One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

Delivery of biomacromolecules via endocytic pathways requires the efficient accumulation of cargo molecules into endosomes, followed by their release to the cytosol. We propose a unique intracellular delivery strategy for bioactive molecules using a new potent macropinocytosisinducing peptide derived from stromal-derived factor (SDF)-1α (SN21). This peptide allowed extracellular materials to enter cells through the activation of macropinocytosis. To provide the ability to release internalized cargoes from endosomes, we conjugated SN21 with membranelytic peptides. The combination of a macropinocytosis-inducing peptide and a membrane lytic peptide successfully delivered functional siRNA and proteins, which include antibodies, Cre recombinase, and an artificial transcription regulator protein having a transcription activator-like effector (TALE) motif. This study shows the feasibility of combining physiological stimulation of macropinocytosis with physicochemical disruption of endosomes as a strategy for intracellular delivery.

A variety of mid-sized and large biomolecules have been used as tools to explore fundamental biological questions. However, such molecules are often cell-impermeable and thus unable to attain sufficient access to the cell interior. This inhibits their ability to yield analytical data about the cell interior or modify the cellular events. We have recently developed a peptide, engineered from a natural hemolytic peptide, named L17E. Substantial cytosolic delivery of biomacromolecules, including antibodies, was attained in the presence of this peptide. In this study, detailed analysis of the modes of action of L17E was conducted, elucidating that a large fraction of the cytosolic translocation of biomacromolecules is accomplished in the presence of L17E within 5 min. L17E stimulates actin polymerization and induces a dynamic structural alteration of cell membranes, resulting in a ruffled appearance. Studies using macropinocytosis inhibitors and proteins that control endosome maturation raise the possibility that the transient permeabilization of ruffled cell membranes, rather than the rupture of endosomal membranes, is the crucial mechanism for facile cytosolic translocation of biomacromolecules in the presence of L17E. Our results provide a distinct concept of intracellular delivery, different from direct translocation through cell membranes or endocytic uptake followed by endosomal escape. This method of permeabilization via membrane ruffling provides a novel concept in intracellular delivery.

Endocytic pathwaysa re practical routes for the intracellular delivery of biomacromolecules.A long with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery.F ocusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes,w e designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues,w hichs erve as as afety catch for preferential permeabilization of endosomal membranes over cell membranes,a nd His-to-Ala substitutions enhance the endosomolytic activity.T he ability of HAad to destabilizee ndosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells.C erebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in am ouse model, thus demonstrating potential applicability of HAad in vivo.

Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer 3 ]w as designed for immunoglobulin G( IgG) delivery into cells.P article-like liquid droplets were generated by mixing Alexa Fluor 488 labeled IgG (Alexa488-IgG) with FcB(L17E) 3 .D roplet contact with the cellular membrane led to spontaneous influx and distribution of Alexa488-IgG throughout cells in serum containing medium. Involvement of cellular machinery accompanied by actin polymerization and membrane ruffling was suggested for the translocation. Alexa488-IgG negative charges were crucial in liquid droplet formation with positively charged FcB(L17E) 3 .B inding of IgG to FcB(L17E) 3 mayn ot be necessary.S uccessful intracellular delivery of Alexa Fluor 594-labeled anti-nuclear pore complex antibody and anti-mCherry-nanobody tagged with supernegatively charged green fluorescence protein allowed binding to cellular targets in the presence of FcB(L17E) 3 .

Accomplishment of efficient intracellular delivery of bioactive peptides and proteins have been reported via conjugation of peptides having membrane permeation ability (i.e., cell‐penetrating peptides [CPPs]), where a physiological uptake system of extracellular materials, endocytosis, often plays a role. When endocytosed, the bioactive peptides and proteins are encapsulated into vesicular compartment named endosomes, and have to escape into the cytosol with the help of tethered CPPs for obtaining the expected bioactivities. Therefore, CPPs having improved endosomolytic activity is necessary. We here introduce an approach to employ hemolytic peptides as a new class of CPPs, which was designed to attenuate their membrane perturbation ability on cell surfaces while recovering the membrane lytic activity under endosomal conditions (i.e., the attenuated cationic amphiphilic lytic [ACAL] peptides). This was realized by introducing negatively charged glutamic acid (Glu) residues into the potential hydrophobic face of the cationic amphiphilic peptides. The applicability of these peptides for CPPs was evaluated through the intracellular delivery of shepherdin, an apoptosis‐inducing peptide.

No abstract

Intracellular delivery of bioactive macromolecules via endocytic pathways has utility in biotechnological and medicinal applications. Various endosomolytic peptides bearing glutamic acid (Glu) residues have been developed with the aim to achieve selective lysis of endosomal membranes without damaging cell membranes (plasma membranes) to release endosome-entrapped macromolecules and obtain their bioactivity. Glu residues on peptides are negatively charged in the extracellular medium, and substitution of this residue onto membrane-lytic peptides prevents its peptide−membrane interaction and its lytic activity. On the other hand, within endosomes, which have a reduced pH of ∼5, Glu is protonated, resulting in the reduction of the hydrophilicity of the peptide, unmasking its lytic activity. Despite this, a limited number of studies have elucidated the optimum positions for Glu substitution. This report investigated the positioning of Glu and the endosomolytic activities of cationic lytic peptides, ponericin-W3, and melittin. By cell-based assays, biophysical analyses, and molecular dynamics simulations, we found that analogues with Glu positioned on the borders between the hydrophobic and hydrophilic faces of the helical structures showed better performance than placing Glu within said faces.