Sílvia Pujals

Homogeneous Conjugation of Peptides onto Gold Nanoparticles Enhances Macrophage Response

Murine bone marrow macrophages were able to recognize gold nanoparticle peptide conjugates, while peptides or nanoparticles alone were not recognized. Consequently, in the presence of conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6, as well as nitric oxide synthase (NOS2) were induced. Furthermore, macrophage activation by gold nanoparticles conjugated to different peptides appeared to be rather independent of peptide length and polarity, but dependent on peptide pattern at the nanoparticle surface. Correspondingly, the biochemical type of response also depended on the type of conjugated peptide and could be correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical nanoparticle conjugate design to either activate the immune system or hide from it in order to reach their targets before being removed by phagocytes.

Peptides conjugated to gold nanoparticles induce macrophage activation

Macrophages that react against pathogenic organisms can also be activated with artificial nanometric units consisting of gold nanoparticles (Au NPs) with a peptide coating. Using bone marrow-derived macrophages, here we show that these cells have the capacity to recognize Au NPs once conjugated to two biomedically relevant peptides, the amyloid growth inhibitory peptide (AGIP) and the sweet arrow peptide (SAP), while they do not recognize peptides or NPs alone. The recognition of these conjugates by macrophages is mediated by a pattern recognition receptor, the TLR-4. Consequently, pro-inflammatory cytokines such as TNF-alpha, IL-1 beta and IL-6, as well as nitric oxide synthase were induced and macrophage proliferation was stopped when exposed to the peptide-conjugated Au NPs. Contamination by lipopolysaccharide in our experimental system was excluded. Furthermore, macrophage activation appeared to be independent of peptide length and polarity. As a result of macrophage activation, conjugated Au NPs were internalized and processed. These results open up a new avenue in the world of adjuvants and illustrate the basic requirements for the design of NP conjugates that efficiently reach their target.

Cytosolic Targeting of Macromolecules Using a pH-Dependent Fusogenic Peptide in Combination with Cationic Liposomes

pH-Sensitive peptides and polymers have been employed as additives to enhance the cytosolic delivery of drugs and genes by facilitating their endosomal escape. However, little attention has been paid to the intracellular fate of these peptides and polymers. In this study, we explored the possibility of utilizing GALA, a pH-sensitive fusogenic peptide, as a cytosol-targeting vehicle. In combination with cationic liposomes, Lipofectamine 2000 (LF2000), the feasibility of this approach for the cytosolic targeting of proteins and nanoparticles was exemplified through the delivery of avidin (68 kDa) and streptavidin-coated quantum dots (15-20 nm) in serum-containing medium. The use of cationic liposomes is critical to enhance the cell-surface adhesion of the GALA conjugates and eventual endosomal uptake. Circular dichroism studies suggest that the GALA can be liberated from cationic liposomes at a reducing pH to form a helical structure and this may eventually lead to disruption of the endosomal membrane to achieve an efficient leakage of the GALA conjugates into the cytosol.

Shuttling Gold Nanoparticles into Tumoral Cells with an Amphipathic Proline-Rich Peptide

Golden bullets: The amphipathic proline‐rich cell‐penetrating peptide sweet arrow peptide (SAP) is able to transport 12 nm gold nanoparticles efficiently into HeLa cells, as observed by three microscopy techniques: transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and transmission X‐ray microscopy (TXM). Multiconjugation to such nanoparticles may provide a convenient method for unifying the key drug properties of high activity, capacity to home onto targets and delivery to therapeutic places of action.

Effect of the attachment of a penetration accelerating sequence and the influence of hydrophobicity on octaarginine-mediated intracellular delivery.

Arginine-rich cell-penetrating peptides (CPPs), including oligoarginine peptides, have been widely used as a tool for intracellular delivery of various molecules with low membrane permeability. We previously reported the enhanced cytosolic entry of arginine-rich CPPs by the attachment of a short peptide segment, the penetration accelerating sequence (Pas). In this study, the importance of hydrophobic sequences, especially phenylalanine residues, in the Pas segment was demonstrated for this enhanced translocation through cell membranes. The advantage of using Pas for intracellular delivery was particularly marked for delivering cargoes with a relatively small molecular weight, such as bioactive peptides. In addition, the results of this study indicate the important roles that the total hydrophobicity of the PasR8 conjugates play in cytosolic translocation and the eventual bioactivity thus attained.

