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Laurence Crombez, Gudrun Aldrian-Herrada, Karidia Konate, Quan N Nguyen, Gary K Mcmaster, Robert Brasseur, Frederic Heitz, Gilles Divita
To cite this version:
Laurence Crombez, Gudrun Aldrian-Herrada, Karidia Konate, Quan N Nguyen, Gary K Mcmaster, et al.. A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.. Molecular Therapy, Cell Press, 2009, 17 (1), pp.95-103. �10.1038/mt.2008.215�. �hal-00345886�
A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.
Laurence Crombez1, Gudrun Aldrian-Herrada1, Karidia Konate1, Quan N. Nguyen2, Gary K. McMaster2, Robert Brasseur3, Frederic Heitz1 and Gilles Divita1*.
1 Centre de Recherches de Biochimie Macromoléculaire, CRBM-CNRS, UMR-5237, UM1- UM2, University of Montpellier, Department of Molecular Biophysics and Therapeutics, 1919 Route de Mende, 34293 Montpellier, France
2 Panomics Inc., 6519 Dumbarton Circle Fremont, CA 94555, USA;
3 Centre de Biophysique Moléculaire Numerique –Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, Belgium.
*To whom correspondence should be addressed; tel : +33 (0)4 67 61 33 92, fax : +33 (0)4 67 52 15 59, e-mail : gilles.divita@crbm.cnrs.fr
Running Title: amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.
Key words: siRNA, Delivery, Cell Penetrating Peptide, primary cell lines
Abstract
RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, siRNA based strategies suffer from their poor delivery and bio-distribution. Cell-Penetrating Peptides have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. In order to improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp-groups that favour cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels.
Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have significant impact on the development of fundamental and therapeutic siRNA-based applications.
INTRODUCTION
Over the past ten years, we have witnessed the emergence of numerous therapeutic approaches based on nucleic acids or analogues affecting expression of target genes [1]. The discovery of RNA interference has raised great hope for many therapies by providing a means to interfere with specific gene products [2,3]. Several reports have revealed that siRNA (small interfering RNA) can be applied to inhibit targets implicated in various diseases including cancer, viral infections and genetic disorders [4-7].
The field of derivate therapeutic applications is in continuous development, however, the major limitation to the clinical application of these methods is the poor permeability of cell membrane to negatively charged nucleic acids and their restricted bio-distribution [5-7].
In order to circumvent these issues, research has focused on the development of efficient delivery strategies and have lead to the design of different viral vectors [4-7] and non-viral approaches for siRNA delivery, including electroporation [8], cationic lipids and polymers [9], nanoparticles [10] and peptides [11,12]. Cell Penetrating Peptides (CPPs) have been shown to significantly improve cellular uptake of various therapeutic molecules both in cultured cells and in vivo [13,14]. Most of the CPPs that have been proposed so far, such as Tat [15], oligo–Arg [16], transportan [17], and penetratin [18], are covalently linked to their cargoes. These CPPs are internalized by cells together with their cargoes essentially through an endocytotic pathway [19-21]. CPPs can be used as potential siRNA carriers and although conjugation strategies using either transportan [22] or penetratin[23] certainly improve their delivery into cultured cells, non-covalent strategies appear more appropriate for siRNA delivery in vivo [11, 12, 24-26]. Short amphipathic CPPs forming non-covalent stable complexes with macromolecular cargoes have been successfully applied to their delivery into mammalian cells [27-29]. In particular the amphipathic peptide MPG efficiently delivers
siRNA in a fully biologically active form into a wide variety of cell lines, including embryonic stem cells [24, 30-33].
Amongst the new CPP-based delivery systems proposed that enable siRNA to cross cell membranes, only a few possess the properties of a therapeutic carrier including high delivery efficiency, absence of cytotoxicity and of immunogenicity. Efficient uptake allows decreasing the dose required to achieve a significant effect, thereby decreasing secondary side effects associated with a lack of specificity. In the present work, we propose a new generation of CPPs, based on a secondary amphipathic peptide CADY, which is able to form stable complexes with siRNA and improves their delivery into challenging suspension and primary cell lines. We demonstrate that CADY promotes target knockdown with sub-nanomolar concentrations of siRNA, without inducing any toxic side effects. We have investigated the mechanism through which CADY mediates cellular uptake of siRNA and show that CADY possesses great potential as a carrier for siRNA-mediated targeting in a therapeutic context.
RESULTS
Design and characterization of CADY.
Delivery of siRNA into suspension cells remains a major challenge for therapeutic applications. Therefore in order to favour cellular uptake of siRNA into challenging cell lines, we designed a new generation of peptide carriers that adopt a helical structure with secondary amphipathic properties. We have previously reported that the chimeric peptide carrier PPTG1 derived from the fusion peptide JTS1 [34], in which all glutamic acids are replaced by lysines or arginines, forms stable complexes with nucleic acids and improves their delivery both in cultured cells and in vivo [35]. Based on PPTG1 sequence as a template, we designed a 20- residue secondary amphipathic peptide, CADY, “Ac-GLWRALWRLLRSLWRLLWRA-
cysteamide”. To improve both interaction of the peptide with siRNA and its ability to interact with the lipid phase of the membrane, Arg and Trp residues were included in the sequence.
