Results and Discussion

The Effect of the assembly process parameters on NP attachment density

The NP attachment density is critical to the efficacy of bacteria-based drug delivery systems, as it determines the amount of therapeutic load carried by each NanoBEADS agent. We first investigated the effect of mixing method (i.e., the type of mechanical mixer employed when mixing the streptavidin-coated NPs and the biotinylated bacterial suspensions, Video S1), assembly volume (i.e., total suspension volume during mixing), and assembly period, as depicted in Table 1, on the formation of Antibody-Biotin-Streptavidin NanoBEADS (ABS NanoBEADS) using 165±11 nm diameter NPs. Informed by our prior work,\cite{Suh_2018}  all NanoBEADS assembly experiments were conducted at the bacteria to NPs ratio 1:100 and biotinylated antibody concentration of 10 µg/mL (see Methods for detail). The number of attached NPs on each bacterium was quantified using Scanning Electron Microscopy (SEM) and reported as the number of attached NPs per projected unit area of the cell surface (#NPs/µm2).
We first evaluated the effect of assembly volume and duration using a vortex mixer (V) for two different assembly volumes of 100 µL (V-100) and 800 µL (V-800) and three different assembly periods of 30, 60, and 90 min. As shown in Figure 3a, assembly volume had no statistically significant effect on the average NP attachment density of ABS NanoBEADS constructed using a vortex mixer, except for the significantly decreased NPs attachment density in the 60 min assembly period, when the volume increased from 100 µL to 800 µL (p<0.01). In the case of 100 µL assembly volume, the NPs attachment density significantly increased by increasing duration from 30 min to 60 min (p<0.01). In contrast, there was no significant difference in the NPs attachment density for V-800-60 comparing to the other two durations of V-800, as shown in Figure 3a, b. Keeping the high throughput biofabrication needs for in vivo experiments and future translational applications in mind, we examined other mixing methods at the higher 800 µL volume and compared the results from V-800 with those of the Belly Dancer® mixer (B-800) and the end-over-end mixer (E-800) for each of the three assembly durations.