A Novel Cell-penetrating Peptide, M918, for Efficient Delivery of Proteins and Peptide Nucleic Acids
Authors: Samir El-Andaloussi, Henrik J Johansson, Tina Holm, Ülo Langel
Journal: Molecular Therapy (2007)

Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently. Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently. IntroductionThe full sequencing of the human genome has led to increasing demands for functional studies of genes and their protein products. For this purpose, either proteins are directly transduced inside cells or one of several oligonucleotide (ON)-based techniques is used. The main techniques to knock down the expression of proteins include the use of antisense oligonucleotides (asONs) and small interfering RNAs.1Crooke ST Progress in antisense technology.Annu Rev Med. 2004; 55: 61-95Crossref PubMed Scopus (389) Google Scholar In 1993, Kole et al. introduced another ON-based platform utilizing asONs to redirect splicing and correct pre-messenger RNAs (pre-mRNAs) by blocking aberrant splice sites, with concomitant sterical blocking of splicosome binding.2Dominski Z Kole R Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides.Proc Natl Acad Sci USA. 1993; 90: 8673-8677Crossref PubMed Scopus (328) Google Scholar These antisense agents have also been used to direct splicing toward certain protein isoforms.3Mercatante DR Bortner CD Cidlowski JA Kole R Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death.J Biol Chem. 2001; 276: 16411-16417Crossref PubMed Scopus (136) Google Scholar,4Taylor JK Zhang QQ Wyatt JR Dean NM Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides.Nat Biotechnol. 1999; 17: 1097-1100Crossref PubMed Scopus (189) Google Scholar Obviously, targeting mRNAs for degradation and controlling pre-mRNA splicing patterns with asONs are attractive ways potentially to treat and manage a variety of diseases.5Sazani P Kole R Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing.J Clin Invest. 2003; 112: 481-486Crossref PubMed Scopus (203) Google ScholarSeveral ON analogs have been developed with improved properties, including peptide nucleic acid (PNA).6Nielsen PE Egholm M Berg RH Buchardt O Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide.Science. 1991; 254: 1497-1500Crossref PubMed Scopus (2809) Google Scholar The inability of these molecules to enter cells remains a major obstacle, so several delivery vectors have been developed.7Patil SD Rhodes DG Burgess DJ DNA-based therapeutics and DNA delivery systems: a comprehensive review.AAPS J. 2005; 7: E61-E77Crossref PubMed Scopus (507) Google Scholar The most frequently used delivery vehicles today are formulas based on cationic liposomes that are highly efficient in vitro and easy to use but are unable to transport uncharged PNAs. Therefore, various peptides, such as receptor ligands and nuclear localization signal sequences, have been conjugated to PNA to promote cellular uptake.8Sun L Fuselier JA Murphy WA Coy DH Antisense peptide nucleic acids conjugated to somatostatin analogs and targeted at the n-myc oncogene display enhanced cytotoxity to human neuroblastoma IMR32 cells expressing somatostatin receptors.Peptides. 2002; 23: 1557-1565Crossref PubMed Scopus (36) Google Scholar,9Brandén LJ Mohamed AJ Smith CI A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA.Nat Biotechnol. 1999; 17: 784-787Crossref PubMed Scopus (348) Google Scholar Another class of molecules that suffer from low bioavailability owing to limited cellular uptake are proteins, and, unlike in the case of ONs, few vectors are available for protein transduction.In the past 10 years a new class of peptides has gained increasing attention. These peptides are usually less than 30 amino acids in length, have a cationic and/or amphipathic nature, and are generally referred to as cell-penetrating peptides (CPPs). These peptides have the ability to translocate rapidly into most mammalian cells carrying various cargo molecules both in vitro and in vivo, as reviewed in ref. 10EL-Andaloussi S Holm T Langel Ü Cell-penetrating peptides: mechanisms and applications.Curr Pharm Des. 2005; 11: 3597-3611Crossref PubMed Scopus (206) Google Scholar. It is not fully established what mechanism is responsible for the uptake of CPPs and their cargoes; however, a number of studies are now emphasizing the role of endocytosis in translocation of CPPs,11EL-Andaloussi S Johansson HJ Lundberg P Langel Ü Induction of splice correction by cell-penetrating peptide nucleic acids.J Gene Med. 2006; 8: 1262-1273Crossref PubMed Scopus (114) Google Scholar,12Fittipaldi A Ferrari A Zoppe M Arcangeli C Pellegrini V Beltram F et al.Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins.J Biol Chem. 2003; 278: 34141-34149Crossref PubMed Scopus (395) Google Scholar,13Richard JP Melikov K Vives E Ramos C Verbeure B Gait MJ et al.Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.J Biol Chem. 2003; 278: 585-590Crossref PubMed Scopus (1462) Google Scholar and in particular macropinocytosis.14Wadia JS Stan RV Dowdy SF Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.Nat Med. 2004; 10: 310-315Crossref PubMed Scopus (1394) Google Scholar,15Nakase I Niwa M Takeuchi T Sonomura K Kawabata N Koike Y et al.Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement.Mol Ther. 2004; 10: 1011-1022Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar,16Magzoub M Sandgren S Lundberg P Oglecka K Lilja J Wittrup A et al.N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis.Biochem Biophys Res Commun. 2006; 348: 379-385Crossref PubMed Scopus (75) Google Scholar Although several CPPs have been developed since the initial discovery of penetratin in 1994 (ref. 17Derossi D Joliot AH Chassaing G Prochiantz A The third helix of the Antennapedia homeodomain translocates through biological membranes.J Biol Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar) and Tat peptide in 1997,18Vives E Brodin P Lebleu B A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.J Biol Chem. 1997; 272: 16010-16017Crossref PubMed Scopus (2034) Google Scholar it remains important to find new peptides that are efficient and less toxic at higher concentrations. In addition, determining the uptake mechanism of CPPs would provide valuable information for the design of new CPPs with improved delivery properties.High cellular uptake of peptides and cargoes does not necessarily correlate with a high biological response, mostly as a result of material being retained in endosomes. One way to assess the "true" delivery efficiency of peptides is to use a positive read-out assay based on splice correction. This assay is based on a splicing reporter system where a plasmid carrying the luciferase cod ing sequence is interrupted by an insertion of intron 2 from the thalassemic β-globin pre-mRNA carrying a cryptic splice site. Unless the aberrant splice site is masked by antisense PNA, the pre-mRNA of luciferase will be improperly processed and no luciferase will be produced. HeLa pLuc 705 cells stably transfected with this plasmid can be used to evaluate various vectors by measuring the luciferase activity in cells.19Kang SH Cho MJ Kole R Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development.Biochemistry. 1998; 37: 6235-6239Crossref PubMed Scopus (252) Google ScholarThe need for new efficient, non-toxic delivery vehicles for proteins, nucleic acids, and their analogs encouraged us to investigate the delivery efficacy of a novel CPP, denoted M918. In addition to assessing the delivery potential of the peptide, we investigate the mechanism of peptide internalization. To quantify the bioavailability of cargo delivered using M918, the abovementioned splicing system was employed, and the results were compared with those for other, well-established CPPs.ResultsM918 internalizes to several different cell lines in a non-toxic fashionM918 is a peptide derived from the tumor suppressor protein p14ARF, amino acids 1–22 with positions 3–8 inverted (Table 1). This peptide was developed as a control peptide in a study where the parent peptide was used to mimic the activity of the ARF protein. To our surprise, M918 showed excellent cell-penetrating properties, and therefore the peptide was further characterized and evaluated in terms of its delivery properties.Table 1Sequences of cell-penetrating peptides (CPPs) and peptide nucleic acids (PNAs)NameAbbreviationSequencePenetratinPenRQIKIWFQNRRMKWKK-NH2Transportan 10TP10AGYLLGK*INLKALAALAKKIL-NH2M918M918MVTVLFRRLRIRRACGPPRVRV-NH2YDEGEYDEGEYDEEGGGE-NH2M705 PNAPNACysKKCCTCTTACCTCAGTTACAKK+-NH2M705 invertedinvPNACysKKCCTCTTACACTCGTTACAKK+-NH2PNAPenetratin, YDEGE, and M918 were fluoresceinyl-labeled on the