Comparative Study on the In Vitro Cytotoxicity of Linear, Dendritic, and

Aug 29, 2012 - Institute of Bioengineering, Laboratory of Cellular Biotechnology, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 6,. CH-1015...
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Comparative Study on the In Vitro Cytotoxicity of Linear, Dendritic, and Hyperbranched Polylysine Analogues Zuzana Kadlecova,† Lucia Baldi,‡ David Hacker,‡ Florian Maria Wurm,‡ and Harm-Anton Klok*,† †

Institut des Matériaux et Institut des Sciences et Ingénierie Chimiques, Laboratoire des Polymères, Ecole Polytechnique Fédérale de Lausanne (EPFL), Bâtiment MXD, Station 12, CH-1015 Lausanne, Switzerland ‡ Institute of Bioengineering, Laboratory of Cellular Biotechnology, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 6, CH-1015 Lausanne, Switzerland S Supporting Information *

ABSTRACT: Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC50 values for the LPL analogues were ∼5−250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (≥3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain ε-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA.

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INTRODUCTION Polycations such as polyethyleneimine (PEI), polylysine, and polyamidoamine dendrimers (PAMAM) are widely used as nonviral vectors for gene delivery.1−6 Nonviral gene delivery starts with complexation of DNA to form a polyplex and ends with intracellular release, nuclear entry, and expression of DNA. Successful gene delivery requires high transfection rates and low toxicity. Many polyplexes are net positively charged and prepared with an excess of the polycation. Free, unbound polycation is also regenerated after the intracellular dissociation of the polyplex.3,7 As a consequence, a fundamental knowledge and understanding of the cytotoxicity of synthetic polycations is critical to the development of efficient gene delivery strategies. The in vitro cytotoxicity of synthetic polycations is usually evaluated using tests that probe the metabolic activity (such as in, e.g., the MTT and MTS assays) or membrane integrity (e.g., LDH release assay or hemolysis) of cells.8−10 Irrespective of chemical composition, the cytotoxicity of synthetic polycations generally increases with increasing concentration,9 molecular weight11 (or generation in the case of dendrimer-based polycations12,13), and charge density.9 Seib et al. compared the cytotoxicity of linear and branched PEI samples of similar © 2012 American Chemical Society

molecular weight (≈20 kDa) and found almost identical IC50 values, suggesting the absence of an influence of polymer architecture.14 Hemolysis10 and LDH release assays15,16 on low and high molecular weight polylysine samples have revealed higher toxicity of the higher molecular weight analogues. While the studies cited above point toward first structure− activity relationships that govern the overall cytotoxicity of synthetic polycations, they do not provide insight into the underlying mechanisms.8 Cell death is a complex process that can proceed via various pathways, which can be differentiated in time and each of which is characterized by specific biochemical or morphological features. Two pathways that have been identified to play a role in polycation induced cytotoxicity are necrosis and apoptosis.8,17−19 Necrosis describes immediate cell death due to catastrophic toxic and traumatic events with massive cell swelling, injury to cytoplasmic organelles, and rapid collapse of internal homeostasis. Apoptosis, or preprogrammed cell death, is an active process that typically only becomes Received: June 18, 2012 Revised: August 24, 2012 Published: August 29, 2012 3127

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Scheme 1. Schematic Representation of the Different Polylysine Analogues Investigated in This Study: (A) Linear Polylysine (LPL); (B) Hyperbranched Polylysine (HBPL); (C) Third Generation Dendritic Polylysine (DPL)

significant after longer exposure times (12−24 h).17 Characteristic features of apoptotic cell death include release of cytochrome C from mitochondria, activation of caspase 3, and mitochondrial membrane permeabilization. Experiments with linear and branched PEI have revealed that both necrotic and apoptotic pathways contribute to the cytotoxicity of these polycations.8,17 Apoptosis has been observed for PAMAM dendrimers20,21 and linear polylysine,15 as well as for PEI alone or complexed with DNA.18 In the case of linear polylysine, low and high molecular weight polylysines were found to initiate apoptosis differently.15 Beyond these examples, however, more comprehensive studies that attempt to establish structure− activity relationships and correlate, for example, polymer composition, molecular weight, and architecture with specific cell death pathways are relatively scarce, but could be of great help for the future development of more efficient gene delivery approaches. This manuscript presents the results of a comparative study on the in vitro cytotoxicity of a library of polylysine analogues that differ with respect to molecular weight and architecture. The investigated polycation library includes linear polylysine (LPL) and dendritic polylysine (DPL), as well as hyperbranched polylysine (HBPL; Scheme 1). Hyperbranched polylysine is structurally related to its dendritic analogue. However, whereas DPLs are perfectly monodisperse macromolecules with a regular, defect-free branched architecture, HBPL is neither monodisperse nor free of defects. In contrast to DPL, which can only be obtained via a laborious multistep synthetic pathway, HBPL is very easily accessible (also on a large scale) via a one-step process.22 Apart from offering the possibility to investigate the influence of structural heterogeneities on the biological properties (in the context of this manuscript: cytotoxicity) of lysine-based polycations, the facile synthetic access to HBPL makes these polycations potentially interesting alternatives to PEI as nonviral gene delivery vectors as was demonstrated recently.23 The experiments discussed in this report have been carried out with CHO DG44 cells, which

