*3.6. Evaluation of the Internalization of B12-ASOs by Epifluorescence Microscopy*

To evaluate the extent of internalized B12-ASO conjugates, compared with the ASOs alone, we used epifluorescence microscopy. An overnight culture of *E. coli* K12 was diluted to an OD<sup>600</sup> of 0.1 in fresh Davis minimum medium. The B12-ASOgapmer, B12-ASOsteric, B12, ASOgapmer, and ASOsteric (all Cy3-labeled) were diluted in sterile distilled H2O and added to the bacterial suspension to a final concentration of 15 and 30 µM per test tube. After 4 h incubation, tubes were centrifuged (3000× *g*, 10 min), and the pellets were resuspended in sterile distilled H2O. To label the bacterial cytosol, 40 ,60 -diamidino-2-phenylindole (DAPI) staining was used. Staining was performed by placing a drop of DAPI (0.5 µg/mL) on top of the dried sample for 5 min. The samples were visualized on a Nikon Eclipse T*i* SR epifluorescence microscope using a Nikon Plan-Apo 100X objective. Ten pictures of each sample were taken randomly, covering all the areas of the sample, using a QImaging Retiga R1 monochromatic camera, and processed with the NIS-Elements Advanced Research. The exposure time and the excitation intensity were maintained throughout the experiments. A G-2A longpass filter (excitation: 535 nm; emission: 580 nm) and a DAPI bandpass filter (excitation: 375 nm; emission: 460 nm) were used. The images obtained using both filters were merged using the Fiji software. Three repeated samples were analyzed for each condition, and three independent experiments were performed.

#### *3.7. Evaluation of the Internalization of B12-ASOs by Bacterial Fractionation 4.7. Evaluation of the Internalization of B12-ASOs by Bacterial Fractionation*

*Antibiotics* **2021**, *10*, x FOR PEER REVIEW 9 of 12

To determine the location of the conjugates in the *E. coli* K12 cells visualized in the previous section and investigate if the observed fluorescence could derive from association to the bacterial envelope, as opposed to intracellular hybridization, the cells were fractionated, and the fluorescence of the outer-membrane fraction and periplasm vs. the fluorescence of the cytosol fraction was measured. To determine the location of the conjugates in the *E. coli* K12 cells visualized in the previous section and investigate if the observed fluorescence could derive from association to the bacterial envelope, as opposed to intracellular hybridization, the cells were fractionated, and the fluorescence of the outer-membrane fraction and periplasm vs. the fluorescence of the cytosol fraction was measured.

QImaging Retiga R1 monochromatic camera, and processed with the NIS-Elements Advanced Research. The exposure time and the excitation intensity were maintained throughout the experiments. A G-2A longpass filter (excitation: 535 nm; emission: 580 nm) and a DAPI bandpass filter (excitation: 375 nm; emission: 460 nm) were used. The images obtained using both filters were merged using the Fiji software. Three repeated samples were analyzed for each condition, and three independent experiments were performed.

An overnight inoculum of *E. coli* K12 was diluted to an OD<sup>600</sup> of 0.1 and grown in Davis minimal medium in the presence of 30 µM of B12-ASOgapmer, B12-ASOsteric, and B12, (all Cy3-labeled) for 4 h. Thereafter, a fractionation protocol (Figure 6) adapted from Banbula et al. [50] was followed. In brief, bacteria were centrifuged (3000× *g*, 20 min), resuspended in 10 mM Tris-150 mM NaCl (pH 7.4), and washed with 50 mM Tris (pH 7.6). To obtain the fraction associated with the outer-membrane (membrane fraction), bacteria were centrifuged (3000× *g*, 20 min) and resuspended in 50 mM Tris buffer solution containing 0.05% Triton X-100 (pH 7.6), for 1 h at room temperature (RT). After new centrifugation (same conditions), the Cy3 fluorescence intensity of the supernatant (membrane fraction) was measured with a fluorometer (BMGLabtech Fluorostar Omega), using 550 nm excitation and 570 nm emission filters. To obtain the fraction associated with the cytosol, the pellet was resuspended in a more astringent buffer containing 50 mM Tris 1% Triton X-100 (pH 7.6), for 1 h at RT. The supernatant resultant from the last centrifugation was removed, and the Cy3 fluorescence of the cytosol (cytosol fraction) was measured. As a control, the same fractionation protocol was applied to bacteria stained only with DAPI, and the fluorescence of the membrane and cytosol fractions was measured using 375 nm excitation and 460 nm emission filters. An overnight inoculum of *E. coli* K12 was diluted to an OD600 of 0.1 and grown in Davis minimal medium in the presence of 30 μM of B12-ASOgapmer, B12-ASOsteric, and B12, (all Cy3-labeled) for 4 h. Thereafter, a fractionation protocol (Figure 6) adapted from Banbula et al. [50] was followed. In brief, bacteria were centrifuged (3000× *g*, 20 min), resuspended in 10 mM Tris-150 mM NaCl (pH 7.4), and washed with 50 mM Tris (pH 7.6). To obtain the fraction associated with the outer-membrane (membrane fraction), bacteria were centrifuged (3000× *g*, 20 min) and resuspended in 50 mM Tris buffer solution containing 0.05% Triton X-100 (pH 7.6), for 1 h at room temperature (RT). After new centrifugation (same conditions), the Cy3 fluorescence intensity of the supernatant (membrane fraction) was measured with a fluorometer (BMGLabtech Fluorostar Omega), using 550 nm excitation and 570 nm emission filters. To obtain the fraction associated with the cytosol, the pellet was resuspended in a more astringent buffer containing 50 mM Tris 1% Triton X-100 (pH 7.6), for 1 h at RT. The supernatant resultant from the last centrifugation was removed, and the Cy3 fluorescence of the cytosol (cytosol fraction) was measured. As a control, the same fractionation protocol was applied to bacteria stained only with DAPI, and the fluorescence of the membrane and cytosol fractions was measured using 375 nm excitation and 460 nm emission filters.

