Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R₇, and R₇W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.     

Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides

The identification of cell-penetrating peptides (CPPs) as vectors for the intracellular delivery of conjugated molecules such as peptides, proteins, and oligonucleotides has emerged as a significant tool to modulate biological activities inside cells. The mechanism of CPP uptake by the cells is still unclear, and appears to be both endocytotic and non-endocytotic, depending on the CPP and cell type. Moreover, it is also unknown whether cargo sequences have an effect on the uptake and cellular distribution properties of CPP sequences. Here, we combine results from quantitative fluorescence microscopy and binding to lipid membrane models to determine the effect of cargo peptide molecules on the cellular uptake and distribution of the arginine-rich CPPs, R7, and R7W, in live cells. Image analysis algorithms that quantify fluorescence were used to measure the relative amount of peptide taken up by the cell, as well as the extent to which the uptake was endocytotic in nature. The results presented here indicate that fusion of arginine-rich CPPs to peptide sequences reduces the efficiency of uptake, and dramatically changes the cellular distribution of the CPP from a diffuse pattern to one in which the peptides are mostly retained in endosomal compartments.     

Newly synthesized peptide,Ara-27, exhibits significant improvement in cell-penetrating ability compared to conventional peptides

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.     

Different membrane behaviour and cellular uptake of three basic arginine-rich peptides

Cell penetrating peptides (CPPs) are peptides displaying the ability to cross cell membranes and transport cargo molecules inside cells. Several uptake mechanisms (endocytic or direct translocation through the membrane) are being considered, but the interaction between the CPP and the cell membrane is certainly a preliminary key point to the entry of the peptide into the cell. In this study, we used three basic peptides: RL9 (RRLLRRLRR-NH(2)), RW9 (RRWWRRWRR-NH(2)) and R9 (RRRRRRRRR-NH(2)). While RW9 and R9 were internalised into wild type Chinese Hamster Ovary cells (CHO) and glycosaminoglycan-deficient CHO cells, at 4°C and 37°C, RL9 was not internalised into CHO cells. To better understand the differences between RW9, R9 and RL9 in terms of uptake, we studied the interaction of these peptides with model lipid membranes. The effect of the three peptides on the thermotropic phase behaviour of a zwitterionic lipid (DMPC) and an anionic lipid (DMPG) was investigated with differential scanning calorimetry (DSC). The presence of negative charges on the lipid headgroups appeared to be essential to trigger the peptide/lipid interaction. RW9 and R9 disturbed the main phase transition of DMPG, whereas RL9 did not induce significant effects. Isothermal titration calorimetry (ITC) allowed us to study the binding of these peptides to large unilamellar vesicles (LUVs). RW9 and R9 proved to have about ten fold more affinity for DSPG LUVs than RL9. With circular dichroism (CD) and NMR spectroscopy, the secondary structure of RL9, RW9 and R9 in aqueous buffer or lipid/detergent conditions was investigated. Additionally, we tested the antimicrobial activity of these peptides against Escherichia coli and Staphylococcus aureus, as CPPs and antimicrobial peptides are known to share several common characteristics. Only RW9 was found to be mildly bacteriostatic against E. coli. These studies helped us to get a better understanding as to why R9 and RW9 are able to cross the cell membrane while RL9 remains bound to the surface without entering the cell.     

Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer

Cell penetrating peptides (CPPs) are short amphipathic and cationic peptides that are rapidly internalized across cell membranes. They can be used to deliver molecular cargo, such as imaging agents (fluorescent dyes and quantum dots), drugs, liposomes, peptide/protein, oligonucleotide/DNA/RNA, nanoparticles and bacteriophage into cells. The utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now clear that CPP can mediate intracellular delivery via both endocytic and non-endocytic pathways. In addition, the orientation of the peptide and cargo and the type of linkage are likely important. In gene therapy, the designed cationic peptides must be able to 1) tightly condense DNA into small, compact particles; 2) target the condensate to specific cell surface receptors; 3) induce endosomal escape; and 4) target the DNA cargo to the nucleus for gene expression. The other studies have demonstrated that these small peptides can be conjugated to tumor homing peptides in order to achieve tumor-targeted delivery in vivo. On the other hand, one of the major aims in molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical agents of malignant transformation. For example, cell penetrating peptide aptamers might disrupt protein-protein interactions crucial for cancer cell growth or survival. In this review, we discuss potential functions of CPPs especially for drug and gene delivery in cancer and indicate their powerful promise for clinical efficacy.     

