A Novel Cysteine-Rich Cell-Penetrating Peptide for Cytosolic Delivery

Feb 14, 2011 - successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire c...
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CyLoP-1: A Novel Cysteine-Rich Cell-Penetrating Peptide for Cytosolic Delivery of Cargoes Deepti Jha,*,† Ritu Mishra,||,† Sven Gottschalk,† Karl-Heinz Wiesm€uller,‡ Kamil Ugurbil,§,† Martin E. Maier,^ and J€orn Engelmann*,† †

High-Field Magnetic Resonance Center, Max Planck Institute for Biological Cybernetics, Spemannstrasse 41, 72076 T€ubingen, Germany ‡ EMC microcollections GmbH, Sindelfinger Strasse 3, 72070 T€ubingen, Germany ^ Institut f€ur Organische Chemie, Universit€at T€ubingen, Auf der Morgenstelle 18, 72076 T€ubingen, Germany

bS Supporting Information ABSTRACT: Cell-penetrating peptides (CPPs) may have implications in biomedical sciences by improving the delivery of a wide variety of drugs through the membrane barrier. CPPs are generally taken up by endocytotic pathways, and vesicular encapsulation is a limiting factor in the area of intracellular targeting. A novel, cationic cysteine-rich CPP, CyLoP-1, has been developed exhibiting distinguished diffused cytosolic distribution along with endosomal uptake at low micromolar concentrations. Comparative uptake analysis with known CPPs showed CyLoP-1 as a promising delivery vector to access the cytosol in a variety of cell types. In addition to the positively charged residues, the presence of cysteines and tryptophans proved to be essential to maintain its functionality. Also, the oxidation status of the cysteines played an important role for the uptake efficiency of CyLoP-1, with the disulfide-containing form being more effective. The distinct feature of CyLoP-1 to enter the cytosol was further explored by the covalent attachment of cargoes of different nature and sizes. In particular, induction of caspase-3 activity (indicating apoptosis) by a CyLoP-1-SmacN7 conjugate proved successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire cytosol already at low micromolar concentrations makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes. Thus, this peptide might prove to be useful for efficient transmembrane delivery of agents directed to cytosolic targets.

’ INTRODUCTION Targeted delivery of substances into cells has long been hampered by the plasma membrane being an efficient barrier to exclude most molecules that are not actively imported by living cells.1 The discovery of cell-penetrating peptides (CPPs), also called protein transduction domains (PTDs), opened up new possibilities for the translocation of larger molecules through the plasma membrane.2-4 Widely used CPPs are the HIV-1 Tat translocation domain, the homeodomain of the Antennapedia protein from Drosophila, the chimeric peptide transportan, and many more.5-8 It has long been questioned which characteristics were necessary for a CPP to exert the translocation function. In general, little structural resemblance has been found among the different families of CPPs. So far, the only feature found consistent is a high content of basic amino acids resulting in a net positive charge.9,10 Thus, it is assumed that CPPs initially bind to negatively charged head groups of lipids or proteins in the plasma membrane11-14 followed by their internalization. Several findings in living and fixed cells have been reported suggesting multiple pathways involved in internalization, and the exact translocation mechanism is still an active area of investigation. Advances in recent years imply uptake mechanisms that are predominantly endocytotic.15-17 r 2011 American Chemical Society

Application of CPPs is an expanding field, and their use as potential transduction vectors has been established in a wide range of studies.18-20 CPPs could also play a vital role in intracellular targeting facilitating the interaction with targets, presumably located in the cytosol. However, their use as cytosolic or even nuclear targeting agents can be compromised by endosomal entrapment, which has been seen with most of the cargo conjugates of CPPs used so far.21,22 Localization in the nucleus or in the cytosol of these previously investigated peptides was observed together with endosomal trapping.23 However, high extracellular concentrations and elongated incubations (required in the presence of serum) result in increased cell death (unpublished results from our laboratory). The novel CPP described in this study was derived from the natural cationic polypeptide crotamine, which is one of the main toxins in the venom of the South American rattlesnake Crotalus durissus terrificus.24 Crotamine has been shown to be a potential CPP with targeting ability.25-27 It is a 42 amino acid long Received: January 22, 2010 Revised: January 21, 2011 Published: February 14, 2011 319

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polypeptide containing two putative nuclear localization sequence (NLS) motifs, namely, Crot(2-18) and Crot(2739).27 We selected the C-terminal NLS Crot(27-39) as starting sequence for structure-activity relationship (SAR) studies. By Fmoc/tBu solid-phase synthesis, various peptides were synthesized using systematic substitution and/or deletion of amino acid residues, as well as epimerizations. The uptake efficiency and cellular distribution of the synthetic peptides was tested in NIH3T3 mouse fibroblasts at a concentration of 2.5 μM using fluorescence spectroscopy and microscopy. SAR studies resulted in a decapeptide, named CyLoP-1 (Cytosol Localizing Peptide-1). This cysteine-rich peptide exhibited the potential for cytoplasmic distribution besides vesicular localization and proficient cellular uptake already at low micromolar concentrations. Moreover, our studies revealed a conceptual framework for understanding how cytosolic localization is influenced by alterations of the amino acid sequence. CyLoP-1 was also compared with known CPPs for uptake efficiency and distribution. Furthermore, the application of this novel peptide as a carrier was investigated by coupling with cargoes of various sizes.

