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Delivery of siRNA complexed with Palmitoylated #-peptide/#peptoid Cell-penetrating Peptidomimetics: Membrane Interaction and Structural Characterization of a Lipid-based Nanocarrier System Xiaona Jing, Camilla Foged, Birte Martin-Bertelsen, Anan Yaghmur, Kolja M. Knapp, Martin Malmsten, Henrik Franzyk, and Hanne M. Nielsen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00309 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 28, 2015

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Molecular Pharmaceutics

Delivery of siRNA Complexed with Palmitoylated α-peptide/β-peptoid Cell-penetrating Peptidomimetics: Membrane Interaction and Structural Characterization of a Lipid-based Nanocarrier System

Xiaona Jinga, Camilla Fogeda, Birte Martin-Bertelsena, Anan Yaghmura, Kolja M. Knappc, Martin Malmstenb, Henrik Franzykc and Hanne M. Nielsena*

a) Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O, Denmark b) Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden c) Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O, Denmark

*Corresponding author: Hanne Mørck Nielsen, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark, Tel.: + 45 35 33 63 46, fax: 35 33 60 01, e-mail: [email protected]

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ABSTRACT Proteolytically stable α-peptide/β-peptoid peptidomimetics constitute promising cell-penetrating carrier candidates exhibiting superior cellular uptake as compared to commonly used cell-penetrating peptides (CPPs). The aim of the present study was to explore the potential of these peptidomimetics in delivery of small interfering RNA (siRNA) to the cytosol by incorporation of a palmitoylated peptidomimetic construct into a cationic lipid-based nanocarrier system. The optimal construct was selected based on the effect of palmitoylation and the influence of the length of the peptidomimetic on the interaction with model membranes and the cellular uptake. Palmitoylation enhanced the peptidomimetic adsorption to supported lipid bilayers as studied by ellipsometry. However, both palmitoylation and increased peptidomimetic chain length was found to be beneficial in the cellular uptake studies using fluorophore-labeled analogs. Thus, the longer palmitoylated peptidomimetic was chosen for further formulation of siRNA in a dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) nanocarrier system, and the resulting nanoparticles were found to mediate efficient gene silencing in vitro. Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet co-existed with a hexagonal phase, which is important for efficient nanocarrier-mediated endosomal escape of siRNA ensuring cytosolic delivery. The present work is a proof-of-concept for the use of α-peptides/β-peptoid peptidomimetics in an efficient delivery system that may be more generally exploited for the intracellular delivery of biomacromolecular drugs.

KEYWORDS siRNA delivery, cell-penetrating peptide, peptidomimetic, palmitoylation, nanocarrier, cryo-TEM, SAXS; drug delivery; nanomedicine ACS Paragon Plus Environment

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INTRODUCTION RNA interference (RNAi), mediated by small interfering RNA (siRNA) in the cell cytoplasm, is an efficient, natural cellular defense mechanism that enables post-transcriptional silencing of gene expression. The RNAi process constitutes a promising approach for future nanomedicines based on chemically synthesized siRNA, and therefore this principle has been extensively investigated since its Noble prize-winning discovery.1 However, efficient delivery systems are required in order to ensure sufficient delivery of exogenous siRNA into the cytoplasm of the target cells due to its unfavorable physicochemical properties arising from the relatively large molecular size and high overall negative charge. In addition, siRNA has a short half-life in vivo due to enzymatic degradation and renal excretion. These characteristics hamper efficient migration from the administration site through tissues to the target cells as well as the final permeation of the cell membrane to reach the cytoplasm of the individual cells.2 Non-viral vectors such as lipid-based carriers3,4 that encapsulate siRNA are comparatively less efficient than viral vectors, but preferred due to a better safety profile. Thus, several methods for encapsulation of siRNA in lipid-based drug delivery systems targeting specific cell compartments have been reported.4 Several studies have reported on attempts to utilize cell-penetrating peptides (CPPs) in lipid-based drug delivery systems to facilitate siRNA delivery because these relatively small peptides are able to condense siRNA into complexes and to interact with cellular membranes and efficiently promote intracellular delivery of therapeutic biomacromolecules.5,6 A prominent example is the oligoarginine (Rn)-modified, siRNA-containing liposomes, which have been shown to mediate gene silencing in lung tumor cell lines.7 To improve the encapsulation efficiency and stability of nucleic acids in colloidal carriers, non-covalent pre-condensation with polycationic CPPs prior to encapsulation has proved to be a successful approach for formulation of plasmid DNA.8 However, pre-condensation is inefficient for siRNAs due to the ACS Paragon Plus Environment

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shorter nucleotide chain length that generally do not allow formation of serum-stable complexes. Thus, additional association forces such as hydrophobic interactions are needed for complex stabilization. An example comprises cholesterol-modified nona-D-arginine (R9) that forms stable complexes with siRNA, and which was successfully tested both in vitro and in vivo for cancer therapy9. Moreover, siRNA complexed with stearoylated transportan-10 modified with a trifluoromethylquinoline analogue enhanced endosomal escape and was found efficient in vitro and in vivo.10 Stearoylated octaarginine (R8)11,12 and stearoylated octahistidine (H8) 13, as well as stearoylated GALA14 have also been used for siRNA complexation in combination with various liposomal envelope coatings. However, the resulting particles have often not been thoroughly characterized with respect to overall structure and biophysical properties, although such studies are recognized as important for careful interpretation of uptake studies and further rational improvement of siRNA delivery systems.15 When using peptide-based nanocarriers, the metabolic stability of the applied CPPs is a crucial determinant for an efficient delivery of associated or encapsulated cargoes.16,17,18 Previously, proteolytically stable α-peptide/β-peptoid peptidomimetics were shown to be taken up by cells four times more efficiently than Tat47-57,19 as determined by intracellular delivery of a fluorophore cargo. Also, CPP backbone stabilization may explain the improved effect of stearoylated oligoarginine analogues modified with unnatural amino acids.18 In the present study, the capacity of a proteolytically stable class of peptidomimetics to deliver siRNA was investigated by incorporating a palmitoylated analogue in a colloidal lipid-based delivery system. Initially, the effects of palmitoylation and chain length of the peptidomimetics (Figure 1) on their membrane adsorption and cellular uptake were assessed. This allowed selection of the most efficient peptidomimetic for formulation of siRNA with fusogenic lipids into a nanocarrier system that was capable of inducing efficient and specific silencing of enhanced green fluorescent protein (EGFP) in vitro.


