High content analysis of cytotoxic effects of pDMAEMA on
human intestinal epithelial and monocyte cultures
Lee-Anne B. Rawlinson, Peter J. O’Brien, David J. Brayden*
UCD School of Veterinary Medicine and
UCD Conway Institute,
University College Dublin, Dublin 4, Ireland.
*
For correspondence:
David Brayden, Ph.D.
Room 214, Veterinary Sciences Building, School of Agriculture, Food
Science and Veterinary Medicine, UCD, Belfield, Dublin 4, Ireland
Tel: +353 1 716 6013
Fax: +353 1 716 6219
Email: david.brayden@ucd.ie
1
Abstract
Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is a cationic polymer
with potential as an antimicrobial agent and as a non-viral gene delivery vector. The aim
was to further elucidate the cytotoxicity of a selected pDMAEMA low molecular weight
(MW) polymer against human U937 monocytes and Caco-2 intestinal epithelial cells
using a novel multi-parameter high content analysis (HCA) assay and to investigate
histological effects on isolated rat intestinal mucosae. Seven parameters of cytotoxicity
were measured: nuclear intensity (NI), nuclear area (NA), intracellular calcium ([Ca2+]i),
mitochondrial membrane potential (MMP), plasma membrane permeability (PMP), cell
number (CN) and phospholipidosis. Histological effects of pDMAEMA on excised rat
ileal and colonic mucosae were investigated in Ussing chambers. Following 24-72 hours
exposure, 25-50 µg/ml pDMAEMA induced necrosis in U937 cells, while 100-250 µg/ml
induced apoptosis in Caco-2. pDMAEMA increased NA and NI and decreased [Ca2+]i,
PMP, MMP and CN in U937 cells. In Caco-2, it increased NI and [Ca2+]i, but decreased
NA, PMP, MMP and CN. Phospholipidosis was not observed in either cell line.
pDMAEMA (10 mg/ml) did not induce any histological damage on rat colonic tissue and
only mild damage to ileal tissue following exposure for 60 min. In conclusion, HCA
reveals that pDMAEMA induces cytotoxicity in different ways on different cell types at
different concentrations. HCA potential for high throughput toxicity screening in drug
formulation programmes.
Key words: pDMAEMA, high content analysis, cytotoxicity assays, Caco-2 intestinal
epithelia, antimicrobial polymers
2
1. Introduction
Poly(2-(dimethylamino ethyl)methacrylate) (pDMAEMA) is a mucoadhesive
polymer, that is cationic in acidified media or when quaternised with an alkylating agent
[1, 2]. It is being investigated as a potential antimicrobial coating on medical devices [35] and as a non-viral gene delivery vector [6, 7]. It induces cytotoxicity in U937 human
monocyte-like cells, but less cytotoxicity in human Caco-2 intestinal epithelial cells [3].
Intravenous delivery of 5.1 mg/kg pDMAEMA was fatal to rat, but 2.1 mg/kg was well
tolerated [8]. In order to eventually use pDMAEMA in a clinical setting, its cytotoxic
potential should therefore be further investigated with improved assays since in vitro
cytotoxicity data from standard assays is equivocal and lacks predictive power.
High Content Analysis (HCA) is a novel technology that allows quantitative
analysis of each cell at sub-cellular microscopic resolution using a selection of multicoloured fluorescence-based non-toxic dyes [9]. It allows combinations of numerous
fluorescent probes to be used concurrently to investigate different parameters of
cytotoxicity following acute and chronic exposure at varying concentrations [10, 11].
The information obtained and throughput is more than an order of magnitude higher than
conventional cytotoxicity assays. For example, a 96-well plate is read in 30 min with a
large proportion of cells in each well analysed. Multiple biochemical, functional and
morphological parameters are measured on individual cells and there is a higher
sensitivity of cytotoxicity detection as indicated by the lower IC50 values for HCA
compared to the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. HCA has
recently been validated against in vivo human toxicity data for hundreds of marketed
drugs (10-13). Additional advantages of HCA over conventional cell-based cytotoxicity
3
assays include detailed data on numerous cell physiological and morphological
parameters measured kinetically up to 72 hours, measurements on individual cells, and
mechanistic information on elucidating the induced pathophysiology.
HCA has also been used to investigate the influence of siRNA on cell cycling
[14], to screen for activators of DNA damage [15], as well as in the study of visionrelated diseases to investigate neurite outgrowth in retinal ganglion cells [16]. The assay
used in this study was based on the multi-parameter cytotoxicity assay protocol
developed by O’Brien et al. [11]. This assay allows concurrent analysis of 6 parameters
of cell cytotoxicity (nuclear intensity (NI), nuclear area (NA), intracellular calcium
([Ca2+]i), mitochondrial membrane potential (MMP), plasma membrane permeability
(PMP) and cell number (CN)). HCA therefore provides high sensitivity (predictive
detection of known cytotoxic agents) and high specificity (predictive correlation with
human toxicity) compared to conventional cytotoxicity end-point assays [11]. By
combining flow cytometry, intracellular fluorescence probes and image analysis of
multiple parameters, extensive data is provided to examine sub-lethal effects, which can
relate to in vivo toxicological outcomes.
HCA was also used here to investigate the potential of pDMAEMA to induce
phospholipidosis. Drug-induced phospholipidosis is a lipid storage disorder that results
in the accumulation of phospholipids in lysosomes in tissues throughout the body due to
inhibition of phospholipid breakdown [17]. Evidence of drug-induced phospholipidosis
in pre-clinical studies can lead to delays or removal of drugs from development [18].
Despite this, there are over 50 marketed pharmaceuticals that are known to induce
phospholipidosis [18]. Many of these molecules are cationic amphiphilic drugs, which
4
are generally characterised by a structure that is comprised of a hydrophobic region and a
hydrophilic side chain with a charged amine group [19]. As pDMAEMA has a similar
structure with charged amine groups, it is possible that it may also induce
phospholipidosis.
Due to the variation in pDMAEMA cytotoxicity between red blood cells and
human cell lines [3], it is possible that the range of different cell types in intact tissue may
afford differential protection. In addition, the mucus layer that is present in many
epithelial tissues may reduce toxicity [20]. To investigate this, freshly excised rat ileal
and colonic intestinal epithelial tissue was incubated for 60 min with pDMAEMA in
Ussing-type chambers. This allowed for a fast and simple assessment of the histological
effects of pDMAEMA on whole tissue. Using HCA, we report that high concentrations of
a relatively low molecular weight pDMAEMA polymer induced necrosis in U937 cells,
while apoptosis was induced in Caco-2 cells. pDMAEMA did not induce
phospholipidosis in either cell line. The cytotoxicity induced in U937 cells was seen at
lower concentrations than in Caco-2 cells. pDMAEMA induced only minimal
histological damage to rat intestinal tissue in vitro, and isolated intestinal tissue mucosae
was clearly more resilient to the polymer than cell cultures.
5
2. Materials and methods
2.1. Materials
All cell culture and HCA reagents were obtained from Invitrogen Corporation
(CA, USA), unless otherwise stated. Cell lines were obtained from the American Tissue
Type Culture Collection (ATCC). Adult male Wistar rats (210–300 g) were obtained
from the Biomedical Facility, UCD. All other general chemicals and reagents used were
of analytical grade and were obtained from Sigma-Aldrich Company Ltd. (Dorset, UK),
unless otherwise stated.
2.2. Synthesis of pDMAEMA
pDMAEMA was synthesized by atom transfer living radical polymerisation
(ATRP) in accordance with our previous descriptions [2, 3]. The molecular weight and
polydispersity index of pDMAEMA were 12.8 kDa and 1.16, respectively, as determined
by size exclusion chromatography (SEC-HPLC) [3]. pDMAEMA had high purity and
minimal levels of catalyst residue and we used the specific polymer that was described
and characterised in [3]
2.3. HCA assay of pDMAEMA against U937 and Caco-2 cells
The HCA cytotoxicity assay was based on the methods of O’Brien et al. [11]. For
HCA, either phenol red-free supplemented DMEM or RPMI media was used. Caco-2
epithelial cells (ATCC: HTB-37, passage numbers 65-69) were cultured in DMEM
containing GlutaMAXTM, supplemented with 10 % foetal bovine serum (FBS), 1 % nonessential amino acids (NEAA) and 1 % penicillin/streptomycin (Pen-Strep). U937
6
monocytes (ATCC: CRL-1593.2, passage numbers 12-14) were cultured using RPMI
medium supplemented with FBS, NEAA, Pen-Strep and 1 % L-glutamine. All cells were
grown in a humidified 37°C incubator with 5 % CO2 in air. The Caco-2 cells were
seeded in 96-well cell culture plates at a density of 500 cells/well and incubated for 24
hours at 37°C with 5 % CO2. After incubation, Caco-2 cell media was replaced with 50
µl fresh media in each well. U937 cells were seeded in 96-well plates by adding 50 µl
cells at a density of 5000 cells/well; these were incubated for 60 min to allow cells to
settle. 100 µl pDMAEMA–containing solutions were added to all wells (at 1.5 times
final concentration) and plates were further incubated for 24, 48 or 72 hours. Plate lids
were left on during acquisition to prevent evaporation. Media alone was added as
untreated controls for the 24 to 72 hour incubations. Dye cocktails were made up (Table
1). Positive control treatments were mixed with the dye cocktails (Table 2) and added to
selected wells after incubations, 30 min prior to data acquisition.
After 24-72 hour incubations with or without pDMAEMA, 50 µl of dye cocktail
was added to wells. Plates were then incubated for 30 to 40 min and images were
acquired on the In Cell® 1000 High Content Analyzer (GE Healthcare, UK), using a 10 X
objective. Ten random fields were viewed per well and concentrations of pDMAEMA
were repeated in triplicate. Experiments were repeated on at least three separate
occasions.
Fluorescence of the dyes were monitored at excitation and emission wavelengths
respectively of: (1) 360 nm and 460 nm for Hoechst 33342; (2) 480 nm and 535 nm for
Fluo 4-AM; (3) 535 nm and 600 nm for TMRM; and (4) 620 nm and 700 nm for
7
TOTO®-3. Exposure times were varied between experiments to optimise image quality.
However, typical exposure and hardware autofocus (HWAF) values were: (1) 100 ms and
11.1 µm for Hoechst 33342; (2) 250 ms and -1.1 µm for Fluo 4-AM; (3) 300 ms and -2.1
µm for TMRM; and (4) 200 ms and -0.6 µm for TOTO®-3. After acquisition of the
images, the data was analysed using In Cell® 1000 Workstation software (GE Healthcare,
UK) using multi-target analysis. Table 3 summarises the analysis settings with a glossary
of In Cell® 1000 analysis terms. For [Ca2+]i and MMP analyses, values were calculated
minus background readings.
2.4. HCA phospholipidosis assay of pDMAEMA
Evidence for phospholipidosis includes intracellular accumulation of
phospholipids and lamellar bodies in cells. To accurately view lamellar bodies, electron
microscopy is normally used [27]. The accumulation of phospholipids also correlates to
induction of phospholipidosis as visualised by Nile red [28]. The HCA phospholipidosis
assay used was based on the method of Halstead et al. [29]. U937 cells were selected to
test for phospholipidosis as they have been found to be sensitive for evaluating this
parameter [30]. In addition, lamellar bodies that are associated with phospholipidosis can
be found in intestinal epithelial cells [31], so Caco-2 cells were also used. 100 µl of
Caco-2 cells were seeded in 96-well cell culture plates at a density of 5 x 104 cells/ml.
Plates were incubated at 37°C in 5 % CO2 for 24 hours. After incubation, Caco-2 cell
media was replaced with 100 µl fresh media. For U937 cells, wells were seeded with 100
µl of cells at a density of 2 x 105 cells/ml and incubated for 60 min to allow cells to settle.
100 µl pDMAEMA (2 times final concentration) was added and the plates further
8
incubated for 24, 48 or 72 hours. Erythromycin (403 µM (250 µg/ml)) was added as a
positive control [32] and Triton X-100 (0.01%) was added as a negative control [33].
After the 24 to 72 hour incubations the plates were centrifuged at 1000 x g for 5 min and
the media removed. 150 µl dye solution, containing 1 µg/ml Nile red (Sigma) and 0.8
µM Hoechst 33342, was added to wells. The plates were further incubated for 30 min
and images were acquired using a 40 x objective as described above. Fluorescence was
monitored at respective excitation and emission wavelengths of: (1) 360 nm and 460 nm
for Hoechst 33342; (2) 535 nm and 600 nm for Nile red. Exposure and HWAF values
were set at, respectively: (1) 1500 ms and 5 µm for Hoechst 33342; (2) 100 ms and -0.1
µm for Nile red. After acquisition of the images, the data was analysed as described in
Table 4.
2.4. Gross histological effects of pDMAEMA on rat intestinal tissue
Maintenance and welfare of animals were in accordance with the “Principles of
Laboratory Animal Care” (National Institutes of Health publication number 85-23,
revised in 1985). Rats were sacrificed by cervical dislocation. Intestinal tissue was
exposed in each species via a mid-ventral abdominal incision. Segments were excised
and immediately immersed in freshly prepared oxygenated Krebs–Henseleit solution
(KH) at room temperature. Proximal ileal and colonic tissue were immediately excised,
dissected along the line of the mesentery and washed with warm oxygenated buffer.
Four-inch tissue segments were then pinned mucosal side up on a cork-board. Mucosal
sheets were mounted in modified Ussing chambers within 15 min of euthanasia, as
previously described [34]. The area of tissue exposed in the Ussing chamber window
9
was 0.63 cm2 and the apical and basolateral compartments contained 1 and 5 ml KH,
respectively. KH was gassed via a flexible polyethylene tube with 95 % O2:5 % CO2 on
the basolateral side only, as apical gassing led to evaporation. Temperature was
maintained at 37°C by immersing the rack of chambers in a heated water bath.
Immediately after mounting tissue, pre-warmed KH with or without pDMAEMA at
selected concentrations or with Triton X-100 (0.1 %, as a positive cytotoxic control), was
added to the apical compartment and the tissue incubated for 60 min.
2.5. Preparation of intestinal tissue for microscopic examination
Tissue for histology was fixed in 10 % buffered formalin and routinely processed
into paraffin wax. 4 µm sections were cut and stained in Harris's haematoxylin and eosin
(H & E) for general morphological assessment. Slides were viewed using a Nikon
(Japan) light microscope and images were captured using a Micropublisher 3.3 RTV
camera (QImaging, Surrey, BC, Canada) with Image-Pro 6.2 software (Media
Cybernetics, Inc., Silver Spring, MD, USA), using a 10 x objective.
2.6. Statistical analysis
Statistical analyses were carried out using one-way ANOVA with Bonferroni
post-hoc tests. The significance level was set at α = 0.05 (95 % confidence intervals).
10
3. Results
3.1. HCA: Cytotoxicity measurements of pDMAEMA in U937 and Caco-2 cells
pDMAEMA is more cytotoxic to U937 monocytes than to Caco-2 intestinal
epithelial cells by MTT analysis [3]. To investigate this further, HCA was utilised here to
concurrently measure 6 parameters from 4 different fluors in the presence and absence of
pDMAEMA over time. The parameters tested were: (1) nuclear area (NA), (2) nuclear
intensity (NI), (3) intracellular calcium ([Ca2+]i,) (4) mitochondrial membrane potential
(MMP), (5) plasma membrane permeability (PMP) and (6) cell number (CN). For
illustration, representative fused images of the separate U937 and Caco-2 cell HCA
assays are shown in response to response to the calcium ionophore, ionomycin (20μM)
(Fig. 1). The In Cell® software allows for three colours to be fused. Blue is the cell
nucleus (stained with Hoechst 33342). Red is the MMP (stained with TMRM). Green is
[Ca2+]i (stained with Fluo-4-AM). pDMAEMA was then assessed in both cell types using
concentrations from 0.01 to 5000 µg/ml for 24, 48 and 72 hours. Results are presented as
percent values compared to 100 % negative control.
The concentrations of pDMAEMA that induced significant effects compared to
untreated cells for the cytotoxicity parameters are summarised in Table 5. pDMAEMA
induced disruption to both PMP and MMP at concentrations of 25-50 µg/ml in U937
cells, while 50-100 µg/ml decreased [Ca2+]i following 24-72 hours exposure. PMP and
[Ca2+]i were disrupted at 100-250 µg/ml concentrations of pDMAEMA at 24, 48 and 72
hours in Caco-2, while NI was increased (100-250 µg/ml) and MMP was disrupted with
an IC50 of 550 µg/ml. In general, most parameters show increased changes in parameters
with increasing time of exposure to pDMAEMA and with increasing concentrations.
11
Fig. 2 shows concentration-response curves of the effect of pDMAEMA on the
cell lines after 72 hours in respect of the cytotoxicity parameters. IC50 values from the
cytotoxicity parameters are also described (Table 6A). The IC50 values for pDMAEMA
against U937 cells were generally lower than those against Caco-2 cells for the
cytotoxicity parameters.
When cells were treated with increasing concentrations of pDMAEMA, NA
decreased in Caco-2, while in U937 it increased. NI and PMP were increased in both cell
lines with pDMAEMA treatment. [Ca2+]i greatly increased in Caco-2 cells when treated
with pDMAEMA, whereas U937 cells showed only a slight increase followed by a
decrease with increasing pDMAEMA concentration. As pDMAEMA concentration
increased, MMP and CN increased and then decreased. Table 6B gives the IC50 values of
pDMAEMA against U937 and Caco-2 cells over time. The concentrations shown
represent the lowest IC50 values of the 6 cytotoxicity parameters tested. Against U937
cells, pDMAEMA demonstrated cytotoxic effects at concentrations of 25-50 µg/ml. In
Caco-2 cells, pDMAEMA demonstrated cytotoxic effects at concentrations of 100-250
µg/ml. In Caco-2 the IC50 values decreased with time, while in U937 there was a small
increase in the IC50 values at 72 hours. This increase may be due to an increase in the
number of cells and closer packing due to faster growth of U937, which may hinder
polymer access.
The positive control agents used in the HCA assay induced the expected activity
in each cell type, i.e. ionomycin increased intracellular calcium, FCCP decreased the
12
MMP and Triton X-100 induced increased the PMP (data not shown). Some of these
agents however caused effects on parameters other than their intended effect. For
example, in addition to induction of increased [Ca2+]i, ionomycin (20µM) increased
MMP in both cell lines (data not shown). This may be due to a mild stimulatory effect of
the drug on protective biochemical processes, similar to what was observed with
pDMAEMA. In addition to its effect on increasing PMP, Triton X-100 (0.2 %) decreased
[Ca2+]i (data not shown). This may be related to the increased membrane permeability
allowing dye leakage.
3.2. HCA: Phospholipidosis induction
Representative fused images of the U937 and Caco-2 cell phospholipidosis assays
are shown (Fig. 3). The nucleus was stained red by Hoechst 33342 and the phospholipids
were stained by green by Nile red. Compared to untreated U937 and Caco-2 cells in Fig.
3A and C, there was an increase in green fluorescence when U937 and Caco-2 were
exposed to the positive control, erythromycin (250 μg/ml) at all time points.
Erythromycin induces an increase in lysosomal phospholipids by inhibiting the activity of
lysosomal phospholipases [32]. There were occasional cells in control images that
showed increased green/phospholipids fluorescence that represent naturally occurring
dying cells. There was no significant increase in phospholipids in either the U937 or the
Caco-2 cells exposed to pDMAEMA (1-100 µg/ml) for 24 to 72 hours (Fig. 4).
Concentrations of pDMAEMA that did not cause cytotoxicity were used in this assay
because higher concentrations would have lead to permeabilisation of the cell membranes
and dye/phospholipid loss. The negative control, Triton X-100, did not induce a
13
significant increase in phospholipid accumulation. There was some decrease in
fluorescence with Triton X-100 and pDMAEMA (10 µg/ml), however this may be related
to the ability of these molecules to permeabilise plasma membranes or lysosomes,
therefore allowing dye leakage.
3.3. Histological effects of pDMAEMA on rat ileum and colon
After 60 min incubation in the horizontal diffusion chambers, control rat ileal
tissue sections showed an intact mucosal epithelium with homogeneous distribution of
villi. pDMAEMA did not cause any disruption to the epithelial surface of rat ileum at
concentrations up to 0.1 mg/ml after 60 min (data not shown). At a concentration of 1
mg/ml, there was some evidence of mucosal sloughing (Fig. 5B) and at 10 mg/ml, the
intestinal villi were somewhat disrupted (Fig. 5C). However, the lower layer of cells was
unaffected and maintained a normal epithelial surface. None of the concentrations of
pDMAEMA tested induced toxicity in comparison to the complete aberration caused by
0.1 % Triton X-100 (Fig. 5D).
With treatments of 1 mg/ml (Fig. 6B) and 10 mg/ml pDMAEMA (Fig. 6C) for 60
min, there was no disruption of the rat colonic epithelial layer, nor sloughing of cells,
compared with control tissue (Fig. 6A). Treatment of rat colonic tissue with 0.1 % Triton
X-100 caused complete aberration of the epithelial surface (Fig. 6D). There was some
oedema visible in all tissue samples, which may be due to the stretching of the tissue in
the horizontal chambers.
14
4. Discussion
HCA analysis allows the combination of fluorescent probes to be used
concurrently to investigate different parameters of cytotoxicity [9-13]. Separate
fluorescence signals can be detected simultaneously using HCA and dyes that have
overlapping fluorescence may be differentiated if their cellular locations do not overlap
[35]. By incorporating three to four fluorescent dyes, up to eight markers of cytotoxicity
have been successfully investigated concurrently [10]. These include nuclear
morphology, plasma membrane integrity, mitochondrial function, intracellular calcium
and cell proliferation [10, 11]. Use of HCA in the development of novel antimicrobial
agents may increase the speed by which these molecules are assessed and processed to
the clinical trials stage of development. To date, in vitro assays including MTT and
lactate dehydrogenase have been commonly used to assess cytotoxicity of pDMAEMA
[3, 36, 37, 38]. Although useful indicators, they do not give much information about the
processes of cytotoxicity nor early stage events. Other fluorescent-based tests using flow
cytometry and confocal microscopy may provide additional information about the
gradual process of cytotoxicity induced by a polymer, nor their mechanism. They are
also time consuming, costly and labour intensive. HCA overcomes these problems by
providing a high throughput process that allows testing of numerous cytotoxicity markers
concurrently.
Data from MTT analysis has suggested variable cytotoxic effects of pDMAEMA
in different cell types [3]. Therefore, the cytotoxic-inducing aspects of this polymer were
investigated by HCA. NA was found to increase in U937 cells and decrease in Caco-2
cells upon exposure to pDMAEMA. An increase in NA may be associated with nuclear
15
swelling as a result of premature senescence [39-41]. Cellular shrinkage and
condensation of the chromatin are markers of apoptosis [42]. The decrease in NA in
Caco-2 cells may be an indication of apoptosis and the swelling of the U937 nuclei may
indicate necrosis. The increased [Ca2+]i observed in Caco-2 cells after treatment with
pDMAEMA may also be an indicator of apoptosis [43]. In U937 cells, [Ca2+]i decreased
upon treatment with pDMAEMA. This may be related to the increased plasma
membrane permeability (PMP), which may cause dye release from the cell thereby
masking an increase in calcium. In addition, the nuclear intensity (NI) of both cell types
increased with increasing concentrations of pDMAEMA. This may also be due to an
increase in the PMP allowing more dye into the cell. pDMAEMA appeared to induce
apoptosis in Caco-2 cells but not in U937 cells, and it has previously been hypothesised
to be inducing cell death in these cells through a necrotic process as detected by flow
cytometry [38]. Our data supports this conclusion, although caution should be taken
when discussing the cytotoxicity of this polymer. Jones et al. [38] found a late-stage
decrease in MMP in U937 cells. They hypothesized that, due to the fact that disruption
of MMP is an early indicator of apoptosis and also a late stage event, the reduction in
MMP was an indicator of necrosis. The data presented here supports this conclusion, as
the concentrations that induced a decrease in MMP in U937 cells were similar to those
that produced other cytotoxic effects. This indicates a late-stage necrotic event.
However, in Caco-2, the MMP was decreased at concentrations significantly lower than
those that induced other cytotoxicity effects, including PMP and this is evidence further
supporting a process of apoptosis in Caco-2.
16
Hormesis is a process produced by some drugs where a biphasic effect is seen at
high versus low concentrations of drugs [11, 44]. This process is characterised by low
concentration stimulation and high concentration inhibition and has been found to occur
with parameters such as MMP and CN [11]. In U937, both parameters were increased at
low pDMAEMA concentrations. At higher concentrations however, there was a
decrease in MMP, but no change in CN. Hormesis may be a reaction of the cells to subcytotoxic concentrations of drug whereby the cells try to overcompensate for its presence
by increasing their biochemical protective mechanisms [45]. In Caco-2 cells, hormesis
was also evident with CN increasing at low concentrations of pDMAEMA, then
decreasing at high concentrations. For MMP, a multiphasic reaction to pDMAEMA was
seen in Caco-2 cells. At all time points there was a decrease in MMP at lower
concentrations, followed by recovery, then a decrease again as the concentration of
pDMAEMA increases. The reason this occurs is unclear, but it may be that a dual
mechanism of action is occurring, i.e., at low concentrations pDMAEMA induces an
apoptotic process of cell death, while at high concentrations a necrotic process is induced.
Modifications of pDMAEMA have also been found to reduce cytotoxicity. These
include PEGylation [6], reduction of terminal thiol groups [46] and complex formation
with n-carboxyethylchitosan [47]. Complexation of pDMAEMA with DNA also
improves transfection efficiency, as did copolymer formation with methyl methacrylate
[48]. It is known that altering the MW of pDMAEMA impacts on cytotoxicity: low MW
pDMAEMA (43 kDa) is considered less toxic than high MW pDMAEMA (915 kDa)
[49]. The MW of the pDMAEMA used here was also quite low (12.8 kDa).
Deciphering these relationships using HCA will be the subject of future investigations of
17
pDMAEMA polymers of different MW, in co-polymer formats and in complexes with
DNA and siRNA.
pDMAEMA itself has not been tested by oral administration in vivo. However,
methyl methacrylate, a component of pDMAEMA, has been found to be non-toxic by
oral administration in vivo in rats [50]. It was rapidly hydrolysed by serum
carboxylesterases resulting in rapid degradation of methacrylic acid to less toxic
compounds. Due to the variation in effect of pDMAEMA on different cell lines, its effect
on whole tissue was assessed. Against rat colonic tissue, pDMAEMA did not cause any
histopathology, while against rat ileal tissue there was a degree of sloughing of cells at
high concentrations. Fluorescent hostasol-conjugated pDMAEMA has previously been
used to visualise interactions of the polymer with rat intestinal tissue [2]. In stomach,
caecum and colon tissue, pDMAEMA was retained in the surface mucus layer of the
tissue, while in ileum, it penetrated into the intestinal crypts. This varying distribution
may explain the reason for the increased histological damage found here in ileal versus
colonic tissue. It is possible that the small amount of damage seen with pDMAEMA in
vitro on rat intestinal ileal tissue may be diminished in vivo. Absorption enhancers have
typically been found to be cytotoxic in vitro, while they produce reversible damage in
vivo, possibly due to the presence of mucus, or to the large capacity for the epithelium to
repair itself [51].
18
5. Conclusions
In summary, using HCA analysis, pDMAEMA induced cytotoxicity with pooled
IC50 values of 25-50 µg/ml against U937 cells and 100-250 µg/ml against Caco-2 cells
over 72 hours. The cytotoxicity induced by lower concentrations of pDMAEMA against
U937 compared with Caco-2 cells reflects similar results found by MTT assay [3], but
the HCA assay detected cytotoxicity at lower concentrations than MTT analysis. It also
provided more information about the state of the cells and the mechanism of cell death
induced by the polymer. Against isolated rat intestinal mucosa tissue, in vitro, however,
pDMAEMA caused no histopathology on rat colon and only limited disruption of the
ileal villi at high concentrations. Therefore, although pDMAEMA may not ultimately be
suitable for intravenous use, it may have potential as an oral or topical mucoadhesive
antibacterial formulation, bearing in mind the high concentrations required for such an
effect [3].
Acknowledgements
This study was funded by Science Foundation Ireland Strategic Research Cluster
Grant 07/SRC/B1154. We thank Eamonn Fitzpatrick from the UCD School of
Agriculture, Food Science and Veterinary Medicine for processing and staining histology
samples. We also thank David Haddleton at the University of Warwick for synthesis and
characterisation of pDMAEMA [3].
19
References
[1] V. Butun, S.P. Armes, N.C. Billingham, Synthesis and aqueous solution properties of
near-monodisperse tertiary amine methacrylate homopolymers and diblock
copolymers, Polymer 42 (2001) 5993-6008.
[2] A.J. Limer, A.K. Rullay, V.S. Miguel, C. Peinado, S. Keely, E. Fitzpatrick, S.D.