is an industrially relevant cell line that is used for the production of recombinant proteins.24 The results of this study, however, may also be relevant for other applications of these nonviral vectors including, for example, basic studies on the expression, regulation, and function of genes and proteins, as well as possible therapeutic applications.

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MATERIALS AND METHODS

Materials. Nα,Nε-Di-(tert-butoxycarbonyl)-L-lysine dicyclohexylammonium salt was obtained from Iris Biotech GmbH (Marktredwitz, Germany). L-Lysine hydrochloride was obtained from BASF AG (Ludwigshafen, Germany). Hydroxybenzotriazole (HOBt·H2O) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were obtained from Novabiochem (Dietikon, Switzerland). Benzylamine, 4-dimethylaminopyridine (DMAP), and triphosgene were obtained from Sigma Aldrich (Buchs, Switzerland) and used as received. All other solvents and reagents were purchased from commercial suppliers and were used as received. Triethylamine (Sigma Aldrich) was distilled over KOH under a flow of nitrogen prior to use. THF and methylenechloride were purified by passage over alumina and copper columns and DMF dried by passage over molecular sieves using a Pure Solv 400 solvent purification system. Ultrahigh quality water with a resistance of 18.2 MΩ·cm (at 25 °C) was obtained from a Millipore Milli-Q gradient machine fitted with a UV lamp and 0.22 μm filter. A stock solution of tetramethylene rhodamine-labeled dextran (dextran-TMR, molecular weight 40000 g·mol−1, Invitrogen AG, Basel, Switzerland) was prepared in PBS at a concentration of 1 mg·mL−1. Solutions of linear PEI (Polysciences, Eppenheim, Germany) were prepared in Milli-Q water at 1 mg·mL−1. The pH of this solution was adjusted to 7 with 0.1 M HCl and filtersterilized. The number-average molecular weight and polydispersity (Mw/Mn) of this PEI sample were 9800 g·mol−1, respectively, 2.9, as determined by GPC. Characterization. NMR Spectroscopy. NMR spectra were recorded on a Bruker Avance 400 spectrometer at room temperature. Chemical shifts are reported relative to residual nondeuterated solvent. For each measurement, 15 mg of dry polymer was dissolved in 1 mL of D2O. Spectra were recorded with a relaxation time (d1) of 10 s by averaging 128 scans. 3128