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Figure 1. Chemical structures of α-peptide/β-peptoid peptidomimetics and R8 used in the present study.

EXPERIMENTAL SECTION Materials Solvents, α-amino acid building blocks and coupling reagents were obtained from IrisBiotech (Marjtredwitz, Germany). Rink amide resin for solid-phase synthesis of peptidomimetics was obtained from Sigma-Aldrich Chemie (Steinheim, Germany). Palmitoylated (Pam) Lys and R8 was acquired as Fmoc-Lys(Pam)-OH from Bachem (Bubendorf, Switzerland) while R8 and N-terminally carboxyfluorescein (CF)-labelled R8 (CF-R8) of >98% purity were purchased from GenScript (Piscataway, NJ, USA). Ac-Lys(Pam)-R8 and CF-Lys(Pam)-R8 of >98% purity were provided by NMI Technologietransfer (Reutlingen, Germany). Palmitoyl-oleoyl phosphatidylcholine (POPC), palmitoyl-


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oleoyl phosphatidylglycerol (sodium salt) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) were all from Avanti Polar Lipids (Alabaster, AL, USA). 2´-O-Methyl modified dicer substrate asymmetric duplex siRNAs directed against EGFP and negative control firefly luciferase (FLuc) were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) as dried, annealed, purified and desalted duplexes and re-annealed, as recommended by the supplier, in the IDT duplex buffer consisting of 30 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 100 mM potassium acetate, pH 7.5. The siRNAs had the following sequences and modification patterns: EGFP, sense 5'pACCCUGAAGUUCAUCUGCACCACcg-3', antisense 5'-CGGUGGUGCAGAUGAACUUCAGGGUCA-3', FLuc sense 5'pGGUUCCUGGAACAAUUGCUUUUAca-3', antisense 5'UGUAAAAGCAAUUGUUCCAGGAACCAG-3', where lower case letters represent 2´deoxyribonucleotides, underlined capital letters represent 2´-O-methylribonucleotides, and p represents a phosphate group. RNase-free diethyl pyrocarbonate (DEPC)-treated Milli-Q water was used for all buffers and dilutions related to experiments with siRNA. HEPES buffer pH 7.4 was from AppliChem (Darmstadt, Germany), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-phenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was provided by Promega (Madison, WI, USA), and phenazine methosulphate (PMS) was from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade.

Synthesis of peptidomimetics Non-labeled α-peptide/β-peptoid peptidomimetics (1 and 2, Figure 1) and palmitoylated compounds (6 and 7, Figure 1) were synthesized on Rink amide resin by standard Fmoc solid-phase synthesis using the appropriate dimeric building blocks20,21 and Pam-OSu (5 equiv. together with 5 equiv. DIPEA). In compounds 8-10 a palmitoyl chain was introduced via commercial Fmoc-Lys(Pam)-OH using PyBOP ACS Paragon Plus Environment

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as coupling reagent followed by N-terminal modification by acetylation or introduction of CF. The fluorophore (CF) in compounds 3-5 and 8-10 was introduced as previously described.19,22. Following cleavage from the resin, all constructs were purified by reversed-phase preparative high pressure liquid chromatography (HPLC). The identity of the compounds was verified by high-resolution mass spectrometry (HR-MS), and the purity was determined using analytical HPLC (> 95% at 220 nm). Target compounds were stored dry at −20°C until used. Analytical VP diode array detection was performed on a Shimadzu HPLC system consisting of an SCL-10A VP controller, an SIL-10AD VP autoinjector, an LC-10AT VP pump, an SPDM10A VP DAD, and a CTO-10AC VP column oven, using a Phenomenex Luna C18(2) column (150×4.6 mm; 3 µm) eluted at a rate of 0.8 mL/min. Injection volumes were 5-10 µL of a 1 mg/mL solution and separations were performed at 40 °C. The system was controlled by Class VP 6 software. Eluents A (H2O/MeCN/TFA 95:5:0.1) and B (MeCN/H2O/TFA 95:5:0.1) were employed for gradient elution. Preparative HPLC separations were carried out on a Phenomenex Luna C18(2) column (250×21.2 mm; 5 µm) using an Agilent 1100 system consisting of two preparative-scale pumps, an autosampler and a multiple-wavelength UV detector. The eluents A and B were employed with a flow rate of 20 mL/min; injection volumes were 300-900 µL. HR-MS spectra were obtained on a Bruker MicroTOF-Q LC mass spectrometer equipped with an electrospray ionization source.

