Thiostrepton in Phospholipid Micelles: Development of Scalable Production Method and In Vivo Evaluation

For over two decades, our laboratory has explored the use of sterically stabilized micelle (SSM) as a safe and practical nanocarrier for the delivery of water-insoluble drugs and amphiphilic peptides. This nanocarrier is composed of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), a biodegradable and biocompatible ingredient that self-assembles in aqueous media above the micellar concentration forming a nanocarrier with a hydrophobic core and a PEGylated hydrophilic shell. However, our group has identified two important limitations with this nanocarrier: (1) loss of drug activity upon encapsulation of charged amphiphilic antibiotics in SSM and (2) lack of scalability of the conventional hydrophobic drug encapsulation method for industrial purposes. To address these challenges, we selected the hydrophobic non-charged antibiotic thiostrepton (TST) as a better antimicrobial drug candidate for delivery in SSM and developed a novel scalable drug encapsulation method using co-solvent freeze-drying. Our hypothesis was that TST could be encapsulated into SSM using a scalable co-solvent freeze-drying method to provide a stable, safe, and effective nanomedicine against resistant staphylococcal pneumonia and sepsis. By systematically altering key process parameters of the co-solvent freeze-drying method, we identified ideal process conditions to generate an optimized stable TST-SSM nanomedicine with drug loading similar to the thin-film hydration method (5 drug molecules per micelle). TST-SSM nanomedicine was 4-8-fold more active than free TST dissolved in 4% DMSO against various Gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA) USA300. This result was because SSM protected TST from degradation at the bacterial media. We evaluated the antibiotic activity of TST-SSM in a murine model of MRSA pneumonia but found no significant changes possibly due to the interference of the anti-inflammatory activity of TST which inhibits the immune response required for efficient bacterial clearance. On the other hand, TST-SSM exhibited significant improvement in the survival and inflammatory markers of mice with polymicrobial sepsis induced by cecum ligation and puncture. These results are likely due to the reported TLR-9 inhibitory activity of TST which reduces the exacerbated host inflammatory activity characteristic of sepsis. In conclusion, we developed a new TST-SSM nanomedicine using a scalable co-solvent freeze-drying method that is suitable for large scale production and can facilitate the clinical translation of TST-SSM nanomedicine for the treatment of sepsis and possibly other immunological disorders.