PROJECT SUMMARY/ABSTRACT
Bacterial biofilms are responsible for most human infections, causing tens of thousands of deaths and billions in
medical costs per year. Topical biofilms alone cause significant harm to patients by growing on open wounds,
skin lesions, burn injuries, or diabetic ulcers, and elsewhere. Biofilms are notoriously difficult to eradicate, in
large part because of the extracellular polymeric substance (EPS), a self-produced extracellular matrix in which
biofilm bacteria reside. The EPS benefits bacteria in many ways, including mediating quorum sensing, providing
nutrients, and blocking transport of antibiotics and host immune response. The ability to actively penetrate the
EPS and deliver anti-bacterial cargo where it is most needed would bypass many of these protections and could
thus have a transformative impact on the remediation of biofilms. First introduced in 2004, artificial self-propelled
particles (SPPs) can propel themselves through complex biological media and deliver cargo to specific locations.
Thus, SPPs hold significant potential for biomedical applications such as biofilm remediation. However, SPPs
must overcome significant challenges in the form of biocompatibility, tracking, and control to be viable for clinical
use. Here, we propose to leverage the burgeoning field of DNA nanotechnology to develop urease-powered
DNA-origami-based self-propelled particles (DNA-SPPs) for biofilm remediation. As a model organism, we focus
on the well-studied pathogen Pseudomonas aeruginosa. Aim 1 of this study will quantify the dependence of
DNA-SPPs’ locomotion on local urea concentration and pH and elucidate the extent to which they perform
chemotaxis in urea gradients. Aim 2 will test the hypothesis that if DNA-SPPs are decorated with glycosyl
hydrolase enzymes (which are widely used to disrupt the biofilm matrix, specifically in the case of P. aeruginosa),
they will degrade the biofilm matrix as they move through it, weakening the protection the EPS normally provides
to bacteria. The success of Aim 2 will be marked by greater efficacy of a model antibiotic (ceftazidime, which has
demonstrated efficacy at treating P. aeruginosa biofilms) administered topically. In Aim 3, we will load ceftazidime
directly onto DNA-SPPs using a pH-sensitive motif (e.g., I-motif) that undergoes structural changes in response
to pH decrease, thus releasing cargo only in acidic regions. By correlating the delivered payload to the pH
distribution, we will confirm the ability of DNA-SPPs to deliver cargo preferentially in acidic regions, where hard-
to-reach bacteria tend to cluster. Finally, we will assess the combinatorial benefits of the approaches in Aims 2
and 3 by using DNA-SPPs to both increase the biofilm’s permeability and to deliver antibiotics deep inside the
biofilm. The major output of this study will be design criteria for DNA-based enzyme-powered SPPs to disrupt
and deliver cargo in extracellular matrix (ECM) environments, which could have a major impact on the treatment
of biofilms, and will lay the foundation for a customizable platform technology applicable to a wide range of ECM-
mediated diseases.