HPLC retention time (RT) and HR-MS data: Compound 123: analyt. RT = 20.00 min (10-60% B in 30 min); (m/z) [M+5H]5+ obsd. = 402.0623 (calcd. = 402.0617, ∆M 1.4 ppm). Compound 223: analyt. RT = 20.68 min (10-60% B in 30 min); (m/z) [M+6H]6+ obsd. = 443.2883 (calcd. = 443.2878, ∆M 1.1 ppm). Compound 3: analyt. RT = 22.10 min (10-60% B in 30 min); prep. HPLC gradient: 10-40% B in 20 min; (m/z) [M+4H]4+ obsd. = 418.9805 (calcd. = 418.9801, ∆M 0.9 ppm). Compound 4: analyt. RT = 22.69 min (10-60% B in 30 min); prep. HPLC gradient: 10-40% B in 20 min; (m/z) [M+5H]5+ obsd. = 465.2692 (calcd. = 465.2690, ∆M 0.4 ppm). Compound 5: analyt. RT = 22.40 min (10-60% B in 30 ACS Paragon Plus Environment

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min); prep. HPLC gradient: 10-40% B in 20 min; (m/z) [M+6H]6+ obsd. = 495.9602 (calcd. = 495.9601, ∆M 0.2 ppm). Compound 6: analyt. RT = 20.18 min (20-100% B in 30 min); prep. HPLC gradient: 10-60% B in 20 min; (m/z) [M+5H]5+ obsd. = 441.3053 (calcd. = 441.3054, ∆M 0.2 ppm). Compound 7: analyt. RT = 19.27 min (20-100% B in 30 min); prep. HPLC gradient: 10-60% B in 20 min; (m/z) [M+6H]6+ obsd. = 475.9901 (calcd. = 475.9897, ∆M 0.8 ppm). Compound 8: analyt. RT = 21.93 (20-100% B in 30 min); prep. HPLC gradient: 10-70% B in 20 min; (m/z) [M+4H]4+ obsd. = 510.8118 (calcd. = 510.8119, ∆M 0.1 ppm). Compound 9: analyt. RT = 20.41 (20-100% B in 30 min); prep. 10-60% B in 20 min; (m/z) [M+5H]5+ obsd. = 538.5317 (calcd. = 538.5315, ∆M 0.3 ppm). Compound 10: analyt. RT = 19.43 (20-100% B in 30 min); prep. HPLC gradient: 10-60% B in 20 min; (m/z) [M+6H]6+ obsd. = 557.0099 (calcd. = 557.0102, ∆M 0.5 ppm).

Preparation of liposomes for ellipsometry Anionic, unilamellar liposomes (POPC:POPG, 80:20, molar ratio) for ellipsometry studies were prepared by using the thin film method as described previously.24,25,26 Briefly, in order to obtain small unilamellar vesicles (SUVs) for deposition onto supported silica surfaces, a dry lipid film was first hydrated with 10 mM HEPES buffer (pH 7.4) to a final lipid concentration of 6.5 mM. The formed large multilamellar vesicles (LMVs) were subjected to eight freeze-thaw cycles and then extruded using a LipoFast Basic extruder (Avestin, Mannheim, Germany) 31 times through a 30 nm polycarbonate membrane (Whatman, Kent, UK).

Ellipsometry The adsorption of peptidomimetics and control peptides (Figure 1) to supported lipid bilayers was studied by null ellipsometry using an Optrel Multiskop (Optrel, Kleinmachnow, Germany) equipped with a 100 mW argon laser as previously described. 24,25 The measurements were carried out at 532 nm at an angle of incidence of 67.52° in a 5 mL cuvette under stirring. The adsorption of lipid as well as of ACS Paragon Plus Environment

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peptidomimetics and peptides was monitored by measuring the changes in amplitude and phase of light reflected at the adsorbing surface, and the final adsorbed amount (Γ) was calculated.24 Prior to the adsorption study, the silica surfaces (Okmetic, Vantaa, Finland) for ellipsometry were prepared from polished silicon slides, which were oxidized to obtain a 30 nm thick oxide layer. Poly-L-lysine (Sigma– Aldrich, Seelze, Germany) was pre-adsorbed to the surfaces prior to lipid deposition to avoid peptidomimetic/peptide adsorption directly onto the silica substrate and to minimize the risk of lipid bilayer defects. The supported anionic lipid bilayer was formed by the liposome adsorption method26 using 30 nm POPC:POPG liposomes. After lipid bilayer formation, peptidomimetic/peptide was added to obtain an initial total concentration of 0.01 µM in the cuvette. This initial injection was followed by three subsequent additions to achieve final concentrations of 0.03, 0.06 and 0.1 µM. In all cases, the adsorption was monitored for approximately 45 min. All measurements were performed at least in duplicates.

Cell culture HeLa WT cells (ATCC, Manassas, VA, USA) were maintained in complete Eagle's minimal essential medium (EMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) from Fisher Scientific (Hampton, NH, USA). The complete EMEM contained Minimum Essential Medium Eagle (MEM) , 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1 mM non-essential amino acids (all from SigmaAldrich) and 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA, USA). The human non-small cell lung carcinoma cell line H1299 (ATCC, CRL-5803) and H1299 cells stably expressing EGFP (EGFPH1299)27 were maintained in RPMI 1640 Medium (Fisher Scientific) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Sigma-Aldrich), 0.2 mg/ml geneticin (Invitrogen), and 10% (v/v) fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria). Cells were cultured at 37 °C in an atmosphere of 5% CO2 and 95% O2.


