Selective procaryotic/eukaryotic effect of eudragit e100 aqueous dispersions

ROMERO VL, ; MANZO RH; ALOVERO FL.

Córdoba

Congreso; II edición RICIFA; 2012

Universidad Nacional de Córdoba

IntroductionIt is known that cationic compounds which exhibit nonspecific antimicrobial activity and affect the integrity and/or the bacterial membrane function must be evaluated for selective activity (1). Recently, we demonstrate that bactericidal activity of fluoroquinolones is enhanced in drug-containing Eudragit E100 aqueous dispersions (Eu-OFX) against P.aeruginosa (2). This effect is attributed to the cationic polymer ability of disorganize the outer membrane of P.aeruginosa although it does not kill bacteria. (3). Also, there are reports indicating that high concentrations of Eudragit E100 has eukaryotic membrane-destabilizing properties (4).Anti-yeast activity is one of the tests commonly used to distinguish the membrane-active entities (1). In order to demonstrate the selective action of Eudragit E100 on bacterial cells, the effect of a wide range of concentrations of this cationic polymer in Eu-OFX was comparatively evaluated on Candida albicans and P.aeruginosa by flow cytometry.Materials and methodsEu-OFX systems were prepared as previously reported (3). Fluoroquinolone-resistant P.aeruginosa clinical isolate and Candida albicans ATCC 10231 were used. Culture overnight was adjusted in saline to an absorbance of 0.3 at 625 nm. Aliquots were exposed to Eu-OFX (polymer/drug concentrations range: 149/32 to 4768/1024 µg/mL), ofloxacin, Eu (polymer concentrations range:149-4768 µg/mL) or saline (control). The mixtures were incubated at 37°C and samples were taken after 1 and 3 h. Aliquots centrifuged (8000 rpm - 2 min) were washed with saline. Propidium iodide (PI) was dissolved in ethanol and further diluted in deionized water. Twenty µL were added to 180 uL aliquots of the recovering cultures (final dye concentration 10 µg/mL). After 5 min in the dark at room temperature, mixtures were acquired on a BD FACS Canto II (BD Biosciences, CA. USA) equipped with a 488nm argon-ion laser. Forward-scatter (FSC-A), side-scatter (SSC-A) and fluorescence signals were collected in logarithmic scale. At least 10000 events were recorded for each sample, and all experiments were conducted in duplicate on separate days.ResultsPI is a fluorescent probe known for its ability to stain cells with altered membranes (5). After 1 hour exposure to Eu-OFX, only 8% of the C. albicans cells were fluorescent while 95% of P. aeruginosa cells were stained with PI. At longer time-exposure the percents of fluorescent cells increased up to 20% and 99% for C.albicans and P.aeruginosa, respectively. The polymer-drug concentrations required to get the maximum effect observed against yeasts was 8 times higher than those used against bacteria.A heterogeneous bacterial size distribution (FSC-A) and changes in granularity (SSC-A) were observed while C.albicans population remained similar to control cells.Cultures exposed to drug-free polymer (Eu) exhibit similar behavior to those exposed to Eu-OFX. By contrast, free ofloxacin did not induce any measurable change over the wide range of concentrations evaluated. ConclusionsThe slight alterations in the yeasts cell integrity in contrast with the highly fluorescent P.aeruginosa cells both exposed to Eu-OFX or free-drug polymer are indicative of selective toxicity of Eudragit E100 dispersions. These results are promissory regarding the potential applicability of this polymer as an adjuvant or enhancer in topical pharmaceutical preparations fluoroquinolones.