| Summary: | Biofilm-associated infections are one of the largest health threats, causing over half a million deaths and costing society billions of dollars each year. Biofilms are associated with 65-80% of human infections and are implicated in antibiotics resistance. Biofilms are sessile, extracellular matrix-enclosed microbial masses that survive hostile environments. This self-produced matrix a viscous meshwork of extracellular polymeric substances (EPS) that attenuates antibiotic penetration due to its mechanical and physicochemical properties. The standard of care for biofilm infections is prolonged, high-dose antibiotics treatment, which are mostly ineffective and result in harmful side-effects. Coupled with a dry antibiotic pipeline, there is a need for new strategies to effectively deliver high concentrations of antibiotics through the biofilm.
Nanoparticle (NP)-based drug delivery systems represent an exciting solution for biofilm eradication. NPs can be loaded with antibiotics, targeted the site of interest, and deliver high local therapeutic concentrations. Furthermore, next-generation NP systems can be designed to imbue desired surface functionalities to enhance efficacy. Indeed, the layer-by-layer (LbL) platform technology, in which oppositely charged polymers are sequentially adsorbed onto charged colloidal NP templates, offers a unique fabrication method to assemble modular, uniform panels of distinct NPs surface chemistries. This thesis investigates three distinct NP surfaces designed to enhance desired cellular or matrix interactions for therapeutic benefit.
The first part of this thesis describes the development of a tunable family of pH-responsive LbL NPs with enhanced penetration and permeation throughout biofilms. Key design considerations were that positively charged NPs bind the EPS matrix and facilitate permeation; however, they are toxic and rapidly cleared in-vivo. To conserve the positive surface charge while mitigating the accompanying toxicity and clearance, a panel of pH-responsive chargereversing polymers that respond to the acidic biofilm microenvironment were synthesized. When layered onto LbL NPs, increased charge-reversal rate resulted in increased biofilm penetration and accumulation throughout the biofilm. Moreover, tobramycin loaded charge-reversing NPs resulted in a three-fold reduction of bacterial burden of P. aeruginosa biofilm as compared to free drug, demonstrating a potential high efficacy treatment.
The second part of the thesis describes the investigation of interactions of distinct NP surfaces with three main biofilm components, the alginate, psl, or pel polysaccharides. A new panel of varied surface chemistries LbL NPs was assembled with readily available polymers and interactions with the biofilm produced by inducible alginate, psl, or pel P. aeruginosa mutants was analyzed by confocal microscopy. NPs with carboxylated surfaces showed an increased colocalization with biofilms produced by mutants expressing alginate as compared to those with sulfated surfaces. These results are clinically significant, as alginate is well-characterized to be overexpressed in the airways of Cystic Fibrosis patients infected with P. aeruginosa. These results lay the foundation for designing new NP drug delivery vehicles capable of potentially increasing antimicrobial delivery throughout the biofilm via controlled biofilm interaction.
The penultimate chapter describes investigations of how surface presentation of cellular binding epitopes can be optimized for the eukaryotic cellular association. While not related to biofilm interactions, this study examined novel strategies to control the bio-nano interface with rational design of NP surfaces. These studies demonstrated that a bottlebrush polymer architecture layered onto NPs binds cell surface receptors more avidly as compared to covalently conjugated and traditionally layered NP constructs. The bottlebrush structure affords binding ligands physical distance from the surface, thereby maximally presenting them to the cell. The thesis ends with the description of optimization of two in vivo models of biofilm-associated infection, namely excisional wounds and airways, for testing the efficacy of systemically administered charge-switchable NPs.
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