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Cellular uptake of peptidomimetics/peptides studied by flow cytometry HeLa cells were seeded in 24-well tissue culture plates (surface area 1.9 cm2/well) in 1 mL complete EMEM containing 10% (v/v) FBS, 1×105 cells per well, and were incubated for 24 h prior to use. The cells were washed with phosphate-buffered saline (PBS) and subsequently incubated with pre-mixed samples containing a total peptidomimetic/peptide concentration of 1 µM in complete EMEM with FBS for 60 min at 37 °C. Afterwards, the cells were washed with PBS and detached by trypsin-EDTA treatment, and 1 mL ice-cold PBS containing 10% (v/v) FBS was subsequently added to each well. The suspended cells were transferred to tubes, centrifuged (1076 × g, 4 °C, 5 min), and resuspended twice in PBS, after which the cells were resuspended in ice-cold PBS containing 10% (v/v) FBS and 1 µg/mL propidium iodide (PI, Invitrogen), and analyzed. The cells were kept on ice prior to analysis carried out immediately after the last resuspension. Acquisition and analysis of 10,000 cells per sample were done with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) using the CellQuest software (Becton Dickinson). Dead cells were gated out based on their light scattering properties as well as PI fluorescence, and the mean fluorescence intensity (MFI) of viable EGFP single positive cells was determined. Cells exposed to complete RPMI with 10% (v/v) FBS were used as negative control. Experiments were performed in triplicate.

Preparation of an siRNA-loaded nanocarrier Peptidomimetic-modified, siRNA-loaded liposomes were prepared according to a protocol adapted from studies with stearoyl R8,11 but with some modifications of the method. In brief, the siRNA was complexed with 7 in RNase-free water at different peptidomimetic nitrogen-to-siRNA phosphate (N/P) ratios (0.2 to 5), and the particle size distribution of the resulting complexes was measured by using dynamic light scattering (DLS) as described below. An N/P ratio of 2 resulted in the smallest and most monodisperse complexes, and this ratio was used in the subsequent formulation steps. A dry lipid film ACS Paragon Plus Environment

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composed of DOPE (450 nmol) and CHEMS (100 nmol) was hydrated with 1 mL of siRNA:7 complex dispersion (1.5 µM siRNA) at 50 °C for 1 min, followed by sonication in a Branson 2510 bath sonicator (Branson, Danbury, CT, USA) for 1 min. The heating-sonication cycle was repeated in total three times. The dispersion was then vortexed for 3 min and heated for 1 min, and the vortexingheating cycle was repeated in total three times, followed by sonication for 1 min. Finally, an aqueous solution containing 5.5 nmol of 7 was slowly added, and the dispersion was mixed by gently tapping on the tube. The mixture was annealed for 30 min at room temperature before further use.

Size and zeta-potential The average particle size distribution and PDI were analyzed by DLS using the photon correlation spectroscopy technique. The surface charge of the particles was estimated by analysis of the zetapotential (laser-Doppler electrophoresis). The measurements were performed in triplicate at 25 ºC using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser and 173º detection optics. For viscosity and refractive index, the values of pure water were used. Malvern DTS v.5.10 software (Malvern Instruments) was used for data acquisition and analysis.

Cryo-TEM Morphological evaluation was carried out by cryo-transmission electron microscopy (cryo-TEM) using a Philips CM120 BioTWIN transmission electron microscope (Philips, Eindhoven, The Netherlands). Samples for cryo-TEM were prepared under controlled temperature and humidity conditions within an environmental vitrification system. A small droplet (5 µL) of sample was deposited on a Pelco Lacey carbon filmed grid. After carefully spreading the drop excess liquid was removed with a filter paper forming a thin (10-500 nm) sample film. Then, the samples were immediately immersed into liquid ethane at -180 °C. The vitrified samples were subsequently transferred in liquid nitrogen to an Oxford CT3500 cryo holder connected to the electron microscope. The sample temperature was always kept ACS Paragon Plus Environment

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below -180 °C. All observations were made in the bright field mode and at an acceleration voltage of 120 kV. Digital pictures were taken with a Gatan Imaging Filter 100 CCD camera (Gatan, Pleasanton, CA, USA).

SAXS Synchrotron small-angle X-ray scattering (SAXS) measurements were performed at beamline I911-4 (MAX-lab, Lund, Sweden) using a 49-period, 3.5 T multipole-wiggler producing a high-flux photon beam with a wavelength of 0.91 Å. The scattering was recorded using a MAR165 CCD detector. With the applied instrumental setup, the effective q-range was 0.0103 to 0.65 Å-1. Silver behenate (CH3(CH2)20-COOAg) with a known d-spacing of 58.380 Å was used to calibrate the angular scale of the measured intensity.28 Before the measurements, the sample was up-concentrated by centrifugation using a Vivaspin 2 column (Satorius Stedim Biotech, Goettingen, Germany) to a final theoretical lipid concentration of 10 mg/mL. The sample was measured in a quartz glass capillary (diameter 1.5 mm) and thermostated in a custom-built sample holder block of brass connected to a circulating water bath (Julabo, Seelbach, Germany). The temperature was set to 25 ºC, and the sample exposure time was 300 s.

The 2D scattering data were azimuthally averaged, normalized by the incident radiation intensity and the sample exposure time, and corrected for background and detector inhomogeneities using the software BioXTAS RAW.29 The radially averaged intensity I is given as a function of the scattering vector q (q = 4π sinθ /λ, where λ is the wavelength and 2θ is the scattering angle). For indexing of the mesophases and calculation of the corresponding unit lattice parameters, the reflection laws for the lamellar and inverse hexagonal phases were applied.30

Viability assay ACS Paragon Plus Environment

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Wild type H1299 cells were seeded in 96-well plates at a density of 10,000 cells/well and incubated at 37 °C as described above for 24 h prior to treatment with siRNA-loaded nanocarriers dispersed in 100 µL RPMI 1640 medium containing 10% (v/v) FBS at siRNA concentrations ranging from approximately 3.75 to 480 nM. After 24 h incubation (37 °C, 100 rpm), the growth medium was removed, and the cells were washed three times with Hanks Balanced Salt Solution (HBSS, Invitrogen). Freshly prepared MTS/PMS reagent in HBSS (100 µL) was added to each well, and then incubation was continued for 1 h. The reagent consisted of 240 µg/mL MTS and 2.4 µg/mL PMS. Cells incubated with sodium dodecyl sulfate (SDS) 0.2% (w/v) were used as positive control, while untreated cells were used as a negative control. The cell viability was determined by measuring the absorbance of enzymatically formed formazan at 492 nm. The obtained results were corrected for particle scattering and normalized to the positive and negative controls and given as mean values of three replicates.

