Encapsulation of Bacteriophage in Liposome Accentuates Its Entry in to Macrophage and Shields It from Neutralizing Antibodies

<div><p>Phage therapy has been a centre of attraction for biomedical scientists to treat infections caused by drug resistant strains. However, ability of phage to act only on extracellular bacteria and probability of interference by anti-phage antibodies <i>in vivo</i> is considered as a important limitation of bacteriophage therapy. To overcome these hurdles, liposome were used as delivery vehicle for phage in this study. Anti-phage antibodies were raised in mice and pooled serum was evaluated for its ability to neutralize free and liposome entrapped phage. Further, ability of phage and liposome-entrapped phage to enter mouse peritoneal macrophages and kill intracellular <i>Klebsiella pneumoniae</i> was compared. Also, an attempt to compare the efficacy of free phage and liposome entrapped phage, alone or in conjunction with amikacin in eradicating mature biofilm was made. The entrapment of phage in liposome provided 100% protection to phage from neutralizing antibody. On the contrary un-entrapped phage got neutralized within 3 h of its interaction with antibody. Compared to the inability of free phage to enter macrophages, the liposome were able to deliver entrapped phage inside macrophages and cause 94.6% killing of intracellular <i>K</i>. <i>pneumoniae</i>. Liposome entrapped phage showed synergistic activity along with amikacin to eradicate mature biofilm of <i>K</i>. <i>pneumoniae</i>. Our study reinforces the growing interest in using phage therapy as a means of targeting multidrug resistant bacterial infections as liposome entrapment of phage makes them highly effective <i>in vitro</i> as well as <i>in vivo</i> by overcoming the majority of the hurdles related to clinical use of phage.</p></div

Comparison of effect of neutralizing antibodies on phage and liposome entrapped phage.

<p>Comparison of effect of neutralizing antibodies on phage and liposome entrapped phage.</p

Bacterial count (CFU/ml) on different days of incubation of a 7 day biofilm of <i>K</i>. <i>pnuemoniae</i> B5055 in a microtiter plate following treatment with plain liposomes (empty lipoosmes as control), antibiotic (amikacin at 40 μg/ml), non-entrapped phages (MOI = 1) and liposome entrapped phages (MOI = 1).

<p>All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions.</p

Biodistribution of liposome entrapped phage and unentrapped phage in spleen of BALB/c mice (n = 3).

<p>All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions</p

Bacterial count (CFU/ml) on different days of incubation of a 7 day biofilm of <i>K</i>. <i>pnuemoniae</i> B5055 in a microtiter plate following treatment with plain liposomes (empty lipoosmes as control), antibiotic (amikacin at 40 μg/ml), non-entrapped phages (MOI = 1) and liposome entrapped phages (MOI = 1).

<p>All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions.</p

Percent bacterial uptake and killing by mouse peritoneal macrophages at different time intervals.

<p>All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions. SEM represent the 95% confidence interval. Confidence interval is calculated as SEM * 3.18. Raw data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153777#pone.0153777.t002" target="_blank">Table 2</a>.</p

Bacterial count (CFU/ml) on different days of incubation of a 7 day biofilm of <i>K</i>.<i>pneumoniae</i> following treatment with phage + antibiotic (P+A) and liposome entrapped bacteriophage + antibiotic (LP+A) in a microtiter plate.

<p>All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions. A- Amikacin (40 μg/ml), P- Phage, LP- Liposome entrapped phage.</p

Comparison of percent killing of intracellular bacteria when treated with phage and liposome entrapped phage.

<p>Comparison of percent killing of intracellular bacteria when treated with phage and liposome entrapped phage.</p

Particle size distribution and PDI of liposomal formulation of phage.

<p>Particle size distribution and PDI of liposomal formulation of phage.</p

Identification of miR-34 regulatory networks in settings of disease and antimiR-therapy: Implications for treating cardiac pathology and other diseases

<p>Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated in settings of heart disease, and inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy. Under chronic cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identification of transcription factor (TF)-miRNA regulatory networks has added complexity to understanding the therapeutic potential of miRNA-based therapies. Here, we sought to determine whether antimiR-34 targets secondary miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identified differentially regulated miRNAs in hearts from mice with cardiac pathology due to transverse aortic constriction (TAC), and focused on miRNAs which were also regulated by antimiR-34. Two clusters of stress-responsive miRNAs were classified as “pathological” and “cardioprotective,” respectively. Using ChIPBase we identified 45 TF binding sites on the promoters of “pathological” and “cardioprotective” miRNAs, and 5 represented direct targets of miR-34, with the capacity to regulate other miRNAs. Knockdown studies in a cardiomyoblast cell line demonstrated that expression of 2 “pathological” miRNAs (let-7e, miR-31) was regulated by one of the identified TFs. Furthermore, by qPCR we confirmed that expression of let-7e and miR-31 was lower in hearts from antimiR-34 treated TAC mice; this may explain why targeting the entire miR-34 family is more effective than targeting miR-34a alone. Finally, we showed that Acsl4 (a common target of miR-34, let-7e and miR-31) was increased in hearts from TAC antimiR-34 treated mice. In summary, antimiR-34 regulates the expression of other miRNAs and this has implications for drug development.</p