Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity

AbstractCell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being “retro-inversion” (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides

Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity

AbstractCell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being “retro-inversion” (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20μM peptide concentration within 4h while parent l-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides

Direct Translocation as Major Cellular Uptake for CADY Self-Assembling Peptide-Based Nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability

Characteristics of Cell-Penetrating Peptide/Nucleic Acid Nanoparticles

Nucleic acids are highly promising candidates for the treatment of various genetic diseases. However, due to the large size and negative charge, nucleic acids are not efficiently taken up by cells, and thus, their clinical potential remains limited so far. Therefore, various delivery vehicles have been designed to assist the cellular uptake of nucleic acids. Among these, cell-penetrating peptides (CPPs) have gained increasing popularity as efficient and nontoxic delivery vectors. CPPs can be coupled to nucleic acids either by covalent or noncovalent association. Noncovalent coupling, which is based on the formation of nanoparticle-like nanocomplexes (NP), has received much attention in recent years, and the number of studies employing the strategy is explosively increasing due to the high therapeutic potential. However, the properties of CPP/nucleic acid NPs have not been characterized in sufficient detail yet. We performed a comprehensive analysis of the size and morphology of nucleic acid nanoparticles with novel transfection peptides, PepFects (PFs) and NickFects (NFs), using negative staining transmission electron microscopy (TEM). In addition, we examined whether the attachment of fluorescence or (nano)gold label to nucleic acid affects the nanocomplex formation or its morphology. We demonstrated that transportan-10-based new generation CPPs from PF and NF families condense nucleic acids to NPs of homogeneous size and shape. The size and shape of assembled nanoparticles depend on the type of the complexed nucleic acid and the sequence of the used peptide, whereas the label on the nucleic acid does not influence the gross characteristics of formed NPs

Distribution of CPP-Protein Complexes in Freshly Resected Human Tissue Material

pharmaceutical

Divalent Metal Ions Boost Effect of Nucleic Acids Delivered by Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs) are promising tools for the transfection of various substances, including nucleic acids, into cells. The aim of the current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed from CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed the effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed by the addition of the ions, without increasing cytotoxicity. We propose a novel strategy for preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, with the aim of increasing CPP-mediated transfection efficiency and the likelihood of using CPPs in clinics

Arginine-Rich Cell-Penetrating Peptides Require Nucleolin and Cholesterol-Poor Subdomains for Translocation across Membranes

Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesicles (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy-dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nucleolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently