Conclusion
The NanoBEADS is a bacteria-based bio-hybrid drug delivery system designed to utilize self-propelling motile bacteria to enhance the interstitial delivery of nanomedicine. As is the case for all biohybrid delivery systems, the effectiveness of NanoBEADS hinges upon maximizing its NP load without affecting its viability and minimal effect on its motility. It is also imperative that the NanoBEADS construction process produces a repeatable distribution of NP load densities (i.e., therapeutic load) to facilitate the use of such a system in translational applications. Thus, a systematic investigation of the effects of the assembly process parameters, linkage chemistry, and NP size on NanoBEADS properties (i.e., NP attachment density and repeatability, growth rate, and swimming speed) was carried out. We selected biotin-streptavidin linkage chemistry for this study due to its prevalence in constructing bacteria-based biohybrid microrobots \cite{Akin_2007}\cite{Traore_2014}\cite{Kazmierczak_2014}\cite{Nguyen_2016}\cite{Alapan_2018}\cite{Kojima_2012}\cite{Huter1999}\cite{Buss2020}\cite{Hiratsuka2005}\cite{Singh2017}\cite{Carlsen2014}. Furthermore, the peritrichously flagellated S. Typhimurium was selected due to the common use of Gram-negative bacteria (E. coli, S. marcescens, etc.) as well as other flagellated bacteria in the construction of bacteria-based microrobots. The selected PLGA nanoparticles of two different sizes represent a good model for the polymeric nanoparticles used in constructing biohybrid microrobots. For the ABS NanoBEADS, using an end-over-end mixer with a total mixing volume of 800 µL for 60 min assembly period (E-800-60) produced the highest and most repeatable NP attachment density without affecting the viability of the bacteria. In the case of BS NanoBEADS, where streptavidin-functionalized nanoparticles were attached to the bacteria using biotin that was physisorbed to the bacteria, the attachment density decreased by more than 70%, compared to the ABS NanoBEADS case. The optimal NanoBEADS construction strategy reported herein is extensible to other NP materials and sizes (Figure S4). The motility speed is adversely affected with the attachment of larger particles at high density, but the attachment of NPs smaller than 120 nm did not affect the motility speed even at high attachment density (Figure S4). Irrespective of NP size, reduction in attachment density restored the growth rate to the control bacteria levels. However, growth was unaffected for the smaller 40 nm gold NPs even at the high NP attachment density of 21 gold NPs/µm2 (Figure S5).
Altogether, we posit that the new knowledge in the effect of assembly parameters and linkage chemistry on bacteria-nanoparticle assembly outcomes and the effect of NP size on key bacterial behaviors of motility and growth in bacteria-based biohybrid systems will facilitate the design and development of more efficacious bacteria-mediated delivery systems for a variety of applications. The exact quantitative values for attachment density or changes in motility speed and growth rate are likely to depend on the choice of bacteria, nanoparticle, and linkage chemistry. Nonetheless, the observed trends, such as the effect of assembly parameters on nanoparticle attachment density and repeatability, are expected to be generalizable (Figure S5). Similarly, the reported trends for the effect of linkage chemistry on nanoparticle attachment density or the effect of nanoparticle size and density on motility speed and growth rate can inform the design of process parameters and nanoparticle selection in other bacteria-based biohybrid systems.
Acknowledgements
This project was partially supported by the National Science Foundation (CAREER award, CBET-1454226) and the Institute for Critical Technology and Applied Science (ICTAS) at Virginia Tech.
Supporting Information
Supporting Information is available from the Wiley Online Library.