Seven residues of PPTG1 were changed, Phe3, Leu7, Leu18 and Lys4, Lys8, Lys11 being mutated into Trp and Arg, respectively. CADY was synthesized with a cysteamide group at its C-terminus and was acetylated at its N-terminus, so as to stabilize the carrier/cargo complex and to improve its potency to cross cell membranes, as previously reported for other carriers [24, 27]. As shown in Fig. 1a, CADY CD-spectrum is characterized by a minimum at 205 nm, a pronounced shoulder at 221nm and a maximum at 190 nm, which reveals that CADY adopts an alpha-helical structure stabilized in the presence of DOPG lipid vesicles (Fig. 1a). Moreover, 3D models of CADY calculated using Peplook software [36] reveal an helical conformation which is responsible for the secondary amphipathicity of CADY, which leads to exposure of Trp-groups on one side, of charged residues on the other, and of hydrophobic residues on yet another (Fig. 1b,c,d).
CADY forms stable complexes with siRNA
The ability of CADY to interact with siRNA was investigated using a small interfering RNA (21mer) targeting GAPDH as reference. siRNAs were incubated with CADY at different molar ratios and the formation of CADY/siRNA complexes was monitored in agarose gel- shift assays and by fluorescence spectroscopy. As shown in Fig. 2a, formation of stable CADY/siRNA complexes is initiated at a low molar ratio of CADY/siRNA (1/1), and at a molar ratio of 15/1 siRNA molecules are entirely associated with CADY, as no free siRNA is detected on the agarose gel. The inability of CADY/siRNA complexes obtained at a molar ratio equal to or greater than 40/1 to enter the agarose gel suggests the formation of high molecular weight complexes or aggregates, as previously documented for other CPPs. The formation of siRNA/CADY complexes was also evaluated by intrinsic fluorescence
spectroscopy, using the four Trp-residues of CADY as internal probes to monitor the interaction with siRNA. The binding of siRNA to CADY induced 42% quenching of fluorescence, associated with a 7 nm blue-shift of the emission spectrum from 345 nm to 338 nm (Fig. 2b and insert). CADY presents high affinity for siRNA and fitting of the titration curve yields an apparent dissociation constant of 15.2 ± 2 nM. Binding saturation was reached at a molar ratio ranging between 5/1 and 10/1, suggesting that several molecules of CADY interact with the siRNA molecule, in agreement with agarose gel results. Interestingly, no shift in the emission spectrum associated with an increase in diffusion was observed with higher molar ratios (40/1) (Fig. 2b insert), suggesting that high molecular weight complexes reported in agarose gel assays are not aggregates but bona fide complexes. We have investigated the impact of the interaction of CADY with siRNA on its stability to serum nuclease. The siRNA were complexed with increasing CADY molar ratios and then incubated for 24 h in the presence of 50% serum (Fig. 2c). In the absence of CADY or in the presence of low molar ratios (1/1 to 10/1), siRNAs are rapidly degraded by serum. In contrast, no significant degradation was observed at high molar ratios (40/1 and 80/1) and the stability of siRNA to serum within CADY particles increased with the ratio of CADY, indicating that CADY protects the siRNA. That siRNAs retain their integrity when complexed to CADY is consistent with the high affinity of CADY for siRNA and the formation of a particle of peptides surrounding the siRNA.
CADY promotes efficient cellular uptake of siRNA into mammalian cells
We next evaluated the potency of CADY to deliver siRNA into cultured cells using fluorescently labelled siRNA molecules (FAM-siRNA). A fixed concentration of siRNA (20 nM) was complexed with CADY at different molar ratios ranging from 10/1 to 80/1 and internalization was monitored by FACS. CADY-mediated cellular uptake of siRNA is
extremely rapid and similar results were obtained for short (30 min) or long (4h) incubation times. As reported in Fig. 3, cellular uptake efficiency was directly related to the molar ratio of CADY/siRNA. Although a 10/1 ratio was sufficient to deliver siRNA into almost every cell, the intracellular fluorescence intensity increased with the CADY/siRNA ratio and doubled between 10/1 and 80/1 ratios. The fluorescence peaks observed at ratios 10/1 and 20/1 are symmetric, revealing a homogeneous cellular distribution of siRNA. In contrast, at a greater ratio (80/1) the increase in fluorescence intensity is associated with a broader peak;
mainly due to heterogeneity in the size of siRNA/CADY complexes and the consequent heterogeneous cellular incorporation of FAM-siRNA, in perfect correlation with results obtained by fluorescence spectroscopy and gel-shift assay.
CADY-mediated siRNA delivery into mammalian cells induces target knockdown.
