Amyloid-like Self-Assembly of a Hydrophobic Cell-Penetrating Peptide and Its Use as a Carrier for Nucleic Acids

Abstract

Cell-penetrating peptides (CPPs) are a topical subject potentially exploitable for creating nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P, proline; F, phenylalanine; V, valine; Y, tyrosine; L, leucine; and I, isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity and a capacity of nuclear localization, and its small size readily hints at its suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form noncovalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rodlike crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms noncovalent complexes with nucleic acids that retain β-sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that the propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.

Data-Mining Unveils Structure-Property-Activity Correlation of Viral Infectivity Enhancing Self-Assembling Peptides

“…The strong correlation observed for µm-sized aggregation further emphasizes a fundamental common principle for infectivity enhancement of peptides: The hydrophobic surface properties may not only be a driving force for peptides to form fibrils, [72,73] increase fibril-fibril association [42,74] which leads to µmsized aggregates, but also a prerequisite to bind and capture viral particles. An interplay between hydrophobicity, electric charge, and anions in buffer is well-known to control the formation of hierarchical structures, such as amorphous aggregates or amyloid fibrils.…”

Molecular Structure and Antibacterial Activity of Degradation Products from Cephalexin Solutions Submitted to Thermal and Photolytic Stress

“…Droplets from this bacterial suspension were spread on the surface of freshly cleaved mica, and fixed with 4 % PFA following protocols used before by our group. [45][46][47] Liquid chromatography coupled to mass spectroscopy with electrospray ionization (LC/ESI-MS): The samples were characterized by LC/ESI-MS to identify the main byproducts in the samples. The experiments were performed on a Waters Alliance 2695 Separations Module coupled with a Waters 2996 PDA detector and a Waters 3100 Mass Detector.…”

“…To reduce imaging issues related to the presence of components from the broth, droplets from the culture were suspended in saline solutions. Droplets from this bacterial suspension were spread on the surface of freshly cleaved mica, and fixed with 4 % PFA following protocols used before by our group [45–47] . Liquid chromatography coupled to mass spectroscopy with electrospray ionization (LC/ESI‐MS) : The samples were characterized by LC/ESI‐MS to identify the main byproducts in the samples.…”

Materialistic Interfaces with Nucleic Acids: Principles and Their Impact

“…[ 58 ] Additionally, the self‐assembly of peptide‐nucleic acids noncovalent complexes exhibited remarkable amyloidogenic features. [ 59 ] It was identified that co‐delivery of apoptotic peptide and DNA sequences as apoptosis‐associated therapy was capable of killing resistant breast cancer cells. [ 60 ] The integration of FAM‐labeled DNA and paclitaxel‐locked nucleic acid (LNA) monomer allowed the assembly and formation of spherical nanomicelles.…”