Microfluidic development of a pneumolysin-responsive liposomal platform for selective treatment of Streptococcus pneumoniae

Abstract

The bacterium Streptococcus pneumoniae has become a leading cause of meningitis, sepsis, and bacterial pneumonia worldwide, with increased prevalence of antibiotic-resistant serotypes only serving to exacerbate the issue. This imposes a significant public health threat and growing economic burden, spurring research into alternative treatments. One effective route to bypass resistance development is through the targeting of virulence factors essential for bacterial pathogenesis, comprising cellular structures, regulatory systems, and molecules produced by the bacterium. The main factor responsible for colonization and immune response escape in pneumococcal infections is the secreted molecule pneumolysin, which is a subset within a family of related toxins that form transmembrane pores in biological membranes through cholesterol recognition and binding. The conserved activity and structure of pneumolysin between all observed S. pneumoniae serotypes, along with its requirement for pathogenicity, has made this molecule an attractive target for vaccination, diagnostic, and sequestration platforms, but not yet as a facilitative agent for therapeutic treatment. The present work aimed to take advantage of pneumolysin’s dependencies through the fabrication of cholesterol-rich liposomes, a natural mimic of the external surface of mammalian cells, to elicit pore formation and subsequent release of an encapsulated peptide. A custom microfluidic system was manufactured to enable rapid and consistent liposomal synthesis, with key formulation parameters optimized through a Box-Behnken response surface design. The vesicles themselves were comprised of naturally-derived phospholipids and varying levels of cholesterol (30/45/60/75 mol%), which displayed desirable physical properties and high encapsulation rates of the bacteriocin nisin. A liposomal cholesterol content above 45 mol% was determined necessary to facilitate interactions with both purified pneumolysin toxin and S. pneumoniae culture, as demonstrated through enhanced nisin release and a reduction in hemolytic rates upon exposure of the toxin with cholesterol-rich vesicles. Antibacterial testing highlighted the ability of the developed platform to elicit a potent and specific bactericidal response in vitro against S. pneumoniae when compared to a control strain, Staphylococcus epidermidis. It further improved viability of a fibroblast cell line upon S. pneumoniae challenge, outperforming free nisin via the synergistic impact of simultaneous bacterial clearance and pneumolysin neutralization. This study indicates that cholesterol-rich liposomes hold promise as a treatment platform against pneumococcal infections, with potential for expansion to other pathogenic bacteria known to produce similar cholesterol-dependent toxins.