N-terminus. Bold letters represent nucleobases. The four underlined bases indicate the sequence within the PNA that is inverted to generate the inverted PNA (invPNA). For the building of conjugates, M918 and penetratin were synthesized with N-terminal Cys(Npys) and TP10 orthogonally on Lys7. Conjugates are denoted CPP–PNAs and illustrated as follows: CPPCys-S-S-CysPNA. *Carboxyfluorescein was coupled to the ɛ-amino group of lysine7 in TP10. +Attachment of carboxyfluorescein on ɛ-amino group of c-terminal lysine. Open table in a new tab First, the cellular uptake of M918 was assessed in a quantitative uptake assay. The results clearly show that M918 is readily internalized into several cell lines (Figure 1b) and to a higher extent than the commonly used CPPs penetratin and TP10 in HeLa cells (Figure 1a) and Chinese hamster ovarian (CHO) cells (data not shown). YDEGE peptide was used as a negative control, displaying negligible uptake levels (Figure 1a). Furthermore, to exclude the possibility that the observed uptake was a result of plasma membrane disturbance, a lactate dehydrogenase (LDH) leakage assay was performed. Figure 1c shows that none of the tested peptide concentrations (5–25 μmol/l) had any noticeable effect on membrane integrity in any cell line tested, in contrast to the positive control, TP10, which induced massive membrane leakage at a 25 μmol/l concentration.Even though the peptide does not affect membrane integrity, it might exhibit other toxic side effects on cells. Therefore a wst-1 assay was carried out to determine whether the peptide influences cellular proliferation. In accordance with the LDH leakage data, no significant effect on proliferation after 72 hours was observed with M918 in any cell line using the same concentration range (Figure 1d). Staurosporine (1 μmol/l), used as a positive control, reduced proliferation significantly. The high quantitative uptake observed for M918 in different cell lines could be explained by peptide aggregation in cellular membranes rather than internalized peptides. Therefore, the uptake was analyzed qualitatively using confocal microscopy. Clearly, neither in human breast cancer MCF-7 cells nor in HeLa cells could any clusters of peptides be detected in the plasma membrane (Figure 2a and c). However, in astrocytoma cells, Hifko, some membrane associated peptides were observed (Figure 2b). Most internalized material appears to reside in vesicles, and low or negligible amounts in the cell nucleus (Figure 2a–c). In HeLa cells, a slight diffuse staining of the cytoplasm was observed (Figure 2c).Figure 2Confocal microscopy of M918 displays mainly vesicular localization inside cells. (a) MCF-7, (b) Hifko, and (c) HeLa cells were seeded in NUNC 8 chambers 2 days before the experiment to reach 60% confluence. After 30 minutes' treatment with 1 μmol/l fluoresceinyl-labeled M918 peptide in HEPES Krebs Ringer (HKR), cells were washed three times with HKR before analysis with confocal microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)M918 promotes protein transductionAvidin and streptavidin were chosen as model proteins for transduction experiments as they have been widely used to quantify delivery and have the advantage of binding biotin with high affinity. In the first set of experiments, peptides were simply mixed with streptavidin (Figure 3a) or avidin (Figure 3c) to evaluate whether CPPs translocate proteins as non-covalent complexes. Figure 3a shows that all three peptides promote cellular uptake of fluorescein isothiocyanate–labeled streptavidin at 5 μmol/l peptide concentrations (25× molar excess), with M918 being the most efficient vector. When 1 μmol/l peptides were used, however, no improvement in delivery of streptavidin was observed with any peptide (data not shown). In contrast, when 1 μmol/l biotinylated peptides that form four nearly irreversible bonds with streptavidin were used, the uptake levels were increased significantly (Figure 3b) compared with incubation with TP10 and M918. Interestingly, penetratin results in equally low uptakes independent of conjugation strategy (Figure 3a and b). When we assessed the quantitative uptake of avidin, negligible levels of internalized protein were observed with any peptide using the co-incubation strategy (Figure 3c), whereas a significant increase in cellular