Intracellular distribution of ampiciUin in murine ...

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Hank's balanced salt solution (HBSS; Gibco Laboratories, France) into the peritoneal cavity of .... (Figure 3) with (3H)ampicillin-loaded nanoparticles led to the complete disappearance of the intact bacteria. .... Williams & Wilkins,. Baltimore.
Journal of Antimicrobial Chemotherapy (1996) 37, 105-115

Intracellular distribution of ampiciUin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles Olivier Balland', Huguette Pinto-Alphandary*, Annie Viron*, Edmond Puvion*, Antoine Andremonf J and Patrick Couvreur*

The intracellular distribution of (3H)ampicillin-loaded polyisohexylcyanoacrylate nanoparticles was studied in murine macrophages (peritoneal cells and the J774 cell line) infected by Salmonella typhimurium C5, using ultrastructural autoradiography. Ampiciliin penetration and retention into the cells obviously increased by means of nanoparticles. After short-term (2—4 h) treatment with the nanoparticle formulation, numerous intracellular bacteria were seen to be in the process of destruction. The tritium labelling was located in the cell cytoplasm and inside vacuoles in which bacteria undergoing degradation were often present. After long-term (12 h) treatment, numerous spherical bodies (d: 100 nm to 500 nm) and larger forms (2 /im) were seen in the vacuoles. Radioactivity was mainly found to be localized on the spherical bodies, indicating marked damaging action of the ampiciliin on the bacterial walls. The targeting of ampiciliin therefore allowed its penetration into the macrophages and vacuoles infected with S. typhimurium.

Introduction

Infections caused by intracellular bacteria constitute a challenge for current antimicrobial therapies because of the requirement that antibiotics reach therapeutic concentrations at the site of infection. Thus, many antibiotics which are active in vitro are often inactive against intracellular bacteria, due to their poor penetration into the cells or to their inactivation by lysosomal enzymes (Van Den Broek et al., 1986). One approach to overcoming this problem consists of using drug carriers capable of enhancing the intracellular delivery of antimicrobial agents (Desiderio & Campbell, 1983; Swenson et al., 1990; Couvreur, Fattal & Andremont, 1991). Recently, we investigated the intracellular uptake of ampicillin-loaded nanoparticles in infected murine macrophages (Balland et al., 1994), and found that (3H)ampicillin Tel.: ( + 33-X1H0793690; Fax. ( + 33-HIHO793705.

0305-7453/96/010105 + 11 $12.00/0

105 X; 1996 The British Society for Antimicrobial Chemotherapy

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"Laboratoire de Pharmacie Galenique, CNRS, URA 1218, Faculte de Pharmacie, 92296 Chdtenay-Malabry Cedex; bUnite de Biologie et Ultrastructure du Noyau, UPR 272, Institut de Recherches Scientifiques sur le Cancer, BP n° 8, 94801 Villejuif Cedex; 'Laboratoire d^Ecologie Microbienne, Institut Gustave Roussy, 94805 Villejuif Cedex; d Laboratoire de Microbiologie, Faculte de Pharmacie, 92296 Chdtenay-Malabry Cedex, France

