Bactericidal Activity of Liposomal Form of Lytic Mycobacteriophage D29 in Tuberculosis Infection PDF

DOI 10.1007/s10517-020-04887-6 Bulletin of Experimental Biology and Medicine, Vol. 169, No. 3, July, 2020 IMMUNOLOGY AND MICROBIOLOGY 361 Bactericidal Activity of Liposomal Form of Lytic Mycobacteriophage D29 in Cell Models of Tuberculosis Infection In Vitro M. B. Lapenk...

DOI 10.1007/s10517-020-04887-6 Bulletin of Experimental Biology and Medicine, Vol. 169, No. 3, July, 2020 IMMUNOLOGY AND MICROBIOLOGY 361 Bactericidal Activity of Liposomal Form of Lytic Mycobacteriophage D29 in Cell Models of Tuberculosis Infection In Vitro M. B. Lapenkova, Yu. S. Alyapkina, and M. A. Vladimirsky Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 169, No. 3, pp. 334-337, March, 2020 Original article submitted September 12, 2019 The use of lytic mycobacteriophages to treat tuberculosis under conditions of acquired resis- tance to anti-tuberculosis drugs is one of the most practical ways to improve the effectiveness of therapy and reduce the spread of this disease. We studied the efficacy of antimycobacte- rial action of mycobacteriophage D29 encapsulated into 400-nm liposomes in cell models of tuberculosis infection in vitro. The antimycobacterial action of lytic mycobacteriophage D29 used in free or liposome-encapsulated forms was demonstrated on cell models of intracel- lularly infected RAW264.7 macrophages and tuberculous granuloma formed by human blood mononuclear cells. The experiments demonstrated pronounced advantage of liposomal form of mycobacteriophage according to the criteria of their penetration into macrophages and lysis of Mycobacterium tuberculosis in culture. Key Words: mycobacteria tuberculosis; mycobacteriophage; macrophages; mononuclears; liposomes The global plan of radical (by 90%) reduction of es into treatment protocols because they are highly the incidence of tuberculosis by 2035 announced by specific and can overcome drug resistance of MBT WHO greatly depends on the development of innova- [3,4,6,8]. However, the use of mycobacteriophages tive treatment methods that can essentially shorten the in the therapy of tuberculosis is impeded by some duration of treatment of this disease and reduce the hurdles, the major challenges being mycobacterio- spread of drug resistance to the basic anti-tuberculosis phage delivery into the areas of infiltrative tuberculo- drugs. sis inflammation and the risk of formation of specific The biological nature of Mycobacterium tubercu- antibodies that could neutralize the phage particles losis (MBT) with 24-h DNA doubling time determines. In light of this, the use of mycobacteriophages the duration of antibiotic therapy and appearance of encapsulated into liposomes seems promising [7,9]. drug-resistant MBT strains. The duration of chemo- Bactericidal effect of lytic mycobacteriophage therapeutic courses in case of multiple drug resistance D29 against MBT was previously demonstrated by us (resistance to isoniazid and rifampicin) attains 1.5-2.0 on the model of transplantable line of macrophages years, which reduces patient’s compliance to anti-tu- infected with MBT in vitro. berculosis therapy. Here we studied the bactericidal action of myco- A fundamentally new approach to the problem of bacteriophages D29 encapsulated into 400-nm lipo- tuberculosis is inclusion of lytic mycobacteriophag- somes on cell models of tuberculosis infection in vitro. National Medical Research Center for Phthisiopulmonology and Infec- MATERIALS AND METHODS tious Diseases, Ministry of Health of the Russian Federation, Mos- cow, Russia. Address for correspondence: [email protected]. Preparation of lytic mycobacteriophages. We used M. A. Vladimirsky lytic mycobacteriophage D29 kindly provided by Prof. 0007­-4888/20/16930361 © 2020 Springer Science+Business Media, LLC 362 Bulletin of Experimental Biology and Medicine, Vol. 169, No. 3, July, 2020 IMMUNOLOGY AND MICROBIOLOGY Gr. Hatful (Pittsburgh Bacteriophage Institute, USA). macrophages were scrapped from the wells in a volume Mycobacteriophages were expanded during culturing of 200 µl per well, twice rapidly frozen and thawed, of M. smegmatis (MC2 155) in a liquid Middlebrook and transferred to the dishes (100 µl per dish) with 7Н9 culture medium with 10% OADC and 1 mM Middlebrook 7Н10 culture medium added with OADC CaCl2 , which allowed obtaining phage prepara- Growth Supplement. The cultures were analyzed in 3 tions with a concentration of 1010-1011 plaque-forming weeks. Penetration of liposomal and