Stimuli-responsive, hydrolysable layer-by-layer nanoparticles enhance biofilm penetration

Bacterial infections are a continuing medical threat, as they affect 17 million people every year, cause 550,000 deaths, and cost the healthcare system billions of dollars each year, leading to warning calls about a “post-antibiotic era” if we are unable to address current recalcitrance, and resistance, mechanisms. One of these mechanisms is the formation of biofilms: communal organizations of bacteria surrounded by the dense and viscous extracellular polymeric substance (EPS). The EPS comprises a heterogeneous matrix of secreted polysaccharides, proteins, lipids, and extracellular DNA, which together control the permeation of nutrients and other materials, such as antimicrobial therapeutics, through the biofilm. As a result, antimicrobials must be administered at exorbitant doses to ensure that a lethal concentration accumulates. Nanoparticles (NPs) represent a promising strategy to more efficiently penetrate and distribute antimicrobial cargo within the matrix, thereby reducing their required dosage. Recent studies have elucidated a key design criterion for these NPs, whereby they must exhibit a positive, cationic charge in order to effectively penetrate through the matrix. However, cationic NPs are toxic and rapidly cleared from the bloodstream when administered systemically, thereby disadvantaging their use in systemic drug delivery treatments. In order to mitigate these orthogonal requirements, we have synthesized an anionic polymer with hydrolysable amide side-chains responsive to moderate acidic pHs (6.5), known to be present within the biofilm. After hydrolysis, the resulting amine provides a net positive charge, resulting in a cationic polymer. By modifying the polymer backbone, as well as the stoichiometry of side-chain reactivity partners, we can tune the cleavage rates, ranging from hours to days. To synthesize functionalized NPs, we utilize the layer-by-layer (LbL) particle assembly method, in which oppositely charge poly-ions are sequentially adsorbed onto a charged colloidal substrate. We can then probe the biofilm penetration of NP-surfaces layered with these charge-converting polymers both 1) in vitro, with Transwell penetration assays and microscopy, and anticipated 2) in vivo, using a chronic infected diabetic ulcer mouse model. From these experiments, we identify structure-property relationships that can be used to maximize the delivery of nano-encapsulated antimicrobials throughout biofilms.