The potency of CADY to deliver active siRNA molecules into cells was investigated by monitoring knockdown of GAPDH, at both mRNA and protein levels (Fig. 4). U2OS cells were incubated with different siRNA concentrations ranging from 0.5 to 20 nM complexed with CADY at a molar ratio of 40/1 or 80/1. CADY/siRNA complexes were formed at a siRNA concentration of 20 nM, then in order to obtain a homogeneous solution of CADY/siRNA complexes, lower concentrations of siRNA were prepared by serial dilution in 0.5 x PBS. GAPDH mRNA level was quantified 24 h after transfection using the quantitative Quantigene assay (Fig. 4a) and knockdown of GAPDH protein levels was evaluated 48 hours after transfection by Western blot analysis (Fig. 4b&c). CADY-mediated siRNA delivery results in a marked siRNA concentration-dependent, downregulation of GAPDH mRNA. A maximal mRNA silencing effect of 95% was obtained with 20 nM siRNA and an IC50 value of 2.1 ± 0.5 nM. Reduction of GAPDH protein is inversely proportional to siRNA concentrations and a complete knockdown of the target protein was obtained for a 10 nM
siRNA concentration complexed to CADY at either 40/1 or 80/1 molar ratio. Depending on the ratio used, 50% of GAPDH protein knockdown was obtained for a siRNA concentration of 0.5 nM and 2.1 nM for molar ratios of 80/1 and 40/1, respectively (Fig. 4 b&c). Taken together, these experiments show a direct correlation between mRNA and protein knockdown and reveal that the biological effect of CADY-delivered siRNA is dependent on the carrier/cargo ratio and on the siRNA concentration.
To determine whether there was a link between siRNA uptake (20 nM) and side effects, the cytotoxicity of CADY was evaluated on U2OS cells with different CADY/siRNA ratios using the MTT assay. As reported in Fig. 4d, CADY/siRNA molar ratios from 20/1 to 80/1 did not affect cell viability for 24 hours at 37°C and at the siRNA concentrations (20 or 40 nM) and molar ratio (20/1 or 40/1) required for an optimal silencing response, no cytotoxicity was observed. At a lower ratio (ratio 10/1) cell viability decreased by only 10 %.
The ability of CADY to deliver siRNA into mammalian cells was then evaluated using a siRNA targeting p53. Cells were incubated with 40 nM of siRNA targeting p53 associated with CADY at a 40/1 molar ratio and the level of p53 expression was evaluated by Western blotting over a period of 5 days following transfection. As reported in Fig 4e, 40 nM of siRNA/CADY induced 97% knockdown of p53 protein after 48 and 72 h, and the reduction in p53 protein levels could be maintained at about 60% for 5 days. Taking into account that the turnover of p53 protein is approximately 10 hours, these results demonstrate that CADY/siRNA particles remain highly stable within cells, again confirming the high potency of CADY for delivery of active siRNA.
CADY-mediated siRNA delivery into challenging cell lines
CADY technology was then challenged against suspension and primary cell lines that have been reported as difficult to transfect, such as suspension THP1 and primary HUVEC and
3T3C cell lines. CADY/siRNA complexes formed at a molar ratio of 40/1 were used and the siRNA dose-response associated GAPDH knockdown was followed at both protein and mRNA levels. As reported in Fig. 5, CADY-mediated siRNA delivery induced an important knockdown of GAPDH at both the mRNA and protein level in all cell lines tested.
Remarkably, the mRNA level was reduced by more than 80% with 20 nM siRNA, irrespective of the cell type, and IC50 values of about 3.4 ± 0.5 nM, 1.5 ± 0.1 nM and 1.2 ± 0.1 nM were estimated for THP1 (Fig. 5a), primary HUVEC (Fig. 5b) and 3T3C (Fig. 5c) cells.
Monitoring the level of cyclophilin B mRNA as control of cell viability, showed no significant toxicity associated with siRNA/CADY, and viability was greater than 80% in all cell lines (Fig. 5).
Cell entry mechanism of CADY/siRNA complexes
In order to better understand the cellular uptake mechanism of CADY/siRNA particles, the CADY-mediated cell-entry of a fluorescently-labelled siRNA was investigated in the presence of different inhibitors of the endocytosis pathway. Cells were treated with amiloride, known to inhibit cell entry associated with macropinocytosis [20, 21], nocodazole for the clathrin-mediated pathway [37] and cyclodextrin for caveolin- and microdomain-associated processes [20]. The cellular uptake of fluorescently-labelled siRNA was determined by flow cytometry and correlated with GAPDH siRNA associated biological response. Results were normalized with a control experiment performed in the absence of inhibitors. As reported in Fig. 6a, none of the inhibitors altered cellular uptake of siRNA/CADY complexes.
Cholesterol depletion by methylcyclodextrin (5 mM) resulted in a weak (3 to 5%) increase in siRNA incorporation. Different concentrations of amiloride were tested (from 1 - 5 mM) and the major increase (about 26%) in CADY-mediated siRNA delivery was obtained with 5 mM amiloride. Monitoring siRNA-associated GAPDH knockdown after treatment with the
different inhibitors also revealed a concentration-dependent increase in the efficiency associated with amiloride treatment up to 16 % for a concentration of 2.5 mM (Fig. 6b) correlating with the improvement in cellular uptake of the fluorescently-labelled siRNA..