uptake was seen when 1 μmol/l biotinylated peptides were used. As with streptavidin, so with biotinylated peptide M918 is the most potent delivery vector, closely followed by TP10.Figure 3CPPs promote protein transduction. HeLa cells (200,000 cells/well) were seeded in 12-well plates 1 day before treatment. (a, b) Streptavidin (SA) or (c, d) avidin (0.2 μmol/l) was either (a, c) incubated for 30 minutes in 50 μl 0.9% NaCl with 5 μmol/l peptide or (b, d) incubated with 1 μmol/l biotin-labeled peptide enabling formation of four nearly irreversible bonds to proteins, and added to HeLa cells for 90 minutes in 500 μl serum-free Dulbecco's modified Eagle's medium. Cells were then treated as in Figure 1. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM, n = 3). FITC, fluorescein isothiocyanate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Internalization of M918 is mediated by endocytosis independently of glycosaminoglycansThe high uptake observed with M918, together with the confocal microscope data showing punctual distribution of peptide inside cells, encouraged us to elucidate the internalization mechanism of the peptide. Here we show that when cells are treated and incubated with M918 at 4 °C, the cellular uptake is decreased significantly in HeLa cells (Figure 4a). Similar results were obtained in CHO cells and with penetratin and TP10 in both cell lines (data not shown). In addition, adding 0.2 mol/l sucrose or 75 μmol/l chloroquine decreased internalization of fluoresceinyl-labeled M918 significantly (Figure 4a). Rhodamine-labeled transferrin, used as a positive control, displayed a significant decrease in uptake after treatment with inhibitors (Figure 4a). To elucidate further the endosomal route used by peptides, a co-localization assay was performed between rhodamine-labeled dextran or transferrin and fluoresceinyl-labeled M918. As seen in Figure 5, the peptide localizes with both markers in MCF-7 cells. Similar results were obtained in CHO and Hifko cells, whereas little localization was observed with transferrin in HeLa cells (data not shown).Figure 4M918 internalizes to cells via endocytosis independent of glycosaminoglycans on the cell surface. (a) HeLa cells (200,000 cells/well) were seeded in 12-well plates 24 hours before the experiment and were incubated with 5 μmol/l fluoresceinyl-labeled M918 or 25 μg/ml rhodamine-labeled transferrin for 1 hour in 500 μl serum-free Dulbecco's modified Eagle's medium and pre-treated for 30 minutes with 75 μmol/l chloroquine (cq), 0.2 mol/l sucrose, or low temperature. Cells were then treated as in Figure 1. (b) Chinese hamster ovary (CHO) and CHO2242 cells treated as in a. Uptake of the three peptides in CHO cells was set to 100%. The values represent the mean of at least three independent experiments performed in triplicate (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (a) Analysis of variance Dunnett's, (b) t-test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5M918 partially co-localizes with both transferrin and dextran in MCF-7 cells. (a) Brightfield image, (b) M918, (c) endocytosis markers, dextran or transferrin, (d) composite. MCF-7 cells (20,000) seeded 2 days before the experiment in NUNC 8 chambers were treated with 1 μmol/l fluoresceinyl-labeled peptide for 30 minutes together with 1 mg/ml of the fluid-phase endocytosis marker rhodamine-labeled dextran or 25 μg/ml of the clathrin-mediated endocytosis marker rhodamine-labeled transferrin. Cells were washed three times with HEPES Krebs Ringer before analysis by confocal microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Several reports have pointed out the necessity of the presence of glycosaminoglycans (GAGs) on the cell surface for the uptake of CPPs.20Marty C Meylan C Schott H Ballmer-Hofer K Schwendener RA Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes.Cell Mol Life Sci. 2004; 61: 1785-1794PubMed Google Scholar,21Console S Marty C Garcia-Echeverria C Schwendener R Ballmer-Hofer K Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans.J Biol Chem. 2003; 278: 35109-35114Crossref PubMed Scopus (365) Google Scholar According to Figure 4b, both penetratin uptake and TP10 uptake are decreased significantly in GAG-deficient CHO cells as compared with wild-type CHO cells (Figure 4b). To our surprise, the uptake of M918 was