We next examined the effect of NP size and attachment density on the growth of NanoBEADS. While NanoBEADS can be stored in media that do not support growth in vitro, they are expected to operate in vivo for days\cite{Suh2019} and are observed to grow in the nutrient-rich tumor microenvironment.\cite{Suh2018} As NanoBEADS grow, the attached NP load is divided amongst the daughter cells, leading to a more effective distribution of the NPs within the tumor.\cite{Suh2019} Furthermore, many tumor-targeting bacteria, including S. Typhimurium VNP20009 used in this study, have instinct and immune-mediated antitumor effects\cite{Song2018} \cite{Zhou2018} in addition to serving as a delivery vector for the nanomedicine attached to their outer membrane. Thus, intratumoral bacterial growth augments the therapeutic effect of fixed nanomedicine payload. The doubling times of both NanoBEADS variants at both NP sizes were measured and compared to the doubling time of the bacteria (control). All experiments were performed in a mammalian cell culture medium, given the ultimate application of the NanoBEADS platform as a cancer drug delivery system. For the optimal assembly parameters, i.e., E-800-60, we first investigated if coating of the outer membrane or the mechanical agitation of the assembly process affects the growth rate. We found no statistically significant difference in the average doubling time of unmodified bacteria, biotinylated antibody-coated bacteria, and biotin-coated (physisorbed) bacteria, suggesting that surface modification did not affect the bacterial growth rate (Figure S2). However, bacteria subjected to mechanical agitation experienced during the E-800-60 assembly process (in the absence of NPs) resulted in a statistically significant (p<0.05) increase in the average doubling time of the bacteria from 49±3 min to 59±4 min, which may be attributable to the mechanical stress. Next, we measured the doubling time of the NanoBEADS constructs and compared them to the doubling time of the mechanically-treated (E-800-60) bacteria as the baseline control (Figure 5f). The average doubling time of the 165 nm and 121 nm ABS NanoBEADS were 95±8 min and 104±2 min, both of which were significantly longer (p<0.05) than the control doubling time. The average doubling time of the 165 nm and 121 nm BS NanoBEADS were significantly shorter (p<0.05) than their ABS NanoBEADS counterparts at 64±0.2 min and 54±5 min. Interestingly, no significant difference between the doubling time of BS NanoBEADS and control bacteria was observed. We attribute the significantly longer doubling time of the ABS NanoBEADS (p<0.05) to the higher NP attachment areal density, as shown in Figure 5a. NP size did not significantly affect the NanoBEADS doubling time within each variant type (p>0.05). Altogether, our results suggest that NP attachment density and not the NP size affects the NanoBEADS doubling time for the range of NP size tested. The observed trend in the effect of NP size on growth rate was conserved when the closely comparable B-800-60 assembly parameters were used (Figure S3b). Lastly, we note that the dependency of doubling time on NP attachment density may become less significant with particle size reduction. We observed that the doubling time of the ABS NanoBEADS constructed with significantly smaller 40 nm gold particles at comparable NP attachment density to 165 nm PLGA particles was comparable to that of control (Figure S4, S5).   

Effect of NP size and attachment density on NanoBEADS motility

We next assessed the effect of NP size and attachment density on motility by measuring each NanoBEADS variant's swimming speeds with small or large NP attached and compared the speeds with that of bacteria as the baseline (control). Before characterizing NanoBEADS motile behavior, we first evaluated how chemical and mechanical processes that comprise the NanoBEADS construction process affect the bacteria motility in the absence of NPs. The average speeds of the bacteria cultured under standard microbiological conditions (control), mechanically-treated bacteria without antibody coating or nanoparticle attachment (control, E-800-60), antibody-coated bacteria, and mechanically-treated (E-800-60) antibody-coated bacteria without particles are shown in Figure 6a. Mechanical agitation decreased the average speed of bacteria from 9.2±5.6 µm/s to 4.8±3.9 µm/s (p<0.05). However, the choice of mixing method did not have a statistically significant effect on the bacteria motility speed (Figure S3c). Antibody coating of bacteria had a similar statistically significant reducing effect on the bacteria motility speed to 4.5±1.5 µm/s (p<0.05). The combination of mechanical agitation and antibody coating did not result in any further statistically significant degradation in speed (4.7±2.1 µm/s). We attribute the reduction in swimming speed to the fragile structure of the flagella in S. Typhimurium VNP20009 cheY+, which can be disrupted through mechanical agitation.  \cite{Broadway_2017}
We next examined the motility speed of both NanoBEADS variants constructed using the E-800-60 assembly parameters and attached with 165 nm or 121 nm NPs, as shown in Figure 6b and S3d. To assess the effect of NP size, we compared the motility speed within each variant category. We did not observe a statistically significant difference between the 121 nm ABS NanoBEADS average swimming speed (4.4±1.0 µm/s) and the 165 nm ABS NanoBEADS (4.0±1.2 µm/s). It appears that the contribution of the larger 165 nm NPs to the drag force is comparable to the contribution of the larger quantity of the smaller 121 nm particles (Figure 5e). In the case of BS NanoBEADS, the NanoBEADS with the smaller 121 nm NPs have a lower average speed (4.2±0.5 µm/s) than NanoBEADS with 165 nm NPs (4.5±1.4 µm/s), but the difference is not statistically significant. To assess the effect of linkage chemistry on motility speed, we compared the motility speed of the two NanoBEADS variants at each particle size. In the case of NanoBEADS with 165 nm NPs, BS NanoBEADS have a significantly higher average speed than ABS NanoBEADS (p<0.01), presumably due to the lower NP attachment density. Whereas, for the NanoBEADS with the smaller 121 nm NPs, both NanoBEADS variants have similar average speed, which was not significantly different from the average speed of antibody-coated E-800-60 control bacteria.