When cells were treated with nocodazole, a 22% increase in GAPDH knockdown efficiency was observed. In contrast cholesterol depletion by methylcyclodextrin (5 mM) resulted in a weak (7 %) inhibition of transfection efficiency.
DISCUSSION
It is currently admitted that siRNA molecules hold amazing therapeutic potential for the development of new drugs interfering with various diseases [5, 7, 9, 38]. However, siRNA- based therapies, as most nucleic acid-based strategies, have to face limitations associated with poor cellular uptake and bio-distribution. Therefore, although several non-viral methods have been reported to improve siRNA delivery [5-7] optimizing stability and cellular uptake of siRNA molecules into primary cell lines remains a major challenge. In the present study, we report the design and characterization of a new amphipathic peptide carrier, CADY, which promotes siRNA delivery into challenging cell lines. We have demonstrated that CADY meets four major requirements (i) binds reversibly and forms stable complexes with short nucleic acids, (ii) crosses the cell membrane of a broad spectrum of cell types, including primary, suspension and adherent cell lines, (iii) selectively and rapidly delivers siRNA into the cytoplasm and (iv) promotes a significant siRNA-associated knockdown of the target.
CADY is a secondary amphipathic peptide derived from PPTG1 peptide which has been reported to improve delivery of plasmid DNA into both cultured cells and in vivo, but not of small nucleic acids [35]. The sequence of PPTG1 was optimized in order to specifically interact with small nucleic acids (siRNA) and to improve the stability of the peptide/small nucleic acid interactions, in particular, by replacing with Trp and Arg, seven residues
throughout the sequence of PPTG1. Arginines have been shown to both improve electrostatic cell surface interactions and to enhance cellular entry of CPPs [16, 40]. Tryptophan plays an essential role in the cellular uptake of CPPs [18, 27, 41, 42] mainly due to their ability to interact with lipid/cholesterol molecules within the membrane. Moreover, in the case of non- covalent peptide-based strategies, Trp-residues are involved in stabilization of carrier/cargo complexes [27, 42]. Secondary amphipathic peptides are generated by the conformational state which allows positioning of hydrophobic and hydrophilic residues on opposite sides of the molecule. CADY adopts a helical conformation in solution and molecular modelling reveals that the lowest energy structural model corresponds to an amphipathic helical conformation stabilized by the stacking and exposure of Trp-residues on one side, of Arg and Lys-charged residues on the other and of hydrophobic residues on yet another (Fig. 1d). The spatial conformation of CADY improves its solubility and its interaction with siRNA.
The high affinity of CADY for siRNA (Kd :15.2 nM) is essentially associated with electrostatic interactions involving Arg residues, but most likely also implies Trp residues that may interact with the minor groove of siRNA molecules. It was recently shown that Trp of the p19 protein that controls siRNA silencing in plants interacts with and makes an end- capping interaction with the 5’end of siRNA [43]. The combination of electrostatic interactions and of hydrogen bonding leads to the formation of stable CADY/siRNA particles which protect siRNA molecules without affecting their silencing activity. The stability of these complexes was validated by the fact that no serum nuclease degradation and a long term silencing response was observed, with 60% knockdown of the target gene after 5 days.
CADY/siRNA complex formation and consequent siRNA delivery are directly associated with the peptide/siRNA molar ratio. Both fluorescence titration and agarose gel-shift experiments demonstrate that several molecules of CADY interact with siRNA, but that although all siRNA molecules are complexed at a CADY/siRNA molar ratio of 10/1, optimal
cellular uptake and biological response are achieved at a molar ratio greater than 20/1, suggesting that control of peptide/siRNA molar ratio is essential to obtain stable and biologically efficient particles in culture medium, as already reported for other non-covalent peptide carriers [42, 44]. Indeed using fluorescent siRNA we have demonstrated that cellular uptake increases with the molar ratio but that at a ratio above 40/1, internalized levels of siRNA per cell were more heterogeneous.
CADY constitutes an interesting tool for ex-vivo siRNA delivery and can be applied to a large number of challenging cell lines. We have shown that CADY-mediated siRNA delivery is associated with a significant knockdown of the target gene at both mRNA and protein levels, in adherent, suspension and primary cell lines, with IC50 values in the low nanomolar range. Furthermore, one of the advantages of CADY/siRNA particles is their high stability, which allows for serial dilution, whilst maintaining constant the peptide/siRNA molar ratio, and enables to achieve significant knockdown of the target at sub-nanomolar siRNA concentrations. In addition, CADY does not show any acute and long-term toxicity on transfected cells at any molar ratio applied, other than a slight effect on cell viability observed at a 10/1 ratio which might be attributable to particle size at this ratio. Indeed biophysical studies have revealed that particle size of CADY/siRNA complexes depends on their molar ratio and that at low ratios, particles tend to aggregate. Regarding these results and siRNA delivery experiments, complex stability and size are clearly critical parameters, which affect the cellular uptake mechanism, and consequently determine the outcome of the biological response.