from the resin by reagent K for 3 h followed by precipitation in MTBE and centrifugation. Preparation of Randomly Oxidized CyLoP-1. CyLoP-1 was oxidized as described by Spetzler and Meldal.29 In brief, peptide synthesized on resin was cleaved with 95% aq TFA for 1 h at RT. DMSO was added (TFA/DMSO, 5:1 v/v) at 0 C for 30 min then stirred at RT for 1 h followed by precipitation by DEE and purification. Excess of cleavage cocktail was added to obtain preferentially intramolecular disulfide bridges and to reduce the formation of the higher molecular weight oligomers. Due to the three cysteine groups in CyLoP-1, a mixture of different oxidation products with randomly distributed disulfide bridges was obtained (like in the slow oxidation process under physiological conditions). PNA Synthesis. An antisense 12-mer PNA was used to target mRNA of the red fluorescent dsRed protein.30 PNA peptide conjugation was achieved by continuous Fmoc synthesis (see above). The peptide was manually synthesized on rink amide MBHA resin at 0.2 mmol/g scale by continuous solid-phase peptide synthesis followed by the Fmoc-Lys(Dde)-OH as a linker for the further coupling of the fluorophore. The PNA chain was elongated by regular coupling of the respective PNA monomers, HATU, DIPEA (1:0.9:2) for 1 h, followed by acetylation at each step. Regular washing with DMF/NMP, DCM, methanol, DCM, and DMF/NMP was done to ensure the removal of the reacting reagents from the reaction vessel. FITC was attached to the linker lysine after the removal of Dde by 2% hydrazine in DMF. On the completion of the PNA peptide conjugate synthesis, the resin was thoroughly washed by DMF, DCM, and methanol, dried, and cleaved by reagent K for 3 h. Purification and Analytical Characterization. All peptides and conjugates were precipitated with MTBE. Precipitates were collected by centrifugation and resuspended in cold MTBE two times. The pellet was dissolved in water and tert-butyl alcohol (1:4) with 2% acetic acid and then lyophilized. Selected samples were purified by semipreparative RP-HPLC (Varian PrepStar Instrument, Australia) equipped with PrepStar SD-1 pump heads. UV absorbance was measured using a ProStar 335 photodiode array detector at 214 nm for peptides and 260 nm for PNA conjugates at RT using water/0.1% TFA (solvent A) and acetonitrile/0.1% TFA (solvent B) on a Varian Polaris C18-Ether column (21.2 mm diameter, length 250 mm, particle size 5 μm). A gradient from 20% B to 90% B in 25 min was applied. The product containing fractions were defined by analytical RP-HPLC on a Varian Polaris C18-Ether column (4.6 mm diameter, length 250 mm, particle size 5 μm). Mass spectrometry (ESI-MS) was performed on an Agilent 1100 series LC/MSD trap system (Agilent, Germany): Nebulizer, 20.0 psi; dry gas, 5.0 L/min; dry temperature, 250 C). The purified product was dissolved in water and tert-butyl alcohol (1:4) with 2% acetic acid and then lyophilized. Peptides containing cysteines are prone to oxidative formation of disulfide bonds, which could be formed either intramolecularly, resulting in a cyclic peptide, or intermolecularly, forming oligomers or aggregates. As most/all of our CPPs were cysteinerich peptides, air oxygen had to be avoided whenever possible during synthesis and storage of the peptides. After lyophilization, peptides were stored under nitrogen at acidic pH. Aqueous stock solutions for internalization studies were aliquoted and stored at -80 C. Dilutions for cell studies were freshly prepared for each experiment from these stock solutions. Cell Culture. The NIH-3T3 embryonic mouse fibroblast cells (DSMZ, Germany), C6 rat glioma cells (a kind gift from Prof. B. Hamprecht, University of T€ubingen, T€ubingen, Germany), PANC-1 human pancreatic carcinoma cells and HeLa human

’ EXPERIMENTAL PROCEDURES Materials. Solvents were of peptide synthesis grade. DMF, DCM, DEE, MTBE, TFA, FITC, and methanol were purchased from Acros Organics (Belgium). Protected Fmoc-amino acids as well as resin were obtained from Novabiochem (United Kingdom). Fmoc/Bhoc protected monomers for PNA synthesis were obtained from Link Biotechnologies (Scotland) and 2-(1-H-7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) from Applied Biosystems (Germany). Peptide Synthesis. Peptides were prepared by fully automated solid-phase peptide synthesis using the Fmoc/tBu-strategy and R-Fmoc-(ε-Boc)-lysine-TCP resin. The resin was distributed as 15 μmol aliquots into filter tubes, positioned in the format of a microtiter plate on valve blocks. Fmoc deprotection was carried out two times, 10 min each, with 30% piperidine in DMF (300 μL). Nine washing steps were done with DMF (300 μL). Fmoc-amino acids (0.5 M) were dissolved with HOBt (0.5 M) in DMF. Fmoc-amino acids were introduced into the reaction vessels in a two-step procedure using a 7-fold molar excess of the respective Fmoc-amino acid. After the first coupling step with Fmoc-amino acids (200 μL), DIC (3 M in DMF, 50 μL) for 1 h, the coupling reagents were filtered off and the resin was washed once with 200 μL DMF. The second coupling step of Fmoc-amino acids (100 μL) with TCTU (0.5 M in DMF, 200 μL) was done for 1 h. After washing four times with 400 μL DMF, Fmoc deprotection was carried out two times, 10 min each, with 30% piperidine in DMF (300 μL). Nine washing steps were done with DMF (300 μL). All peptides were then elongated with Boc-L-Lys(Fmoc)-OH followed by Fmoc deprotection. The ε-amino group of N-terminal lysine was manually labeled with FITC (4-fold excess) mixed with triethylamine (1:2) in DMF overnight. Completion of thiourea formation was monitored by the Kaiser Test.28 Peptides were washed with DMF, DCM, and methanol (4  each), dried, and cleaved from the resin with Reagent K (500 μL). A similar synthesis was performed for CyLoP-1 elongated by Penetratin and SmacN7 peptides. Each coupling was followed by capping to block the unreacted active sites. Fmoc-Lys(Dde)-OH was introduced as a linker between CyLoP-1 and cargo (penetratin and SmacN7) for further coupling of the fluorophore. On completion of the synthesis, the conjugate was cleaved 320

