Bioadhesive Bacterial Microswimmers for Targeted Drug Delivery in the Urinary and Gastrointestinal Tracts

Bacteria‐driven biohybrid microswimmers (bacteriabots), which integrate motile bacterial cells and functional synthetic cargo parts (e.g., microparticles encapsulating drug), are recently studied for targeted drug delivery. However, adhesion of such bacteriabots to the tissues on the site of a disease (which can increase the drug delivery efficiency) is not studied yet. Here, this paper proposes an approach to attach bacteriabots to certain types of epithelial cells (expressing mannose on the membrane), based on the affinity between lectin molecules on the tip of bacterial type I pili and mannose molecules on the epithelial cells. It is shown that the bacteria can anchor their cargo particles to mannose‐functionalized surfaces and mannose‐expressing cells (ATCC HTB‐9) using the lectin–mannose bond. The attachment mechanism is confirmed by comparing the adhesion of bacteriabots fabricated from bacterial strains with or without type I pili to mannose‐covered surfaces and cells. The proposed bioadhesive motile system can be further improved by expressing more specific adhesion moieties on the membrane of the bacteria.


Bacterial strains and cultivation
E. coli MG1655 which is used to fabricate the bacteriabots was purchased from Coli Genetic Stock Center (Yale University, New Haven, USA). The bacterial strain without pili (ECM1, E. coli MG1655ΔfimA-H) was obtained from Prof. Luis Ángel Fernández (Spanish National Center for Biotechnology, Madrid, Spain). The strain, which has a deletion in the operon encoding type 1 pili, is derived from E. coli MG1655 33 .
The bacterial strains, which are used for the fabrication of bacteriabots, were cultivated on swarm plates to show the highest motility. Both strains were cultured overnight in LB broth at 37 °C, 200 rpm. For E. coli MG1655, 25 μL of the bacterial culture was inoculated on the surface of a swarm plate and incubated overnight at 30 °C. The bacteria from the edge of the swarm plate were used to inoculate the tryptone broth which was incubated until it reached to OD 600 0.2. For ECM1 strain, due to lack of pili, the bacteria was directly transferred to tryptone broth and incubated at 37 °C until it reached to OD 600 0.3.

Growth analysis of the bacteria at different pH conditions
The growth of E. coli MG1655 at different pH conditions were investigated using BioTek Synergy 2 Multi-Mode microplate reader. Briefly, single colonies of the bacteria were inoculated into each well of 96-well plate which contained LB media with pH 5.7, pH 6.0 and pH 7.4. Then, the plate was incubated with a slow shaking at 30 °C and absorbance at 600 nm was collected for every 10 min.

Viability analysis of the bacteria at different pH conditions
The viability of E. coli MG1655 at different pH conditions was investigated using Molecular Probes LIVE/DEAD ® BacLight ™ bacterial viability kit. The experimental was performed as provided by the company with slight changes. Briefly, E. coli MG1655 was cultured overnight in different LB media with pH 5.7, pH 6.0 and pH 7.4 at 30 °C, 200 rpm. 5 mL of the bacterial cultures were concentrated by centrifugation at 10000 x g for 10 min.
After that, the pellets were resuspended in 1 mL of 0.85% (w/v) NaCl, and 500 µL of these suspensions were added into new tubes which contained 10 mL of 0.85% (w/v) NaCl. The samples were incubated for 1h at room temperature by mixing in every 15 min. At the end of the incubation period, the samples were collected by centrifugation at 10000 x g for 10 min, and then washed 2 times with 0.85% (w/v) NaCl. 1 mL of each bacterial suspensions were mixed and incubated with 3 µL of the dye, which was prepared by combining equal volume of Component A and Component B, at room temperature for 15 min. Finally, 5 µL of the stained bacterial suspensions were investigated with Nikon Eclipse Ti-E spinning disk confocal microscope using 60x oil immersion objective.

Motility analysis of the bacteria at different pH conditions
The motility of E. coli MG1655 at different pH conditions was analyzed using an inhouse MATLAB tracking code. Briefly, 25 μL of the bacterial culture was inoculated on the surface of a swarm plate and incubated overnight at 30 °C. Then, 25 µL of the bacteria from the edge of the swarm plate was inoculated into different LB media with pH 5.7, pH 6.0 and pH 7.4, and incubated until they reached to OD 600 0.2. After that, the bacterial cultures were 10 times diluted in their respective motility buffers which had different pH conditions (pH 5.7, pH 6.0 and pH 7.4). Finally, the bacterial motility was checked under Leica DMi8 inverted microscope in micro-chambers using 40x water immersion objective and the videos, used for the analysis, were recorded at 50 fps.