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uptake increased 9-fold in the J774 cell line and 20-fold in peritoneal macrophages. Significant intracellular retention of ampicillin was observed, despite a partial efflux from the cell. Transmission electron microscopy showed that nanoparticles were actively endocytosed by peritoneal macrophages (Pinto-Alphandary et al., 1994). In cells infected with Salmonella typhimurium, nanoparticles were found to be present either in isolated form or closely associated with bacteria inside phagosomes or phagolysosomes. Although the carrier has been visualized, little is known about the behaviour of the ampicillin. If the mechanism by which ampicillin-nanoparticles exert their action is by direct contact with the bacteria, then colocalization in the same compartment is a prerequisite. If it is by releasing ampicillin from the nanoparticles, this would not require colocalization, as ampicillin can diffuse through the cell to bacteria located at other sites in the cells. Both possibilities should be considered. The purpose of the present study was to clarify the distribution of ampicillin inside the intracellular compartments of infected macrophages, by the use of ultrastructural autoradiography. Materials and methods ( H)arnpicillin -bound nanoparticles (3H)ampicillin-bound nanoparticles were prepared by emulsion polymerization of isohexylcyanoacrylate monomer (Sopar, Belgium), to form polyisohexylcyanoacrylate (PIHCA) as described previously (Henry-Michelland et al., 1987; Fattal et al., 1991). (3H)ampicillin trihydrate (kindly provided by SmithKline Beecham, UK) with a specific radioactivity of 1.02 x 10" Bq/mole, was used instead of unlabelled ampicillin. ('H)ampicillin-nanoparticles were separated from the polymerization medium by ultracentrifugation for l h at 110,000^. The amount of (3H)ampicillin bound to the nanoparticles was determined by counting in heonic fluor scintillation liquid using a Packard Tri Carb 2000 CA analyser (Packard Instruments, USA). For a concentration of 2 mg/mL of ampicillin, 90 ± 4% of (3H)ampicillin was bound to the nanoparticles (i.e., 1.8 mg ampicillin/10 mg PIHCA nanoparticles/mL). Particle diameter, estimated by laser light scattering (Nanosizer Coulter N4MD, Coultronics, France), was 240 ± 36 nm (n = 4). The concentration of radioactivity in the final nanoparticle suspension (2 mg of (3H)ampicillin/mL) was 504 kBq/mL. Macrophage monolavers Resident murine peritoneal macrophages were obtained by injection of 5 mL of Hank's balanced salt solution (HBSS; Gibco Laboratories, France) into the peritoneal cavity of OF1 mice aged 6-8 weeks (IFFA-CREDO, France). Cells were washed twice in HBSS and resuspended at a concentration of 2 x 106 cells/well in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine. Two mL of macrophage suspension were placed in 35-mm tissue culture dishes (Falcon, Becton Dickinson Labware, USA) and incubated for 2 h at 37'C in a 5% CO2 incubator. The monolayers were then washed with fresh culture medium to remove non-adherent cells and further incubated for two days before use. The murine continuous cell line J774 (kindly provided by Dr Desnottes, Rhone-Poulenc Rorer, France), was maintained in the same culture medium under the conditions described above.

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IntraceUular distribution of nanoparticle ('H)ampicillin

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Bacterial strain S. typhimurium strain C5 (kindly provided by Dr Popoff, Institut Pasteur-Paris, France) was grown in brain-heart infusion broth (Difco Laboratories, USA) at 37°C, harvested in the logarithmic phase, and washed twice in phosphate buffered saline (PBS). The bacteria thus obtained were opsonized by incubation for 30 min at 37°C with a subagglutinating concentration of anti-Salmonella serum (O: 4,5; Diagnostics Pasteur, France). The opsonized bacteria were washed in PBS and adjusted to 2 x 107/mL in the culture medium (RPMI 1640 supplemented with 10% FCS). Before macrophage infection, bacterial concentrations were determined by optical density at 620 nm and by counting in a Petit Salumbeni chamber (Preciss, France) and further confirmed by counting colony-forming units on Mueller-Hinton agar plates.

Macrophage infection and treatment

Specimen preparation for electron microscopy After 2, 4, or 12 h treatment with free or nanoparticle-bound ('H)ampicillin, infected macrophages were washed three times with PBS. The cells were fixed with 1.6% glutaraldehyde (Sigma, France) in 0 . 1 M cacodylate buffer pH 7.3 for l h at 4°C, postfixed with 2% OsO< in 0.2 M cacodylate buffer pH 7.3 for 1 h at 18°C, and carefully washed. Macrophages were then gently scraped off the culture dishes with a cell scraper and centrifuged. The pellet was dehydrated through graded ethanol solutions and embedded in Epon (Fullam, France). For routine examinations, ultrathin sections 800 A thick were placed on formvar-coated grids (Fullam, France) and stained with 2% uranyl acetate and 0.2% lead citrate.

Ultrastructural autoradiograph v Autoradiography was carried out on preparations of peritoneal or J774 macrophages infected with S. typhimurium and treated with (3H)ampicillin-nanoparticles under the conditions described above. Sample processing. Ribbons of ultrathin sections were mounted on formvar-coated grids. Coating. The grids were covered with nuclear emulsion (Ilford L4) using the loop method (Bouteille, 1976) in a dark room at a constant temperature of 30°C, in an atmosphere of 50% humidity. This emulsion of silver bromide crystals was diluted in distilled water (1:4, v/v) by slow stirring in a 45°C water bath for 1 h. After storage overnight at 4GC, the emulsion was again stirred for 1 h at 45°C under the above degree of humidity. The grids were placed on the top of metal poles. The "loop method" consists of carefully depositing on the surface of each grid, a monolayer of crystals

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Culture dishes contained 2 x 10* macrophages (peritoneal or J774) in RPMI supplemented with 10% FCS. Cells were infected with opsonized S. typhimurium at a ratio of 20 to 30 bacteria per macrophage. Macrophages were then incubated for 30 min at 37°C to allow optimal phagocytosis, washed three times to remove extracellular bacteria, and treated with either (3H)ampicillin-loaded nanoparticles or free (3H)ampicillin, at a final concentration of 10/