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Bioconjugate Chemistry epithelial cervical cancer cells (ATCC, USA) were grown as monolayers in Dulbecco’s Modified Eagle Medium (DMEM), with the exception of HeLa cells kept in Minimal Essential Medium (MEM), supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 μg/mL streptomycin, and 100 U/mL penicillin (all from Biochrom AG, Germany) at 37 C with 10% CO2. Confluent cultures were split using trypsin/EDTA 0.05/ 0.02% (w/v) in phosphate-buffered saline (PBS; Biochrom AG, Germany). The CCL-11 mouse fibrosarcoma cells (ATCC, USA) were cultured in an antibiotic-free mixture of NCTC 135 medium with 10% heat-inactivated donor horse serum at 37 C with 5% CO2. Subculturing was performed two times a week by trypsinization. Determination of Concentrations of Fluorescently Labeled Peptides and Cargo Conjugates. In-house synthesized peptides were dissolved in water (10 mM by weight) and pH was adjusted to 6.7 with diluted solutions of HCl and NaOH. These peptide-stock solutions were diluted 1:100 in DMEM to determine the exact peptide concentration. Absorbance of the solutions was measured with a multiplate reader (BMG Labtech, Germany) at 485 nm using ratiometric correction of turbidity at 690 nm. The concentrations of the stock solutions were then calculated assuming the molar extinction coefficient ε(fluorescein, 485 nm) = 81 000 L mol-1 cm-1. All further dilutions were done according to this calculated concentration. Cellular Uptake by Fluorescence Spectroscopy. NIH-3T3 fibroblast cells were seeded (6  103 cells/well) in 96 well microplates and grown for 24 h. Cells were then incubated with different peptides and cargo conjugates for 18 h at 37 C with 10% CO2. To estimate cell number, cellular nuclei were made visible by incubating with the nuclear stain Hoechst 33342 (Fluka, Germany) for 30 min.31,32 Afterward, incubation with cold Trypan Blue (0.05% (w/v), Sigma, Germany) in PBS was done for 3 min to quench extracellular fluorescence.33 Following repeated washes with Hanks’ buffered saline (Biochrom AG, Germany), FITC fluorescence (ex 485 nm/em 530 nm) and Hoechst 33342 fluorescence (ex 346 nm/em 460 nm) were recorded in a multiplate reader. Intracellular FITC fluorescence intensity was corrected for the cell number (division by the Hoechst fluorescence intensity). The results (multiplied by 1000) are expressed as so-called “corrected fluorescence units” (corr. f.u.). Intracellular Localization by Microscopy. The same cells (see above) were subjected to microscopy studies without fixation using a Zeiss Axiovert 200 M microscope (Zeiss, Germany) with a LD Plan NeoFluor 40 objective. Cellular localization and distribution of the peptides were observed by exciting with blue light (470/40 nm) and a filter set to 525/50 nm. Manual observations of at least three independent experiments were compiled to conclude if the peptide shows diffused, vesicular, or both types of uptake. Apart from FITC fluorescence, the nuclear labeling by Hoechst 33342 was observed at 460/50 nm and Trypan Blue fluorescence was viewed at 645/75 nm. Furthermore, phase contrast images of the same areas were taken to verify if the cells maintained their normal morphology in the presence of peptides. Determination of Free Thiols. Free thiols were determined using Ellman’s reagent (5,50 -dithiobis-(2-nitrobenzoic acid), DTNB) as described elsewhere.34 In brief, DTNB stock solution was prepared with a final concentration of 50 mM sodium acetate and 2 mM DTNB in Milli-Q water. Tris solution (1 M) was prepared and pH was adjusted to 8. Test solution was prepared by adding 160 μL of water, 20 μL of DTNB solution, 10 μL of

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Tris solution, and 10 μL of the peptides (0.5 mM) in a microtiter plate. Samples were thoroughly mixed and absorbance at 412 nm was measured after 5 min. Polyacrylamide Gel Electrophoresis (PAGE). Peptide solutions, culture medium samples with CyLoP-1, and cell lysates after incubation with CyLoP-1 were analyzed by gel electrophoresis under conditions optimized for small peptides35,36 using a vertical Mini-Protean system (Bio-Rad Laboratories, Munich, Germany) with the exception that no SDS was used in running and sample buffer and the sample buffer was prepared without βmercaptoethanol. The bands were imaged for FITC fluorescence in unfixed gels. Afterward, the gels were fixed with glutaraldehyde and the peptides/proteins in the gel were stained with silver by a procedure adapted from Blum et al.37 Determination of Caspase-3 Activity after Incubation with CyLoP-1-SmacN7. HeLa cells were seeded (5  105 cells/flask) in 25 cm2 cell culture flasks and grown for 72 h. After incubation with peptide conjugates for 18 h at 37 C with 10% CO2, cells were collected by scraping, washing with ice-cold PBS, and then lysed for 30 min at 0 C in the lysis buffer (150 mM NaCl, 1 mM dithiothreitol (DTT), 1% (v/v) Triton X-100). To inhibit protease activity, 1% (v/v) of a commercially available mix of protease inhibitors ProteaseArrest (G-Biosciences, Maryland Heights, MO, USA) was added. Protein content of the supernatant was determined by a standard Bradford protein assay. The activity of caspase-3 was analyzed by follow-up of the cleavage of 20 μM of the caspase-3 substrate Ac-DEVD-[7amino-4-methylcoumarin] (AMC) (Upstate, Lake Placid, NY) in a reaction mixture containing the samples (adjusted to 50 μg protein) in the following assay buffer: 100 mM HEPES, 200 mM NaCl, 2 mM EDTA, 20% (v/v) glycerol, 0.2% (w/v) CHAPS, 10 mM DTT, and pH adjusted to 7.4. Increase in fluorescence intensity due to cleavage of Ac-DEVD-AMC and the release of the fluorescent AMC was recorded over 60 min at 37 C with a multiplate reader (ex 346 nm/em 460 nm). The change in fluorescence intensity over time (ΔFU/s) was calculated. Cellular Uptake in Different Cell Lines. NIH-3T3 mouse fibroblast, CCL-11 mouse fibrosarcoma, C6 glioma cells, or PANC1 (human pancreatic carcinoma cells) were treated with CyLoP-1 at 2.5 μM in 96 well plates. The incubation was performed for 18 h followed by nuclear staining, Trypan Blue quenching, and HBSS washing. Fluorescence spectroscopy and microscopy was performed on the treated cells as described above. Statistical Analysis. Data were expressed as the means and standard errors of the mean (SEM) for the various statistical groups (three or more experiments with six replicates each). The two mean values of interest were tested for statistically significant differences by unpaired Student’s t test using GraphPad Prism v 5.02 for Windows (GraphPad Software, USA). When comparing more than two groups or means, a one-way analysis of variance (ANOVA) with Bonferroni’s post tests for multiple comparisons was performed.

