Confocal microscopy as a promising tool to predict the antimicrobial efficacy of chitosan nanodroplets.

Hypoxia, inflammation, impaired skin tissue remodeling, and microbial infections are common features of chronic wounds. Chitosan-shelled and 2H,3H-decafluoropentane-cored nanodroplets have been developed as an innovative and nonconventional platform of gas nanocarriers and have proven effective in delivering oxygen to hypoxic tissues, restoring normoxia-like levels of inflammatory and matrix-remodeling molecules. Since additional antimicrobial properties have been reported for chitosan, in the present work oxygen-loaded nanodroplets (OLNDs) and oxygen-free nanodroplets (OFNDs) were challenged for their antimicrobial activity against four common infectious agents of chronic wounds: methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans, and Candida glabrata. Complementary analysis by confocal microscopy was also performed to study the physical interaction between nanodroplets and cell walls/membranes. Bacterial and fungal growths were monitored upon incubation with/without OLNDs/OFNDs for increasing times (2, 3, 4, 6, and 24 h). Both OLND and OFND formulations displayed spherical morphology, cationic surfaces, and diameters in the nanometer range (<750 or 350 nm, respectively). According to cell counts, OLNDs and OFNDs displayed long-term antimicrobial efficay against S. pyogenes, C. albicans, and C. glabrata, significantly inhibiting baterial or fungal growth up to 24 h. In contrast, nanodroplets promoted only short-term cytostatic effects against MRSA, inhibiting bacterial growth up to 6 h. Further analysis by confocal microscopy showed OLNDs and OFNDs internalisation by S. pyogenes, C. albicans, and C. glabrata already after 3 h of incubation. In contrast, OLNDs and OFNDs were not internalised by MRSA, even after 24 h. However, nanodroplet adhesion to the bacterial wall was clearly observed. Based on these data, long-term antimicrobial efficacy of nanodroplets appears to be dependent on cellular internalisation, whereas short-term efficacy associates with adhesion to the cell wall. Therefore, preliminary analysis by confocal microscopy might be employed as a promising tool to predict the degree of antimicrobial efficacy of chitosan nanodroplets over time.