Porous crystalline frameworks underpin technologies ranging from molecular separations to drug delivery, yet most established examples rely on abiotic components such as metal ions and organic linkers. Metal-organic frameworks, MOFs, and covalent organic frameworks, COFs, offer well-defined pore geometries but often require organic solvents or elevated temperatures for assembly, and their biocompatibility can demand additional post-synthetic modification. Peptide-based frameworks promise a biocompatible alternative, but the conformational flexibility of most peptides resists the structural regularity needed to form extended porous architectures.
Researchers in the Merg Group at the University of California, Merced, published in Nature Communications, addressed this limitation by leveraging the intrinsic rigidity of collagen triple helices. The team synthesized amphiphilic collagen-mimetic peptides, aCMPs, by conjugating fatty acid chains of varying length to the N-terminus of CMP333, a charge-segregated collagen-mimetic peptide comprising three Pro-Arg-Gly, three Pro-Hyp-Gly, and three Glu-Hyp-Gly triads. The four aCMP variants, designated C6333, C8333, C10333, and C12333, were prepared via Fmoc SPPS on a microwave-assisted synthesizer, purified by reverse-phase HPLC, and self-assembled by heating aqueous solutions to 90 °C and slow-cooling to room temperature in 20 mM MES buffer at pH 6.0.
Conjugation of a C12 dodecanoyl chain to CMP333 shifted assembly morphology from multi-layer nanosheets to monodisperse crystalline particles approximately 150 nm in diameter. Transmission electron microscopy revealed triangular-shaped mesoporous channels, and fast Fourier transform analysis of individual crystals confirmed a hexagonal lattice with interplane distances of approximately 9.5, 5.6, and 4.9 nm. Small- and wide-angle X-ray scattering on C12333 assemblies produced Bragg peak ratios of 1:√3:2:√7:√12, consistent with a hexagonal-like lattice, while the analogous non-lipidated CMP333 retained its known tetragonal nanosheet packing. C10333 and C8333 likewise formed porous crystalline frameworks, with hub-to-hub distances of 10.6 nm and 9.4 nm respectively, slightly contracted relative to the 10.9 nm value calculated for C12333. Circular dichroism confirmed that all three aCMPs retain collagen triple-helical character, with cooperative thermal unfolding between 66 °C and 74 °C. A scrambled derivative that lacked the Gly-at-every-third-position requirement for triple helix formation failed to produce porous frameworks under identical conditions, establishing the triple helix as a structural prerequisite.
Fluorescence studies using the solvatochromic probe Prodan revealed that the hydrophobic cores of C8333 frameworks adopt a gel-like, more ordered lipid phase, while C12333 frameworks harbor a liquid-crystalline, more mobile environment, a trend corroborated by Nile Red partitioning and by root mean square fluctuation analysis from all-atom molecular dynamics simulations. Encapsulation experiments with doxorubicin showed a 174% increase in doxorubicin fluorescence at 593 nm at a 1:10 doxorubicin:aCMP molar ratio in C8333 frameworks compared with free doxorubicin in buffer, attributed to partitioning into the lipophilic domains and reduced collisional quenching by water. Molecular dynamics simulations of a proposed hub-and-spoke packing model, in which collagen triple-helix spokes radiate from solvent-accessible hydrophobic hubs in a hexagonal arrangement, converged after 100 ns and revealed an average of approximately 7.83 salt bridges per triple helix, with roughly twice as many inter-unit as intra-unit contacts. Overlay of the simulated assembly unit onto filtered cryo-TEM images of C8333 crystals confirmed agreement between the computational model and experimental lattice dimensions.
The aCMP framework platform demonstrates that covalently combining a lipid chain with a charge-segregated collagen-mimetic peptide is sufficient to redirect self-assembly from two-dimensional nanosheets toward three-dimensional mesoporous crystals. The modular nature of both the alkyl chain length and the CMP sequence design offers a route to systematically tune pore dimensions and internal chemistry, expanding the architectural vocabulary of peptide-based materials toward the predictive design principles already established for MOFs and COFs. Future work aims to extend stability to physiological ionic strength via post-assembly modification and to establish quantitative design rules for mapping aCMP sequence parameters onto pore geometry and guest-encapsulation capacity.