’ RESULTS AND DISCUSSION Structure-Activity Relationship Studies. The sequence of the tridecapeptide Crot(27-39) (peptide 1, KMDCRWRWKCCKK) included three cysteine residues, tryptophans, aspartic acid, methionine along with six basic amino acids (arginine and lysine). The positively charged amino acids were unaltered because of their essential role in membrane adherence.38 Cysteine residues in peptide sequences are known to form intra- and intermolecular 321

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Table 1. Sequences of 38 Synthesized Peptides, the Calculated and Observed Molecular Mass (MþH)þ of the Conjugates (KFITC or k-FITC at N-terminus), and Intracellular Fluorescence Intensity (corr. f.u.) Values in NIH-3T3 Cells after a Labeling Period of 18 h at 2.5 μMa intracellular fluorescence intensity [corr. f.u.]

molecular weight

a

peptide

sequence

expected (MþH)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

KMDCRWRWKCCKK Crot (27-39) MDCRWRWKCCKK DCRWRWKCCKK CRWRWKCCKK (CyLoP-1) RWRWKCCKK KMDCRWRWKCKK KMDCRWRWKKK KMDRWRWKKK KDCRWRWKCCKK KCRWRWKCCKK KRWRWKCCKK MDCRWRWKXCKK DCRWRWKXCKK DCRWRWKCXKK CRWRWKXCKK CRWRWKCXKK RWRWKXCKK MDCRWRWKXXKK DCRWRWKXXKK CRWRWKXXKK RWRWKXXKK CRWRWKCSKK SRWRWKCCKK SRWRWKCSKK SRWRWKSCKK CRWRWKSSKK SRWRWKSSKK CRFRWKCCKK CRWRFKCCKK CRFRFKCCKK crwrwkcckk KCCKWRWRCK kcckwrwrck CrWRWKCCKK CRwRWKCCKK CRWrWKCCKK CRWRwKCCKK CrwrwKCCKK

2288.7 2160.5 2029.3 1914.3 1809.1 2185.6 2082.4 1979.3 2157.5 2042.4 1939.3 2142.5 2011.3 2011.3 1896.2 1896.2 1793.1 2124.5 1993.3 1878.2 1775.0 1898.2 1898.2 1882.1 1882.1 1882.1 1866.1 1875.2 1875.2 1836.2 1914.3 1914.3 1914.3 1914.3 1914.3 1914.3 1914.3 1914.3

þ

þ

observed (MþH)

mean

SEM

% of CyLoP-1

2288.6 2162.0 2029.9 1914.9 1811.3 2186.4 2084.7 1979.6 2159.2 2043.0 1940.8 2142.0 2013.0 2011.6 1897.0 1896.6 1793.1 2124.8 1993.8 1880.0 1776.2 1899.0 1898.6 1881.9 1882.4 1882.0 1866.0 1875.0 1875.8 1836.8 1914.8 1914.9 1914.9 1914.1 1913.9 1915.0 1914.0 1915.0

3176 3344 1909 4050 1511 1516 1202 100 1043 1583 1226 1937 1741 943 2044 1347 1211 1256 872 1390 405 2172 1867 572 475 415 193 2656 2476 2569 2109 1713 1449 1529 1229 1304 1412 1488

271 374 272 322 256 181 218 50 137 246 120 335 427 313 408 171 529 187 554 199 109 597 367 94 77 109 89 320 382 197 503 148 295 163 40 345 456 360

78 83 47 100 37 79 30 2 26 39 30 48 43 23 50 33 30 31 22 34 10 54 46 14 12 10 5 66 61 63 52 42 36 38 30 32 35 37

X, R-amino-n-butyric acid. D-Amino acids are represented in lower case. In some cases, the molecular ion was calculated from the divalent ion.

bridges and hence promote oligomerization.39 Cysteine, methionine, and tryptophan may cause side reactions such as racemization, oxidation, and alkylation.40-43 Therefore, our studies were focused on the functional significance of these particular residues in order to achieve a sequence optimized for cell penetrating properties, as well as for easy production and long shelf life. The peptides were synthesized on preloaded TCP resin using N-Fmoc protection. To ensure minimal racemization, Fmoc-amino acids were coupled by DIC/HOBt in DMF with 5 min preactivation.44 Peptides were elongated with Boc-L-Lys(Fmoc)-OH for the coupling of FITC at the ε-amino group of this lysine. All peptides were produced in parallel on an automated peptide synthesizer and analyzed by HPLC and ESI-MS. The peptides were tested for their uptake efficiency in the crude form. Deletions. In order to determine the optimal length for cell penetrating properties, deletion of amino acid residues of peptide 1 was done one by one from the N-terminus [peptides 2-5] and randomly [peptides 6-11]. As expected, differences in the uptake behavior and cytosolic distribution were observed with the removal of amino acids one by one from the N-terminus or randomly in the sequence (Table 1, for a complete list of