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Gel Permeation Chromatography (GPC). Samples of polycations were dissolved at a concentration of 5 mg·mL−1 in 0.1 M aqueous NaHCO3. Analyses were carried out on a Viscotek TDA 300 system equipped with two Shodex OHpak columns (SB-803 HQ, SB-804 HQ) as well as a HB-G guard column. As eluent, 0.1 M NaHCO3 was used at a flow rate of 0.5 mL·min−1 at 25 °C. For each analysis, a sample volume of 100 μL was injected and sample elution was monitored simultaneously with a viscometer and differential refractive index detector. Elution times were converted into molecular weights using a universal calibration curve, which was constructed using poly(ethylene oxide) standards with a narrow molecular weight distribution.25 Mass Spectrometry. Samples for mass spectrometry were dissolved in methanol. Electrospray ionization mass spectrometry experiments (ESI-MS) were performed on a Waters Ultima API quadrupole timeof-flight mass spectrometer (Q-TOF) fitted with a standard Z-spray source and operating in the positive ionization mode. Data were analyzed using MassLynx 4.1 software. Osmolality Measurement. Osmolalities were determined using a multiosmette freezing point osmometer (Precision Systems Inc., Natick, MA) that was calibrated with a range of standards with osmolality values of 100, 500, 1000, 1500, and 2000 mOsm·kg−1 (Precision Systems Inc. Standard Solutions, Fisher Scientific). Osmolality measurements were carried out with the samples LPL1, DPL1, and DPL6, which were dissolved in the cell culture medium at their EC50 concentration. The osmolality of ProCHO5 cell culture medium (Lonza AG, Verviers, Belgium) was determined as well. Reported values represent the mean of the three independent experiments. Synthesis. Hyperbranched Polylysine (HBPL). HBPL was synthesized via a modification of a previously published protocol.22 First, L-lysine hydrochloride (27.45 g, 0.15 mol) dissolved in 55 mL Milli-Q water was neutralized by addition of KOH (8.4 g, 0.15 mol). Then, the aqueous solution of lysine was heated to 150 °C for 48 h under a stream of N2. After that, the crude polymerization product was first dialyzed (Snakeskin Dialysis Tubing, Thermo Fisher Scientific, Lausanne, Switzerland, molecular weight cut off: 3000 g·mol−1) against water to remove excess salt and unreacted monomer. The dialyzed product was subsequently freeze-dried and fractionated over a Sephadex G75 gel filtration column (GE Healthcare, Glattbrugg, Switzerland). To this end, the column was loaded with 50 mL of a 2 mg·mL−1 HBPL solution in 0.01 M HCl and subsequently eluted with 0.01 M HCl. Fractions of 20 mL were collected and lyophilized. The final product was stored as powder at −20 °C. Isolated yield after fractionation 16 g. 1H NMR (400 MHz, D2O, 298 K): 4.18 (br, 1H, COCH(R)NαH, dendritic unit), 3.91 (br, 1H, COCH(R)NαH, αlinear unit), 3.39 (br, 1H, COCH(R)NαH, ε-linear unit), 3.30 (br, 1H, COCH(R)NαH, terminal unit), 3.18 (m, 2H, -CH2-NεH2, dendritic unit and ε-linear unit, ε-terminal unit), 2.97 (m, 2H, -CH2-NεH2, αlinear unit), 2.85 (m, 2H, -CH2-NεH2, α-terminal unit), 1.92−1.3 (br m, 6H, -CH2-). 13C NMR (100.6 MHz, D2O, 298 K): 176.23 (C(O)NH), 55.14 (COCH(R)NαH), 38.77 (-CH2-NεH2), 34.73 (-CH2CH2−NεH), 28.87 (NαH-CH−CH2-), 22.78 (-CH2-CH2-CH2-NεH2). Nα,Nε-Di-(tert-butoxycarbonyl)-L-lysine. Nα,Nε-di-(tert-butoxycarbonyl)-L-lysine dicyclohexylammonium salt (6 g, 0.011 mol) was neutralized in ethyl acetate (EtAc; 25 mL) by addition of 6 mL of 1 M H2SO4. After the addition of the acidic solution a clear solution was obtained, which was further diluted with 35 mL of H2O and 35 mL of EtAc. Then, the two phases were separated and the aqueous phase washed two times with EtAc. Finally, the combined EtAc extracts were washed with Milli-Q water, dried over MgSO4, filtered, and concentrated under vacuum. Isolated yield 3.5 g, 90% yield. 1H NMR (400 MHz, DMSO, 298 K): 7.0 (NεH, 1H), 6.75 (NαH, 1H), 3.82 (CαH, 1H), 2.87 (CεH2, 2H), 1.60 (CβH2, 2H), 1.40 (CγH2, CδH2, tBu, 3×CH3, 22H). Nε-(tert-Butoxycarbonyl)-L-lysine N-Carboxyanhydride. Nα,Nε-Di(tert-butoxycarbonyl)-L-lysine (3.2 g, 0.009 mol) was dissolved in 200 mL THF in a sealed round-bottom flask equipped with a Teflon stirrer and nitrogen inlet. Triphosgene (0.89 g, 0.003 mol) was dissolved in THF (20 mL) and added under a N2 flow. Freshly distilled