The cellular uptake mechanism of CPPs is most often associated with the endosomal pathway and dependent on the cargo, cell line and local concentration of CPPs. We have investigated the mechanism through which CADY/siRNA enter cells and found that the use of inhibitors of the endosomal pathway barely affect cellular uptake and the associated
biological response. Moreover, we propose that the helical conformation of CADY is involved in the interactions with the cell membrane and in the process of cell penetration itself. JTS1, INF1 or KALA peptides have the tendency to adopt a helical structure at low pH, inducing the formation of an amphipathic helix with a strong potency to destabilize membranes and favour release from the endosome [34]. In contrast, CADY, like PPTG1, adopts a helical structure independently of the pH and cellular uptake was only slightly modified by inhibition of the vacuolar proton pump [35], suggesting that the mechanism of these peptide is less sensitive to pH and only partially involves the endosomal pathway.
Although we cannot exclude another endosomal mechanism, it seems that CADY uptake is associated with its ability to interact with the cell membrane and that aromatic Trp residues play a major role by interacting with membrane components. Lack of toxicity associated with the helical conformation suggests a temporary interaction of CADY/siRNA complexes with the cell membrane, which is not the case for other helix-forming peptides that interact with membranes such as mellitin [45]. The helical conformation has a major impact on CPP cellular uptake and improves their release from endosomes, as reported for penetratin, transportan, proline-rich peptides and calcitonin-derived peptides [46].
We have demonstrated that CADY/siRNA complexes enter cells rapidly and independently of the endosomal pathway. Similar data have been reported for other CPPs which exhibit a tendency to form complexes with membranes or at high concentrations [47].
We suggest that particle organization upon interaction with the cell membrane,together with GAG clusterization may induced a local high concentration of CPP [47], which, together with the Trp-residues present in the sequence, favour membrane penetration and rapid cellular entry. There is a critical balance between stability of the particle, CPP concentration and their ability to interact with the lipid phase of the membrane, as already reported for other CPPs.
Alterations in this equilibrium by small changes in peptide sequence may trigger particle
aggregation and induce cell entry via a different pathway, as already reported for MPG or penetratin, when amino acid modifications in the membrane-binding domain of the peptide are made [48]; In the case of MPG 4 mutations in the hydrophobic domain suffice to make the peptide more toxic and to promote cellular uptake through the endosomal pathway [49].
In conclusion, CADY is the first secondary amphipathic peptide which allows the formation of stable complexes with siRNA via non covalent interactions and that efficiently promotes cellular uptake of suspension and challenging cell lines. Taking together these results, with the fact that CADY is not toxic and its ability to support a sustained silencing response with low nanomolar concentrations of siRNA, suggests that this peptide carrier is extremely efficient for silencing different target genes. Hence the peptide-based nanoparticle CADY-system constitutes a promising technology for siRNA delivery for therapeutic purposes. Its development for in vivo applications should reduce the dosage of therapeutic molecules, thereby limiting off-target effects and associated toxicity.
MATERIALS AND METHODS
Peptide synthesis and siRNA. CADY peptide (Ac-GLWRALWRLLRSLWRLLWRA-cya), was synthesized by solid-phase peptide synthesis synthesized using AEDI-expensin resin with (fluorenylmethoxy)-carbonyl (Fmoc) continuous (Pionner, Applied Biosystems, Foster city, CA) as described previously [31]. CADY was purified by semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC; C18 column Interchrom UP5 WOD/25M Uptisphere 300 5 ODB, 250 mm x 21.2 mm) and identified by electrospray mass spectrometry and amino acid analysis [31]. The siRNA and fluorescently labelled siRNA (5’
FAM) were obtained from Eurogentec (Belgium). The different sequences are, for anti- GAPDH: 5′-CAUCAUCCCUGCCUCUACUTT-3′ (sense strand), and for targeting p53: 5′-
GGAAAUUUGCGUGUGGAGUTT-3′ (sense strand) [50]. The stock solution of siRNA was prepared at 5 µM concentration in 50 mM Tris, 0.5 mM EDTA buffer.
Cell culture and CADY-mediated siRNA transfection. The human osteosarcoma U2OS, THP-1 monocytes and mouse 3T3C cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). HUVEC cells were obtained from Panomics Inc (USA).
All media were from Gibco (VA, USA). U2OS and 3T3C cells were maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS). THP-1 and HUVEC suspension cell lines were cultured in RPMI-1640 Medium supplemented with 10% FCS and 0.05 mM 2-mercaptoethanol. CADY peptide was resuspended at a concentration of 2 mg/ml (0.3 mM) in water containing 2% DMSO and sonicated for 10 min. CADY/siRNA complexes were formed by adding CADY peptide (100 µM stock solution) into siRNA (5 µM stock solution) and incubated 30 min. at 37°C. For siRNA delivery, 150 000 cells plated in a 35 mm dish, the day before transfection were overlaid with the 200 µl of preformed complexes, incubated for 3-5 min, then 400 µl of DMEM were added. After 4 hours incubation 1 ml of media containing 16% FCS was added in order to obtain a final FCS concentration of 10%.