not reduced significantly, although a minor decrease was observed. Again, the high uptake observed in GAG-deficient CHO cells could be a result of membrane aggregation, but most peptides are evenly distributed in vesicles throughout the cells as judged by confocal microscopy (data not shown).Cell-penetrating antisense PNA induces splice correction dose-dependentlyIn an attempt to quantify the delivery efficacy of CPPs, antisense PNA targeting luciferase pre-mRNA, described in the Introduction, was used. When cells were treated for 1 hour with peptides covalently conjugated via a disulphide bridge to fluoresceinyl-labeled PNA and luciferase expression was measured 16 hour later, all three peptides promoted splicing dose-dependently, but with varying efficacy (data not shown). Again, M918 appears to be the most potent vector, conferring a 15-fold increase in splicing compared with 10- and 5-fold increases for TP10 and penetratin, respectively, over untreated cells at 5 μmol/l concentrations (Figure 6a). Interestingly, there is a correlation between uptake and splicing in the very same cell lysate using M918 (Figure 6b). Neither an inverted PNA sequence conjugated to M918 nor free PNA induces significant splicing at 10 μmol/l concentrations (Figure 6a). These results clearly indicate that the system is specific and that CPPs are responsible for the cellular uptake of PNA, as naked PNA is unable to promote splicing significantly.Figure 6M918-PNA enters cells via macropinocytosis and dose-dependently induces splice correction. HeLa pLuc 705 cells (60,000 cells/well) were seeded 48 hours before the experiment in 24-well plates, after which cells were treated with peptide–PNA conjugates for 1 hour in serum-free Dulbecco's modified Eagle's medium followed by 16 hours in serum medium. (a) Splice correction after treatment with 5 μmol/l peptide–PNA conjugates presented as fold increase in splicing over untreated cells. The controls for specificity and delivery efficacy of M918 are 10 μmol/l inverted PNA (invPNA) and PNA, respectively. (b) Uptake and splicing after treatment with different M918-PNA concentrations presented as relative light units (RLU)/mg. (c) Cells were pre-treated with 4 μmol/l cytochalasin D, 50 nmol/l wortmannin, or pre-maintained at 4 °C for 30 minutes and treated at for 1 hour with 5 μmol/l M918-PNA. RLU/mg values obtained with conjugate only were set to 100% splice correction. (d) Cells were treated as in a with M918-PNA at the indicated concentrations together with 75 μmol/l chloroquine (cq). The values represent the mean of three independent experiments each performed in triplicate (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (c) Analysis of variance (ANOVA) Dunnett's, (d) ANOVA Bonferroni. PNA, peptide nucleic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lysosomotrophic agent improves splicing several-fold and macropinocytosis inhibitors block M918-mediated splicingTo confirm the involvement of endocytosis in uptake of M918, as suggested by the data for endocytosis inhibitors and co-localization studies, the splice correction assay was performed at 4 °C. As seen in Figure 6c, splicing is decreased significantly compared with cells treated at 37 °C, further emphasizing the uptake to be, at least partially, endocytic. Furthermore, when cells were pre-treated with the endocytosis inhibitors cytochalasin D and wortmannin, splicing was reduced significantly compared with cells treated with conjugate only (Figure 6c). These data clearly indicate the involvement of macropinocytosis in the uptake of M918.The major drawback with endocytosis of CPPs and other compounds is the concomitant entrapment of molecules inside endosomes.14Wadia JS Stan RV Dowdy SF Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.Nat Med. 2004; 10: 310-315Crossref PubMed Scopus (1394) Google Scholar,22Abes S Williams D Prevot P Thierry A Gait MJ Lebleu B Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates.J Control Release. 