triethylamine was added dropwise (0.8 g, 0.008 mol). The reaction mixture was stirred overnight at room temperature. Then, the reaction mixture was cooled down to −20 °C and the precipitated triethylamine hydrochloride salts were removed by filtration. After removal of the THF in vacuo, the remaining product was redissolved in EtAc. The solution was again cooled down to 4 °C and washed once with cold water and twice with 5% w/w aqueous NaHCO3 solution. After partial evaporation of the EtAc under vacuum, the final product was precipitated by addition of cold hexane. This precipitation cycle was repeated five times. Finally, the precipitate was isolated by centrifugation. Isolated yield 1.53 g, 62%. MS (ES+) m/z = 295, 1 (100%) [M + Na]+. 1H NMR (400 MHz, DMSO, 298 K): 9.0 (NαH, 1H), 6.79 (NεH, 1H), 4.4 (CαH, 1H), 2.87 (CεH2, 2H), 1.7 (CβH2, CγH2, 4H), 1.4 (tBu, 3CH3, 9H), 1.2 (CδH2, 2H). 13C NMR (100.6 MHz, D2O, 298 K): 173 (-C(O)-CH-NH), 156 (tBu-O-C(O)), 153 (NH-C(O)-O), 78 (tBu), 58 (Cα), 39 (Cε), 31 (Cδ), 28 (Cβ), 20 (Cγ). Linear Polylysine (LPL). First, 5 mL of a 0.2 g·mL−1 solution of Nε(tert-butoxycarbonyl)-L-lysine N-carboxyanhydride in methylenechloride was injected into the polymerization reactor via a septum. The methylenechloride was subsequently evaporated under vacuum and replaced by 5 mL of a 1:5 (v/v) mixture of DMF and THF. The reactor was then immersed in an oil bath and thermostatted at 35 °C. After complete dissolution of monomer, the calculated volume of benzylamine was added via syringe. The solution was stirred for 3 days, after which the polymerization was stopped by precipitation into a 10fold excess of cold diethyl ether. Subsequent deprotection of the polymer was accomplished with 70 mL of TFA to afford a polymer solution of approximately 1 mg·mL−1 of poly-Nε-(tert-butoxycarbonyl)-L-lysine under vigorous stirring and nitrogen flux for 2 h. After that, the reaction mixture was precipitated in a 10-fold excess of diethylether. To exchange the trifluoroacetate counterions against chloride, the precipitate was dissolved in 0.1 M HCl (0.1 g·mL−1) and dialyzed against 0.1 M HCl for 2 days. Final product was obtained by freeze-drying. Number-average degrees of polymerization obtained from 1H NMR spectroscopy were determined by comparison of the integrals of the CεH2 group of the monomer units at 2.8 ppm and the initiator-derived aromatic resonance at 7.4 ppm. 1H NMR (400 MHz, D2O, 298 K): 7.4 (benzyl, H, 5×H), 4.2 (CαH, 1H), 2.8 (CεH2, 2H), 2.0−1.0 (CβH2, CγH2, CδH2, 6H). 13C NMR (100.6 MHz, D2O, 298 K): 175 (-C(O)-CH-NH), 129 (benzyl), 58 (Cα), 39 (Cε), 31 (Cβ), 28 (Cδ), 22 (Cγ). Dendritic Poly(L-lysine) (DPL). First, Nα,Nε-di-(tert-butoxycarbonyl)-L-lysine dicyclohexylammonium salt (15 g, 0.028 mol) was neutralized to afford Nα,Nε-di-(tert-butoxycarbonyl)-L-lysine, as described above. For the first generation dendrimer (1.4 g, 0.012 mol), hexamethylenediamine was dissolved in DMF at 0.2 g·mL−1. HOBT·H2O (3.56 g, 0.023 mol), HBTU (8.8 g, 0.023 mol), and DMAP (3.42 g, 0.028 mol) were dissolved separately in DMF at a concentration of 0.2 g·mL−1 and charged together with a solution of Nα,Nε-di-(tert-butoxycarbonyl)-L-lysine (8 g, 0.023 mol) in DMF at 0.2 g·mL−1 into a round-bottom flask and allowed to react for 24 h. After that, DMF was evaporated and the product dissolved in 50 mL of EtOH at 60 °C. Purification procedure consisted of multiple precipitations from ethanol into a 10-fold excess of cold aqueous citrate solution (10%) and after that into 10-fold excess NaOH (0.1 M). Subsequently, the product was precipitated from 70 °C EtAc to ice cold EtAc to remove any unreacted monomer from dendrimer due to differential solubility of Nα,Nε-di-(tert-butoxycarbonyl)-L-lysine and dendrimer in cold EtAc. The deprotection of the first-generation dendrimer was accomplished by dissolving the tert-butoxycarbonyl protected dendrimer in TFA at a concentration of 1 mg·mL−1. After 2 h, the solution was precipitated in a 10-fold excess of cold diethylether. To exchange the trifluoroacetate counterion against chloride, the precipitate was dissolved in 0.1 M HCl (0.1 g·mL−1) and dialyzed against 0.1 M HCl for 2 days. The final product was obtained by freeze-drying. Isolated yield 3.3 g (78%). Higher generation dendrimers were synthesized in a similar manner. With increasing generation number the reaction time was gradually increased. The reaction time for the second and third generation dendrimer was 2 d. The reaction time for the fourth and fifth generation dendrimer was 5 3129

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30, and 40 μg·mL−1 in respect to the cell culture. After incubation with the polycation for 1, 2, or 3 h, 100 μL aliquots of cell culture were sampled, washed in cold PBS, and stained. Apoptotic and dead cells were detected by Annexin-V binding and PI nuclear incorporation (BD Biosciences, Allschwil, Switzerland), respectively. FITC-Annexin V was diluted at a concentration of 100 μg·mL−1 in a binding buffer (BD Biosciences) and cells were resuspended in 10 μL of this solution and incubated for 30 min under shaking at room temperature. After that, 0.1 mL of PI solution was added at a final concentration of 3 μM and incubated in the dark at room temperature for 5 min. Cells were washed twice with PBS and diluted with PBS to a final volume of 2 mL and subsequently analyzed by flow cytometry. Negative control consisted of stained fresh cell culture. As a positive control, cells were incubated with Camptothecin for 4 h at a final concentration of 10 μM. Mitochondrial Membrane Potential Analysis. The mitochondrial membrane potential (ψm) was analyzed by epifluorescence microscopy and flow cytometry using the ψm-sensitive dye 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Invitrogen AG, Basel, Switzerland). Cells were incubated with the appropriate polycation for up to 4 h at a final concentration of 10, 30, and 40 μg·mL−1 under the same conditions as described above for the phosphatidylserine detection. Cell aliquots of 1 mL were collected at defined time points (0.5, 1, 2, and 4 h), washed in cold PBS, and resuspended in 5 mL of PRoCHO5 containing 1 mM JC-1. Following incubation at 37 °C for 20 min, cells were extensively washed with PBS and analyzed. The emission maxima of JC-1 monomers and aggregates are 527 and 590 nm, respectively. As a positive control, cells were exposed to Camptothecin as described above. Cells were analyzed by fluorescence microscopy. Cells stained with JC-1 were visualized using a Zeiss Axiovert 200 M inverted microscope equipped with a Zeiss AxioCam MRm camera and Axiovision software. The number of cells presenting depolarized mitochondria was determined by flow cytometry. Detection of Cytochrome C and Caspase 3 by Indirect Immunocytochemistry. Immunocytochemistry with primary antibodies against cytochrome C and p-20 subunit of caspase 3 followed by fluorescently labeled secondary antibodies was used to qualitatively asses later stages of apoptosis. Aliquots (5 mL) of cell culture containing 2 × 106 cells·mL−1 were incubated with the appropriate polylysine solution at a final concentration of 10, 30, and 40 μg·mL−1 for 12 h. After that, cells were washed with cold PBS and fixed with paraformaldehyde solution (37%, Sigma-Aldrich) at a final concentration of 4% v/v. Fixed cells were washed again with PBS and permeabilized with 0.1% Tween solution in PBS for 5 min at 4 °C. Nonspecific binding sites were blocked by incubation with 10 wt % BSA solution in PBS at room temperature for 30 min. Cytochrome C was detected by goat polyclonal IgG against cytochrome C (Santa Cruz Biotechnology, Santa Cruz, CA) dissolved at a concentration 1:500 (v/v) in PBS. After three washing steps with PBS, cells were incubated for 45 min in the dark at room temperature with IgG Alexa Fluor 488 donkey antigoat antibody at a dilution of 1:500 in PBS (Invitrogen AG, Basel Switzerland). After that, cells were washed three times with PBS and nuclei were stained by 10 μL of 4,6-diamidino-2phenylindole (DAPI) prepared as a solution in PBS at 10 mg·mL−1 (Sigma Aldrich). Slides were mounted using Vectashield mounting medium (Vector Laboratories) and examined using a Leica SP5 WLL confocal microscope equipped with a 63× HCX Plan Apochromat oil immersion objective lens and a white laser excitation source. Caspase-3 subunit p20 was detected by rabbit polyclonal IgG (Santa Cruz Biotechnology) dissolved at 1:500 (v/v) in PBS following the identical immunostaining protocol and incubation times described above for Cytochrome C. Alexa Fluor 488 labeled donkey antirabbit IgG (Invitrogen AG) secondary antibody was used at 1:500 (v/v) in PBS. Enzymatic Degradation. Polylysine samples HBPL3, DPL6 and LPL5 were dissolved in a 0.1 M NH4HCO3 (pH 8) digestion buffer at a concentration of 2 mg·mL−1 and heated to 37 °C. A suspension of immobilized trypsin (Immobilized TPCK Trypsin, Thermo Fisher Scientific, Lausanne, Switzerland) was prepared according to the manufacturer’s instructions. Then, 1 mL of polymer solution was