Structural characterization of CADY. Circular Dichroïsm (CD): CD spectra were recorded on a Jasco 810 (Tokyo, Japan) dichrograph in a quartz cell with an optical path of 1 mm for peptide in aqueous solutions. The DOPG phospholipid (dioleoylphosphatidylglycerol) was purchased from Avanti Polar Lipids (Alabaster, AL).
Molecular modelling: 3D models of CADY were calculated using PepLook software [36].
Briefly, a set of 99 3-D models were selected as the conformations of lower energy among the 10*6 structures calculated by the PepLook iterative Boltzman-stochastic procedure using
CADY sequence, the CBMN semi-empirical Force Field and a set of backbone angles calibrated for optimal structure prediction [36].
Intrinsic fluorescence spectroscopy. Fluorescence experiments were performed at 25°C, using a SPEX-PTI spectrofluorimeter in a 1 cm path-length quartz cuvette, with a band-pass of 2 nm for excitation and emission. The binding of siRNA to CADY was monitored by fluorescence microscopy in phosphate buffer. A fixed concentration of CADY (200 nM) was titrated by increasing siRNA concentrations from 1 to 500 nM. Intrinsic Trp-fluorescence was excited at 290 nm and emission spectra recorded from 310 to 400 nm. Data were fitted, using a quadratic equation (GraFit, Erithacus Software) as described previously [41].
Serum nuclease protection assay.
Naked siRNA (50 fmol) or CADY/siRNA complexes formed as described above were incubated at 37°C in DMEM containing 50% FCS. After 24h, the reaction was stopped by adding Proteinase K (200 µg/ml) for 1 h at 37°C allowing also the digestion of CADY peptide. Then siRNA was extracted with phenol:chloroform and analyzed on a 1% agarose gel (w/v).
Quantification of mRNA. mRNA quantification was performed using QuantiGene® 2.0 Reagent System (Panomics Inc., CA, USA) directly on cell lysates without mRNA purification or amplification according to the manufacturer’s instructions. Experiments were performed in triplicate and the signal obtained for the siRNA-targeted gene was normalized to the control housekeeping gene of cyclophilin-B.
FACS analysis. U2OS cells were transfected with appropriate CADY/siRNA concentrations for 4 h at 37°C. Cells were then washed with PBS and trypsinized for 5 min, washed and then resuspended in PBS containing RNAse A (0.1 mg/ml). FITC fluorescence was immediately measured by flow cytometry (BD FACS-Calibur System; Beckton Dickinson, France). For each sample fluorescence was acquired on 10,000 cells.
Western blotting. Transfected cells were harvested by trypsination, washed in PBS and lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.1% NP40, and 0.1% deoxycholate, including protease inhibitors: 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM PMSF (phenyl-methylsulfonyl fluoride). Cell lysates were kept on ice for 30 minutes, mixed every 5 min., and centrifuged at 4°C for 15 min. at 10,000g.
Supernatants were collected and protein concentrations were determined using the Bradford assay. Cell extracts (30 µg) were separated by SDS-PAGE on a 12.5 % SDS-polyacrylamide gel. After electrophoresis, samples were transferred onto nitrocellulose in a semi dry transfer apparatus for 1 hour at 1 mA per cm2 . Monoclonal mouse primary antibodies anti-GAPDH (6C5) and anti-p53 (DO-1) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit anti-actin antibody was obtained from Sigma-Aldrich (France) and sheep secondary anti-mouse HRP-linked whole antibody from Amersham (France).
Cytotoxicity. The cytotoxicity of CADY/siRNA complexes was investigated on U2OS cells.
30,000 cells plated in 24-well plates the day before transfection, were incubated with 20 nM of siRNA complexed with CADY at a molar ratio ranging from 10/1 to 80/1 for four hours prior to addition of medium containing a final 10% concentration of FCS. Cytotoxicity was measured 24 hours later by colorimetric MTT assay (Sigma, Germany). Cell culture medium
was removed and replaced with PBS containing 2.5 mg/ml of MTT for 4 h. Results shown correspond to the average of 3 separate experiments.
Agarose gel-shift assay. The siRNA targeting GAPDH gene was incubated for 30 min. at 37°C in 0.5x PBS with different concentrations of CADY corresponding to a molar ratio ranging between 1 and 80/1. The preformed complexes were analyzed by electrophoresis on agarose gel (1% w/v) stained with ethidium bromide.