synthesized peptides see also Table S1 in the Supporting Information). CyLoP-1 [CRWRWKCCKK, peptide 4] showed highest uptake efficiency and the most homogeneous distribution in the cytosol. Interestingly, this shorter peptide 4 showed an approximately 20% more efficient uptake compared to peptide 1 (Table 1, Figure 1A) and exhibited uniformly distributed cytoplasmic fluorescence along with endosomal, vesicular fluorescence (Figure 1B). Therefore further mutations were carried out in order to optimize the amino acid composition and stereochemistry in CyLoP-1. Effect of Substitution of Cysteine Residues in CyLoP-1. The significance of cysteines for internalization in peptide 1 (KMDCRWRWKCCKK) and CyLoP-1 (peptide 4, CRWRWKCCKK) was determined by substituting cysteine residues with R-amino-n-butyric acid (Abu, X) or serine. Serine and Abu are isosteric analogues of cysteine; therefore, they are often used for cysteine replacement in structure-activity relationship studies.45,46 Substitution of one to three cysteine(s) by serine(s) in CyLoP-1 [peptides 22-27] revealed that the replacement of cysteines correlated with a loss in uptake efficiency. Total exchange led to a nearly complete loss of uptake and cytosolic diffusion (Figure 2A). Similar results were 322

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Figure 1. Internalization efficiency of peptide 1 and CyLoP-1 (A) and intracellular distribution of K(FITC)-coupled CyLoP-1 (B) in NIH3T3 cells. Cells were incubated with the fluorescently labeled peptides at 2.5 μM for 18 h. Corr. f.u. - corrected fluorescence units; data are represented as mean ( SEM. K(FITC) was coupled to peptides at the N-terminus. The bright punctate and encapsulated FITC fluorescence was categorized as vesicular uptake, while fluorescence distributed in the entire cell with similar intensity was designated as diffused. The bar represents 20 μm.

Figure 2. Influence of cysteine replacement on internalization of CyLoP-1 analogues (A) and influence of amino acid enantiomers and of the L-inverse and D-inverse peptides on the internalization efficiency (B). NIH-3T3 cells were incubated with 2.5 μM of fluorescently labeled peptides for 18 h. Corr. f.u., corrected fluorescence units; data are represented as mean ( SEM; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 significantly different compared to CyLoP-1 (ANOVA, Bonferroni’s Multiple Comparison Test). K(FITC) or k(FITC) was coupled to peptides at the N-terminus. D-amino acids are represented in lower case.

obtained for Abu-substituted peptides (Table 1). Furthermore, deleting single cysteine residues one by one [peptides 6-8] led to a decrease of uptake as well (Table 1). Substitutions with serine or Abu reduced the cellular uptake and cytosolic distribution. These findings were in concordance with the results obtained by deletion of cysteines. Thus, the number and location of all cysteine residues in the peptide play an important role in maintaining the carrier function. Oxidation Status of CyLoP-1 under Physiological Conditions. Peptides containing cysteines are prone to oxidative formation of disulfide bonds, which could be formed either intramolecularly, resulting in a cyclic peptide, or intermolecularly, forming oligomers or higher aggregates. Indeed, this could only be avoided if the peptide is stored lyophilized and at low pH. When the peptide was dissolved and pH was adjusted to physiological conditions, slow oxidation took place resulting in the formation of intra- as well as intermolecular disulfide bridges (as can be shown by mass spectrometry and gel electrophoresis, see Supporting Information, Figure S1, S2). The disulfide nature of the oxidation products could be proven by the disappearance of the band for the dimeric form after reduction of the sample with β-mercaptoethanol prior to PAGE (Figure S2 C). By using controlled chemical oxidation to form selectively monomeric CyLoP-1, as well as CyLoP-1 containing predominantly intermolecular disulfides, it could be shown that under physiological conditions a complex mixture of mainly monomers and dimers was formed (Figure S3). In addition, small amounts of higher oligomers could be detected. The evaluation of free cysteine groups in freshly prepared samples of the reduced peptide and the chemically oxidized form by the Ellman’s assay34 resulted in the expected ratio of 3:1 (Figure S4). Effect of Oxidation Status of CyLoP-1. Interestingly, there are several indications that the oxidized form is taken up better by cells and was distributed homogenously throughout the cells

compared to the reduced form. Fluorescence microscopy and spectroscopy measurements displayed a significantly higher uptake of the chemically oxidized forms of CyLoP-1 (data not shown). The analysis of incubation medium after contact with cells for 18 h by gel electrophoresis showed the preferential disappearance of the oxidized dimeric form of CyLoP-1 compared to incubation in the absence of cells (Figure S3). These experiments also proved that the complete peptide could be detected in serum-containing medium after the relative long incubation time of 18 h in the absence and presence of cells. However, a time-dependent increase of the degradation product Lys-FITC could be observed, which was more prominent in the presence of cells (Figure S3 A,B). This might indicate an uptake mechanism in which cell-surface thiols (extrafacial protein thiols, EPTs) are involved. Recent studies reported on the direct interaction of thiol/disulfide containing peptides and imaging probes with these EPTs, hence modifying and in some cases enhancing the uptake into cells.47,48 Interestingly, the imaging probes were internalized into the entire cytosol and complete endosomal entrapment was avoided48 like it was also observed for CyLoP-1. The proposed mechanism is the internalization of the EPT-bound compound together with the corresponding (yet unknown) protein and subsequent cleavage of the disulfide bond under the reductive conditions in the cytosol. It can be therefore hypothesized that CyLoP-1 is taken up at least partially by such a mechanism. As discussed by Aubry et al.,47 this might require the presence of an oxidized form 323

