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Cell penetrating peptides (CPPs) are generally defined as short positively charged peptides, containing 5–30 amino acids. Based on their physicochemical properties, they are classified as three main groups, namely hydrophobic, amphipathic, and hydrophilic. They are capable of interacting with the cell membrane without inducing serious toxicity, and they can carry cargo molecules across the membrane. Cargo molecules could be different therapeutics which makes CPPs valuable in the field of drug delivery into living cells. Nowadays, CPPs are considered as potential parts of therapeutics against several diseases. Despite similarities in their primary structure, the interactions of CPPs with a cell membrane may vary a lot. This is even more complicated when the CPP is bound to the cargo molecule. The mechanism(s) of their cellular uptake and endosomal escape have not been completely resolved. Understanding the mechanism of membrane interaction will help us designing a CPP with enhanced, selective cargo delivery, hopefully resulting in better disease treatments. So far energy independent direct membrane penetration and energy-dependent endocytosis have been suggested as two main mechanisms of cellular entry for CPPs, and both may be applicable for the same CPP-complex, depending on the conditions. In order to understand which mechanism is associated with a particular CPP’s cellular uptake in a particular cell (sometimes including endosomal escape), different biological and biophysical methods and strategies have been applied. In this chapter, we will address several biophysical methods, such as fluorescence spectroscopy, circular dichroism (CD) spectroscopy, dynamic light scattering, and NMR. We also review different membrane model systems which are suitable for the biophysical studies. These include large unilamellar phospholipid vesicles (LUVs), which are the most commonly used in the lipid–peptide interaction studies. Detergent micelles and mixed micelles (bicelles) ar

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.

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

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.

A series of cell-penetrating PepFect peptide analogues was developed by substitutions of the galanin-derived N-terminal sequence. Histidine modifications were incorporated in order to make the peptides pH-responsive. The peptides were all able to form non-covalent complexes with an oligonucleotide cargo by co-incubation in buffer. The complexes were characterized by dynamic light scattering and circular dichroism, and an assay to evaluate the peptide-cargo affinity was developed. Cellular bioactivity was studied in HeLa cells using a luciferase-based splice correction assay. In addition, the membrane interactions of the peptides in large unilammelar vesicles was studied using a calcein leakage assay. The effects of substitutions were found to be dependent of the non-modified, C-terminal sequence of the peptides; for analogues of PepFect 3 we observed an increase in membrane activity and bioactivity for histidine-containing analogues, whereas the same modifications introduced to PepFect 14 lead to a decreased bioactivity. Peptides modified with a leucine/histidine sequence were found to be pH responsive, complexes formed from these peptides were small at pH 7 and grew under acidic conditions. The most promising of the novel PepFect 3 analogues, PepFect 132 has a significantly higher bioactivity and membrane activity than the parent peptide PepFect 3.

Despite many studies made on cell-penetrating peptides (CPPs), the mechanism of their cellular uptake and endosomal escape has not been completely resolved. This is even more unclear when the CPP is bound either covalently or non-covalently to the cargo molecules. To answer remaining questions, we require a combination of different methods, model systems, and experiments since there is no single method which could give a complete answer to all questions. Biophysical investigations of CPPs have a significant impact on CPP research considering their molecular mechanisms of action. In this chapter, we present different membrane model systems suitable for biophysical studies as well as the basic practical aspects underlying several common biophysical methods and experiments. The methods include fluorescence spectroscopy, circular dichroism spectroscopy, and dynamic light scattering and concern peptide-membrane interactions and vesicle model membrane leakage. We have also described the potential and limitations of biophysical studies on the CPP-membrane interactions and their impact on our understanding of how CPPs mediate the transport of cargoes into living cells.

Cell-penetrating peptides with the ability to escape endosomes and reach the target are of great value as delivery vectors for different bioactive cargoes and future treatment of human diseases. We have studied two such peptides, NickFect1 and NickFect51, both originated from stearylated transportan10 (PF3). To obtain more insight into the mechanism(s) of peptide delivery and the biophysical properties of an efficient vector system, we investigated the effect of different bioactive oligonucleotide cargoes on peptide-membrane perturbation and peptide structural induction. We studied the membrane interactions of the peptides with large unilamellar vesicles and compared their effects with parent peptides transportan10 and PF3. In addition, cellular uptake and peptide-mediated oligonucleotide delivery were analyzed. Calcein leakage experiments showed that similar to transportan10, NickFect51 caused a significant degree of membrane leakage, whereas NickFect1, similar to PF3, was less membrane perturbing. The results are in agreement with previously published results indicating that NickFect51 is a more efficient endosomal escaper. However, the presence of a large cargo like plasmid DNA inhibited NickFect's membrane perturbation and cellular uptake efficiency of the peptide was reduced. We conclude that the pathway for cellular uptake of peptide complexes is cargo dependent, whereas the endosomal escape efficacy depends on peptide hydrophobicity and chemical structure. For small interfering RNA delivery, NickFect51 appears to be optimal. The biophysical signature shows that the peptide alone causes membrane perturbation, but the cargo complex does not. These two biophysical characteristics of the peptide and its cargo complex may be the signature of an efficient delivery vector system.

Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

Single-stranded DNA (ssDNA) is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide PDYN (prodynorphin) fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4 degrees C, and some also at 37 degrees C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA.

Voltage-gated K+ channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a “paddle” domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the Kv channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic–hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

Several effects of the endogenous opioid peptide dynorphin A (Dyn A) are not mediated through the opioid receptors. These effects are generally excitatory, and result in cell loss and induction of chronic pain and paralysis. The mechanism(s) is not well defined but may involve formation of pores in cellular membranes. In the 17-amino acid peptide Dyn A we have recently identified L5S, R6W, and R9C mutations that cause the dominantly inherited neurodegenerative disorder Spinocerebellar ataxia type 23. To gain further insight into non-opioid neurodegenerative mechanism(s), we studied the perturbation effects on lipid bilayers of wild type Dyn A and its mutants in large unilamellar phospholipid vesicles encapsulating the fluorescent dye calcein. The peptides were found to induce calcein leakage from uncharged and negatively charged vesicles to different degrees, thus reflecting different membrane perturbation effects. The mutant Dyn A R6W was the most potent in producing leakage with negatively charged vesicles whereas Dyn A L5S was virtually inactive. The overall correlation between membrane perturbation and neurotoxic response [3] suggests that pathogenic Dyn A actions may be mediated through transient pore formation in lipid domains of the plasma membrane.

Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms., Multidisciplinary BIO, VINNOVA-SAMBIO