Mechanisms of Peptide Amphiphile Internalization by SJSA-1 Cells in Vitro

Abstract

Self-assembly of peptide amphiphiles into nanostructures makes them attractive for a variety of applications in drug and peptide delivery. We here report on the interactions of micelles composed of a palmitoylated, pro-apoptotic peptide derived from p53 tumor suppressor protein with a human cancer cell line. Characterization of self-assembly in aqueous buffered solutions revealed formation of elongated rod-like micelles above a critical micelle concentration. Our results however demonstrate that monomers instead of micelles are internalized, a finding which correlates with the dynamic nature of the assemblies and the non-covalent interactions that hold them together. Internalization is shown to occur via adsorption-mediated, energy-dependent pathways, resulting in accumulation of the material in endocytic vesicles. We conclude that palmitoylation of peptides is an efficient way to increase peptide permeability inside SJSA-1 cells and that increased micelle stability would be required for intact micelle internalization.Synthetic peptides derived from human proteins, or identified via powerful new technologies, have emerged as potential therapeutics for various diseases, acting as signals, promoters or inhibitors of cellular functions. Frequently, their site of action is intracellular and therefore they are required to cross cell membranes in order to reach their target. Among the strategies explored in order to attain this goal, the one receiving the most attention is modification with peptide sequences that are able to translocate inside cells, widely referred to as cell penetrating peptides (CPPs) 1, 2 . A variety of different CPPs have been identified, ranging in activity and mechanism of action. An alternative strategy has been the covalent attachment of a hydrophobic tail onto the peptide of interest 3-6 . Borrowed from nature, where cells link hydrophobic moieties onto proteins to efficiently localize them on cellular membranes 7 , this mechanism has proven a simple, yet effective way to transport peptides inside the cell and thus enhance their activity 8, 9 . Peptide amphiphiles or lipopeptides, as the resulting constructs are usually termed, have a higher affinity for lipid bilayers than the parent peptides 9, 10 and are able to insert into the cell membrane by virtue of their hydrophobic tail. Following this first step toward internalization, the subsequent uptake mechanism and the final destination of lipopeptides remain a subject of investigation. Attaching a hydrophobic tail onto a peptide additionally has implication on its biodistribution. Depending on the type of lipid tail, binding to certain biomolecules in vivo can be promoted in order to aid their transport to sites of interest 11 or act as a drug reservoir in the blood 12 . Moreover, lipopeptides exhibit improved stability against As an example of a pro-apoptotic signal we here selected a peptide derived from the binding site of tumor suppressor p53 to the MDM2 protein 26 . The 16-mer peptide from the N-terminus of p53 (p53 [14][15][16...

Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix

“…This is consistent with previous results that palmitoylation of proapoptotic peptides can affect micelle stability and cell uptake. 56 The results show that there was higher osteogenic differentiation of encapsulated hMSCs when the BMP-2 peptide was dissolved in the hydrogel matrix as compared with the peptide that was conjugated (Fig. 4), which was similar to the previously reported lower activity of BMP-2 protein attached to immobilized heparin.…”

Peptide Amphiphile Micelles Self-Adjuvant Group A Streptococcal Vaccination

“…They have been shown capable of delivering biologically active peptides for a variety of applications including angiogenesis (19,20), osteogenesis (21,22), neurogenesis (23,24), atherosclerosis treatment (25), cancer therapy (26,27), and islet transplantation (28,29). Also, peptide amphiphile micelles are comprised of a high concentration of peptide (30), inhibit peptide degradation (31), and can greatly increase peptide intracellular delivery (32). Recent research by the Tirrell Group has shown that peptide amphiphile micelles that display a tumor-specific cytotoxic T cell epitope can function as a self-adjuvanting vaccine delivery system capable of inducing a tumor-suppressing immune response when given prophylactically (33).…”

General discussion

“…2 Peptide nanobres might also pierce the lipid cell membrane in the same manner as another class of brillar nanostructures, carbon nanotubes, to access the intracellular environment 3,4 due to their similarity in shape.…”

“…1 The non-linear glycopolypeptides presented here have the ability to spontaneously self-assemble into spherical micelles or in disk-like structures in water, a property that is not observed with their linear counterparts. 2,3 One can hypothesize that these disklike structures may bring some advantages in terms of bioactivity and we are currently developing drug delivery systems to be tested in vitro and in vivo. Professor Stupp remarked: Is there a possibility of controlling the polydispersity of the polysaccharide given that its volume fraction should affect the type of supramolecular packing observed?…”