Improved effect of amikacin-loaded poly ( D , L-lactide-co-glycolide ) nanoparticles against planktonic and biofilm cells of Pseudomonas aeruginosa

Purpose. Amikacin is one of the most effective antibiotics against Pseudomonas aeruginosa infections, but because of its high toxicity, the use of this antibiotic has been clinically limited. In the present study, amikacin was successfully loaded into a new formulation of nanoparticles (NPs) based on poly(D,L-lactide-co-glycolide) 50 : 50 in order to enhance the treatment efficacy. The synthetized amikacin-loaded PLGA nanoparticles with high drug loading and stability were used to eliminate P. aeruginosa cells in planktonic and biofilm states. Methodology. P. aeruginosa PAO1 biofilm susceptibility studies were done using the minimum biofilm eradication concentration assay. The association of fluorescently labeled amikacin-loaded nanoparticles (A-NPs) with mouse monocyte macrophage cells (RAW 264.7), and the nanoparticles ability to interact and eradicate the bacterial cells even in the form of biofilms, was investigated using Flow cytometric studies and confocal laser scanning microscopy. Results. Flow cytometric studies showed that these NPs were able to interact with planktonic and biofilm bacterial cells. Moreover, following 1 h of incubation of A-NPs with 1-day-old biofilm, it was found that particles penetrate through the entire biofilm thickness. Live/dead fluorescent staining followed by CLSM analysis showed that the A-NPs were more effective than free drug in biofilm eradication. Conclusion. The good antibacterial and antibiofilm activities of A-NPs, in addition to their ability to enter macrophages without any cytotoxicity for these cells, make them a potential candidate to treat P. aeruginosa infections. INTRODUCTION Pseudomonas aeruginosa is one of the most important opportunistic respiratory pathogens, especially in immunocompromised individuals and patients suffering from cystic fibrosis or burn wound infections [1], due to several mechanisms of resistance (e.g. outer membrane permeability and the capability to form biofilms) [2–4]. Biofilms produced by Pseudomonas cause chronic infections because they show a higher resistance to the host immune system recognition and effector processes, such as phagocytosis, than planktonic cells do [5]. In addition, bacteria living in biofilms undergo several shifts in gene regulation, which cause significant phenotypic changes leading to a broad resistance to antibiotic therapies, among others, lower membrane permeability and metabolic activities, increased expression of b-lactamases, upregulation of efflux pumps and dormancy [6–10]. Furthermore, the bacteria are embedded in a self-produced polymeric matrix that can prevent the penetration of antibiotics. A mature biofilm contains different newly created micro-habitats characterized by sharp gradients that must be taken into account, since small variations in growth factors, dissolved oxygen or hydrogen ion concentration can affect the antibiotic uptake [11]. Aminoglycosides are one of the most effective families of antibiotics generally used in the treatment of P. aeruginosa infections; in particular, amikacin is the second drug of choice for cystic fibrosis centres and nosocomial infections [12, 13]. Received 17 November 2016; Accepted 9 January 2017 Author affiliations: Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran; Department of Microbiology and Parasitology, University of Navarra, Pamplona, Spain; Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Pamplona, Spain. *Correspondence: Carlos Gamazo, cgamazo@unav.es