Introduction
Biohybrid or cell-mediated drug delivery systems, also referred to as biohybrid microrobots, consist of nanocarriers conjugated with host cells,\cite{Stephan_2010}\cite{Mooney_2014}\cite{Huang_2015}\cite{Villa_2016}\cite{Choi_2007}\cite{Roger_2010} attenuated pathogens,\cite{Akin_2007}\cite{Traore_2014}\cite{Suh_2018} or generally regarded as safe (GRAS) microorganisms,\cite{Xie_2017} which are harnessed as living machines for transport of therapeutic loads (for a recent comprehensive review, see ref\cite{Bastos-Arrieta2018}\cite{Schmidt_2020}). In contrast to the traditional systemically administrated therapeutics, bio-hybrid microrobotic systems for drug delivery are capable of active and targeted delivery using a variety of motility mechanisms (i.e., actuation for self-propulsion) and receptors (i.e., sensors) that enable the cells to process and respond to external signals from their environment, including other cells. The innate actuation and sensing mechanisms powered by chemical energy harvested from the environment provide a distinct advantage to biohybrid microrobots, compared to fully synthetic microrobotic systems that typically rely on external electrical, magnetic, optical, or acoustic sources of energy for controlled actuation. Thus, biohybrid microrobots have considerable potential for targeted delivery of drugs, genes, mRNA, proteins, imaging contrast agents, and radioactive seeds to specific target locations accessible vascularly, orally, or even interstitially. In particular, bacteria possess unique properties of high speed (up to ~50 body lengths per second) self-propulsion through blood,\cite{Gekle2016} mucus,\cite{Celli2009} and tissue,\cite{Toley2012} biased migration or taxis in response to a variety of stimuli (e.g., chemotaxis,\cite{Adler_1966} aerotaxis,\cite{Taylor_1999} magnetotaxis,\cite{Faivre_2008} phototaxis,\cite{Bhaya_2004}\cite{Taylor_1975} and pH-taxis\cite{Kihara_1981}), and can be genetically manipulated to produce attenuated or auxotrophic strains,\cite{Clairmont_2000}\cite{Low_1999}\cite{Zhao2005} which support safe and selective colonization of bacteria in vivo.\cite{Toso_2002}\cite{Heimann_2003}\cite{Nemunaitis_2003}\cite{Le_2012}\cite{Roberts_2014}\cite{Schmitz_Winnenthal_2018}\cite{Le_2015}\cite{Basu_2018} Altogether, the advantages of targeted accumulation, deep penetration through self-propulsion, and straightforward genetic manipulation, make bacteria an ideal candidate for targeted therapeutic delivery.
Bacteria-based bio-hybrid drug delivery systems are comprised of live bacteria, for sensing and controlled transport, and abiotic micro- or nano-particles as cargo. Construction of an effective bio-hybrid drug delivery system requires an attachment mechanism that is stable in vivo and is amenable to interstitial transport, yields sufficient and repeatable cargo attachment density for a predictable therapeutic outcome, and does not render the bacteria non-motile. Electrostatic interactions, \cite{Behkam_2008}\cite{van_Loosdrecht_1990} hydrophobic interactions,\cite{van_Loosdrecht_1990} covalent binding,\cite{Xie_2017}\cite{Felfoul_2016}\cite{Taherkhani_2014} bioaffinity interactions,\cite{Traore_2014}\cite{Kazmierczak_2014}\cite{Nguyen_2016}\cite{Alapan_2018} antibody-antigen interactions,\cite{Xu_2012} or a combination thereof,\cite{Akin_2007}\cite{Traore_2014}\cite{Alapan_2018}\cite{Kojima_2012} have been used to attach nanoparticles (NP) to bacteria. Although various attachment methodologies have been explored, a systematic investigation of the effect of the conjugation chemistry and the assembly process parameters on the NP attachment density and repeatability has not been attempted before. Furthermore, the effect of NP load size and quantity on bacterial motility and growth is rarely explored. In this work, we used our previously developed bacteria-based bio-hybrid platform, known as Nanoscale Bacteria-Enabled Autonomous Drug Delivery System (NanoBEADS),\cite{Traore_2014} to investigate the aforementioned questions. Two linkage chemistries were separately utilized to attach poly(lactic-co-glycolic acid) (PLGA) NPs to the tumor-targeting S. Typhimurium VNP20009 cheY+ bacteria, as shown in Figure 2. The effect of assembly process parameters of mixing method, volume, and duration on the NP attachment density and repeatability was investigated. Subsequently, for the two best performing sets of assembly parameters, the effect of linkage chemistry and NP size on NP attachment density, viability, growth rate, and motility of NanoBEADS was studied. We found the linkage chemistry most significantly affected the NP attachment density. For each of the two binding mechanisms tested, the assembly process parameters also influenced NP attachment's areal density and repeatability. Furthermore, the increase in the NP load-carrying capacity led to an increase in doubling time and a reduction in NanoBEADS motility speed in an NP size-dependent manner.