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Bioconjugate Chem. 2010, 21, 1850–1854
Noncationic Dipeptide Mimic Oligomers As Cell Penetrating Nonpeptides (CPNP) Lubomir L. Vezenkov,†,⊥ Marie Maynadier,‡,⊥ Jean-Franc¸ois Hernandez,† Marie-Christine Averlant-Petit,§ Olivier Fabre,§ Ettore Benedetti,| Marcel Garcia,‡ Jean Martinez,*,† and Muriel Amblard*,† Institut des Biomole´cules Max Mousseron, UMR5247 CNRS, Universite´s Montpellier 1 et 2, 15 avenue Charles Flahault, 34000 Montpellier, France, IRCM, Institut de Recherche en Cance´rologie de Montpellier, INSERM U896, Universite´ Montpellier 1, CRLC Val d’Aurelle Paul Lamarque, Montpellier, F 34298, France, UMR7568 Laboratoire de Chimie-Physique Macromole´culaire (LCPM), 1 Rue Grandville, BP 20451 54001 Nancy Cedex, France, and Istituto di Biostrutture e Bioimmagini CNR and Dipartimento di Chimica Biologica, Universita` degli Studi di Napoli Federico II, Napoli, 80134, Italy. Received April 28, 2010; Revised Manuscript Received July 12, 2010
Small oligomers of constrained dipeptide mimics have been synthesized as new vectors for intracellular delivery. They are highly internalized by cells and delivered to the lysosomes by an energy-dependent pathway. This new class of vectors referred to as cell penetrating nonpeptides (CPNP) possess the distinctive feature of being noncationic.
INTRODUCTION The delivery of drugs into cells remains a major limitation in several therapies. Peptide vectors referred to as cellpenetrating peptides (CPPs) have emerged as promising tools for the intracellular delivery of bioactive cargoes (drugs, peptides, siRNA...) (1-3). Several classes of CPPs have been reported; most of them are basic or amphipathic peptides (4-8) and often display high R-helical propensity (9). Hydrophobic segments from protein signal sequences or membrane translocating sequences (MTS), as well as a noncharged polyproline peptide, were also described for cell membrane transport (10, 11). Non-natural folded oligomers termed foldamers (12, 13) have emerged as an important family of versatile frameworks with the first evidence of their ability to cross the cell membrane. A large number of guanidinium rich foldamers including β-peptides (14) and peptoids (5) have been reported to deliver drugs or labeled compounds into cells. Other oligomers constructed from monomers that differ significantly from R- or β-amino acids, possessing predictable helical structure and amphipathic character were also found to efficiently enter into cells (15-17). Thanks to the possibility to control their secondary structure and their resistance against protease degradation (18), structured oligomers constitute a privileged platform for designing cell penetrating nonpeptide compounds (CPNP). Alternatively, it has been shown that sterol derivatives could enhance molecular uptake (19, 20). Taking into account these considerations, our aim was to develop a novel class of small molecules as potential vectors to mediate cell penetration. They are based on constrained dipeptide mimic oligomers. A few years ago, we showed using molecular modeling that oligomerization of constrained β-turn dipeptide mimics could induce helix-like structures (21). Among * To whom correspondence should be addressed. E-mail: jean.martinez@ univ-montp1.fr (J.M.);
[email protected] (M.A.). † UMR5247 CNRS, Universite´s Montpellier. ‡ IRCM, Universite´ Montpellier. § UMR7568 Laboratoire de Chimie-Physique Macromole´culaire (LCPM). | Universita` degli Studi di Napoli Federico II. ⊥ These authors contributed equally to this work.
the different motifs selected by this program, we particularly focused on (3-S)[amino]-5-(carboxymethyl)-2,3-dihydro-1,5benzothiazepin-4(5H)-one (DBT) described as a β-turn mimic (22, 23), supposedly a favorable feature to induce structured oligomers. DBT oligomers are neither polycationic nor amphipathic oligomers unlike the majority of described vectors. By virtue of their hydrophobicity, their expected ability to achieve helical type structure and resistance against protease degradation, we hypothesized that DBT oligomers should be good candidates as translocating agents. Short oligomers were synthesized and their cellular uptake and subcellular localization studied.
EXPERIMENTAL PROCEDURES Oligomers. They were synthesized on Rink amide resin following solid phase peptide synthesis methodology and labeled at their N-terminus with fluorescein isothiocyanate (see Supporting Information). The monomeric units Fmoc-DBT-OH and Fmoc-LBT-OH were prepared as previously described (24). Cell Preparation and Treatment with Oligomers. We used 1 mM stock solutions of compounds dissolved in DMSO that were further diluted with serum-free medium to obtain a final concentration of 10-5 M. All compounds were protected from light during the experiments, and their fluorescence values were regularly verified. Human breast cancer MDA-MB-231 cells were maintained in monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50 µg/mL gentamycin at 37 °C in 5% CO2. One day prior to the experiment, cells were harvested using trypsin in phosphate-buffered saline (PBS), centrifuged, and resuspended with culture medium. The suspension containing 106 cells/mL was transferred into plastic 24-well plates (Becton Dickinson, Le Pont De Claix, France) (200 µL/well) and incubated overnight until they reached >90% confluency. On the day of the experiment, the culture medium was removed, and the cells were washed once with phenol red-free DMEM. Then, 200 µL of DMEM containing 10% FBS and 10-5 M of compounds was dispersed on cells. Experiments were carried out in triplicate for each compound. To determine the amount of compounds assimilated by cells, including membrane-bound fractions, cells were washed twice with PBS. To determine the amount of internalized compounds, cells were washed with PBS
10.1021/bc1002086 2010 American Chemical Society Published on Web 09/03/2010
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Scheme 1. Synthesis of Fluorescein Labeled Oligo-(D- and L-BT)a
a
The scheme is given for the D series.
and then treated for 5 min with 30 µL of a solution containing 0.5 mg/mL trypsin. After trypsinization, cells were resuspended in phenol red-free DMEM and centrifuged for 5 min at 800g. Adherent cells or cell pellets were lysed in 200 µL of cell culture lysis reagent (Promega, Charbonnie`res, France). After 30 min of incubation at room temperature, 200 µL of distilled water was added before analysis on a Gemini XS spectrofluorimeter (Molecular Devices, CA, USA), with the excitation wavelength adjusted to 485 nm and emission measured at 530 nm. All fluorescence values were normalized according to the fluorescence intensity of each compound at 10-5 M in lysis buffer in the presence of 2 × 105 cells. Kinetics of Internalization. One day before the experiment, 2 × 106 MDA-MB-231 cells were seeded on plastic 24-well plates. The cells were then washed with DMEM and incubated for increasing times from 1 to 24 h with 10-5 M oligomer at 37 °C. At the indicated time, cells were treated by trypsin and intracellular fluorescence measured as described above. Visualization of Fluorescent Oligomers by CLSM. The day prior to the experiment, MDA-MB-231 cells were seeded onto bottom glass dishes (World Precision Instrument, Stevenage, UK) at a density of 106 cells per square centimeter. On the day of the experiment, cells were washed once and incubated in 1 mL phenol red-free medium containing fluorescent labeled compounds at a concentration of 10-5 M for 3, 16, and 24 h. Thirty minutes before the end of incubation, cells were loaded with Hoechst 33342 (Invitrogen, Cergy Pontoise, France) for nuclear staining at a final concentration of 5 µg/mL. For membrane labeling, a Vybrant lipid-raft labeling kit (Invitrogen) was used as described by the manufacturer. For endosome labeling, 20 min before the end of the experiment, 35 mg/mL of transferrin (Invitrogen) was added to the culture medium. For lysosome labeling, 3 h before the end of the experiment, 50 nM of lysotracker red DND-99 (Invitrogen) was added to phenol redfree DMEM. Before visualization, cells were washed gently with phenol red-free DMEM. Cells were then scanned with a LSM 5
LIVE confocal laser scanning microscope (Carl Zeiss, Le Pecq, France), with a slice depth (Z stack) of 0.67 µm.
