Insights into Sub-20 nm Nanocarriers (3-helix Micelles) at the Molecular and Cellular Level

The focus of this dissertation is centered around the development of the peptide-PEG-lipid amphiphile based nanocarrier system named 3-helix micelles (3HM). 3HM have been physically characterized as a model system of a new class of self-assembling materials and introduced to biological settings with promising results. In order to thoroughly understand the behavior of 3HM as a nanomedicine and develop them further toward preclinical and clinical studies, advancing the understanding of the interactions 3HM have with biomolecules and living cells is critical. In light of the challenges the drug delivery field currently faces with clinical translation, systematic studies detailing how 3HM micelles behave, from alone as a newly developed material to increasingly-complex environments, will provide foundational knowledge for nanocarrier design and development. This work forms specific understanding of the co-assembly of 3HM with a model small molecule drug and compatible amphiphiles. It then progresses knowledge of the toxicity, transcriptional effects, and cellular trafficking of 3HM formulations in the context of glioblastoma, one of the most prominent drug delivery challenges./nThe pathology of many diseases, including glioblastoma, necessitates 10-30 nm drug delivery systems. At this length scale, the structural and energetic contributions of ~1-2 nm small molecules become integral to the behavior of the nanocarrier itself. The structure-function relationships between internal structure, stability, and release behavior of 3HM formulated with doxorubicin was studied as a model system of nanomaterial-drug co-assembly./nThe molecular architecture of 3HM is chemically modular and homologous to many biomolecular and synthetic amphiphiles. Co-assembling 3HM with DSPE-PEG and GM1 ganglioside, which has clinical potential for Parkinson’s disease, resulted in uniform and stable mixed micelles. The resultant mixed micelles systems displayed tunable drug loading and physical stability. Distinct phase behavior of the lipid core of the mixed 3HM was characterized as a function of composition. These studies are promising to expand the range of therapeutic and excipient molecules that are compatible with 3HM./nTowards preclinical development of 3HM for glioblastoma treatment, the biomolecular and cellular effects of 3HM formulations, with and without doxorubicin, were evaluated using target glioblastoma cell and mouse models. Human patient-derived glioblastoma cells were susceptible to 3HM-doxorubicin treatments in vitro. Transcriptional analysis and in vivo toxicity studies indicate that pure 3HM do not induce any adverse molecular or systemic reactions. Quantification of effective 3HM-doxorubicin concentrations and exposure times inform future efficacy studies./nFinally, the kinetics and mechanism of cellular internalization of 3HM were studied with patient-derived glioblastoma cells. 3HM were found to undergo multiple modes of endocytosis, with clathrin-mediated endocytosis being the most heavily involved. Inhibition experiments suggest the low density lipoprotein receptor is responsible for a majority of the initiation of 3HM uptake, likely through protein corona interactions./nThe understanding developed in this work serves as a critical intermediary between the in-depth physical understanding of 3HM and its promising biological behavior. Details of 3HM interactions at the cellular level substantiate pursuing its development as a clinically-relevant nanocarrier system. Approaches to nanomedicine characterization established here will accelerate future 3HM design and formulation efforts targeted towards other drug delivery scenarios. Key conclusions of this work inform imminent research endeavors to rationally design preclinical formulations with controlled pharmacokinetics.

Advisor: Xu, Ting