Glycoprotein biosynthesis in myeloma cells. Characterization on nonglycosylated immunoglobulin light chain secreted in presence of 2-deoxy-D-glucose.

The biosynthesis and secretion of a glycosylated, K-type immunoglobulin light chain (K-46) was studied in a mouse myeloma tumor, mineral oil plasmacytoma-46B. Viable single cell suspensions were prepared from excised tumors and optimal conditions were established for incorporation of amino acid and carbohydrate precursors into the protein synthesized and secreted by the cells. The glucose analog, 2-deoxy-D-glucose, was utilized as an inhibitor of glycosylation to determine the role of glycosylation in the biosynthesis, intracellular transport, and export of the protein from the cell. It was determined that 6 mM 2-deoxyglucose prevents the incorporation of glucosamine, mannose, and galactose into secreted protein, but permits the incorporation of leucine at approximately 40% of control values. The nonglycosylated protein, secreted in the presence of 2-deoxyglucose, was characterized as a nonglycosylated form of K-46 light chain by the following criteria: (a) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, (b) reactivity of the nonglycosylated protein with antisera prepared against native, fully glycosylated, K-46 light chain, (c) analysis of the protein by gel filtration techniques, (d) behavior of the protein on lectin-derivatized Sepharose, and (e) analysis of tryptic peptides derived from the protein. We have concluded that 2-deoxyglucose-inhibited cells synthesize and secrete the normal polypeptide chain of K-46 devoid of its carbohydrate side chain indicating that glycosylation is not an essential step in the biosynthesis, intracellular transport, or export of this protein that is normally synthesized and secreted in a glycosylated form. Under conditions of 2-deoxyglucose inhibition, the nonglycosylated form of K-46 light chain constitutes a significantly greater proportion of accumulated intracellular protein, suggesting that the biosynthesis of the polypeptide chain of K-46 light chain proceeds at a nearly normal rate, but that the absence of the carbohydrate side chain of the protein retards, but does not prevent, the intracellular transport of the protein and its export from the tumor cell.     

Cellular uptake and biophysical properties of galactose and/or tryptophan containing cell-penetrating peptides

Glycosylated cell penetrating peptides (CPPs) have been conjugated to a peptide cargo and the efficiency of cargo delivery into wild type Chinese hamster ovary (CHO) and proteoglycan deficient CHO cells has been quantified by MALDI-TOF mass spectrometry and compared to tryptophan- or alanine containing CPPs. In parallel, the behavior of these CPPs in contact with model membranes has been characterized by different biophysical techniques: Differential Scanning and Isothermal Titration Calorimetries, Imaging Ellipsometry and Attenuated Total Reflectance IR spectroscopy. With these CPPs we have demonstrated that tryptophan residues play a key role in the insertion of a CPP and its conjugate into the membrane: galactosyl residues hampered the internalization when introduced in the middle of the amphipathic secondary structure of a CPP but not when added to the N-terminus, as long as the tryptophan residues were still present in the sequence. The insertion of these CPPs into membrane models was enthalpy driven and was related to the number of tryptophans in the sequence of these secondary amphipathic CPPs. Additionally, we have observed a certain propensity of the investigated CPP analogs to aggregate in contact with the lipid surface.     

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.     

PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells

Cell-penetrating peptides (CPPs) constitute a family of peptides with the characteristic ability to cross biological membranes and deliver cargo into the intracellular milieu. Several CPPs have been proposed for delivery of polypeptides and proteins into cells through either of two strategies: covalent or complexed in a non-covalent fashion. Members of the PEP family are primary amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes. CADY is a secondary amphipathic peptide which has been demonstrated to deliver short nucleic acids, in particular siRNA with high efficiency. Here we review the characteristics of the PEP and CADY carriers and describe a novel derivative of CADY termed CADY2, which also presents sequence similarities to Pep1. We have compared Pep1, CADY and CADY2 in their efficiency to interact with and internalize short fluorogenic peptides and proteins into cultured cells, and provide evidence that CADY2 can interact with proteins and peptides and deliver them efficiently into living cells, similar to Pep1, but in contrast to CADY which is unable to deliver any peptide, even short negatively charged peptides. This is the first study to investigate the influence of the cargo on the interactions between PEP and CADY carriers, thereby providing novel insights into the physicochemical parameters underlying interactions and cellular uptake of peptides and proteins by these non-covalent CPPs.     

Peptide internalization enabled by folding: triple helical cell‐penetrating peptides

Cell‐penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30‐mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen‐like folding domains. The CPP domains are hexa‐arginine (R₆) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline–hydroxyproline–glycine (POG [proline‐hydroxyproline‐glycine])_n repeats that form a collagen‐like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell‐penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Translocation of cell‐penetrating peptides into Candida fungal pathogens

Cell‐penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)₃K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration‐dependent manner, and an additional peptide (TP‐10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)₃K, and TP‐10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.     

Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures

The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.     

Biosynthesis of glycosylated human lysozyme mutants.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.     

Design of a Tumor Homing Cell-Penetrating Peptide for Drug Delivery

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.     

综述: 细胞穿透肽及其结构改造在siRNA传递中的应用

小RNA药物应用于临床的主要技术瓶颈在于如何高效、低毒地将小RNA分子传递到它发挥功能的场所．基于细胞穿透肽在小RNA透皮给药的临床应用中所取得的进展,本文系统评述了近年来细胞穿透肽在小RNA的体内、体外传递方面的研究动态,分析了细胞穿透肽的结构改造对肽/小RNA复合物转染进入细胞发挥功能的影响,展望了细胞穿透肽作为小RNA的体内药物传递载体的发展方向．     

Conformationally constrained peptides for drug delivery

Peptides have shown great potential in acting as template for developing versatile carrier platforms in nanomedicine, aimed at selective delivery of drugs to only pathological tissues saving its normal neighbors. Cell‐penetrating peptides (CPPs) are short oligomeric peptides capable of translocating across the cell membrane while simultaneously employing multiple mechanisms of entry. Most CPPs exist as disordered structures in solution and may adopt a helical conformation on interaction with cell membrane, vital to their penetrative capability. Herein, we report a series of cationic helical amphipathic peptides (CHAPs), which are topologically constrained to be helical. The peptides were tested against cervical and breast cancer cells for their cell penetration and drug delivery potential. The cellular uptake of CHAP peptides is independent of temperature and energy availability. The activity of the peptides is biocompatible in bovine serum. CHAPs delivered functional methotrexate (MTX) inside the cell as CHAP‐MTX conjugates. CHAP‐MTX conjugates were more toxic to cancer cells than MTX alone. However, the CHAP‐MTX conjugates were less toxic to HEK‐293 cells compared with the cancer cells suggesting higher affinity towards cancer cells.     

Real‐time monitoring of antibody glycosylation site occupancy by in situ Raman spectroscopy during bioreactor CHO cell cultures

The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time‐ and labor‐intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real‐time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and nonglycosylated mAbs. By comparing variable importance in projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed‐harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and nonglycosylated mAbs could be successfully and simultaneously estimated in real time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on‐line optimization of mAb quality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:486–493, 2018     

Transduction of peptides and proteins into live cells by cell penetrating peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.