3-Helix micelles stabilized by polymer springs

Abstract

Despite increasing demands to employ amphiphilic micelles as nanocarriers and nanoreactors, it remains a significant challenge to simultaneously reduce the particle size and enhance the particle stability. Complementary to covalent chemical bonding and attractive intermolecular interactions, entropic repulsion can be incorporated by rational design in the headgroup of an amphiphile to generate small micelles with enhanced stability. A new family of amphiphilic peptide-polymer conjugates is presented where the hydrophilic headgroup is composed of a 3-helix coiled coil with poly(ethylene glycol) attached to the exterior of the helix bundle. When micelles form, the PEG chains are confined in close proximity and are compressed to act as a spring to generate lateral pressure. The formation of 3-helix bundles determines the location and the directionalities of the force vector of each PEG elastic spring so as to slow down amphiphile desorption. Since each component of the amphiphile can be readily tailored, these micelles provide numerous opportunities to meet current demands for organic nanocarriers with tunable stability in life science and energy science. Furthermore, present studies open new avenues to use energy arising from entropic polymer chain deformation to self-assemble energetically stable, single nanoscopic objects, much like repulsion that stabilizes bulk assemblies of colloidal particles.

Figure 1

(a) Chemical structure of the monomeric subunit composed of the peptide (1coi), double C16 tails (grey) and PEG chains (black). Schematic drawing of the designed amphiphilic trimeric subunit where the headgroup contains a 3-helix bundle with the polymer covalently attached to the exterior. When micelles form, the PEG chains are confined in close proximity and are compressed in comparison to its solution conformation. (b) Picture of Christmas tree stand where three screws push in to hold the tree in place. The same design principle was utilized to stabilize the 3-helix micelle by means of entropic repulsion.

Figure 2

(a) Vitreous ice cryogenic TEM of 1coi-dC16-PEG2K at 1 mg/ml in 25 mM phosphate buffer at pH 7.4; (b) Negatively stained TEM of 1coi-dC16-PEG2K at 1 mg/ml in 25 mM phosphate buffer at pH 7.4; (c) SAXS of 1coi-dC16-PEG2K at 5 mg/ml in 25 mM phosphate buffer. Fitting of the data (solid line) to a core-shell spherical form factor yields a core diameter of ~5.6 nm, a shell thickness of ~ 4.6 nm, and polydispersity of ~7%. (d) Concentration dependent SAXS of 1coi-dC16-PEG2K in 25 mM phosphate buffer. Scattering profiles at scattering vector q < 0.08 Å⁻¹ can be fitted using the micelle form factor for all samples studied.

Figure 3

FRET spectra of a mixture of 1coi-dC16-PEG2K micelles encapsulating DiO and DiI FRET pair dyes. After 44hrs, minimal fluorescence change due to energy transfer was observed, indicating the absence of cargo leakage. The excitation wavelength is 450 nm and the emission spectrum was collected between 475 nm and 650 nm.

Figure 4

Fitting of fluorescence recovery data into first-order exchange kinetics. [labeled peptide] = 15 μM; [non-labeled peptide] = 600 μM. Data was fitted into equation $I(t)=I(\infty )+[I(\mathit{0})-I(\infty )][f{{e^{-k}}_{\mathit{1}}}^t+(\mathit{1}-f){{e^{-k}}_{\mathit{2}}}^t]$. The fast rate constant, k₁ is attributed to the dilution of the labeled micelles upon the addition of the non-labeled micelles, leading to an equilibrium shift toward fluorescently labeled unimers. The slower rate constant, k₂ represents the rate of monomer desorption from labeled micelles followed by rapid incorporation into the non-labeled micelles and can be used to compare the kinetics stability of different micelles.

Figure 5

CD spectra of amphiphilic peptide-polymer conjugates with different headgroups. Peptide concentration: 200 μM. 1coi-dC16-PEG2K forms a coiled-coil alpha helix. SingleHelix-dC16-PEG2K and Scmb-dC16-PEG2K form a mixed alpha helix and random coil with 34% and 20% helicity, respectively.

Figure 6

Fitting of fluorescence recovery data into first-order exchange kinetics. (a) Scmb-dC16-PEG2K. (b) SingleHelix-dC16-PEG2K. [labeled peptide] = 15 μM; [non-labeled peptide] = 600 μM. Data was fitted into equation $I(t)=I(\infty )+[I(\mathit{0})-I(\infty )][f{{e^{-k}}_{\mathit{1}}}^t+(\mathit{1}-f){{e^{-k}}_{\mathit{2}}}^t]$. The fast rate constant, k₁ is attributed to the dilution of labeled micelles upon the addition of non-labeled micelles, leading to an equilibrium shift toward fluorescently labeled unimers. The slower rate constant, k₂ represents the rate of monomer desorption from labeled micelles followed by rapid incorporation into the non-labeled micelles and can be used to compare the kinetics stability of different micelles. Thus, for Scmb-dC16-PEG2K and SingleHelix-dC16-PEG2K, data acquired after 20 min were chosen to be fitted into the equation as the new equilibrium is reached.

Figure 7

DSC thermograms of (a) Scmb-dC16-PEG2K (b) SingleHelix-dC16-PEG2K (c) 1coi-dC16-PEG2K. Sample concentration: 200 μM in phosphate buffer (25 mM, pH=7.4). The endothermic peaks result from aliphatic chain melting in the hydrophobic core. The difference in melting temperatures observed indicates the effect of headgroup protein secondary and tertiary structure on the hydrophobic core packing.

Figure 8

Stability of micelles with different headgroups by time-resolved FRET. (a) Scmb-dC16-PEG2K micelles encapsulating DiO and DiI FRET pair dyes. (b) 1coi-dC16-PEG2K micelles encapsulating DiO and DiI FRET pair dyes. (c) 1 to 1 hybrid micelles of 1coi-dC16-PEG2K and Scmb-dC16-PEG2K encapsulating DiO and DiI (d) Plot of normalized FRET versus time.