and 8 d for the sixth generation dendrimer. ESI and 1H NMR mass spectra of all dendrimers are included in the Supporting Information (Figures S1 and S2). Cell Culture, Cytotoxicity, and Degradation Studies. Cell Culture. Suspension cell cultures of CHO DG44 cells26 were maintained in square-shaped glass bottles (250 mL) in serum-free ProCHO5 cell culture medium supplemented with 13.6 mg·L−1 hypoxanthine, 3.84 mg·L−1 thymidine, and 4 mM glutamine (SAFC Biosciences, St. Louis, MO), as described by Muller et al.27 Cell cultures were maintained in an ISF-4-W orbital shaker at 110 rpm (Kühner AG, Birsfelden, Switzerland) at 37 °C in a 5% CO2 atmosphere. Flow Cytometry. Flow cytometric analysis was performed using a Cyan ADP (Beckman Coulter, Fullerton, CA) flow cytometer equipped with three lasers (405/488/635 nm) using Summit 4.2 software (BeckmanCoulter). All samples were filtered prior to flow cytometry analysis. A total of 50000 cells were acquired for each analysis. Data were further processed with Flow Jo Software. Results are represented as the mean from three independent experiments, performed in triplicate, and error bars indicate standard deviations. The mean values of triplicates were used for statistical analysis. In Vitro Assessment of Acute Cytotoxicity. In vitro cell viability was measured by Viacount Reagent (Guava Technologies, Millipore, Zug, Switzerland). On the day of the experiment, cells were centrifuged and resuspended in 2 mL of ProCHO5 medium at a density of 2 × 106 cells·mL−1 in TubeSpinbioreactor 50 tubes (TPP, Trasadingen, Switzerland). Then, the appropriate polylysine sample was added as a solution in Milli-Q water to afford a polymer concentration in the culture medium that was varied from 10−1 to 10−8 M. After 1 h, a 200 μL aliquot of cell suspension was withdrawn and incubated with 180 μL Viacount Reagent in a 5 mL round-bottom polypropylene tube for 5 min at room temperature. After that, the sample was diluted with 1 mL PBS and analyzed by flow cytometry. Each experiment was carried out as three independent replicates. The EC50 was calculated from the dose−response curve as the concentration of polylysine that results in 50% cell death after 1 h incubation with polylysine.28 Assessment of Cell Membrane Permeability. Aliquots (5 mL) of cell culture containing 2 × 106 cells mL−1 were incubated for 1 h with the polycation solution in Milli-Q water (1 mg·mL−1) at a final concentration of 20, 30, and 40 μg·mL−1 with respect to the cell culture. After that, cells were washed three times with ice cold PBS. Washing was carried out by centrifugation of the cell suspension at 200 g for 5 min, followed by decantation of the supernatant and resuspension of the cells in 10 mL of PBS. Then, cells were resuspendend at 4 °C in 1 mL of a 1 mg·mL−1 PBS solution of tetramethylene rhodamine-labeled dextran solution. After incubation for 15 min at 4 °C with shaking, followed by three washing steps in ice-cold PBS, cells were analyzed for the presence of dextran-TMR by flow cytometry using 488 nm excitation light and filter FL2 to detect dextran-TMR emission at 580 nm. As positive and negative controls, cells were exposed to a 0.1 wt % Triton X-100 solution in PBS, respectively, 100 μg·mL−1 PEG5000 for a period of 1 h. Experiments were performed in three independent triplicates. Standard errors were within 15% of the mean values for all the replicates. The data of membrane permeabilization assay are presented as a heatmap, which was generated by HeatMap Builder 1.0 software.29 The heatmap represents the percentage of cells with a permeabilized membrane for a particular sample and at a particular concentration as a single square at a corresponding gray scale. The Heatmap was generated using data set normalized sorting and linear color morphing algorithm with a grayscale color coding. Detection of Phosphatidylserine by Annexin V-FITC and Propidium Iodide (PI). Number of apoptotic cells was determined by detecting phosphatidylserine (PS) translocation. Simultaneous staining with propidium iodide was used to identify cell population with compromised cell membrane. In these cells, Annexin V could have stained PS localized at the inner leaflet of the membrane and would not represent the translocation of PS as a hallmark of apoptosis. Aliquots (5 mL) of cell culture containing 2 × 106 cells mL−1 were incubated with the polycation solution at a final concentration of 10, 3130

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Table 1. Structural Characteristics of the Linear Polylysine (LPL) Samples Used in This Study sample

[M0]/[I0]a

tb (h)

Mn/thc (g·mol−1)

Mn/NMRd (g·mol−1)