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of CyLoP-1 with enhanced reactivity toward protein thiol groups. The oxidizing extracellular conditions as well as the long incubation time facilitate the generation of such intra- or intermolecular species but also of mixed disulfides with components of the medium or serum, as can be seen by gel electrophoresis (Figure S2, S3). A further evidence for the importance of disulfide bridges for the internalization process is the preferential removal of the oxidized forms of CyLoP-1 from the culture medium in the presence of cells indicating their faster and more effective cellular uptake (either directly or via EPTs) compared to the reduced peptide. Gel electrophoresis of cell lysates and cytosolic extracts after incubation of cells for 18 h displayed still intact fluorescently labeled monomeric peptide aside of an significantly increased amount of the degradation product LysFITC (data not shown). Thus, the cleavage of the Lys residue used to link cargo molecules was enhanced inside the cells. Nevertheless, intact peptide was also present in the cytosol even after the long-term incubation indicating that the observed diffuse cytosolic fluorescence is in part due to the intact CyLoP-1. Because of the reducing conditions in the cytosol, most probably only reduced CyLoP-1 could be detected. However, the fluorescence spectroscopy as well as microscopy data presented here, display only the intracellular internalized contribution of CyLoP-1 and not some extrafacial membrane-bound peptide because of the quenching of the extracellular fluorescence with Trypan Blue. In an initial experiment in which we treated the cells after 18 h labeling with CyLoP-1 with the reducing agent tris-(2-carboxyethyl) phosphine (TCEP, not taken up by cells) in addition to the quenching, we could not observe any further decrease of our fluorescence intensities or changes in the microscopy images (data not shown). Effect of Tryptophan Substitution on Internalization. The presence of tryptophans in CPPs has been demonstrated to participate in membrane permeation. Tryptophans are reported to destabilize the membrane once the peptide is adhered to the surface.49 To examine these effects in CyLoP-1, replacement of either one or both tryptophans by phenylalanine49 was investigated [peptides 28-30]. A decreased uptake (Table 1) and almost complete disappearance of cytosolic diffused fluorescence was observed. Our results suggest that the number of tryptophans in CyLoP-1 is of high significance, because a lower cytosolic distribution and decrease of uptake is observed after substitution of tryptophan. The high intracellular delivery and distribution by peptides 1 and 4 might be the combined effect of both charge and hydrophobicity. Effect of Stereochemical Modifications on Uptake. The CPP-based delivery approach is potentially hampered by the instability of the vector to endogenous peptidases.20 A possible solution to overcome this hurdle could either be the use of the Dform instead of the naturally occurring L-amino acids, the use of β-peptides50 or peptoids20 to enhance vector stability. In this concern, we studied further analogues of CyLoP-1 to evaluate the influence of the stereochemistry of the peptide backbone (enantiomeric purity of CyLoP-1 was about 95% as shown by GC-MS analysis on chiral phase), as well as the sequence alignment. Incorporation of D-amino acids and reversal of the sequence (inverse isomers) of the peptide has been shown to increase the transmembrane delivery of the Tat peptide.20 Therefore, the D-isomer [peptide 31] and L-inverse as well as D-inverse isomers of CyLoP-1 [peptide 32, peptide 33] were synthesized. Unexpectedly, these analogues showed reduced uptake by about 50% (Figure 2B). In addition, the Arg-Trp-

Figure 3. Concentration-dependent cellular internalization of CyLoP-1 into NIH-3T3 cells measured by fluorescence spectroscopy. Cells were incubated with fluorescently labeled CyLoP-1 at increasing concentrations in complete medium for 18 h. Data are represented as mean ( SEM.

Arg-Trp domain of the peptide sequence seemed to be a key feature in this CPP paradigm, as indicated by the results of the initial screening [peptides 54-60] (Table S1, Supporting Information). Thus, the effects of configurational changes were examined with restriction to this region [peptide 34-38] in CyLoP-1. As a result, the internalization was reduced to about 40% accompanied with a decrease in cytoplasmic diffusion as compared to CyLoP-1 (Figure 2B). These results demonstrated that stereochemistry has a clear effect on cellular uptake of CyLoP-1. Unlike most of the known CPPs, CyLoP-1 proved to be most efficient in its natural L-amino acid form. Concentration Dependence and Toxicity Studies. The effect of concentration on cellular uptake was evaluated by applying various concentrations of CyLoP-1 peptide on NIH3T3 cells (Figure 3). A concentration-dependent increase in cellular delivery was observed up to 5 μM of treatment concentration. At higher treatment concentrations (i.e., 9 μM), a precipitate of the peptide was observed sticking on the cellular membrane (data not shown). The precipitated peptide also slightly reduced the number of viable cells at concentrations above 5 μM mainly due to the large local excess of CyLoP-1. No cytotoxic effect of the peptide was detected by Hoechst 33342 nuclear DNA staining31,32 and Trypan Blue staining at lower concentrations. Also, the cellular morphology was comparable to the control cells, in support of the previous observation. Comparison with Well-Known CPPs. The carrier ability of CyLoP-1 was compared to other well-characterized CPPs, namely, Tat, penetratin, and octaarginines. Although a vast list of CPPs has come up over the last two decades, the selection of these CPPs was made mainly due to their amino acid composition. D-Tat49-57 (rkkrrqrrr) and D-octaarginine (rrrrrrrr) on one hand are highly cationic, whereas penetratin (RQIKIWFQNRRMKWKK) and CyLoP-1 (CRWRWKCCKK), apart from being positively charged, are also rich in hydrophobic residues. In comparison to CyLoP-1, lower cellular internalization was observed for these wellstudied CPPs at 2.5 μM during long-term incubation (Figure 4). In addition, all the CPPs other than CyLoP-1 displayed predominantly vesicular distribution with almost no cytosolic fluorescence. No change of the cellular distribution was observed for shorter incubation periods (data not shown). It has been reported that at concentrations above 10 μM cytosolic distribution was seen for these peptides after short-term incubations.23 On the other hand, CyLoP-1 was proficiently taken up by cells even at a labeling concentration of 2.5 μM particularly showing cytosolic distribution along with vesicular uptake. The CPPs prepared from D-amino acids 324

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Figure 4. Comparison of internalization efficiency of CyLoP-1, Antp, D-Tat 49-57, D-Tat 57-49, and D-R8. NIH-3T3 cells were incubated with 2.5 μM of the fluorescently labeled peptides for 18 h. Data are represented as mean ( SEM; ***p < 0.001 significantly different compared to CyLoP-1 (ANOVA, Bonferroni’s Multiple Comparison Test). K(FITC) or k(FITC) was coupled to peptides at the N-terminus. D-Amino acids are represented in lower case.

