Development of DNA Nanoparticles with Properties for Enhanced Biofilm Uptake

Abstract

Microbial biofilms account for up to 80% of all bacterial infections. They are described as small structural communities of bacteria which are embedded in a self-produced extracellular (EPS) matrix. This EPS matrix poses a substantial challenge for antimicrobial treatment by hindering traversal of antimicrobial agents. Nanotechnology based drug delivery systems (DDS) offer a promising solution to the low antimicrobial efficacy of free drug molecules via encapsulation into optimized carriers to enhance penetration. DNA nanotechnology has drawn considerable interest given the high biocompatibility, excellent structural control and ease of carrier modification, but have properties associated with low biofilm penetration. As such, this project seeks to develop DNA nanoparticles with properties for enhanced biofilm penetration. A series of DNA nanoparticles were prepared solely via thermal annealing processes or a combination of thermal annealing and polymer coating to achieve four unique carriers with different properties. Characterization of the nanoparticles was performed via dynamic light scattering (DLS). Biofilm penetration of the nanoparticles was evaluated using an in-house dsDNA quantification method, confocal microscopy (CLSM) and fluorescent spectroscopy. Biofilm penetration, biofilm inhibition and effect of the optimized formulation on mature biofilms was tested using crystal violet staining and isothermal microcalorimetry (ICM). The toxicity of the nanoparticles was evaluated against HaCaT cells. The two modified nanoparticles NMC and NNPChi had an average size of 22,3 ± 1,3 nm and 297,0 ± 2,9 nm and exhibited a zeta potential of -24,2 ± 3,2 mV and +30,9 ± 1,0 mV respectively. Both nanoparticles showed high biofilm penetration when compared to the control formulation (52,5 ± 10,4 nm and -21,5 ± 3,5 mV). From these, the micellar formulation NMC was chosen for drug loading with polymyxin B (PMB) due to its ideal morphology, small size and affinity to DNA. The optimized PMB loaded formulation significantly inhibited P. aeruginosa biofilm growth after co-incubation for 16h and had a significant effect on the time to peak (+444 ± 50 minutes) and relative metabolic rate (20 ± 5%) of mature biofilms after 2h. Toxicity studies on all formulations revealed negligible toxicity. In conclusion, cationic surface modification and conjugation of a hydrophobic moiety significantly increased the biofilm penetration of DNA nanoparticles. The optimized drug loaded formulation demonstrated promising efficacy against P. aeruginosa biofilms.