Mn/GPCe (g·mol−1)

Mw/GPCe (g·mol−1)

Mw/Mne (−)

LPL1 LPL2 LPL3 LPL4 LPL5

40 40 50 150 200

72 72 72 78 65

5120 5120 6400 19200 25600

800 2500 4400 14500 19600

2400 7000 11000 18700 16300

3300 11300 14300 26100 43100

1.4 1.6 1.3 1.4 2.6

a

Initial ratio of monomer to initiator. bPolymerization time. cTheoretical number-average molecular weight determined from the initial [M]/[I] ratio and conversion. dNumber-average molecular weight determined by 1H NMR spectroscopy. eNumber-average molecular weight (Mn), weightaverage molecular weight (Mw) and polydispersity (Mw/Mn) determined by SEC.

Table 2. Structural Characteristics of Dendritic Polylysine (DPL) Samples Used in This Study sample DPL1 DPL2 DPL3 DPL4 DPL5 DPL6

Mn/tha (g·mol−1) 372 885 1909 3959 8058 16266

Mb (g·mol−1) 373.35 443.35 478.18 660.64 1009.01 2323.19

Mc (g·mol−1) ([M ([M ([M ([M ([M ([M

+ + + + + +

Mnd (g·mol−1)

Mwd (g·mol−1)

Mw/Mnd (−)

1200 3100 5600 12000

1300 4000 7200 18000

1.1 1.3 1.3 1.5

+

H] ) 372.55 2H]2+) 443.35 4H]4+) 478.37 6H]6+) 660.83 8H]8+) 1008.25 7H]7+) 2323.30

a

Theoretical molecular weight. bExperimental molecular weight from ESI mass spectrometry. Most mass spectra revealed multiple charged species. Only the most prominent signals are listed. cm/z theoretical. dNumber-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity (Mw/Mn) experimentally determined by SEC. incubated with 0.25 mL of trypsin suspension at 37 °C on a shaker. Aliquots of polymer were collected 8 h postincubation, frozen in liquid nitrogen, and lyophilized. Products of enzymatic degradation were analyzed by SEC.

peaks that could be due to a small fraction of structural defects, which are difficult to completely exclude using a divergent synthetic strategy. Hyperbranched polylysine (HBPL) was prepared in a one-step reaction from L-lysine hydrochloride.22 After dialysis to remove excess salt and unreacted monomer, a series of HBPL samples covering a range of molecular weights was obtained by size exclusion chromatographic fractionation of the reaction product. In contrast to DPL, which is nearly monodisperse and structurally uniform, HBPL is polydisperse and has a randomly branched structure. Acute Cytotoxicity. The acute cytotoxicity of the polylysine analogues was evaluated by assessing cell viability 1 h after exposure to the polycations. Cell viabilities were quantified by flow cytometry using the Viacount assay. For each polycation, the EC50 value was determined by evaluating cell viability over a range of concentrations varying from 10−8 to 10−1 M. The EC50 values for all the investigated polylysine analogues are summarized in Figure 1. Figure 1 indicates for all three investigated polylysine analogues a decrease in EC50 value with increasing polymer molecular weight. At equivalent molecular weights, however, the EC50 values of the DPL and HBPL samples were at least five times lower as compared to the corresponding LPL sample. DPL and HBPL samples of similar molecular weight did not reveal significant differences in EC50 value. The EC50 values of the DPL samples ranged from 62 ± 9 mM to 33 ± 7 μM. To understand the origin of the acute cytotoxicity of the polylysine analogues, the effect of hyperosmolality on cell viability was investigated. To this end, osmolalities of selected polylysine samples were determined in cell culture medium at their respective EC50 concentration (Table S1). Whereas the osmolality of DPL6 at its EC50 concentration was similar to that of the cell culture medium (≈320 mOsm·kg−1), solutions of LPL1 and DPL1 at their EC50 had significantly higher osmolalities of 609 and 663 mOsm·kg−1, respectively. For suspension cell culture, significant changes to cell viability were observed already at 410 mOsm·kg −1 .31 To investigate exclusively the effect of osmotic pressure on cell viability, a

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RESULTS AND DISCUSSION Polymer Library Design and Synthesis. Scheme 1 represents the structures of the different polylysine analogues that were prepared to investigate and compare the effects of polycation molecular weight and architecture on in vitro cytotoxicity. For each of these analogues, a series of compounds with different molecular weights was prepared. The characteristics of all the samples investigated in this study are listed in Tables 1−3. Linear polylysine (LPL) was synthesized via ringTable 3. Structural Characteristics of the Hyperbranched Polylysine (HBPL) Samples Used in This Study sample