**Figure 6.** Fractionation protocol adapted from Bandula et al. [50]. A series of washing steps with a Triton X-100 gradient allows the isolation of the membrane and the cytosol fractions. **Figure 6.** Fractionation protocol adapted from Bandula et al. [50]. A series of washing steps with a Triton X-100 gradient allows the isolation of the membrane and the cytosol fractions.

#### *4.8. Statistical Analysis 3.8. Statistical Analysis*

For the evaluation of the statistical significance, the two-way analysis of variance test (ANOVA) followed by Sydak's multiple comparisons was used. A *p*-value of *p* ≤ 0.05 was considered statistically significant.

#### **4. Conclusions**

In summary, this innovative investigation discloses the challenges that need to be overcome before B<sup>12</sup> -mediated ASO internalization is considered realistic toward tackling

the challenge of antimicrobial resistance. This strategy is based on the uptake of the micronutrient B12, which seems to be insufficient to act as an efficient trojan-horse for ASOs designed to inhibit the expression of an essential bacterial gene. In the future, it would be relevant to assess the concentration of internalized ASOs needed to efficiently knock down bacterial genes and inhibit bacterial growth. In addition, improving the bioavailability of vitamin B<sup>12</sup> by modifying the conjugates and choosing better adapted bacterial targets would be important for successful translation from in vitro to in vivo application.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics10040379/s1, Figure S1: Analytic RP-HPLC trace and MALDI-MS on B12-ASOgapmer and B12-ASOsteric. The top panel shows the retention time (in minutes) of the B<sup>12</sup> conjugates, and the bottom panel shows the mass (m/z) of each B<sup>12</sup> conjugate.; Figure S2: Interaction of Cy3 labeled ASOs, B12, and B<sup>12</sup> conjugates with *E. coli* K12, after 4 h incubation at a concentration of 15 µM. Bacteria are counterstained with DAPI. Images are representative of three independent experiments (using duplicates in each). Scale bar represents 5 µm; Figure S3: Most of the conjugated (B12-ASOsteric) and unconjugated B<sup>12</sup> (B12) are prevented from internalization into *E. coli* cytosol since they remain at the outer-membrane; the percentages in the periplasm are only residual. The isolation of the periplasm was performed after the isolation of the OM fraction and was based on the fractionation protocol by Malherbe et al. 2019. *E. coli* cells were washed in spheroplast buffer (0.1 M Tris-NaCl, 500 mM sucrose, 0.5 mM EDTA, pH 8.0) followed by resuspension in distilled water and incubation for 15 s on ice. The osmotic shock occurred after the addition of MgSO<sup>4</sup> (final concentration 20 mM). DAPI was used as a control, majorly localizing at the cytosol, as expected. Statistical differences are indicated when appropriate in \* (*p* ≤ 0.0001, \*\*\*\*); Table S1: Characterization of the synthesized conjugates, including their HPLC retention times (tR) and molecular masses, as well as the yield of the respective conjugation reactions.

**Author Contributions:** Conceptualization, S.P., R.S.S., N.F.A., and J.W.; methodology, S.P., R.Y., P.T.J., J.W., N.F.A., and R.S.S.; experimental, S.P., M.G., and R.Y.; statistical analysis, S.P.; writing—original draft preparation, S.P. and R.S.S.; writing—review and editing, J.W.; N.F.A., P.T.J., M.G., and R.Y.; supervision, R.S.S., N.F.A., and J.W.; funding acquisition, N.F.A. and J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by Fundação para a Ciência e Tecnologia, PhD grant SFRH/BD/ 118018/2016; the Project UID/EQU/00511/2019-Laboratory for Process Engineering, Environment, Biotechnology and Energy–LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project "LEPABE-2-ECO-INNOVATION"–NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); and the European Union's Horizon 2020 research and innovation program under grant agreement No 810685; Biomolecular Nanoscale Engineering Center (BioNEC), a VILLUM center of excellence, funded by VILLUM FONDEN, grant number VKR18333.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article and in the supplementary material.

**Acknowledgments:** Joan Hansen and Tina Grubbe from BioNEC are thanked for technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