In vitro gene silencing activity The gene silencing activity of freshly prepared siRNA-loaded nanocarriers was evaluated in vitro essentially as previously described31,32 using the EGFP-H1299 cells. The cells were seeded in 24-well tissue culture plates in RPMI1640 medium at a density of 8×104 cells per well (corresponding to 4.2 × 104 cells/cm2). After 24 h of growth, the proliferating cells were washed twice with PBS and incubated with 300 µL of pre-mixed samples (nanocarriers and control) in RPMI 1640 medium containing 10% (v/v) FBS. Cells exposed (for 24 h) to RPMI 1640 medium with 10% (v/v) FBS without the nanocarrier were used as the negative control. With the number of cells seeded, non-confluency was evident at the time for addition of the nanocarrier, i.e. after 24 h of culturing as well as after additional 24 of incubation with the nanocarrier, and the level of EGPF expression in the cells was maintained during the experiment (Figure S1 and S2, Supplementary Information). As a positive control, Lipofectamine 2000 (Invitrogen), which is a well-known transfection reagent, but not a potential nanocarrier for in vivo applications, was used in amounts recommended by the manufacturer with 120 nM siRNA. After ACS Paragon Plus Environment

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incubation of the cells with the nanocarriers for 24 h at 37 °C, the cells were harvested, washed and analyzed by flow cytometry as described above.

Statistics Statistically significant differences were assessed by analysis of variance (ANOVA) at a 0.05 significance level, followed by the Tukey´s post test.

RESULTS AND DISCUSSION Palmitoylation enhances adsorption to supported lipid bilayers, while chain length is important for non-lipidated peptidomimetics The initial interaction between CPPs/peptidomimetics and membranes is crucial for their subsequent intracellular translocation. 33,34,35 Therefore, the amounts of membrane-adsorbed α-peptide/β-peptoid peptidomimetics, displaying alternating cationic and hydrophobic residues, and with different chain length and N-terminal modification (palmitoylation or acetylation, Figure 1), were assessed by ellipsometry. Lipid bilayers of a composition (POPC:POPG, 80:20 molar ratio) representative for the eukaryotic plasma membrane were used, and the longest compound (2) afforded an adsorbed molar amount per area that was almost two-fold higher than that of its shorter counterpart 1 and 30 times higher than the adsorbed molar amount of R8 at a concentration of 0.1 µM, even though 2 and R8 carries the same net positive charge (Figure 2A). However, a similarly strong dependency on oligomer length was not observed when the N-termini were covalently modified with a palmitoyl moiety (compounds 6 and 7, Figure 2B). Overall, the palmitoylated peptidomimetics (Figure 2B) showed a much higher degree of adsorption (∼10-fold) as compared to the non-palmitoylated constructs (Figure 2A), while the adsorption of palmitoylated R8 was dramatically increased (equaling that of 6 and 7) as compared to its non-lipidated counterpart (Figure 2B).


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Figure 2. Adsorbed amount (Γ) of selected α-peptide/β-peptoid peptidomimetics of different length and with acetyl (A) or palmitoyl (B) N-terminal modifications onto supported POPC:POPG (80:20) bilayers. R8 and palmitoylated octaarginine (Pam-R8) were included as reference CPPs. Values represent the mean ± SD (n ≥ 2). *designates non-detectable adsorption.

This demonstrates that palmitoylation of the peptidomimetics within this size range greatly enhances and dominates their propensity for adsorption to immobilized lipid membranes, increasing the adsorbed amount by at least one order of magnitude as compared to the corresponding non-lipidated oligomers. The immobilization of POPC:POPG bilayers to the poly-L-lysine-coated silica surface was not hampered by the addition of the palmityolated compounds (results not shown). Due to the dominating influence of lipidation, the effect of the peptidomimetic length is insignificant, and hence the importance of the aromatic residues and the net amount of positive charges for membrane interaction previously reported36 appears negligible in this experimental setup testing the peptidomimetics in solution. The critical micelle concentration of 7 is above 10 µM, as determined by isothermal titration ACS Paragon Plus Environment

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calorimetry (data not shown), which is much higher than the highest concentration used in the ellipsometry studies. Thus, the distribution of individual palmitoylated molecules at the immobilized lipid bilayer is not in competition with extensive micelle formation.

Both palmitoylation and chain length of the peptidomimetics influence cellular uptake Previous studies have shown that membrane adsorption of α-peptide/β-peptoid peptidomimetics often is directly correlated to their level of cellular uptake.36 This tendency was corroborated in the present study for non-palmitoylated peptidomimetics with systematically varied chain length as the CF-labeled analogues of compounds 1 and 2 (Figure 2A) exhibited improved cellular uptake with increasing chain length (i.e. 4 and 5, Figure 3). Noticeably, the contribution from the hydrophobic β-peptoid residues in the short cationic peptidomimetic 3 resulted in an uptake comparable to that of the CF-R8 despite a significantly higher net positive charge of the latter (i.e. +4 vs. +8). The ratios of uptake of the CFlabeled peptidomimetics relative to the CF-R8 correspond well to previous results with similar αpeptide/β-peptoid peptidomimetics displaying either lysine or homoarginine as cationic moieties. 36,37

Figure 3. Quantitative flow cytometric analysis of the cellular uptake of peptidomimetics and reference R8 peptides. HeLa cells were incubated for 60 min with 1 µM of CF-labeled peptidomimetics or CF-R8


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at 37 °C. The cellular mean fluorescence intensity data represent mean ± SD (n =3). Data has been normalized to the mean fluorescence intensity of CF-R8. Significant differences from CF-R8 are indicated by * p < 0.05, *** p < 0.001.

