Cell-Penetrating Peptidomimetics: Membrane Interaction and Intracellular Drug Delivery
Xiaona Jing
The remarkable progress of life science research has altered the concept of what constitutes pharmaceuticals today. Currently, there is great interest in treating various diseases with biopharmaceuticals, and this has prompted extensive efforts to achieve efficient and safe drug delivery systems of bioactive macromolecules (for example proteins and nucleic acid-based drugs). Such biopharmaceuticals have to pass one or more lipid membranes to reach their pharmacological target site that is often localized in the cytoplasm of cells. The challenge is that the size and the hydrophilic nature of biomacromolecules are unfavorable for their permeation of biological membranes resulting in very limited bioavailability.
The identification and exploitation of cell-penetrating peptides (CPPs) during the last two decades represents a promising approach to overcome membrane barriers inspired by the discovery of naturally occurring CPPs like Tat and penetratin. However, the inherent sensitivity of peptides to degradation by proteases has been shown to hamper the high delivery potential of some peptide carriers, and therefore their stabilization is an important challenge in the future design of CPPs. Peptide backbone modifications usually confer significantly reduced sensitivity to proteolytic enzymes - an approach adopted for several peptidomimetics, such as β-peptides and peptoids intended for use as carriers. Recently, enhanced cellular uptake and membrane destabilizing properties were found for a series of novel α-peptide/β-peptoid peptidomimetics with superior proteolytic stability.
In the present study, the effects of various physicochemical properties of peptidomimetics on membrane interaction and cellular uptake are investigated for the design of future cell-penetrating peptidomimetics. Furthermore, the capability of these α-peptide/β-peptoid peptidomimetics to assist the delivery of a nucleic acid-based biomacromolecule cargo, small interfering RNA (siRNA), formulated in a particulate delivery system was investigated.
It was shown that adsorption of α-peptide/β-peptoid peptidomimetics to anionic lipid bilayers was favoured not only by increased length, positive charge and hydrophobicity of the peptidomimetic, but also by the presence of guanidinium groups and side group chirality. Thermodynamic studies suggested that the adsorption process was exothermic and the interaction was enthalpy-driven. Quantitative cellular uptake of carboxyfluorescein-labeled peptidomimetics with physicochemical variations showed an overall close correlation between the adsorbed amount on anionic model membranes and the cellular uptake. In particular, lipidization of peptidomimetics showed enhancement both on membrane adsorption and cellular uptake, though to different extents. Above all, both the adsorbed amount on membrane and the amount of cellular uptake of all guanidinylated peptidomimetics were much higher than the reference CPP octaarginine. The clear positive correlation found between the cellular uptake and anionic model membrane adsorption indicates that the initial interaction with the membrane is of key importance for the uptake for these peptidomimetics,
The surface hydrogen bonding abilities of the peptidomimetics determined by molecular simulations correlated well to the level of membrane adsorption and cellular uptake, indicating the importance of hydrogen bonding for membrane interaction and cellular uptake. Moreover, the intramolecular hydrogen bonding capability correlated to their mean residue ellipticity obtained from the circular dichroism studies, suggesting that intramolecular hydrogen bonding constitutes a major structural determinant for peptidomimetic folding.
Finally, the potential of alternating sequences of peptidomimetics to deliver siRNA to the cytoplasm was explored by incorporating a palmitoylated peptidomimetic construct into siRNA-loaded lipid-based nanocarriers. Small angle X-ray scattering analysis showed that the majority of the lipids in the nanocarrier were organized in lamellar structures, yet co-existing with a hexagonal phase, which has been shown to be an important factor for efficient, nanocarrier-mediated endosomal escape and cytosolic siRNA delivery. Cryogenic transmission electron microscopy confirmed the existence of multilamellar, onion-like spherical vesicles, potentially with the siRNA intercalated between the lipid bilayers. The resulting siRNA-loaded nanocarriers mediated efficient gene silencing at a concentration well-tolerated by cells. This is the first proof-of-concept that α-peptides/β-peptoid peptidomimetics with alternating structures are efficient cell-penetrating peptidomimetics that can be exploited further for intracellular delivery of biomacromolecular drugs.
Furthermore, this interdisciplinary project also provided several interesting methodological inspirations to address cell-penetrating peptidomimetics investigation. Together, the findings demonstrated that thorough molecular understanding from membrane interaction and cellular activity profiling are important for the future design of cell-penetrating peptidomimetics. Moreover, the detailed morphology and structural investigation of the resulting formulations is essential in order to explain their mechanism of action.