extraliposomal units (PFU, i.e. lysis zones on the lawn of agar cul- mycobacteriophages into macrophages was assessed ture of M. smegmatis). Isolation and purification of with quantitative PCR. phage particles of M. smegmatis bacterial lysates were Peripheral blood mononuclears were isolated by carried out by ion exchange chromatography on a Q centrifugation in a Ficoll-Paque density gradient and Sepharose column. washed in physiological saline and RPMI-1640 me- Preparation of liposomal lytic mycobacterio- dium, and then cultured in the same medium with phages. Purified mycobacteriophage preparation with growth supplements in a CO2 incubator at 37°C and a concentration of ≥109 PFU/ml in phage buffer (3 ml) 5% CO2 in 24-well plates (500 µl/well, 1.0-1.2×106 was shaken in a flask for 5-10 min after evaporation in cell/ml). a rotary evaporator of the phospholipid film consisting The model of tuberculous granuloma was de- of 40 mg egg lecithin (Lipoid), 8 mg cholesterol, and veloped by culturing peripheral blood mononuclears 8.2 mg Tween-80. The obtained suspension was ex- from patients with latent or active tuberculosis at 6:1 truded through a 5-µ filter followed by 20-fold extru- eukaryotes-MBT ratio. sion through 0.4-µ filters. Finally, mycobacteriophage RPMI-1640 medium with the above supplements fraction included into liposomes was separated by 30- was refreshed every 2 days. Cultivation was monitored min centrifugation at 20,000 rpm. under a Leica inverted microscope with a Leica DFC The fraction of liposome-encapsulated D29 was 420 digital camera at 200×. Granulomas were formed evaluated by quantitative real-time PCR of mycobac­ on days 13-15 (Fig. 1). teriophage DNA using D29 primers AGCCG­A­T­­ After formation of granulomas, the medium was CA­G AAGCACGGGC (F) and AGCGG­C TC­T­T­A ­ refreshed, and 50 µl free or liposomal mycobacterio- G­G A­G GGGCC (R) and FAM-labeled probe AG­ phages (109 PFU/ml) were added into every 3 wells; CCA­CGA­ACTCGCGACCCACGG. The fraction of nutrient medium was added to control wells. mycobacteriophages included into liposomes was ≥15% In 24 h, the granuloma cells were washed once Cell models of tuberculosis infection. The lytic with the medium, scraped in a volume of 200 μl, and (antibacterial) effect of liposome-encapsulated D29 subjected to two freezing—thawing cycles. Then the was examined on two cell models: intracellularly in- specimens (100 µl) were plated onto the dishes with fected RAW264.7 (ATCC) macrophage culture and in Middlebrook 7Н10 culture medium added with OADC vitro tuberculous granuloma formed by human pe- Growth Supplement. Two experiments were conducted ripheral blood mononuclears in the presence of MBT. by this protocol. In the first model, RAW 264.7 cells were cul- tured in a CO2 incubator at 37°C in 6-well plates with RPMI-1640 medium supplemented with 20% fetal calf serum, 2 mM L-glutamine, penicillin-streptomycin, Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA), MEM Vitamins, and 1 mM sodium pyruvate (Gibco). The cells were infected over 1 day with growing MBT of the virulent H37Rv strain at a ratio of 15 MBT per macrophage (calcu- lated by quantitative analysis of MTB DNA using the single-copy RegX3 gene). To separate macrophages from non-phagocytized MBT, the macrophages were washed with culture medium and centrifuged in a Fi- coll-Paque density gradient; the interface layer was collected and the cells were washed with physiologi- cal saline and culture medium. Then, macrophages infected with MTB were cultured for 24 h in the same medium with addition of 100 µl free D29 (108 PFU/ ml), 100 µl liposome-encapsulated D29 (108 PFU/ml), Fig. 1. Formation of granuloma with destructive focus in the center. Light microscopy, ×200. or 100 µl culture medium (control wells). After that, M. B. Lapenkova, Yu. S. Alyapkina, and M. A. Vladimirsky 363 RESULTS TABLE 1. Antimycobacterial Action of Free and Liposomal Mycobacteriophage D29 on the Model of Intracellularly The results of assessment of the antimycobacterial Infected Macrophages (m±SE) action of free or liposomal mycobacteriophages on Mean number the model of intracellularly infected RAW 264.7 mac- Group of MBT colonies rophages are presented in Table 1. The antibacterial Control MBT 62.0±2.6 effect of liposome-encapsulated D29 was significantly higher than that of free mycobacteriophage. Free mycobacteriophage D29 17±1** Quantitative real-time PCR showed that the gene Liposomal mycobacteriophage D29 7.0±0.3* copy number assessing amount of DNA in the phages Note. *p