Liposome delivery of ciprofloxacin against intracellular Francisella tularensis infection

Abstract

The effect of liposome delivery on the controlled release and therapeutic efficacy of ciprofloxacin against intracellular Francisella tularensis infection in vivo was evaluated in this study. Ciprofloxacin was encapsulated in small unilamellar vesicles by a remote loading procedure using an ammonium sulfate gradient. This procedure produced uniform sized liposomes (100 nm) with an entrapment rate of 90±3.5%. Following administration of unencapsulated or liposome-encapsulated ciprofloxacin by intravenous injection or aerosol inhalation, levels of ciprofloxacin in sera, lungs, liver and spleen were determined using ¹⁴C-ciprofloxacin as radiotracer for ciprofloxacin. Intravenous injection of liposome-encapsulated ciprofloxacin resulted in higher serum levels of drug in serum, as well as increased drug retention in lungs, liver and spleen, compared to that of free encapsulated drug. Aerosol administration of liposome-encapsulated ciprofloxacin by jet nebulization resulted in significantly higher drug levels and prolonged drug retention in the lower respiratory tract compared to the free drug. Aerosol inhalation of liposome-encapsulated ciprofloxacin, given either prophylactically or therapeutically, provided complete protection to mice against a pulmonary lethal infection model of F. tularensis. In contrast, ciprofloxacin given in its free form, was ineffective. These results suggest that liposome encapsulation of ciprofloxacin enhances drug delivery to the primary site of infection and results in increasing therapeutic efficacy against F. tularensis.

Introduction

Infectious diseases caused by intracellular bacteria present a significant challenge to antibiotic therapy. Antibiotic treatment of these types of infections has been associated with high failure and/or relapse rates [1], [2]. Intracellular pathogens, whether obligate or facultative, can hide, reside and multiply within the phagocytic cells of the reticuloendothelial system (RES), and by virtue to their intracellular location, are protected from the actions of the immunological defence cells and of antimicrobial agents [2], [3], [4], [5]. The ineffectiveness of conventional antibiotics against intracellular infections may also be attributable to poor drug penetration, limited drug accumulation in subcellular compartments and/or drug inactivation by acidity in subcellular compartments [3], [4], [5]. These factors may explain why some antibiotics are bactericidal against extracellular bacteria in vitro, but are ineffective in killing intracellular forms of the bacteria [4], [5], [6].

Ciprofloxacin, a fluoroquinolone, is a potent and broad-spectrum antibiotic, and has good antibacterial activity against most gram-negative bacteria and gram-positive cocci. Ciprofloxacin has been shown to have a superior ability to penetrate most tissues compared to other antibiotics [7], [8], [9], [10], accumulates in macrophages [11] and neutrophils [12] and is bactericidal in low pH environment [13]. These attributes contribute partly to ciprofloxacin being the drug-of-choice for the treatment of infectious diseases caused by intracellular pathogens. Furthermore, ciprofloxacin, when orally or intravenously administered, is known to reach such organs as liver, spleen, lungs and lymph nodes [14], which are important infection sites for intracellular bacteria. However, ciprofloxacin does not preferentially accumulate well at these tissues and may therefore not reach high sustain therapeutic levels at these sites.

Liposomes have been shown to be a promising delivery system for a number of antimicrobial drugs including antibiotics [15], [16], [17], [18]. Conventional liposomes are readily taken up by phagocytic cells of the RES system, including macrophages. They therefore constitute a valuable delivery vehicle for targeting high therapeutic doses of antibiotics to those intracellular sites where the parasitic bacteria reside. In addition, the sustained release of antibiotics from the liposomes may prolong the half-lives of these drugs in the body.

The objective of this study was to evaluate the effect of liposome encapsulation on drug accumulation in the primary sites of infection for intracellular bacteria and to compare the therapeutic efficacy of ciprofloxacin and liposome-encapsulated ciprofloxacin against a pulmonary infection caused by an intracellular pathogen, Francisella tularensis. F. tularensis is a facultative intracellular bacterium that can cause tularemia, which can be a potentially fatal human disease if untreated. F. tularensis is a potential biological warfare/bioterrorism agent, and medical and public health management of the infectious disease caused by this bacterial agent is important [19]. Infection by F. tulerensis involves the RES system and leads to bacterial growth within the lungs, liver and spleen [20]. Tularemia in humans can be treated with antibiotics, including streptomycin, gentamicin and chloramphenicol, but even with prolonged daily therapy, relapse and failure rates can range from 0% to 33% [21]. Tularemia in mice can be treated subcutaneously with ciprofloxacin and doxycycline, but to be effective, antibiotic treatment was given twice daily and given 48 h before infection and continued for 5 days post infection. Even with the prolonged antibiotic treatment in these mice, occurrence of relapse was significant [22]. Successful treatment of tularemia using ciprofloxacin depends on high sustain drug concentrations in tissues and intracellular sites where the bacteria reside and multiply. Due to controlled release and intracellular targeting provided by liposome delivery, liposome-encapsulated ciprofloxacin may represent a promising therapeutic drug for the prevention and treatment of intracellular infections, including tularemia.