Figure 5. Fluorescence microscopy images displaying intracellular distribution of CyLoP-1 in CCL-11 mouse fibrosarcoma (A), C6 rat glioma (B), and PANC-1 human pancreatic carcinoma (C) cells. Cells were incubated with fluorescently labeled CyLoP-1 at 2.5 μM labeling concentration for 18 h. The bar represents 20 μm.

peptide after 2.5 μM of CyLoP-1 for 18 h incubation (Figure 5). Nevertheless, peptide-filled fluorescent vesicles were detected in the perinuclear region as well. Similar to NIH-3T3, the intensity and distribution of fluorescence was not equal over the whole cell layer. Conversely, the CCL-11 fibrosarcoma cells distinctly displayed vesicular distribution of CyLoP-1. There was almost no detectable cytosolic localization. After application of higher concentrations, a faint augmentation in cytosolic fluorescence was observed, but most of the peptide was entrapped in vesicles. Thus, the uptake of CyLoP-1 is strongly dependent on cell type, and the cytosolic distribution could vary on the basis of cells being labeled. Uptake Efficiency of Various Cargoes Attached to CyLoP-1. Delivery of a wide range of biomolecules like antisense oligonucleotides, peptide nucleic acids (PNA), or peptides/proteins across the plasma membrane had been a major challenge before the advent of CPPs. Covalent or noncovalent strategies have been employed for CPP-cargo conjugation.52 The delivery efficiency and mechanism may differ on attachment of the cargo to the CPP. In order to address the cargo dependence on the uptake and distribution, CyLoP-1 was covalently conjugated to a panel of cargoes of various molecular sizes at the N-terminus (Table 2). Payload consisted of the fluorophore FITC, a Magnetic Resonance Imaging (MRI) agent (Gd)-DOTA, the bioactive peptide SmacN7, a larger peptide (Penetratin), and a 12-mer PNA molecule. These cargoes are different not only in their sizes, but also in their charge,

demonstrated increased uptake when compared with peptides composed of amino acids in their native L-form.20 However, CyLoP-1, composed of L-amino acids, was the most efficient CPP for long-term labeling. Radis-Baptista and co-workers recently reported other structural simplified sequences by splicing the C- and N-terminal amino acid residues 1-9 and 38-42 of crotamine.51 These sequences contained only one cysteine and retained the ability for nucleolar localization like the original crotamine. About 50% of all cells in culture could be labeled by the treatment with 15 μM of peptide. The proposed uptake mechanism is receptormediated endocytosis, since the all-D-enantiomer was poorly internalized. Whereas the latter behavior is similar to CyLoP-1, its cellular distribution remains quite different, also in comparison to crotamine. Only diffuse fluorescence in the nuclear region and no nucleolar accumulation could be observed for CyLoP-1 unlike for the shorter peptide of Radis-Baptista et al. or whole crotamine in proliferating cells.5,27,51 In addition, all cells in culture were labeled albeit with varying intensity. Effect of Cell Type on Cellular Uptake. In order to address the effect of cell type on uptake efficiency and intracellular distribution, CyLoP-1 was tested on various cell lines obtained from different tissues and species. Similar to NIH-3T3 (mouse fibroblast), PANC-1 (human pancreatic carcinoma cells) and C6 (rat glioma cells) displayed mainly a cytosolic localization of the 325

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Table 2. List of Various Cargoes That Were Attached to CyLoP-1 molecular weight cargo

expected (MþH)þ

conjugate

observed (MþH)þ

FITC

Optical reporter-CyLoP-1

1914.3

1914.9

Gd-DOTA

MR reporter-CyLoP-1

2454.9

2456.5

SmacN7

AVPIAQK K(FITC)-CyLoP-1

2622.1

2623.6

Penetratin (Antp)

Ac-RQIKIWFQNRRMKWKK(FITC)-CyLoP-1

4056.9

2029.9a

PNA

Ac-tccgtgaacggc-K(FITC)-CyLoP-1

5208.4

a

2605.1b 2þ

The detected ions for Penetratin (Antp) and CyLoP-1 conjugate with a lysine linker and FITC were (Mþ2H) = 2029.9, (Mþ3H)3þ= 1353.0, (Mþ4H)4þ = 1015.3, (Mþ5H)5þ = 812.3, (Mþ6H) 6þ = 677.2. They were consistent with the calculated mass of the desired product. b The detected molecular ions for PNA and CyLoP-1conjugate with a lysine linker and FITC were (Mþ2H)2þ= 2605.1, (Mþ3H)3þ= 1738.8, (Mþ4H)4þ = 1304.1, (Mþ5H)5þ = 1044.6. They were consistent with the calculated mass of the desired product.