D-SAP: a new, noncytotoxic, and fully protease resistant cell-penetrating peptide.

Protease resistant cell‐penetrating peptides (CPPs) are promising carriers for drugs unable to cross the cell membrane. As these CPPs are stable in vivo for much longer periods of time compared to other classes of therapeutic peptides, noncytotoxicity is a property sine qua non for their pharmacological development. Described herein is a fully protease resistant CPP that is noncytotoxic at concentrations up to 1 mM. Proteolytic stability was obtained by chiral inversion of the residues of a known self‐assembling CPP—from all L‐amino acids to all D‐amino acids—and then assessed against trypsin and human serum. Circular dichroism studies confirmed the enantiomeric structure of the analogue, and transmission electron microscopy (TEM) studies indicated that the new inverso analogue retains the ability of the original peptide to self‐assemble. The results of uptake experiments indicate that the protease‐stable (that is, D‐amino acid) analogue of the peptide is internalised by cells to the same extent as the protease‐susceptible (that is, L‐amino acid) parent peptide. Also reported herein are the results of studies on the cellular internalisation mechanism of the all‐D analogue, which reveal the steps followed by the peptide upon its entry into the cell.

Novel System to Achieve One-Pot Modification of Cargo Molecules with Oligoarginine Vectors for Intracellular Delivery

There is a growing number of reports showing the usefulness of cell-penetrating peptides (CPPs) including oligoarginines for intracellular delivery of macromolecules. Although the covalent attachment of the CPP segments to the cargo molecules is usually required to ensure effective delivery, conventional methods of conjugation need several manipulation steps that are often time-consuming and laborious. Here, we report a novel approach to allow easy modification of cargo molecules with oligoarginine CPPs. The key feature is the employment of oligoarginines (R8 and R12) equipped with a sulfosuccinimidylsuberyl moiety (BS(3)-R8 and -R12). One-pot modification is achieved simply by mixing BS(3)-R8 and -R12 with cargos in an aqueous buffer. The usefulness of this approach was exemplified through the conjugate formation with Fab fragments of immunoglobulin G, amino-functionalized poly(ethylene glycol)s (amino-PEGs), and amino quantum dots (amino-QDs), yielding an efficient cellular uptake.

Curvature Engineering: Positive Membrane Curvature Induced by Epsin N-Terminal Peptide Boosts Internalization of Octaarginine

Epsin-1 is a representative protein for inducing the positive curvature necessary for the formation of clathrin-coated pits. Here we demonstrate that the N-terminus 18-residue peptide of epsin-1 (EpN18) has this ability per se, as proved by differential scanning calorimetry (DSC) and solid-state NMR. Moreover, it is shown how this positive curvature promotion can be exploited for promoting the direct penetration of a representative cell-penetrating peptide (CPP), octaarginine (R8), through artificial and plasma membranes. This synergistic effect has been used for the efficient delivery of a proapoptotic domain peptide (PAD), which induced high level of apoptosis only when coadministered with R8 and EpN18, thus emphasizing the importance of positive curvature induction for achieving the desired ultimate cargo bioavailability.

Electrochemical investigation of cellular uptake of quantum dots decorated with a proline-rich cell penetrating peptide.

The use of square wave voltammetry to monitor the cellular uptake, in HeLa cells, of quantum dots (QD) decorated with sweet arrow peptide (SAP) is reported. A SAP derivative containing an additional N-terminal cysteine residue (C-SAP) was synthesized using the solid-phase method and conjugated to QDs. The obtained results show that QDs-SAP either interact with the extracellular cell membrane matrix or translocate the bilayer. The first situation, membrane adsorption, is probably a transient state before cellular uptake. Both confocal microscopy and SWV results support the detection of this cellular internalization process. The developed electrochemical investigation technique can provide valuable insights into the study of peptide-mediated delivery, as well as the design and development of nanoparticle probes for intracellular imaging, diagnostic, and therapeutic applications. In addition, the described electrochemical interrogation is low cost, is easy to use, and offers future interest for diagnostics including cell analysis.

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.