RESULTS AND DISCUSSION Design of the Target DBT Oligomers. Oligomers of different lengths were synthesized (see Supporting Information) on a solid support using Rink amide PS resin, by successive addition of N-Fmoc protected DBT (Fmoc-DBT-OH) (24) in the presence of HBTU as the coupling reagent (Scheme 1). In order to track their cellular internalization by fluorescence microscopy, fluorescein isothiocyanate was used to label the oligomers. The fluorescent tag was attached directly to the N-terminus of the poly(DBT) anchored to the resin (JMV2949, JMV2968 and JMV3229). The importance of the configuration and the structure of the oligomers was assessed by the preparation of the LBT derivative (JMV4228) and the D/LBT alternated oligomer (JMV4287), respectively. Cellular Uptake of DBT Oligomers. At first, the cellular uptake in MDA-MB-231 breast cancer cells of the oligomers 1-5 was analyzed by fluorescence emission measurement (Figure 1) and compared to fluorescein labeled octa-arginine (25) (Arg8) as a positive control and carboxyfluorescein (CF) as a negative control. In order to determine the internalized fraction of the compounds, a 5-min trypsin treatment was realized to remove membrane-bound transduction compounds (Figure 1A) (26). It is worth noting that the percentage of membrane-bound oligomers was not as important as in the case of the polycationic Arg8. The highest intracellular fluorescence intensity was found for DBT4 (JMV3229) with a drastic decrease (>4-fold) for DBT3 (JMV2968) and DBT2 (JMV2949) oligomers. Thus, the cellular uptake appeared length-dependent with an increase of internalization with the oligomer size. Moreover, the amount of JMV3229 that was internalized was more significant than that of Arg8 despite the fact that it is uncharged. Even though the increase in efficiency from DBT2 to DBT4 could be associated with an increase of the hydrophobicity, the high difference between DBT3 and DBT4 might be also attributed to a
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Figure 1. Uptake of Arg8 and oligomers 1-5 in MDA-MB-231 cells. Data are the means ( SD from three independent experiments. In B to D, cells were treated by trypsin before fluorescence measurements. (A) Determination of total and internalized compounds after incubating cells for 3 h at 37 °C with 10-5 M 1-5. *p < 0.05, **p < 0.01 relative to Arg8 values with or without trypsin treatment, respectively (Student’s t test). (B) Comparison of fluorescence emission after incubation for 3 h with several concentrations ranging from 10-6 to 5.10-5 M. *p < 0.05, **p < 0.01 relative to Arg8 values at the same concentration (Student’s t test). (C) Kinetics of internalization of JMV3229 and Arg8 at 10-5 M. *p < 0.05, **p < 0.01 relative to Arg8 values at the same time (Student’s t test). (D) Effect of temperature on the cellular uptake of JMV3229 at 3 h. **p < 0.01 relative to Arg8 values (Student’s t test).
progressive organization of the oligomer structure. Compound JMV4228 constructed by oligomerization of an L-benzothiazepinone moiety was as potent as its D-counterpart suggesting that the configuration of the BT moiety was not important and that the CPNPs do not interact with a specific receptor but rather with hydrophobic plasma membrane molecules. Compound JMV4287 constructed from the alternation of D and LBT exhibited a 2-fold decreased uptake. This result could be explained by a change in the oligomer structure. A dose-response analysis was performed to compare the uptake of the BT-oligomers with octa-arginine (Figure 1B). The penetration of all compounds appeared to be dose-dependent. However in contrast to Arg8, uptake of JMV compounds still increased at high doses (5.10-5 M). The kinetics study performed with 10-5 M JMV3229 (Figure 1C) showed that its cellular entry was at least as efficient at 1 h as that of Arg8. Then, JMV3229
Vezenkov et al.