The cellular uptake of lipidated 8, 9 and 10 exhibited a significant, but weak dependence of the peptidomimetic chain length, and palmitoylation generally lead to a ∼2-fold enhanced cellular uptake for the shorter oligomers (Figure 3). However, for the 16-mer peptidomimetic the effect of lipidation was almost absent with respect to the cellular uptake (Figure 3). Thus, the relative uptake enhancement due to palmitoylation decreased with increasing length of the peptidomimetic oligomers. Interestingly, membrane adsorption of the lipidated peptidomimetics (i.e. 6 and 7) and palmitoylated octaarginine (Pam-R8) were up to 10-fold higher than that of the non-palmitoylated counterparts. The aromatic residues in α-peptide/β-peptoids are obviously important for their efficient cellular uptake as both peptidomimetics 1 and 2 displayed significantly higher membrane-adsorptive properties than R8 having the same charge as the longer oligomer 2. In addition, the presence of the large CF moiety may also contribute to the overall membrane-interacting properties of the non-palmitoylated compounds investigated explaining the less distinct effect of palmitoylation in the uptake study involving fluorophore-labeled compounds. The possible contribution of fluorophore labels on cellular uptake of CPPs has recently been reviewed.38 However, the smaller relative differences in uptake of palmitoylated peptidomimetics of varying length as compared to that found for the non-lipidated analogues is clearly caused by the effect of the hydrophobic tail that dominates their membrane interaction rather than the presence of the fluorophore. Overall, the longest palmitoylated peptidomimetic exhibited superior properties in terms of membrane adsorption with its CF-labeled counterpart also giving rise to the highest cellular uptake (Figure 3), and thus compound 7 was considered most suitable for incorporation into the lipid-based siRNA delivery system.


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Preparation and characterization of lipid-based siRNA nanocarrier Peptidomimetic-modified, siRNA-loaded liposomes were prepared according to a protocol adapted from studies on stearoylated R8.11 Thus, construct 7 was pre-complexed with siRNA at different N/P ratios, and based on size measurements of the resulting complexes an N/P ratio of 2 was chosen for further formulation with the lipid envelope. At this N/P ratio, the complexes had an average size of 49.7 ± 3.5 nm (polydispersity index 0.3) and a slightly positive zeta-potential of +9.8 ± 0.4 mV (n = 3). We suggest that these complexes are stabilized by i) positive electrostatic interactions between the anionic siRNA and the cationic peptideomimetic, in combination with ii) hydrophobic interactions involving the palmitoyl moieties and/or the hydrophobic β-peptoid residues. The complexes were incorporated into negatively charged DOPE/CHEMS vesicles, which subsequently were further surface-grafted with 7. The resulting lipid-based nanocarrier particles had an average size of 232.1 ± 6.6 nm (PDI 0.2, Figure 4) and a positive zeta-potential of +41.4 ± 1.8 mV (n = 3). This increase in size as well as in zeta-potential clearly indicates that the peptidomimetic:siRNA complexes associate with the net anionic lipid mixture forming vesicles, which subsequently are coated with cationic 7 added after the hydration step.

Figure 4. A representative volume-based particle size distribution of the siRNA nanocarrier particles.


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Lipid-based siRNA nanocarrier structures consist of unilamellar and multilamellar, onion-like spherical vesicles with co-existing lamellar and inverse hexagonal lipid phases. To examine the structure of the resulting nanocarrier particles in more detail, cryo-TEM and SAXS were performed. Micrographs obtained from cryo-TEM showed that several types of nanostructures coexisted in the formulated sample (Figure 5).


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Figure 5. Representative cryo-TEM images of siRNA-loaded, lipid-based nanocarriers postmodified with palmitoylated α-peptide/β-peptoid peptidomimetics. (A) A general view of the detected nanoparticles. (B) Close up of a multilamellar vesicle. (C) Close up of a multilamellar nanocarrier illustrating the effect of irradiation The scale bars represent 100 nm.

Multilamellar, electron-dense liposomes with onion-like appearance were the dominant nanostructures with a varying number of bilayers (Figure 5), and the interlamellar spacing was roughly estimated from the intensity profile of the micrographs to be approximately 50 Å well in line with reports on condensed lamellar structures composed of alternating layers of cationic lipid bilayers and hydrated oligonucleotides.39 It has previously been suggested that mixing oligonucleotides with cationic lipids may lead to membrane fusion and subsequent lamellar condensation.40 In the present study, liposomes composed of an initial binary mixture of neutral (DOPE) and anionic (CHEMS) lipids were subsequently treated with palmitoylated α-peptide/β-peptoid 7, which is a cationic amphiphile with CPP-like properties. The fatty acid moiety of the highly cationic peptidomimetic 7 may associate with or even insert into the lipid membrane during preparation of the vesicles, as previously reported for cationic lipids39, thereby conferring a positive charge to the final nanocarrier particles. Nevertheless, the closely packed multilamellar structures observed by cryo-TEM show that the electrostatic repulsions between the highly cationic CPP-like lipid components are not destabilizing the vesicle encapsulating siRNA. Thus, it is likely that the negatively charged siRNA in fact is bridging adjacent lipid membrane bilayers thereby reducing the electrostatic repulsion as indicated from other studies concerning nucleotide-containing liposomes.41,42 During the cryo-TEM experiments it was observed that the condensed regions between bilayers were the first to be damaged by the radiation (indicated by the white spots in Figure 5C). Since nucleic acids are more sensitive to radiation damage than lipids, this further infers that the siRNA indeed is located between the lipid bilayers. This additional finding further supports that the initially positively charged siRNA:peptidomimetic complexes subsequently ACS Paragon Plus Environment

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rearrange into multilamellar structures upon mixing with negatively charged lipids and/or during the hydration of the surface instead of simply being covered by the lipid film.

