Efficiency of bactericidal activity of liposomal mycobacteriophages against intracellular Mycobacteria tuberculosis in the model of macrophages RAW 264

Abstract

Introduction: The use of lytic mycobacteriophage D29 incorporated into liposomes may be one way to achieve more efficient TB treatment.

Aim: To study the bactericidal effect of liposomal lytic mycobacteriophage (phage) against intracellular MTB in the model of cultured macrophages.

Methods: RAW 264-7 (ATCC) macrophage cell culture was infected for 24 hours with H37RV MTB at a concentration of 10⁷/ml. Then macrophages were isolated by centrifugation in a Ficoll-Paque density gradient. Phage D29 (minimum 10⁸ PFU) was purified on an ion-exchange Q-sepharose column and then shaken with a phospholipid film consisting of phosphatidylcholine and cholesterol, followed by 20-fold extrusion of the obtained liposomal mycobacteriophage particles using 0.4 microns pores filters. The liposomal inclusion of phage was controlled by analyzing phage DNA by real-time PCR and transmission electron microscopy. The experimental wells of macrophage cells, 3 for each experiment, were analyzed: control, phage and liposomal phage. After 24-hour incubation the macrophages were removed with scrubbers from the surface of the wells and after double freezing and thawing were sown on plates with agar medium 7H10 and cultivated in a CO₂ incubator at 37°C.

Results: Resulting counting of MTB colonies: control - 62±2,6; mycobacteriophage -17±1; liposomal mycobacteriophage -7±0,3. P_1-2 <0,01; P_2-3 < 0,05.

Conclusions: Method of preparation macrophage culture with intracellular MTB infection was presented and the higher bactericidal efficacy of mycobacteriophages included in 400 nm liposomes was shown.