uptake increased up to 16 h to reach a 6-fold higher concentration than Arg8, for which maximal concentration is reached at 1 h. A temperature-dependent analysis performed at 4 and 37 °C (Figure 1D) showed that the cellular uptake exhibited a 3-fold decrease at low temperature, hence suggesting the involvement of an energy-dependent endocytotic pathway. The remaining fluorescence was most probably due to cell membrane-bound oligomers as shown in Figure 2B. Intracellular Distribution of DBT Oligomers. Confocal laser scanning microscopy (CLSM) analyses were also performed in living cells to assess internalization and intracellular distribution. These experiments were associated with a kinetic study of DBT oligomer internalization. Figure 2A shows the internalization of JMV3229 in cellular organelles after 3 h of incubation with the highest accumulation at 16 h and an apparent decrease at 24 h in accordance with the kinetic study reported in Figure 1C. Co-staining with a membrane marker (lipid-raft labeling) indicated that this hydrophobic compound was not held in the lipid membrane. However, at 4 °C, the majority of the compound was colocalized with the membrane marker (Figure 2B) rather than internalized, confirming the temperature effect on the intracellular uptake described in Figure 1D and supporting an energy-dependent internalization mechanism. To gain more insight into JMV3229 internalization, costainings were performed with other fluorescent markers for subcellular components such as the nucleus, endosomes, and lysosomes. The results showed that JMV3229 was mostly colocalized with an endosomal marker at 3 h (Figure 2C) and with a lysosomal marker at 16 h (Figure 2D). Similar data were obtained for the L-counterpart JMV4228 (Supporting Information). This indicated an endosomal uptake of these oligomers followed by a lysosomal accumulation. Altogether, these data indicate the ability of these oligomers to target the endolysosomal pathway. Although most of the initial drug delivery studies aimed to avoid lysosomal addressing to prevent subsequent drug degradation, more recent studies demonstrated the relevant clinical utility to target this compartment for drug delivery in the treatment of lysosomal storage diseases, Alzheimer’s disease, and cancer (20, 27-31). The cytotoxicity of this new class of molecules was also determined in MDA-MB-231 cells using the MTT viability assay (Supporting Information). After a 5 day-incubation, these compounds exhibited no significant effect on cell viability at the 10-5 M concentration used for internalization studies. These results indicated that poly-DBT could be safely used as vectors for cell drug delivery.
CONCLUSIONS The cellular penetration efficiency of the described short DBT oligomers offers a novel class of vectors having the particularity of not being cationic. This feature should open new avenues in the field of compounds able to cross the cell membranes. The drastic increase of the cellular uptake along with the oligomer size could suggest an essential role of structuration and/or hydrophobicity for this class of compounds. Moreover, the decrease in the uptake of the D/LBT alternated oligomer should be also associated with structural features. In order to establish a relationship between their capacity to penetrate into the cell and their structural and physical features, more DBT oligomer derivatives need to be constructed. The structure of the oligomers is currently investigated using NMR spectroscopy and X-ray crystallography. The specific localization of DBT oligomers in endosomes and lysosomes has been evidenced. Studies toward the application of these CPNP as functional drug delivery vectors and the understanding of their translocation pathway are also in progress.
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Figure 2. Confocal microscopy images of living MDA-MB-231 breast cancer cells incubated with JMV3229 compound. Images are representative of at least 3 independent experiments. (A) Distribution of JMV3229 after 3, 16, and 24 h of incubation at 37 °C. (B) Distribution of JMV3229 at 4 °C and colocalization of the oligomers with the membrane marker. (C) Co-localization of JMV3229 with the endosomal marker at 3 h at 37 °C. (D) Co-localization of JMV3229 with the lysosomal marker at 16 h at 37 °C.
ACKNOWLEDGMENT This study was supported by the Agence Nationale de la Recherche (ANR-08-BLAN-0066-01) and ARC grant number 3953. Michel Gleizes is acknowledged for technical assistance with cell cultures. Supporting Information Available: Solid phase supported synthesis and characterization of the different oligomers; description of the MTT assay for cellular toxicity analysis; confocal laser scanning microscopy of JMV3229 and JMV4228. This material is available free of charge via the Internet at http:// pubs.acs.org.
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