GALA along with tags, was detected in the whole-cell lysate of recombinant W3110, indicating that the fusion protein had efficiently been expressed in W3110 cells. Furthermore, the presence of the fusion protein in the OMVs derived from the engineered ∆msbB/∆pagP W3110 samples was confirmed by immunoblotting using anti-6XHis-tag (data not shown) and anti-affibody antibody (Figure 1B). Detection of a reacting band of about 50 kDa further confirmed the successful expression of the ClyA-affi_EGFR-GALA fusion protein in the OMVs.

Next, to verify whether the fusion protein was present on the surface of OMVs, the intact W3110 cells and OMVs were subjected to anti-affibody antibody treatment and analyzed by flow cytometry. Our findings confirmed the presence of affi_EGFR-GALA on the surface of OMVs. In other words, cells and OMVs displaying affi_EGFR were brightly fluorescent, while no fluorescence was detected for the untransformed cells and OMVs (Figure 1C). To validate these findings, we also expressed and then purified the affi_EGFR recombinant protein (16 KDa) as a positive control in BL21 DE3 (Figure 1D).

Taken together, using genetic engineering techniques, the affi_EGFR-GALA was successfully presented on the surface of ∆msbB/∆pagP W3110-derived OMVs via fusion to ClyA.

The identical size distribution of the OMVs and affi_EGFR-OMVs was confirmed by dynamic light scattering analysis (Figure 2B). As reported in Table 1, there was no significant difference (p > 0.05) in terms of polydispersity index (PDI), maximum peak (nm), and Z-average (d.nm) between two types of OMVs. These results were in parallel with the TEM findings confirming that incorporation of a heterologous protein did not alter the size of the affi_EGFR-OMVs. To verify the stability of the OMVs, the frozen OMVs were thawed and the above parameters were determined on three consecutive days at 4°C. As indicated in Table 2, no significant alteration was observed in terms of PDI and Z-average.

Moreover, to demonstrate the colloidal feature of the OMVs solution, the Tyndall effect was evaluated in the samples. The Tyndall effect is the phenomenon indicating the divergence of a light beam when it passes through a colloidal dispersion and a portion of the light is scattered. As it can be seen from Supplementary Figure S1, the PBS buffer sample has no obvious Tyndall effect, while the OMVs solution sample has a strong Tyndall effect confirming the presence of OMVs.

Affi_EGFR-OMVs have to identify EGFR protein in ELISA. For further verification, affi_EGFR protein was used as a positive control. As shown in Figure 3A, both affi_EGFR-OMVs and affi_EGFR interact with the EGF receptor in a concentration-dependent manner; i.e., the higher the affi_EGFR-OMVs concentration, the greater the strength of binding. It is worth mentioning that the graph in Figure 3A finally reaches a saturation plateau. As expected, affi_EGFR was effective even at a low applied concentration. Interestingly, no absorbance was detected for the OMVs even at the highest applied concentrations (Figure 3A). Our calculated K_d values were 86.41 and 2.845 μg/ml for the engineered OMVs and affi_EGFR, respectively. Basd on the K_d values, it is suggested that 1 μg of the affi_EGFR-OMVs includes 32 ng of affi_EGFR corresponding to about 3% of the total protein.

For further validation, an ELISA test was also carried out on the cell lysates. Both affi_EGFR-OMVs and affi_EGFR interacted with MDA-MB-468 cells. A high and reliable concentration (∼EC₉₀) was used for satisfactory results. The results showed that the absorbance values for affi_EGFR and affi_EGFR-OMVs interacting with MDA-MB-468 cell lysate were significantly higher than those for the HEK293T cell lysate. As expected, HEK293T cells did not interact with either affi_EGFR-OMVs, affi_EGFR, and Cetuximab owing to their lack of receptor (Figure 3B). These results are in line with our immunoblotting findings verifying no EGF receptor expression in HEK293T cells (Supplementary Figure S2). Moreover, no significant difference in terms of OD values was detected following incubation of both MDA-MB-468 and HEK293T cells with non-targeted OMVs. In contrast, Cetuximab as a positive control exhibited a high OD value at the concentration applied in MDA-MB-468 cells.

In summary, the above results indicate specific binding of affi_EGFR-OMVs to the cells expressing EGFR receptors due to the presence of the Z_EGFR affibody.

The binding property of the affi_EGFR-OMVs was also compared to that of non-targeted OMVs in MDA-MB-468 and HEK293T cells using flow cytometry (Figures 4A,B). In parallel, affi_EGFR was utilized in this assay as a positive control. As illustrated in the results, the shift in fluorescence intensity for the affi_EGFR-OMVs and affi_EGFR is well associated with the expression level of the receptor on the MDA-MB-468 cell surface; i.e, cells having a higher EGFR expression level revealed a greater shift than the non-EGFR-expressing HEK293T cell line. The non-targeted OMVs exhibited no binding to HEK293T and MDA-MB-468 cells ruling out any nonspecific binding of OMVs to the tumor cells under investigation. These results are in agreement with the ELISA data addressing the specific binding of the affi_EGFR-OMVs to the EGF receptor.

We also found no detection of affi_EGFR-OMVs and affi_EGFR when EGFR-negative HEK293T cells were used since neither a fluorescent shift nor an enhancement in intensity was detected relative to the negative control under the same conditions. Interaction between the engineered OMVs and MDA-MB-468 cells was further investigated by confocal microscopy after co-culturing of the cells with 25 µg of the affi_EGFR-OMVs for 2 h at 4°C. As it can be seen in Figure 5A, the affi_EGFR-OMVs clearly bound to the surface of MDA-MB-468, whereas no binding was observed for HEK293T cells, used as the EGFR deficient cells. The findings of the confocal microscopy were in parallel with those of our previous assays, confirming that the affi_EGFR-OMVs can selectively and effectively target EGF receptors.