Mna (g·mol−1)

Mwa (g·mol−1)

Mw/Mna (−)

HBPL1 HBPL2 HBPL3 HBPL4 HBPL5 HBPL6

1400 8100 21000 46200 83900 146800

2700 14500 44300 101200 232200 352600

1.9 1.8 2.1 2.2 2.8 2.6

a

Number average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity (Mw/Mn) experimentally determined by SEC.

opening polymerization of Nε-(tert-butoxycarbonyl)-L-lysine Ncarboxyanhydride using benzylamine as the initiator and subsequent side-chain deprotection. Samples of different molecular weights were obtained by varying the initial monomer-to-initiator ratio. Dendritic polylysines (DPL) up to the sixth generation were prepared via a previously reported divergent strategy using standard peptide coupling chemistry.30 ESI mass spectra of all DPL samples are included in the Supporting Information (Figure S1). These mass spectra confirm the structure of the polymers but also show minor 3131

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were exposed to solutions of polycations for 1 h at 37 °C, followed by incubation with tetramethylene rhodamine-labeled dextran (dextran-TMR) with a molecular weight of 40000 g·mol−1 at 4 °C for 1 h and flow cytometric analysis. These experiments were carried out with 20−40 μg·mL−1 solutions of the different polylysines, which corresponds to 1−2 μM for a polylysine analogue of 20000 g·mol−1. Whereas the hydrophilic and high molecular weight dextran-TMR is excluded from entering into the cytosol if the cell membrane is intact, diffusion of dextran-TMR across the cell membrane is possible if the cell membrane integrity has been compromised. For each of the three classes of polylysine analogues, three different concentrations (significantly below the EC50 value of the respective sample) were investigated. As negative and positive controls, polyethylene glycol (PEG5000, MW 5000 g·mol−1) and a 0.1 wt % solution of Triton X-100 were used. The heatmap in Figure 2 represents the percentage of dextran positive cells, as determined by flow cytometry, after 1 h incubation. While relatively few ( LPL. Using immunocytochemical analysis, it was further established that exposure of cells to DPL and HBPL polycations resulted in

Figure 6. Immunofluorescent analysis of the distribution of cytochrome C in CHO DG44 cells. Cell nuclei were DAPI stained and appear blue. Images (A) and (D) are negative and positive controls and represent cells that were exposed to 100 μg·mL−1 PEG5000, respectively, 10 μM camptothecine for 12 h. The other micrographs represent CHO DG44 cells that were exposed for 12 h to 20 μg·mL−1 of (B) DPL1; (C) LPL5; (E) DPL6; or (F) HBPL3. Scale bar 10 μm. 3135

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Figure 7. Immunofluorescence detection of caspase 3 activation in CHO DG44 cells upon exposure to PEG5000 (100 μg·mL−1) at t = 0 (A) and after 12 h (B); (C) after treatment with 10 μM camptothecin for 12 h and after exposure for a period of 12 h to a 20 μg·mL−1 solution of (D) LPL5, (E) HBPL3, and (F) DPL6. Cells were stained by DAPI to allow visualization of the cell nuclei (blue). Scale bar is 10 μm.

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higher molecular weight analogues cell membrane damage due to direct interactions between the polycations and the cell membrane were found to play a role. Assessing cytotoxicities over extended periods of time (≥3 h) revealed marked differences between the apoptotic activities of the three classes of polylysine cations. Generally, apoptosis was more pronounced for DPL and HBPL as compared to LPL (at comparable polymer molecular weight). These differences are most likely due to differences in proteolytic stability; whereas LPL was found to be completely degraded in model experiments with trypsin, only partial degradation was observed for DPL and HBPL, which also contain inert ε-peptidic linkages. Taken together, the results of this study provide some first insight into the structure−property relations that describe the in vitro cytotoxicity of polylysine based cations and show that both molecular weight and architecture are important factors. Furthermore, since their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: harm-anton.klok@epfl.ch. Fax: + 41 21 693 5650. Tel.: + 41 21 693 4866. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. Elisa Corbacella (Department of Audiology, University of Ferrara) for useful suggestions and discussions. The authors wish to thank Dr. Laure Menin (EPFL, Mass spectrometry core facility) for help with mass spectrometry. This study was supported by the European Commission NMP4-CT-2006-02556 (Project Nanoear).

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR and ESI mass spectra of the dendritic polylysines. Additional microscopy and flow cytometric data as well as SEC chromatograms of the degradation assay. This material is available free of charge via the Internet at http://pubs.acs.org. 3136

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