Design of 18 nm Doxorubicin-loaded 3-helix Micelles: Cellular Uptake and Cytotoxicity in Patient-derived GBM6 Cells

Abstract

The fate of nanocarrier materials at the cellular level constitutes a critical checkpoint in the development of effective nanomedicines, determining whether tissue level accumulation results in therapeutic benefit. The cytotoxicity and cell internalization of ~18 nm 3-helix micelle (3HM) loaded with doxorubicin (DOX) was analyzed in patient-derived glioblastoma (GBM) cells in vitro. The inhibitory concentration (IC50) of 3HM-DOX increased to 6.2 µg/mL from < 0.5 µg/mL for free DOX in patient derived GBM6 cells, 15.0 µg/mL from 6.5 µg/mL in U87MG cells, and 21.5 µg/mL from ~0.5 µg/mL in LN229 cells. Modeling analysis of previous 3HM biodistribution results predict these cytotoxic concentrations are achievable with intravenous injection in rodent GBM models. 3HM-DOX formulations were internalized intact and underwent intracellular trafficking distinct from free DOX. 3HM was quantified to have an internalization half-life of 12.6 h in GBM6 cells, significantly longer than comparable reported liposome and polymer systems.3HM was found to traffic through active endocytic processes, with clathrin-mediated endocytosis being the most involved of the pathways studied. Inhibition studies suggest substantial involvement of low density lipoprotein receptor (LDLR) in initiating 3HM uptake. Since 3HM surface is polyethylene glycol (PEG)-ylated with no targeting functionalities, protein corona involvement in 3HM recognition is expected. The present work develops insights of the cytotoxicity, pharmacodynamics and cellular interactions of 3HM and 3HM-DOX relevant for ongoing pre-clinical studies. This work also contributes to efforts to develop predictive mathematical models tracking accumulation and biodistribution kinetics at a systemic level.

Designing sub-20 nm self-assembled nanocarriers for small molecule delivery: Interplay among structural geometry, assembly energetics, and cargo release kinetics

“…In vivo, where physiological temperatures ~37°C remain lower than the alkyl tail melting temperature, strong interactions between DOX molecules and the 3HM core is expected to hold true. In combination with tumor transport parameters, 72 cell internalization kinetics, and drug activity time-course, 73 ongoing efforts are being pursued to project 3HM-DOX release postinjection and estimate time-cytotoxicity curves in tumor cells. In this way, recommendations for optimal dosing strategies can be made, which maximizes anti-tumor activity while minimizing side effects.…”