Cellular entry mechanism of CADY. Prior transfection, cells were treated in serum free medium, for 30 min with 5 mM methylβcyclodextrin or for 1 h with either 5mM amiloride or 100 ng/ml nocodazole at 37°C. Cells were then incubated for 30 min with 20 nM of fluorescently-labelled anti-GAPDH siRNA complexed with CADY (molar ratio 40/1) in presence of endocytosis inhibitors. Medium was removed and transfections were performed as described above except that following the 3-5 minute incubation with complexes, DMEM supplemented with the appropriate inhibitor concentration was added. After 30 min of transfection, cells were washed with PBS and recovered by trypsinisation (5 min. Trypsin- EDTA, Sigma). After centrifugation (1200 rpm) cells were resuspended in PBS containing RNAse-A and then analyzed by FACS. For the evaluation of the biological activity of transfected siRNA, after 30 min of transfection in the presence of inhibitors, cells were washed with PBS, trypsinised and GAPDH protein level was evaluated after 48h, by Western blot as described previously.
ACKNOWLEDGEMENTS
This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), by the Agence Nationale de la Recherche (ANR, ANR-06-BLAN-0071-Pepvec4Ther) and by
a grant from Panomics Inc. We thank May Catherine Morris (CRBM-UMR5237-CNRS) for critical reading of the manuscript and all members of the laboratory for fruitful discussions.
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FIGURE LEGENDS
Figure 1: CADY adopts a second amphipathic helical structure. (a). Far-UV Circular dichroïsm spectrum of CADY (0.1 mg/ml) in the presence of DOPG vesicles. (b,c) Molecular modeling of the structure of CADY based on Peplook calculation. Two different orientations of the surface representation of the structure of lowest energy of CADY. Hydrophobic and electrostatic domains of the molecule are in green and orange, respectively. (d) 3D representation of the structure of CADY, Trp-residues are in yellow, charged residues in red and hydrophobic residues in orange.
Figure 2: CADY forms stable complexes with siRNA. (a). The preformed CADY/siRNA complexes were analyzed by electrophoresis on agarose gel (1% w/v) stained with ethidium bromide. siRNA targeting GAPDH gene was incubated with different concentrations of CADY corresponding to a molar ratio ranging between 1 and 80/1. Lane 1, corresponds to siRNA control in the absence of CADY; and Lanes 2-8, to molar ratios of 1, 5/1, 10/1, 15/1, 20/1, 40/1 and 80/1 respectively. (b). CADY/siRNA interaction as monitored by fluorescence spectroscopy. A fixed concentration of CADY (200 nM) was titrated by increasing siRNA concentrations from 1 to 500 nM. Intrinsic Trp-fluorescence was excited at 290 nm and the emission spectra were recorded from 310 to 400 nm (insert). CADY exhibits a characteristic Trp-emission spectrum (Insert: in magenta) and siRNA binding to CADY results in a marked quenching of fluorescence of about 20% and 40% obtained for an siRNA concentration of 10nM (Insert: in blue) and 50 nM (Insert: in green), respectively. Quenching of CADY emission fluorescence upon siRNA binding was monitored at 340 nm. Data were fitted using a quadratic equation (GraFit, Erithacus Software) as described previously [41] and correspond to an average of 3 distinct experiments. (c). CADY/siRNA complexes formed with different
molar ratios from 1/1 to 80/1 (50 fmol of siRNA) were incubated with serum (50 % for 24h) and the CADY associated siRNA protection to serum nucleases was evaluated on agarose gel after peptides and proteins digestion by Proteinase K and a phenol/chloroform extraction.
Figure 3: CADY improves the cellular uptake of fluorescent siRNA in a ratio-dependent manner. CADY/siRNA complexes were formed at different molar ratios (10/20/40/80) using a fluorescently-labelled siRNA and overlaid onto U2OS cells for 4 hours at 37°C. Cells were then washed with PBS and trypsinized and fluorescent cells were immediately counted by flow cytometry. Results are reported for 100 000 cells.
Figure 4: CADY-mediated siRNA delivery into cultured cells. A stock solution of CADY/siRNA (20 nM) was prepared at a molar ratio of 40/1 or 80/1, and then lower concentrations of formulated siRNA (from 20 nM to 0.5 nM) were obtained by serial dilution of the stock solution in PBS. U2OS cell (60 % confluency) were overlaid with preformed complexes for 4 min, then cells were complemented with fresh DMEM supplemented with 10% FCS, and returned to the incubator for 24 h. (a) A CADY/siRNA molar ratio of 40/1 was used. GAPDH mRNA levels were measured 24 h after transfection using QuantiGene® 2.0 Reagent System (Panomics Inc., CA, USA) technology. The results correspond to an average of 4 separate experiments. (b,c) GAPDH protein levels were determined 48 h after transfection by Western blotting using actin as a control for quantification. (d) Cytotoxicity of CADY/siRNA complexes investigated on U2OS cells. 30,000 cells plated in 24-well plates the day before transfection, were incubated with 20 or 40nM of siRNA complexed with CADY at molar ratio ranging from 10/1 to 80/1 and cytotoxicity was measured 24 hours later by colorimetric MTT assay. Results correspond to the average of 3 separate experiments and are compared to non transfected cells incubated in the same conditions. (e) CADY/siRNA p53
complexes were formed at a molar ratio of 40/1 with an siRNA concentration of 20 nM. The kinetics of human p53 protein silencing were assessed by Western blotting for 48 h (lane 1), 72 h (lane 2), 144 h (lane 3) and 168 h (lane 4) after transfection. Lane 5 corresponds to a control after 48 h.