To further study the uptake of the OMVs by the cancer cells, MDA-MB-468 were treated with the non-targeted OMVs and affi_EGFR-OMVs for 2 h at 37°C. Confocal microscopy revealed the cellular entry of the affi_EGFR-OMVs, with red and green fluorescence pointing to the location of cell nuclei and OMVs, respectively, while no fluorescence was observed in the MDA-MB-468 cells treated with the non-targeted OMVs (Figure 5B).

To further investigate the specific binding of the affi_EGFR-OMVs, EGFR-specific ligands, EGF, or Cetuximab was applied to compete with the affi_EGFR-OMVs for binding to EGFR-overexpressing MDA-MB-468 cells. The flow cytometric data verified a specific interaction between affi_EGFR-OMVs and MDA-MB-468 cells. As evidenced in Figure 6A, pretreatment with an excess amount of EGF or Cetuximab heavily blocked affi_EGFR-OMVs binding to the target cells (8.20 and 6.04 MFI, respectively), while no significant fluorescent shift was observed in the case of OMVs. Moreover, co-incubation with 3.25 nM–12.5 μM of EGF resulted in a decrease of fluorescent intensity from 89.43 to 9.2% (Figure 6B). Indeed, these data indicate a gradual reduction in the binding ability of affi_EGFR-OMVs to the receptors with increasing amounts of EGF, further supporting the specific binding to EGF receptors.

We also assessed the target-specific delivery of the affi_EGFR-OMVs. To do it, EGF or Cetuximab pretreated MDA-MB-468 cells were examined with a fluorescence confocal microscope. As evidenced in the data, a significant reduction in green fluorescence intensity was visualized following the pretreatment of MDA-MB-468 cells with an excess amount of the competitors, EGF and Cetuximab (Figure 6C).

Overall, the competition results further demonstrated the target-specific affi_EGFR-OMVs delivery to the cells.

Autophosphorylation of EGFR as the primary transformation that happens upon ligand induction is a crucial characteristic of EGFR activation (Lemmon and Schlessinger, 2010). To examine if the affi_EGFR-OMVs activate EGF receptors, phosphorylation of EGFR was monitored following treatment of MDA-MB-468 cells with affi_EGFR-OMVs and EGF as a positive control. Western blotting results using EGFR phosphorylation site-specific antibody (Tyr1068) exhibited a significant band indicating expression of the phosphorylated EGFR in the EGF-treated samples. In contrast, no phosphorylated protein expression level was detected in the affi_EGFR-OMVs treated cells (Figure 7). These findings confirmed that OMVs presenting affibody target cancer cells with no receptor stimulation effect.

Besides the efficacy of targeting, the safety issue is a pivotal concern in the development of nanomedicine, where a bacterium-derived product is utilized. Since OMVs consist of a large portion of LPS activating toll-like receptors (TLRs), which in turn provokes an inflammatory response (Kuehn and Kesty, 2005), we thus evaluated OMV-induced inflammatory response by assessing TNF-α level and cytotoxicity in vitro. We also performed hemolysis and sterility studies and measured the endotoxin levels as well to assure the biocompatibility of the OMVs.

Findings from the gel clot LAL test showed the absence of a stable solid clot in the test tubes at the end of reactions confirming the lack of LPS for affi_EGFR-OMVs and its lysate at all the concentrations used. The lack of endotoxin activity in the intact OMVs and the OMVs lysate, in which the lipid-A component of LPS is exposed, confirmed the exploitation of the LPS-modified ∆msbB/∆pagP W3110 strain in the current study.

The sterility of the purified OMVs was determined by plating the affi_EGFR-OMVs on LB-agar plates and incubating at 37°C overnight. The absence of the bacterial colonies proved that the OMV products were bacteria-free (Figure 8A).

To validate the hemolytic activity of the affi_EGFR-OMVs, the blood agar plated OMVs were prepared under the standard incubation conditions. No hemolysis was visualized upon plating the affi_EGFR-OMVs and its origin bacteria, while clear zones were seen owing to the β-streptococcus strain culturing as a positive control (Figure 8B). The data from the quantitative liquid hemolytic assay supported our findings and displayed no considerable difference in erythrocyte hemolysis compared to PBS as the negative control (Figure 8C). These observations indicated that ClyA fusion protein is in an inert state and the affi_EGFR-OMVs are safe enough to be used as a nanocarrier.

Our data on the MTT assay on HEK293T and MDA-MB-468 cell lines revealed no significant alteration in terms of viability following transfection with the affi_EGFR-OMVs compared to the PBS-treated cells, while cell viability was reduced to about 50% following Cisplatin treatment as a positive control. In other words, the normal appearance of the cells in both treatments implies no cytotoxic effect associated with the affi_EGFR-OMVs (Figure 8D).

Based on a previous study reporting on the potent stimulatory activity for LPS in the production of TNF-α in THP1 cells, we next investigated the stimulation of the immune system by affi_EGFR-OMVs in these cells and found that the affi_EGFR-OMVs caused a slight increase in TNF-α level in human monocyte THP1 cells compared to cells induced with LPS (Figure 8E).

The desired findings from our in vitro assays pointed out the safety and biocompatibility of the engineered OMVs as a drug carrier in cancer research.