The SAXS study revealed further structural characteristics of the nanocarrier. The scattering pattern at 25 °C (Figure 6A) indicated the coexistence of lamellar (Lα) and inverse hexagonal (HII) phases. Considering the above-mentioned cryo-TEM observations, the predominant reflection most likely corresponds to the first reflection of a lamellar phase. The lamellar lattice spacing was calculated to be 50.4 Å, which is in good agreement with the lipid bilayer thickness of approximately 50 Å as estimated by cryo-TEM as well as with a previous study characterizing cationic lipid:ODN vesicles.39 The three remaining peaks observed in the SAXS pattern (Figure 6A) were consistent with the (100), (110) and (200) reflections for an HII phase. The unit cell parameter of this phase, which corresponds to the distance between adjacent hydrophilic cylindrical nanochannels embedded in a hydrophobic continuous matrix, was calculated to be 75.2 Å close to the value of 74.2 Å found for the fully hydrated pure DOPE at 25 °C.43 The similar unit cell parameter dimensions for pure DOPE and the investigated nanocarriers under full hydration conditions may be attributed to the high DOPE content in the investigated lipidic system. Similarly, it has been reported for various self-assembled systems that the lipid composition is modulating the nanostructures formed.44


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Figure 6: (A) The SAXS diffraction pattern for siRNA-containing lipid-based nanocarriers modified with palmitoylated α-peptide/β-peptoid amphiphiles. (B) The SAXS 2D detector pattern. The signal-tonoise ratio was low for the performed measurements.

Because of its inverted cone-like shape, the nonbilayer-forming lipid DOPE preferably adopts an inverted hexagonal phase HII, that is highly fusogenic.45 However, combining DOPE with the pHsensitive lipid CHEMS results in the formation of a stable lamellar phase and thereby in pH-sensitive liposomes. In an acidic endosomal environment, the shape of the CHEMS molecule will change from a cone-like shape to a cylindrical shape due to protonation of the headgroup with a concomitant loss of bilayer-stabilizing properties. As a result, DOPE may undergo a lamellar-to-hexagonal phase transition ACS Paragon Plus Environment

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at reduced pH, which could promote fusion of liposomes and endosomal membranes leading to destabilization of the endosomes and hence release of e.g. plasmid DNA to the cytoplasm.46 In contrast, for siRNA delivery with cationic liposomes, it has been shown that inclusion of DOPE in the formulation does not promote transfection efficiency of the liposomes, but rather causes increased toxicity and lower target-specific gene silencing.47

A peptidomimetic-modified lipid nanocarrier mediates efficient gene silencing. The transfection efficiency of siRNA:peptidomimetic (7) complex and of peptidomimetic-modified, siRNA-containing lipid-based nanocarriers were examined in H1299 cells stably expressing EGFP, and the silencing effect was measured at the protein level by flow cytometry. Gene silencing measured at the protein level in this cell type has previously been found to correlate to silencing at the mRNA level48.


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Figure 7. EGFP gene knockdown activity of siRNA incorporated into nanocarriers (NC). As negative controls, the cells were treated with medium (column 1) or EGFP siRNA (120 nM) alone (column 2), whereas the positive control was EGFP siRNA (120 nM) complexed with lipofectamine 2000 (LF, column 3). Other negative controls were FLuc siRNA (120 nM) with LF (column 4) and peptidomimetic 7 (160 nM) alone (column 5). Additional controls were EGFP siRNA (120 nM) and 7 (156 nM) complex at an N/P ratio 2 (column 6), and nanocarriers encapsulating the FLuc negative control siRNA (120 nM, column 7). Specific concentration-dependent EGFP silencing was demonstrated at 120 nM (column 8) and 30 nM (column 9) siRNA concentrations. Results denote the means ± SD of triplicate samples. Significant differences from the untreated cells are indicated: *** p < 0.001. Y- axis presents the relative mean fluorescence intensity (MFI) of the transfected EGFP-H1299 cells, which was normalized to the MFI of untreated cells.

The simple complexes of siRNA and lipidated peptidomimetic (EGFP and 7 Complex) did not mediate gene silencing under the applied experimental conditions (Figure 7). In contrast, lipid-based nanocarriers containing palmitoylated peptidomimetic 7 and EGFP siRNA (at 120 nM and 30 nM) showed a significant gene silencing as compared to untreated cells (p < 0.001) with the highest concentration being at the level of the positive control used. No difference in the level of PI-stained cells in samples exposed to the standard transfection reagent LF and the nanocarrier was evident (Figure S3, Supplementary Information). Gene silencing activity of siRNA encapsulated in this novel nanocarrier was dose-dependent (Figure 7). These studies thus demonstrate that the nanocarrier is capable of delivering siRNA to the RNAi pathway in the cytoplasm under the present experimental conditions. The results from the ellipsometry studies quantifying the adsorption of the peptidomimetic to immobilized POPC:POPG lipid bilayers, which was chosen as a model for the plasma membrane indicate that coating the nanocarriers with peptidomimetic is an attractive strategy for promoting the intracellular delivery of siRNA. However, further studies are needed to elucidate the cellular uptake, ACS Paragon Plus Environment

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intracellular trafficking and endosomal escape mechanisms involved in the process and the contributions of the structural characteristics of the carrier and the peptidomimetics, respectively.

