Internal Structure of 15 nm 3-Helix Micelle Revealed by Small-Angle Neutron Scattering and Coarse-Grained MD Simulation

Abstract

3-Helix micelles (3HM) formed by self-assembly of peptide-polymer conjugate amphiphiles have shown promise as a nanocarrier platform due to their long-circulation, deep tumor penetration, selective accumulation in tumor, and ability to cross the blood-brain barrier (BBB) for glioblastoma therapy. There is a need to understand the structural contribution to the high in vivo stability and performance of 3HM. Using selective deuteration, the contrast variation technique in small-angle neutron scattering, and coarse-grained molecular dynamics simulation, we determined the spatial distribution of each component within 3HM. Our results show a slightly deformed polyethylene glycol (PEG) conformation within the micelle that is radially offset from its conjugation site toward the exterior of the micelle and a highly solvated shell. Surprisingly, ∼85 v/v % of 3HM is water, unusually higher than any micellar nanocarrier based on our knowledge. The result will provide important structural insights for future studies to uncover the molecular origin of 3HM's in vivo performance, and development of the nanocarriers.

Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3-Helix Bundle-Forming Peptide-PEG Conjugates

“…This finding is in good agreement with our recent small-angle neutron scattering contrast variation study that showed ~85% of 3HM is composed of water. 26 …”

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

“…29,41,42 The density of brush layer PEG750 on 3HM is ~7 chains per 100 nm 2 based on geometric calculations with previously determined internal structural information of the trimeric peptide headgroup and PEG2K conformation. 13,15 If only considering these chains, the density is below the 20 chains per 100 nm 2 reported as a minimum threshold for circulation prolongation. 39 However, the areas between the PEG750 presented by the peptide is also composed of PEG2K compressed out from the side-conjugated position, which brings it above this threshold.…”

“…13,14,39 The resulting surface thus has two different PEG conformations shown in Scheme 1, (1) lateral correlation of the PEG750, which corresponds to the interhelical distance between α-helical headgroups, either within a trimeric coiled-coil or between trimers (1.2 and 3.5 nm respectively), 15 and (2) a hydrated PEG2K surface filling in the space between trimers. 10, 13,15 PEGylated surfaces (even below this 20 chains per 100 nm 2 threshold) have been reported to attract elevated levels of apolipoproteins to their protein coronas, notably apolipoprotein B-100 (ApoB-100) and apolipoprotein E (ApoE). 29,[40][41][42] These two moieties can be recognized by the LDLR (low density lipoprotein receptor), which is integral to the internalization of low density lipoprotein (LDL) particles.…”

“…17,21 PDX cells were harvested from flank tumors and subsequently cultured in 2D for short passage numbers, where they maintain their pathological heterogeneity (dense, angiogenic, and invasive niches) once re-implanted orthotopically in mice, as shown by Garner et al 22 The PEGylation architecture of 3HM is composed of two chains of different molecular weights and conjugation sites, and may afford behavior in biological contexts different from other homogeneously PEGylated nanocarriers. 6,13,14 As shown in Scheme 1, there is a 0.8 nm brush layer of PEG (MW:750 g/mol, 'PEG750') attached to the end of 3-helix bundle. 10 Side-conjugated PEG (MW:2000 g/mol, 'PEG2K') is presented laterally in a regular geometric arrangement by the peptide coiled-coil structure.…”

“…MD simulations and structural scattering studies published previously suggest the PEG2K are exposed on the surface of the micelles in between the PEG750 brushes. 6,10,13 The resulting patterned surface of 3HM may be pivotal in mediating its interaction with media/serum biomolecules and subsequent biological machinery. The current work explores the cellular fate of 3HM formulations, elucidating their cell uptake mechanisms, kinetics, and cytotoxicity in the context of GBM therapy development.…”