Biological constraints in diseased tissues have motivated the need for small nanocarriers (10-30 nm) to achieve sufficient vascular extravasation and pervasive tumor penetration. This particle size limit is only an order of magnitude larger than small molecules, such that cargo loading is better described by co-assembly processes rather than simple encapsulation. Understanding the structural, kinetic, and energetic contributions of carrier-cargo co-assembly is thus critical to achieve molecular-level control towards predictable in vivo behavior. These interconnected set of properties were systematically examined using sub-20 nm self-assembled nanocarriers known as three-helix micelles (3HM). Both hydrophobicity and the "geometric packing parameter" dictate small molecule compatibility with 3HM's alkyl tail core. Planar obelisk-like apomorphine and doxorubicin (DOX) molecules intercalated well within the 3HM core and near the core-shell interface, forming an integral component to the co-assembly, as corroborated by small-angle X-ray and neutron-scattering structural studies. DOX promoted crystalline alkyl tail ordering, which significantly increased (+63%) the activation energy of 3HM subunit exchange. Subsequently, 3HM-DOX displayed slow-release kinetics (t_1/2 = 40 h) at physiological temperatures, with ~50× greater cargo preference for the micelle core as described by two drug partitioning coefficients (micellar core/shell K_p1 ~ 24, and shell/bulk solvent K_p2 ~ 2). The geometric and energetic insights between nanocarrier and their small molecule cargos developed here will aid in broader efforts to deconvolute the interconnected properties of carrier-drug co-assemblies. Adding this knowledge to pharmacological and immunological explorations will expand our understanding of nanomedicine behavior throughout all the physical and in vivo processes they are intended to encounter.

3-Helix micelles have demonstrated excellent in vitro and in vivo stability. Previous studies showed that the unique design of the peptide-polymer conjugate based on protein tertiary structure as the headgroup is the main design factor to achieve high kinetic stability. In this contribution, using amphiphiles with different alkyl tails, namely, C16 and C18, we quantified the effect of alkyl length on the stability of 3-helix micelles to delineate the contribution of the micellar core and shell on the micelle stability. Both amphiphiles form well-defined micelles, <20 nm in size, and show good stability, which can be attributed to the headgroup design. C18-micelles exhibit slightly higher kinetic stability in the presence of serum proteins at 37 °C, where the rate constant of subunit exchange is 0.20 h(-1) for C18-micelles vs 0.22 h(-1) for C16-micelles. The diffusion constant for drug release from C18-micelles is approximately half of that for C16-micelles. The differences between the two micelles are significantly more pronounced in terms of in vivo stability and extent of tumor accumulation. C18-micelles exhibit significantly longer blood circulation time of 29.5 h, whereas C16-micelles have a circulation time of 16.1 h. The extent of tumor accumulation at 48 h after injection is ∼43% higher for C18-micelles. The present studies underscore the importance of core composition on the biological behavior of 3-helix micelles. The quantification of the effect of this key design parameter on the stability of 3-helix micelles provides important guidelines for carrier selection and use in complex environment.

Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration, and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt % doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-Helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics.