Mechanistic studies of in vitro cytotoxicity of poly(amidoamine) dendrimers in mammalian cells

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<ul><li><p>yyaf Te</p><p>Revised 21 July 2010Accepted 16 August 2010</p><p>PAMAM dendrimersCytotoxicityEC50Reactive oxygen species</p><p>)fro</p><p>dendrimer generation and number of surface amino groups. In this work, using generation G4, G5, and G6c response is furthermore demonstrated for the generation of reactive oxygenand the onset of apoptosis and levels of DNA damage. The results are consistent</p><p>oxidative stress, apoptosis and DNA damage. RO</p><p>Toxicology and Applied Pharmacology 248 (2010) 259268</p><p>Contents lists available at ScienceDirect</p><p>Toxicology and Appl</p><p>.e lized (Esfand and Tomalia, 2001) and are recognized as a unique newclass of synthetic nanostructures. Dendrimers allow the precisecontrol of size, shape and placement of functional groups that isadvantageous for many life science applications. Their systematicallyvariable structural architecture and large internal free volume makethese dendrimers an unique class of molecule for various biomedicalapplications including drug (Na et al., 2006; Yiyun et al., 2005), DNA(Guillot-Nieckowski et al., 2007; Kukowska-Latallo et al., 1996), andsiRNA (Zhou et al., 2006) delivery, and as MRI probes (Dear et al.,2006; Swanson et al., 2008) etc.</p><p>It is reported however that PAMAM dendrimers induce oxidativestress by producing reactive oxygen species (ROS) (Lee et al., 2009)and can lead to a cytotoxic response (Mukherjee et al., 2010; Naha etal., 2009). Although the response is mild, the molecular denition andthe systematic variability of the size and structure of the PAMAMdendrimers render them an ideal material in which to study cytotoxicresponses and elucidate their mechanisms. In both mammalian(Mukherjee et al., 2010) and ecotoxicological (Naha et al., 2009)studies, clear structureactivity relationships have been demonstrat-ed, indicating that the toxicity increases in proportion to the surfacePAMAM dendrimers contain a 2-carbon ewhich the terminal amidoamines are attabranched radial structure having tertiary</p><p> Corresponding author. Fax: +353 1 402 7901.E-mail address: (S.P. Mukh</p><p>0041-008X/$ see front matter 2010 Elsevier Inc. Adoi:10.1016/j.taap.2010.08.016s are the rst completeerized and commercial-</p><p>tions (G0G10) the diameter and the number of surface aminogroups systematically increase ( (PAMAM) dendrimerdendrimer family to be synthesized, charactIncreased lysosomal activityApoptosisDNA damage</p><p>Introductiongenerated levels and timescales are systematically generation dependent (G4bG5bG6). Flow cytometryconrms that with increasing dose, the percentage of healthy and early apoptotic cells decreases, whereasthe late apoptotic and necrotic cell populations increase. This process is again systematically generationdependent. DNA damage as measured using the TUNEL assay further demonstrates a systematic trend, G4,G5 and G6 showing 4.69%, 25.87% and 89.63% DNA breakage respectively. Increases in lysosomal activity attimescales of ~24 h are observed in HaCaT but not SW480 cells upon low concentration PAMAM exposure.Overall, signicant differences are observed between the responses of the dermal cell line, HaCaT, and thecolon cell line, SW480, and it is suggested that these can be understood in terms of differing intrinsicantioxidant levels.</p><p> 2010 Elsevier Inc. All rights reserved.</p><p>primary amino groups on the surface. With the successive genera-thylenediamine core onched yielding a highlyamine branches and</p><p>area or numberIn this stud</p><p>explored in termlysosomal activitween the respAlamar Blue (ABfurther framedactivity, apoptoserjee).</p><p>ll rights reserved.S production is co-located in the mitochondria, and both</p><p>Keywords: with a pathway of localisation of PAMAM dendrimers in the mitochondria leading to ROS production causingAvailable online 22 August 2010dendrimers, this systematispecies, lysosomal activity,Mechanistic studies of in vitro cytotoxicitmammalian cells</p><p>Sourav Prasanna Mukherjee a,, Fiona M. Lyng a, Amaa Radiation and Environmental Science Centre, Focas Research Institute, Dublin, Institute ob Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland</p><p>a b s t r a c ta r t i c l e i n f o</p><p>Article history:Received 1 April 2010</p><p>Poly(amidoamine) (PAMAMcytotoxicological response</p><p>j ourna l homepage: wwwof poly(amidoamine) dendrimers in</p><p>Garcia a, Maria Davoren a, Hugh J. Byrne b</p><p>chnology, Kevin Street, Dublin 8, Ireland</p><p>dendrimer nanoparticles have been demonstrated to elicit a well denedm mammalian cell lines, the response increasing systematically with</p><p>ied Pharmacology</p><p>sev ie locate /ytaapof surface groups.y, the mechanism of PAMAM toxicity is furthers of generation dependant ROS production, cellularty, apoptosis and DNA damage. Relationships be-onses of the standard cytotoxicity assays MTT,) and Neutral Red (NR) assays are also elucidated andwithin the context of ROS production, lysosomalis and DNA damage.</p></li><li><p>260 S.P. Mukherjee et al. / Toxicology and Applied Pharmacology 248 (2010) 259268Materials and methods</p><p>Test materials. Polyamidoamine (PAMAM) dendrimers, G4, G5 andG6, were purchased from Sigma Aldrich Ltd. (Ireland). All the particleshave an ethylenediamine core and PAMAM G4, G5 and G6 haverespectively 64, 128 and 256 functional primary amino groups on thesurface. The molecular weights of PAMAMG4, G5 and G6 are 14,215 Da,28,826 Da and 58,048 Da respectively. The nominal diameters of thePAMAM G4, G5 and G6 dendrimers are 4.5, 5.4 and 6.7 nm respectively(</p><p>Reagents. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) and Neutral Red (NR) were purchased from Sigma AldrichLtd. (Ireland). Alamar Blue (AB)was purchased fromBiosource (UK). Cellculture media and supplements were purchased from Sigma Aldrich(Ireland) and Bioscience (Ireland). 5-(and-6)-carboxy-2,7-dichlorou-orescein diacetate (carboxy-H2DCFDA), LysoSensor Green DND-189,LysotrackerGreen DND-26 and MitoTracker Orange CM-H2TMRoswas purchased from Invitrogen (Ireland). Vibrant apoptosis assay kit YO-PRO-1/propidium iodide was purchased from Molecular Probes,Invitrogen (Ireland). APO-DIRECT KIT was purchased from BDBiosciences Pharmingen (U.K.).</p><p>Cell culture. HaCaT cells, an immortal non-cancerous humankeratinocyte cell line, (kindly provided by Prof. Dr. Boukamp,Heidelberg) and SW480 cells (ATCC, CCL-228), a primary adenocar-cinoma cell line of the colon, were employed for testing. SW480 cellswere cultured in Dulbecco's Modied Eagle's Medium NutrientMixture F-12 HAM with 2 mM L-glutamine supplemented with 10%fetal bovine serum (FBS), 45 IU ml1 penicillin and 45 IU ml1</p><p>streptomycin at 37 C in 5% CO2. HaCaT cells were cultured in thesame cell culture medium under the same conditions with theaddition of 1 g/ml hydrocortisone.</p><p>Preparation of dendrimer solutions. Dendrimer test solutions wereprepared in the respective cell culture media. They were readilysoluble in the media at 37 C and were dispersed uniformly by lowspeed vortex. The concentration ranges used for the cytotoxicityassays with G4, G5 and G6 were 0.0121.1 M, 0.035.2 M and 0.015.168 M respectively for HaCaT cells and 0.017.03 M, 0.035.2 Mand 0.015.168 M respectively for SW480 cells. Concentrationranges were chosen as identied in a previous study of PAMAMdendrimer cytotoxicity (Mukherjee et al., 2010). For ROS, Lysosensor,Lysotracker and Mitotracker studies, the concentrations used forHaCaT cells with G4 were 1.623.2 M, with G5 were 0.55.75 M andwith G6 were 0.53.17 M. For ROS in SW480 cells the concentrationsused for G4 were 0.7210.8 M, for G5 were 0.184.33 M, and for G6were 0.581.87 M. For the apoptosis study the concentrations usedfor HaCaT cells with G4 were 3.2123.16 M, with G5 were 1.075.75 M, andwith G6were 1.023.17 M. In the TUNEL assay, PAMAMG4 was used at concentrations of 3.17 M and 23.16 M, G5 at aconcentration of 3.17 M and 5.75 M, and G6 at a concentration of3.17 M.</p><p>Alamar Blue (AB) and Neutral Red (NR) assays. AB and NR assaysare standard cytotoxicity assays. The AB assay measures the generalcellular metabolism (O'Brien et al., 2000), whereas the NR assaymeasures the lysosomal activity of the cell (Repetto et al., 2008; Yanget al., 2007). For the AB and NR assays, cells were seeded in 96-wellmicroplates (Nunc, Denmark) at a density of 1105 cells/ml in 100 lof respective media containing 10% FBS. After 24 h of cell attachment,plates were washed with 100 l/well PBS and the cells were treatedwith increasing concentrations of each generation of dendrimer,prepared in 5% FBS containing media for 24 h. All incubations were</p><p>performed at 37 C in a 5% CO2 humidied incubator. Six replicatewells were used for each control and test concentrations permicroplate.</p><p>The AB followed by the NR assays were conducted consecutivelyon the same set of plates. The assays were carried out according to themanufacturer's instructions. Briey, control media or test exposureswere removed; the cells were rinsed with PBS and 100 l of AB/NRmedium (5% [v/v] solution of AB and 1.25% [v/v] of NR dye) preparedin freshmedia (without FBS or supplements) were added to eachwell.After 3 h incubation, AB uorescence was measured at the respectiveexcitation and emission wavelengths of 531 nm and 595 nm in aVICTOR3V 1420 Multilabel Counter (Perkin Elmer, USA). Wellshaving only AB and media were used as blanks. After measurement,the wells were then washed with PBS and 100 l of NR xative (50%ethanol, 49% dH2O and 1% glacial acetic acid) were added to each welland the plates were shaken at 240 rpm for 10 min. The NRuorescence was then measured at the excitation and emissionwavelengths of 531 nm and 642 nm respectively in the sameinstrument. For both assays, mean uorescent units for the sixreplicate cultures were calculated for each exposure treatment.</p><p>MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)assay. MTT assay is also a standard cytotoxicity assay and measuresthe mitochondrial metabolism of the cell from its mitochondrialoxidoreductase enzyme activity (Loveland et al., 1992). A parallel setof plates was set up for the MTT assay and seeded and exposed in anidentical manner as described in Alamar Blue (AB) and Neutral Red(NR) assays section. After 24 h of PAMAM dendrimer exposure, themedium for the control or test exposures was removed, the cells werewashed with PBS and 100 l of freshly prepared MTT in media (5 mg/ml of MTT in media [without FBS or supplements]) were added toeach well. After 3 h incubation, the medium was discarded and thecells were rinsed with PBS and 100 l of MTT xative solution(isopropanol with 0.04 N HCl) were added to each well and the plateswere shaken at 240 rpm for 10 min. The absorbance was thenmeasured at 595 nm in a TECAN GENios (Grodig, Austria) platereader.</p><p>Lysosensor, Lysotracker and Mitotracker studies. The HaCaT cellswere seeded at a density of 1106 cells/ml in glass bottom Petridishes for confocal uorescence microscopic study and 1105 cells/ml in 96wellmicroplates for quantitative study using the plate reader,in 10% FBS supplemented media and incubated at 37 C in 5% CO2 for24 h for cell attachment before exposure to the dendrimers. Sixconcentrations of each generation of dendrimer were prepared in 5%FBS supplemented media and the cells were exposed for 24 h.Following the exposure period, the cells were washed twice withpre-warmed PBS (37 C). The Lysosensor/Mitotracker dye solutionwas prepared using concentrations of 2 M and 250 nM respectivelyin pre-warmed PBS. Cells were stained with the dye solution for30 min in a 37 C, 5% CO2 incubator. Following staining, the cells werewashed 3 times with pre-warmed PBS to ensure complete removal ofunloaded dyes. Confocal microscopic images were then taken using aZeiss Confocal Fluorescence Microscope (LSM 510 META, Version 3.2SP2, Carl Zeiss, Germany). For the Lysosensor assay, the excitationwavelength used was 488 nm and the uorescence emission wasdetected using a 505530 nm band pass lter. For the Mitotrackerassay, the excitation wavelength used was 543 nm and the uores-cence emission was detected using a 560 nm long pass lter.Lysosomal activity was also quantied with Lysosensor andLysotracker dye in a TECAN GENios (Grodig, Austria) plate reader.The Lysosensor dye used in this assay is a pH sensitive dye and canonly quantify lysosomes of pH 5.2. It has recently been indicated thatlocalisation of G5 PAMAM dendrimers in lysosomes can cause anincrease in pH, however (Thomas et al., 2009). Thus Lysotracker,independent of the pH, was employed for quantifying cellular</p><p>lysosomal activity. For this study, HaCaT cells were seeded in 96</p></li><li><p>well microplates at a density of 1105 cells/ml in 100 l of respectivemedia containing 10% FBS. Following 24 h cell attachment the cellswere washed with pre-warmed PBS and exposed to 6 differentconcentrations of PAMAM of each generation. After 2, 4, 6 and 24 hexposures, the cells were washed twice with PBS and stained with2 M Lysosensor and 75 nM Lysotracker separately in PBS in two</p><p>concentration of 10 M and the Petri dishes were kept in a 37 C, 5%CO2 incubator for 1 h. Following incubation, the dye solution wasremoved, the cells werewashed twice with PBS andwere treatedwith1 M concentrations of G6 dendrimer, prepared in 5% FBS containingmedia. Following 1 h and 24 h exposures, the cells were washed twicewith pre-warmed PBS (37 C). The Mitotracker dye solution was</p><p>Table 1EC50 concentrations from AB, NR and MTT assays of PAMAM G4, G5 and G6 upon 24 h exposure to HaCaT and SW480 cells (reproduced from Mukherjee et al., 2010).</p><p>Cytotoxicityassays</p><p>EC50 in M [Condence Interval]</p><p>PAMAM G4 PAMAM G5 PAMAM G6</p><p>HaCaT SW480 HaCaT SW480 HaCaT SW480</p><p>MTT assay 3.21 [2.893.52] 1.44 [1.281.6] 1.07 [0.641.5] 0.37 [0.180.55] 1.02 [0.921.13] 1.16 [0.761.56]Alamar Blue assay 16.35 [13.6919.02] 8.3 [7.269.33] 1.89 [1.352.43] 3.12 [2.643.59] 1.30 [11.59] 1.56 [1.231.88]Neutral Red assay 23.16 [16.4329.88] 10.8 [8.5713.03] 5.75 [3.987.52] 4.33 [3.894.76] 3.17 [2.383.96] 1.87 [1.462.27]</p><p>261S.P. Mukherjee et al. / Toxicology and Applied Pharmacology 248 (2010) 259268independent experiments for 30 min in a 37 C, 5% CO2 incubator.Then the cells were washed 3 times with pre-warmed PBS to ensurecomplete removal of unloaded dyes and the measurement wasundertaken in the plate reader at excitation and emissionwavelengths of 488 nm and 535 nm respectively.</p><p>Intracellular reactive oxygen species study. Carboxy-H2DCFDA dyewas used to study the intracellular reactive oxygen species generationin HaCaT cells. The study was performed in black 96-well microplates(Nunc, Denmark), wherein the cells were seeded at a density of1105 cells/ml in 100 l of respective media containing 10% FBS.After 24 h of cell attachment, the cells were washed twice with100 l/well PBS and carboxy-DCFDA dye was added at a concentra-tion of 10 M in 100 l/well and the plates were kept in a 37 C, 5%CO2 incubator for 1 h. Following incubation, the dye solution wasremoved, the cells were washed twice with 100 l/well P...</p></li></ul>


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