A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia

Abstract

Mareike Klinger-Strobel,1,2,* Julia Ernst,3,* Christian Lautenschläger,4 Mathias W Pletz,1,2 Dagmar Fischer,3,5 Oliwia Makarewicz1,2 1Center for Infectious Diseases and Infection’s Control, 2Center for Sepsis Control and Care, Jena University Hospital, 3Department of Pharmaceutical Technology, Friedrich Schiller University Jena, 4Department of Internal Medicine IV, Jena University Hospital, 5Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Jena, Germany*These authors contributed equally to this work Abstract: Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO® 9, propidium iodide, fluorescein). Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(D,L-lactide-co-glycolide) (PLGA)-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA), after which blue fluorescent poly(ethylene glycol)-block-PLGA (PEG-PLGA) particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. Keywords: 7-amino-4-methyl-3-coumarinylacetic acid, PLGA, PEG, confocal laser scanning microscopy, cystic fibrosis, chitosan, hydrodynamic diameter

Keywords

• nanoparticles
• 7-amino-4-methyl-3-coumarinylacetic acid
• Staphylococcus aureus
• Burkholderia cepacia
• PLGA
• PEG
• confocal laser scanning microscopy
• cystic fibrosis