The maximum tolerated concentration of the nanocarrier by the H1299 cells was estimated in a cell viability study involving incubation of the cells with the nanocarrier administered in serum-containing growth medium. Figure 8 depicts the relative cellular viability upon incubation with various concentrations of siRNA present in nanocarrier formulation. It is evident that an siRNA concentration in the formulation up to 120 nM, corresponding to a concentration of 7 in the formulation of 160 nM, was tolerated by H1299 cells.

120 100

Relative viability (%)

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80 60 40 20 0 1

10

100

1000

Concentration of siRNA in nanocarriers (nM)

Figure 8. Effect of the nanocarrier on the viability of H1299 cells. The cell viability after exposure to the NC for 24 h was determined by the MTS/PMS assay. The data is expressed as the mean percentage viability relative to the control +/- SD (n=3).

CONCLUSION


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The designed peptidomimetic-containing DOPE/CHEMS-based nanocarrier incorporating an Nterminal palmitoylated peptidomimetic as targeting moiety via surface modification was found to mediate efficient gene silencing with no indications of cytotoxicity. The liposomal carrier encapsulating siRNA appeared in suspension as multilamellar, onion-like spherical vesicles, and SAXS analysis revealed the co-existence of lamellar structures along with a hexagonal phase, which might be an important factor for efficient, nanocarrier-mediated endosomal escape resulting in cytosolic siRNA delivery. Thus, such lipidated peptidomimetics appear to be promising components in lipid-based vesicles for cytosolic delivery of encapsulated siRNA. As a result of investigations of membrane adsorption and cellular uptake of palmitoylated peptidomimetics with different oligomer lengths it was evident that palmitoylation was a major determinant for membrane adsorption, yet influenced the cellular uptake less. These findings infer further exploration of the capacity of this class of peptidomimetics for delivery of siRNA formulated as colloidal nanocarrier systems.The present work represents the proof-of-concept for utilization of α-peptides/β-peptoid peptidomimetics with alternating sequences as components possessing dual properties as RNA-complexing agents and as efficient cellpenetrating peptidomimetics for nanocarriers that may be exploited in delivery of biomacromolecular drugs to intracellular targets.

ACKNOWLEDGMENT This work was funded by the Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark. In addition, we are grateful to the Swedish Research Council and the European Communities Sixth Framework Programme Integrated Project MediTrans, Contract No. 026668 for financial support. Equipment funding was obtained from The Danish National Advanced Technology Foundation and The Danish Agency for Science, Technology and Innovation. We are grateful to Maria L. Pedersen (Department of Pharmacy, University of Copenhagen) for assistance with cell culturing, Lise-Britt ACS Paragon Plus Environment

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Wahlberg (Department of Pharmacy, Uppsala University) for excellent technical support and Dr. Lovisa Ringstad (Department of Pharmacy, Uppsala University) for scientific advice on ellipsometry. MaxLab is acknowledged for providing beamtime and the instrument for the SAXS studies. The research leading to the SAXS results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 226716.

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254x114mm (96 x 96 DPI)



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Chemical structures of α-peptide/β-peptoid peptidomimetics and R8 used in the present study. 251x176mm (300 x 300 DPI)


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Adsorbed amount (Γ) of selected α-peptide/β-peptoid peptidomimetics of different length and with acetyl (A) or palmitoyl (B) N-terminal modifications onto supported POPC:POPG (80:20) bilayers. R8 and palmitoylated octaarginine (Pam-R8) were included as reference CPPs. Values represent the mean ± SD (n ≥ 2). *designates non-detectable adsorption. 99x100mm (300 x 300 DPI)



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Quantitative flow cytometric analysis of the cellular uptake of peptidomimetics and reference R8 peptides. HeLa cells were incubated for 60 min with 1 µM of CF-labeled peptidomimetics or CF-R8 at 37 °C. The cellular mean fluorescence intensity data represent mean ± SD (n =3). Data has been normalized to the mean fluorescence intensity of CF-R8. Significant differences from CF-R8 are indicated by * p < 0.05, *** p < 0.001. 66x52mm (300 x 300 DPI)


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A representative volume-based particle size distribution of the siRNA nanocarrier particles. 66x53mm (300 x 300 DPI)



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(A) The SAXS diffraction pattern for siRNA-containing lipid-based nanocarriers modified with palmitoylated α-peptide/β-peptoid amphiphiles. (B) The SAXS 2D detector pattern. The signal-to-noise ratio was low for the performed measurements. 82x141mm (300 x 300 DPI)


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EGFP gene knockdown activity of siRNA incorporated into nanocarriers (NC). As negative controls, the cells were treated with medium (column 1) or EGFP siRNA (120 nM) alone (column 2), whereas the positive control was EGFP siRNA (120 nM) complexed with lipofectamine 2000 (LF, column 3). Other negative controls were FLuc siRNA (120 nM) with LF (column 4) and peptidomimetic 7 (160 nM) alone (column 5). Additional controls were EGFP siRNA (120 nM) and 7 (156 nM) complex at an N/P ratio 2 (column 6), and nanocarriers encapsulating the FLuc negative control siRNA (120 nM, column 7). Specific concentrationdependent EGFP silencing was demonstrated at 120 nM (column 8) and 30 nM (column 9) siRNA concentrations. Results denote the means ± SD of triplicate samples. Significant differences from the untreated cells are indicated: *** p < 0.001. Y- axis presents the relative mean fluorescence intensity (MFI) of the transfected EGFP-H1299 cells, which was normalized to the MFI of untreated cells. 120x161mm (300 x 300 DPI)



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Effect of the nanocarrier on the viability of H1299 cells. The cell viability after exposure to the NC for 24 h was determined by the MTS/PMS assay. The data is expressed as the mean percentage viability relative to the control +/- SD (n=3). 135x88mm (300 x 300 DPI)


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Î²-Peptoid

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