(110c) Chemotactic Live Autonomous Drug Delivery Agents With Different Body Geometries

Chemotactic live autonomous
drug delivery agents with different body geometries

Ali Sahari¹, Mahama A. Traore²,
and Bahareh Behkam^{1, 2}

¹School
of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA 24061

²Mechanical
Engineering Department, Virginia Tech, Blacksburg, VA 24061

INTRODUCTION

Bacteria-based
drug delivery carriers are envisioned to significantly improve the targeted
delivery of drug molecules and imaging agents to the regions of interest. These
autonomous drug carriers if directed by chemical cues, normally present in
diseased areas such as tumors, can offer novel solutions for targeted drug
delivery. DrugBots, the potential drug carriers developed by the authors, are
constructed by interfacing various shapes micro/nano-particles with an ensemble
of live engineered bacteria with the purpose of using the bacteria for
actuation, sensing, communication and control. Non-spherical
micro/nanoparticles are recognized to have advantages over their spherical
counterparts such as higher margination and adhesion as well as prolonged
circulation which make them attractive for drug delivery purposes. Gradients of
chemical attractants (such as naturally occurring gradients of disease
biomarkers) can be utilized to control the motion of the bacteria (and hence
DrugBots with spherical and non-spherical body shapes) towards higher
concentrations of chemo-effectors (i.e. disease site). In the work presented
here, the chemotactic response of DrugBots is quantitatively characterized in the
presence of a stable and well-controlled concentration gradient of a
chemo-attractant, established within a flow-free hydrogel-based microfluidic
device. This study focuses on investigating the role of body shape and
chemotaxis in the motile behavior of DrugBots. We established a linear gradient
of L-Aspartic acid for bacteria in a hydrogel-based microfluidic device in
order to characterize the DrugBot transport for two different morphologies of
the DrugBot, namely sphere and elliptical disk. Disks were fabricated here
using a casting and mechanical stretching technique [1]. This non-spherical
shape was selected as it has been shown to exhibit longer half-life circulation
and higher targeting specificity in mouse models [2] which makes them a good
candidate, along with their spherical counterparts, for chemotaxis experiments.

MATERIALS AND METHODS

PEG-DA based Microfluidic device fabrication: A diffusion-based microfluidic device using 700 Da
polyethylene glycol diacrylate (PEG-DA) hydrogel was fabricated through a
one-step UV polymerization process. The three parallel channels separated by the
two porous hydrogel walls enabled us to establish concentration gradients of
chemo-effectors for bacteria (and DrugBots) (see Fig. 1). This was accomplished
by continuous flowing of a chemo-attractant and a buffer solution in the two
outer channels and lateral diffusion of the chemo-attractant through the
hydrogel wall and into the flow-free buffer filled center channel after
assembling the device. The quasi-steady linear gradient in the center channel
was maintained throughout the entire experiment.

DrugBot Construction: Elliptical disk-shaped (ED) polystyrene (PS) particles were
fabricated using a high throughput particle casting and stretching technique
[1]. Motile sub-populations of Escherichia coli (E. coli) MG1655
transformed with pHC60 (GFP-expressing plasmid) were grown in Tryptone Broth to
OD₆₀₀=0.5 at 32 ˚C and incubated with goat polyclonal
anti-lipid A LPS antibody labeled with biotin (10 μg/ml). The two
collections of 6 µm microparticles (spheres and EDs) were coated with
Cy3-streptavidin and were added to the bacterial solution which produced the DrugBots
(see Fig. 2) through self-assembly by utilizing the high affinity between biotin
and streptavidin.

L-Aspartic acid, a known chemoattractant for E.
coli bacteria, was selected to control the movement of the DrugBots in the device.
Spherical and ED-shaped DrugBots were injected into the chemotaxis observation
channel (see Fig. 3) after a concentration gradient of the chemo-effector (0.1
mM/mm) was established. Motile DrugBots were observed using a Zeiss
AxioObserver D1 inverted microscope equipped with a motorized stage and an
AxioCam HSm camera plus a 20× objective and the captured videos were analyzed
using the ImageJ software (NIH, Bethesda, MD). For data, analysis, the width of
the observation channel is divided into two partitions and the spatial
distribution of DrugBots is calculated across the width of the channels in
order to quantify the migration profile of the DrugBots.

RESULTS AND DISCUSSION

Quantitative
investigation of DrugBot chemotaxis in response to concentration gradients of
L-Aspartic acid as well as screening the effect of particle geometry on this
chemotactic response is the main purpose of this study. The fabricated PEG-DA
hydrogel microfluidic device generates a well-controlled chemical gradient
across the chemotaxis observation channel and allows us to measure the
chemotactic response quantitatively. The chemo-attractant gradient reached a
steady state after around 35 min and it was maintained by a slow flow through
the channels at 5 µl min^-1 flow rate. The fabricated spherical and
ED-shaped DrugBots were then introduced into the center channel and every 5
min, 10 second videos were recorded at 20 frames/sec frame rate. In order to
quantify the migration profile of the DrugBots, the spatial distribution of the
DrugBots in the two partitions across the chemotaxis chamber was calculated
using ImageJ software. The results showed that the number of the DrugBots at
the chemo-attractant side increases during the first 20 min of the experiment
implying that there was a distinct bias towards the high concentration of L-Aspartic
acid for both types of DrugBots. Preliminary data analysis shows that spherical
DrugBots have a slightly higher chemotaxis partition coefficient (CPC),
compared to ED-shaped DrugBots after 40 minutes of running the experiment. CPC
is a measure of the strength of the response of the DrugBots to the
chemoattractant gradient. Further statistical analysis is being performed to
determine the statistical significance of the observed trends. Moreover, in
order to ensure that the DrugBot movement is because of the chemo-attractant, a
control experiment was conducted with no chemo-attractant and as expected, the
distribution of the DrugBots remained unchanged (data not shown). The gradient steepness
can also influence the migration of the DrugBots. A steeper gradient is
expected to generally yield a stronger chemotaxis response. However, an
inhibitory effect might be observed if the high chemo-attractant concentration
saturates the bacterial chemotaxis receptors. A thorough study of the effect of
a varying gradient steepness on the chemotactic response of the DrugBots is
currently being performed.

CONCLUSIONS

Bacteria-based
cargo carriers with spherical and ED shapes along with E. coli strain
MG1655 were constructed here. Chemotaxis was exploited to control the movement
of these autonomous drug delivery vehicles introduced into a PEG-DA hydrogel
based microfluidic device. Both types of these carriers, called DrugBots, have
shown a biased movement towards higher concentrations of L-Aspartic acid. These
controllable DrugBots have a great potential to autonomously navigate and carry/deploy
a wide variety of cargos such as therapeutics and contrast agents to
hard-to-reach diseased areas.

Figure 1: Top view of the PEG-DA hydrogel microfluidic device before assembly. Scale bar is 3 µm. Figure 2: Fluorescent DrugBots within the center channel of the microfluidic device. Scale bar is 5 µm. Figure 3: A representative section of the observation channel occupied by chemotactic DrugBots at t=40 min. Scale bar is 100 µm. ,0" src="https://www.aiche.org/sites/default/files/aiche-proceedings/conferences/..." height="192" class="documentimage">

REFERENCES

[1] Champion,
J.A. et al., Making polymeric micro- and nanoparticles of complex shapes,
Proceedings of the National Academy of Sciences, 104:11901 (2007).

[2] Muro, S. et al. Control of
endothelial targeting and intracellular delivery of therapeutic enzymes by
modulating the size and shape of ICAM-1-targeted carriers. Molecular Therapy,
16:1450?1458 (2008).