Interaction of the bacteria with BSA-mannose coated surfaces at different pH conditions
The attachment of the bacteria to BSA-mannose functionalized surfaces at different pH conditions was performed as described before. Briefly, the glass surfaces were functionalized with APTES by incubating them with APTES for 45 min at room temperature, followed by rinsing with isopropanol and performing soft-backing at 120 °C for 5 min. The APTES-treated glass slides were incubated with BSA-mannose solutions (100 μg/mL) for 1 h and rinsed three times with PBS afterward. The suspensions of the bacteria were added to these surfaces and incubated at room temperature for 1 h. The slides were washed three times using 1X PBS and the density of bacteria attached to the surface was calculated after imaging 12 samples using spinning disk confocal microscope.

Toxicity analysis of the bacteriabots
The cytotoxicity of bacteriabots was analyzed using MTT and LDH assays over a broad range of concentration of bacteria, particles, and bacteriabots. For this purpose, the cells were seeded in 96 well-plate at a density of 10 4 cells/well and incubated to reach to 90% confluency. The cells were washed using DPBS buffer. Different concentrations of bacteria, particles, and bacteriabots suspended in RPMI 1640 media were added to the cells and incubated for 4 h at 37 °C. Pure RPMI 1640 was used as positive control and 1% Triton X-100 was added as a negative control. After 4 h, the supernatant was separated and incubated with LDH reagent for 5 min, followed by reading the absorbance at 492 nm using microplate reader (Synergy2, Biotek, Winooski, USA). The cells were incubated with MTT reagent for another 4 h, followed by dissolving cells using DMSO to release the formazan content. The absorbance was read at 550 nm using a plate reader. The absorbance of positive and negative controls was considered as 100% and 0% viability of the cells.

Assessment of the immunogenicity of the bacteriabots
To investigate the effect of the bacteriabots on activation of the complement system, a C3a complement ELISA kit (Quidel, San Diego, USA) was used according to the manufacturer instructions. For this purpose, 100 μL aliquots of normal human serum (NHS, Quidel, San Diego, USA) were incubated with different concentrations of bacteria (with or without pili) and bacteriabots for 30 min at 37 °C. 10 μL of EDTA (220 mM) was added to NHS as a positive control, and 3 μL of cobra venom factor (CVF, Quidel, San Diego, USA) was added to NHS as a negative control. CVF is supposed to activate the whole C3a content. 100 μL of 5000 times diluted samples were added to the 96-well ELISA plate (containing a murine antibody) and the plate was incubated for 1 h at room temperature. The wells were washed 4 times before100 μL of conjugate solution (contains horseradish peroxidaseconjugated polyclonal antibody to C3a) was added to each well. The plate was incubated at room temperature for 1 h and washed afterward. 100 μL of substrate solution (containing 3,3′,5,5′-tetramethylbenzidine and H 2 O 2 ) was added to each well. After incubating the plate at room temperature for 15 min (protected from light), 100-μL stop solution (1 N HCl) was added to each well. The C3a concentration was calculated after reading the absorption of each well at 450 nm using a microplate reader. The results were normalized considering the positive control as 0 ng/mL C3a. Figure S1. The effect of biotin-conjugation type (using long or short spacer arms) on the attachment efficiency of bacteria to particles. Kolmogorov-Smirnov test (nonparametric and unpaired t test) was applied to analyze the results and significant differences (***) were expressed when p<0.05. Error bars indicate standard error of the mean (SEM, n=6). Figure S2. The density of bacteria with or without pili (E. coli MG1655 and E. coli ECM1) attached to different surfaces, functionalized with BSA and BSA-Mannose. Kolmogorov-Smirnov test (nonparametric and unpaired t test) was applied to analyze the results and significant differences (****) were expressed when p<0.05. Error bars indicate standard error of the mean (SEM, n=10).       Figure S10. HTB-9 cells treated with Concanavalin A and exposed to MG1655-based bacteriabots. It can be seen that no particles attached to the cells (scale bar=20 μm). Figure S11. Cytotoxicity of different concentrations of (a) 2.2 µm PMMA particles, (b) E. coli MG1655, and (c) E. coli ECM1 toward HTB-9 cells analyzed by LDH assay. LDH activity was monitored after 4 h incubation of the cells with the samples (n=3, 0.1% Bead=3×10 8 particles/mL). Figure S12. Cytotoxicity of bacteriabots, which were fabricated from 2.2 µm PMMA particles and E. coli ECM1 toward HTB-9 cells, analyzed by (a) LDH and (b) MTT methods. Concentration X is the optimum concentration, which was used in the attachment experiments according to materials and method part.