2006; 110: 595-604Crossref PubMed Scopus (129) Google Scholar On the basis of this knowledge, a splice correction experiment was conducted where M918-PNA was added with 75 μmol/l chloroquine, a lysosomotrophic agent, to HeLa pLuc 705 cells and incubated as previously described. According to Figure 6d, splicing is increased several-fold after treatment with chloroquine as compared with using M918-PNA alone. The effect of chloroquine is, however, not as pronounced when using 5 μmol/l M918-PNA, although it remains significant.DiscussionThe discovery of CPPs has opened a new avenue for delivery of therapeutic molecules both in vitro and in vivo. Most studies using CPPs have utilized the first peptides to be discovered, penetratin and Tat, where the latter peptide has been used in most peptide and protein transduction experiments.23Murriel CL Dowdy SF Influence of protein transduction domains on intracellular delivery of macromolecules.Expert Opin Drug Deliv. 2006; 3: 739-746Crossref PubMed Scopus (95) Google Scholar In 1998 antisense PNA (targeted against the galanin receptor 1) was for the first time administrated in vivo using CPPs.24Pooga M Soomets U Hällbrink M Valkna A Saar K Rezaei K et al.Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.Nat Biotechnol. 1998; 16: 857-861Crossref PubMed Scopus (541) Google Scholar In addition to penetratin, a new CPP, transportan, was utilized in that study and showed remarkable antisense effects when conjugated to PNA via a disulphide bridge. In most cargo-delivery studies using CPPs, the cargo has been covalently linked to peptides; in the case of proteins, the CPP has been expressed as a fusion protein from a plasmid vector or peptides have been linked via disulphide bonds (e.g., to ONs) or peptide bonds.10EL-Andaloussi S Holm T Langel Ü Cell-penetrating peptides: mechanisms and applications.Curr Pharm Des. 2005; 11: 3597-3611Crossref PubMed Scopus (206) Google Scholar However, delivery of ONs and proteins with CPPs has also proven possible by simply mixing peptides with cargo (e.g., the co-incubation strategy).25EL-Andaloussi S Johansson H Magnusdottir A Järver P Lundberg P Langel Ü TP10, a delivery vector for decoy oligonucleotides targeting the Myc protein.J Control Release. 2005; 110: 189-201Crossref PubMed Scopus (61) Google Scholar,26Jain M Chauhan SC Singh AP Venkatraman G Colcher D Batra SK Penetratin improves tumor retention of single-chain antibodies: a novel step toward optimization of radioimmunotherapy of solid tumors.Cancer Res. 2005; 65: 7840-7846PubMed Google Scholar,27Morris MC Chaloin L Mery J Heitz F Divita G A novel potent strategy for gene delivery using a single peptide vector as a carrier.Nucleic Acids Res. 1999; 27: 3510-3517Crossref PubMed Scopus (168) Google Scholar Although several CPPs have shown promising results in terms of transporting bioactive cargoes inside cells, there is generally a correlation between high capability to translocate bioactive agents and unwanted toxic side effects. Hence, it is of the greatest importance to develop CPPs with the ability to internalize different cargo molecules efficiently in a non-toxic fashion.Here we present a novel CPP, denoted M918. To verify the potential of the peptide as a vector, it was compared with two well-characterized CPPs, penetratin and a deletion analog of transportan, TP10.28Soomets U Lindgren M Gallet X Hällbrink M Elmquist A Balaspiri L et al.Deletion analogues of transportan.Biochim Biophys Acta. 2000; 1467: 165-176Crossref PubMed Scopus (229) Google Scholar Penetratin is a 16-amino-acid-long, highly cationic peptide carrying eight positive charges; TP10 is longer (21 amino acids) and less cationic (+5) but more amphipathic. In these terms, M918 has approximately the same length (22 amino acids) as TP10 but shares the cationic nature of penetratin, having seven positively charged amino acids (Table 1). Figure 1a shows that M918 is more readily internalized into cells than TP10 and penetratin. In another study comparing the toxicity of CPPs it was, however, observed that TP10 induces significant membrane leakage in different cell lines at 10 μmol/l concentration,29Saar K Lindgren M Hansen M Eiriksdottir E Jiang Y Rosenthal-Aizman K et al.Cell-penetrating peptides: a comparative membrane toxicity study.Anal Biochem. 2005; 345: 55-65Crossref PubMed Scopus (209) Google Scholar whereas penetratin is non-toxic up to 100 μmol/l concentrations30Jones SW Christison R Bundell K Voyce CJ Brockbank SM Newham P et al.Characterisation of cell-penetrating peptide-mediated peptide delivery.Br J Pharmacol. 2005; 145: 1093-1102Crossref PubMed Scopus (315) Google Scholar (data not shown).In that