Available online at www.sciencedirect.com International Journal of Pharmaceutics 355 (2008) 293–298 Pharmaceutical Nanotechnology Antimicrobial effectiveness of liposomal polymyxin B against resistant Gram-negative bacterial strains Misagh Alipour a , Majed Halwani a , Abdelwahab Omri a , Zacharias E. Suntres a,b,∗ a Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada b Medical Sciences Division, Northern Ontario School of Medicine, Lakehead University, Thunder Bay, Ontario, Canada Received 20 September 2007; received in revised form 20 November 2007; accepted 20 November 2007 Available online 24 November 2007 Abstract Polymyxin B is a polycationic antibiotic effective in the treatment of Gram-negative bacterial infections. Systemic use of polymyxin B has been limited due to its toxicity, most notably nephrotoxicity, ototoxicity, and neuromuscular blockade. Entrapment of antibiotics in liposomes is known to enhance their antimicrobial activities while minimizing their toxic effects. In the present study, polymyxin B was incorporated into liposomes composed of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) or 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC) and Chol. The entrapment efficiency of sonicated liposomes containing DPPC/Chol (32.1 ± 2.43%) was six-fold higher than that of liposomes containing POPC/Chol (5.35 ± 0.32%). On the other hand, the entrapment efficiency of extruded DPPC/Chol liposomes (3.23 ± 0.46%) was about 30% less than that of liposomes composed of POPC/Chol (5.10 ± 0.37%). Incubation of extruded DPPC/Chol liposomes containing polymyxin B in serum at 37 ◦ C resulted in a complete release of the antibiotic into the supernatant after 3 h as compared to 6 h in the case of POPC/Chol liposomes. Spontaneous release of polymyxin B from DPPC/Chol liposomes incubated in saline was significantly higher (66%) than that from POPC/Chol liposomes (24%) after 48 h at 37 ◦ C. With respect to the antimicrobial activities of the liposomal polymyxin B formulations, the MICs of sonicated DPPC/Chol liposomes against Gram-negative strains were generally lower when compared to free polymyxin B. Immunocytochemistry and electron transmission microscopic studies revealed that the penetration of polymyxin B into a resistant strain of Pseudomonas aeruginosa was higher following its administration as a liposomal formulation as compared to its conventional form. The combination of free drug and plain liposomes had an antibacterial activity similar to that of free antibiotic. These data suggest that incorporation of polymyxin B in liposomes could be useful in the management of Gram-negative infections induced by these microorganisms. © 2007 Elsevier B.V. All rights reserved. Keywords: Polymyxin B; Liposomes; Gram-negative bacteria; Antibacterial; Antibiotic 1. Introduction The polymyxins (polymyxin B and colistin) are polypep- tide antibiotics isolated from Bacillus polymyxa and known to have potent bactericidal activity against a broad range of Gram-negative bacteria (Arnold et al., 2007; Horton and Pankey, 1982). In general, the polymyxins exert their bactericidal activ- ity by binding to acidic phospholipids and lipopolysaccharides of bacterial cell membranes, resulting in leakage of intracel- lular components, leading to cell death (Arnold et al., 2007; Cardoso et al., 2007; Clausell et al., 2007). They also have anti- ∗ Corresponding author at: 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada. Tel. +1 807 766 7395. E-mail address: Zacharias.Suntres@Normed.ca (Z.E. Suntres). endotoxin activity (Arnold et al., 2007; Clausell et al., 2007), but concerns arising from adverse effects have restricted their use almost exclusively for the treatment of Gram-negative bacilli infections that are resistant to other preferred antimicrobial agents or in intolerant patients to the preferred antimicrobials (Falagas and Kasiakou, 2006; Lee et al., 2006). Resistance in Gram-negative strains towards polycationic antibiotics, whether mutational or adaptive, is common (Macfarlane et al., 1999; Moskowitz et al., 2004; Kwon and Lu, 2006). Since polymyxin B can interact with the outer membrane of Pseudomonas aeruginosa, possible mechanisms of resistance include cell impermeability and alterations in cell surface (reduction in LPS, outer membrane proteins, and divalent cation levels) (Tsubery et al., 2002; McPhee et al., 2003; Kwon and Lu, 2006). These alterations decrease the binding sites on the cell surface, ultimately reducing cell wall disruption of structure 0378-5173/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijpharm.2007.11.035