Figure 6. Comparison of internalization efficiency of CyLoP-1 cargo conjugates in NIH-3T3 cells. Cells were incubated with 2.5 μM of fluorescently labeled CyLoP-1 cargo conjugates for 18 h. CyLoP-1 (CRWRWKCCKK, MW 1914) with additional K(FITC) at the N-terminus, Gd-DOTAK(FITC)-CyLoP-1, MW 2454, SmacN7-K(FITC)-CyLoP-1 (AVPIAQK-K(FITC)-CyLoP-1, MW 2622), Antp-CyLoP-1 (CH3CO-RQIKIWFQNRRMKWK-K-(FITC)-CyLoP-1, MW 4056), PNA-CyLoP-1 (CH3CO-tccgtgaacggc-K(FITC)-CyLoP-1, MW 5208). Data are represented as mean ( SEM; **p < 0.01, ***p < 0.001 significantly different compared to CyLoP-1 (ANOVA, Bonferroni’s Multiple Comparison Test).

hydrophobicity, or function and are otherwise poorly taken up through the plasma membrane. Successful delivery of cargoes of various sizes attached to CyLoP-1 was assessed after incubation of NIH-3T3 cells with these peptide conjugates at the concentration of 2.5 μM for 18 h. With the increase in molecular size, an overall decrease in the intracellular uptake was observed (Figure 6). More vesicular uptake and decreasing cytosolic distribution were detected, thus promoting endocytosis for high molecular weight cargoes. An exception was the conjugate with SmacN7, which showed only a similar uptake compared to the larger penetratin-CyLoP-1 construct indicating that the nature of the cargo is also important for the internalization. The changes in the uptake and localization pattern concomitant with the increase in the cargo size may also be ascribed to the increase of rigidity caused by the cargo on the CPP construct. There might be different mechanisms responsible for the diffused and the vesicular uptake, the predominance of which depends on the nature of the cargo coupled to CyLoP-1. Cytosolic Delivery of SmacN7-CyLoP-1: Caspase-3 Activation. To verify the capability of CyLoP-1 to transport a biologically active cargo across cellular membranes, we attached the pro-apoptotic peptide sequence AVPIAQK (SmacN7) to CyLoP-1. SmacN7 is derived from the N-terminus of the second mitochondria-derived activator of caspase (Smac) protein which is released from mitochondria in response to apoptotic stimuli and promotes caspase-3 activation in the cytosol.53

The short sequence SmacN7 has been shown to be sufficient to maintain the original function of Smac, which mainly results in increased activity of the executioner caspase-3.54 However, SmacN7 alone is not capable of passing through cellular membranes, and it needs to bind to its cytosolic target protein to exert its pro-apoptotic action.53,54 By measuring the level of active caspase-3 in treated cells, SmacN7 can thus be used to prove the cytosolic delivery and maintenance of functionality of CPPSmacN7 conjugates.23 Treatment of HeLa cells with K(FITC)-CyLoP-1, SmacN7K(FITC), SmacN7-K(FITC)-CyLoP-1, or the corresponding derivative with the Tat-peptide, SmacN7-K(FITC)-Tat, resulted in a statistically significant induction of caspase-3 activity only for the CyLoP-1 derivative (136 ( 10% of control, p < 0.001). CyLoP-1 or Smac-N7 alone as well as the Tat derivative failed to increase the caspase-3 activity (Table S2, Supporting Information). Whereas for SmacN7 no uptake into cells could be observed, the Tat conjugate showed exclusively endosomal uptake under these conditions (data not shown). These results clearly indicate the superior ability of CyLoP-1 to deliver bioactive cargos at low micromolar concentrations to the entire cytosol. Interestingly, the chemically oxidized form of the CyLoP-1-SmacN7 conjugate was also more effective in inducing caspase-3 activity than the corresponding mainly reduced form (data not shown) further indicating the preferential internalization of the disulfide-containing form of CyLoP-1. 326

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Bioconjugate Chemistry In conclusion, a new potent peptidic delivery agent CyLoP-1 was designed and synthesized. Any alteration introduced in the peptide sequence had a direct effect on the intracellular localization profiles, indicating the role played by the chemical properties of the different amino acid side chains. The distribution in the entire cytosol (along with the expected vesicular localization) at concentrations as low as 2.5 μM is a unique feature of CyLoP-1. This is in contrast to the usual long endosomolytic peptide sequences like EB1 or the original crotamine, or fusogenic peptides like HA2 from influenza virus.22,26,55 Increased cellular uptake and cytosolic gain was observed for CyLoP-1 but not for the other well-known CPPs at concentrations of 2.5 μM. Intracellular uptake and distribution of CyLoP-1 was concentration and cell-type dependent. In addition, a cargo dependency was observed when CyLoP-1 was conjugated to cargoes of different sizes and nature. Thus, efficient intracellular delivery and the distribution into the entire cytosol make it an appropriate candidate for cytosolic delivery of cargoes of small sizes. This might expand the scope of targeted delivery approaches but is surely dependent on the nature and size of the attached payload. Though the actual internalization mechanism of peptide and peptide-cargo constructs is still to be unveiled, continued rational design and combinatorial approaches will undoubtedly result in further improvements of peptide CyLoP-1 in the near future.

ARTICLE

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’ ASSOCIATED CONTENT

bS

Supporting Information. Table containing the results of all 60 peptides synthesized for the Structure-Activity Relationship Studies, ESI-MS spectra of reduced and oxidized CyLoP-1, results of the analyses by gel electrophoresis, of the Caspase-3 activation by SmacN7 derivatives, and of the Ellman’s assay. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Corresponding authors. E-mail [email protected], Tel. (þ 49) 7071-601-717. E-mail joern.engelmann@tuebingen. mpg.de, Tel. (þ 49) 7071-601-704, Fax: (þ 49) 7071-601-702. )

Present Addresses

Department of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, England. § Center for Magnetic Resonance Research, Department of Radiology, University of Minnesota, 2021 6th St. SE, Minneapolis, MN 55455, USA.

’ ACKNOWLEDGMENT The authors would like to thank Hildegard Schulz for technical assistance. The authors also thank Dr. Tetsuo Yamane and Dr. Irina Kerkis for initiating our interest in crotamine and sharing their knowledge and experience on this peptide, C.A.T. GmbH & Co, Chromatographie und Analysentechnik KG, T€ubingen, Germany, for the GC-MS analysis. This work was supported by the German Ministry for Education and Research (BMBF), FKZ 01EZ0813, and the Max Planck Society. It was performed in the frame of COST D38 action. 327

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dx.doi.org/10.1021/bc100045s |Bioconjugate Chem. 2011, 22, 319–328