Figure 5: CADY-mediated siRNA delivery into challenging cell lines. CADY was evaluated on three challenging cell lines THP1 (a), primary HUVEC (b) and 3TC3 (c). A stock solution of CADY/siRNA (40 nM) was prepared at a molar ratio of 1/40, and then lower concentrations of formulated siRNA (from 40 nM to 0.6 nM) were obtained by serial dilution of the stock solution in PBS. Cells were plated at 10,000 cells/well in a 96-well plate overnight and then transfected with varying concentrations (0.6 - 40 nM) of human GAPDH siRNA complexed with CADY at a 40/1 molar ratio. Twenty-four hours post transfection, cells were lysed and both GAPDH (grey bars) and cyclophillin B (in red) gene expression were quantified using the QuantiGene® 2.0 Reagent System (Panomics Inc., CA, USA).
Changes in GAPDH mRNA levels were normalized to non transfected cells
Figure 6: Inhibitors of the endosomal pathway do not affect CADY cellular uptake.
Prior transfection, U2OS cells were treated in serum free medium, for 30 min with methylβcyclodextrin or for 1 h with amiloride or nocodazole with the indicated concentrations. Cells were then incubated for 30 min with 20 nM of fluorescently-labelled (a) or non labelled (b) anti-GAPDH siRNA complexed with CADY (molar ratio 40/1) in presence of endocytosis inhibitors. Then the medium was eliminated and rinsed cells were (a) trypsinized, washed and siRNA transfection efficiency was determined by FACS analysis or (b) incubated with fresh 10 % FCS medium for 48h before analysis by Western blot. The
relative transfection efficiency (%) is compared to the transfection (CADY/siRNA) performed in the absence of any inhibitors. Data correspond to an average of 3 separate experiments.
260 240
220 200
180 60000 40000 20000 0 -20000 -40000
260 240
220 200
180 60000 40000 20000 0 20000 40000
a
b c
d
Wavelengths (nm)
Ellipticity (deg.cm2.M-1)
siRNA (nM)
0 10 20 30 40 50 60 70 80 90 100 110
Fluorescence (%)
50 60 70 80 90 100 110
a b
c
siRNA complex Molar ratio
CADY/siRNA 0 1 5 10 15 20 40 80 Molar ratio
CADY/siRNA
Molar ratio CADY/siRNA
siRNA
0 1 5 10 15 20 40 80
- SVF 20
Control
RATIO 10 RATIO 20
RATIO 40 RATIO 80
Counts
128
0 64
32 96
101 102 103 104
100
FL1-H Control
RATIO 10 RATIO 20
RATIO 40 RATIO 80
Counts
128
0 64
32 96
101 102 103 104
100
FL1-H
Figure 3
0 20 40 60 80 100 120
0 0.6 1.3 2.5 5 10 20
relative GAPDH mRNA(%)
siRNA (nM)
co nt ro l 48 H
72 H
14 4 H
16 8 H
p53
GAPDH
Time after transfection
co nt ro l 48 H
72 H
14 4 H
16 8 H
p53
GAPDH
Time after transfection
Molar ratio CAD2/siRNA: 80
Molar ratio CAD2/siRNA: 40 -
GAPDH actin
siRNA (nM) 10 5 2.5 1.25 0.5
GAPDH actin
-
siRNA (nM) 20 10 5 2.5 1.25 0.5 Molar ratio CAD2/siRNA: 80
Molar ratio CAD2/siRNA: 40 -
GAPDH actin
siRNA (nM) 10 5 2.5 1.25 0.5
GAPDH actin
-
siRNA (nM) 20 10 5 2.5 1.25 0.5
a b
c
d e
Molar ratio CADY/siRNA 40/1 Molar ratio CADY/siRNA 80/1
siRNA / CADY (nM)
relative cellviability(%)
Time after transfection
0 20 40 60 80 100 120
20/200 20/400 20/800 20/1600 40/800 control
siRNA (nM)
Relative GAPDH mRNA
0 20 40 60 80 100 120
0 0.6 1.3 2.5 5 10 20 40
a
siRNA (nM)
0 20 40 60 80 100 120
0 0.6 1.3 2.5 5 10 20 40
Relative GAPDH mRNA
b
siRNA (nM)
0 20 40 60 80 100 120
0 0.6 1.3 2.5 5 10 20 40
Relative GAPDH mRNA
c
-5 0 5 10 15 20 25 30
relative transfection efficiency (%
Amiloride (5 mM)
Methylβcyclodextrin (5 mM) Nocodazole
(100 ng/ml)
b
0.5 mM 1 mM 2.5 mM 100 ng/ml
Methylβ- cyclodextrin Nocodazole
Amiloride
5 mM
relative transfection efficiency (%)
-20 -15 -10 -5 0 5 10 15 20 25 30