Carrington, D.J. Brayden, D.M. Haddleton, Fluorescently tagged star polymers by
living radical polymerisation for mucoadhesion and bioadhesion, Functional
Polymers 66 (2006) 51-64.
[3] L.-A. Rawlinson, S.M. Ryan, G. Mantovani, J.A. Syrett, D.M. Haddleton, D.J.
Brayden, Antibacterial effects of poly(2-(dimethylamino ethyl)methacrylate) against
selected Gram-positive and Gram-negative bacteria, Biomacromolecules 11(2010)
443-453.
[4] A. Yousefi Rad, H. Ayhan, U. Kisa, E. Piskin, Adhesion of different bacterial strains
to low-temperature plasma treated biomedical PVC catheter surfaces, J. Biomater.
Sci. Polym. Ed. 9 (1998) 915-929.
[5] S.B. Lee, R.R. Koepsel, S.W. Morley, K. Matyjaszewski, Y. Sun, A.J. Russell,
Permanent, nonleaching antibacterial surfaces. 1. Synthesis by atom transfer radical
polymerization, Biomacromolecules. 5 (2004) 877-882.
[6] Y. Qiao, Y. Huang, C. Qiu, X. Yue, L. Deng, Y. Wan, J. Xing, C. Zhang, S. Yuan, A.
Dong, J. Xu, The use of PEGylated poly [2-(N,N-dimethylamino) ethyl methacrylate]
as a mucosal DNA delivery vector and the activation of innate immunity and
improvement of HIV-1-specific immune responses, Biomaterials 31 (2009) 115-123.
[7] F.J. Verbaan, C. Oussoren, C.J. Snel, D.J. Crommelin, W.E. Hennink, G. Storm,
Steric stabilization of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes
mediates prolonged circulation and tumor targeting in mice, J. Gene Med. 6 (2004)
64-75.
[8] E. Moreau, M. Domurado, P. Chapon, M. Vert, D. Domurad, Biocompatibility of
polycations: in vitro agglutination and lysis of red blood cells and in vivo toxicity, J.
Drug Target. 10 (2002) 161-173.
[9] D.L. Taylor, In: D. L. Taylor, J. R. Haskins and K. A. Guiliano (Eds.), High content
screening: A powerful approach to systems cell biology and drug discovery, Humana
Press Inc, Totowa, NJ, USA, 2007, pp. 3-18.
[10] V.C. Abraham, D.L. Towne, J.F. Waring, U. Warrior, D.J. Burns, Application of a
high-content multi-parameter cytotoxicity assay to prioritize compounds based on
toxicity potential in humans, J. Biomol. Screen. 13 (2008) 527-537.
20
[11] P.J. O'Brien, W. Irwin, D. Diaz, E. Howard-Cofield, C.M. Krejsa, M.R. Slaughter,
B. Gao, N. Kaludercic, A. Angeline, P. Bernardi, P. Brain, C. Hougham, High
concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity
measured in a novel cell-based model using high content screening, Arch. Toxicol. 80
(2006) 580-604.
[12] P.J O’Brien, M. Canelas-Domingos, Use of high content analysis in toxicologic
clinical pathology for identification and monitoring of translational safety biomarkers.
Int. Drug Discovery 1 (2009) 17-21.
[13] J.J. Xu, P. V. Henstock, M. C. Dunn, A. R. Smith, J. R. Chabot, D. de Graaf,
Cellular imaging predictions of clinical drug-induced liver injury, Tox. Sci. 105
(2008) 97-105.
[14] J.L. Stilwell, Y. Guan, R.M. Neve, J.W. Gray, In D. L. Taylor, J. R. Haskins and K.
A. Giuliano (Eds.), High content screening: A powerful approach to systems cell
biology and drug discovery, Humana Press Inc, Totowa, NJ, USA, 2007, pp. 353-365.
[15] B. Zhang, X. Gu, U. Uppalapati, M.A. Ashwell, D.S. Leggett, C.J. Li, High-content
fluorescent-based assay for screening activators of DNA damage checkpoint
pathways, J. Biomol. Screen. 13 (2008) 538-543.
[16] J.B. Kerrison, D.J. Zack, In D. L. Taylor, J. R. Haskins and K. A. Giuliano (Eds.),
High content screening: A powerful approach to systems cell biology and drug
discovery, Humana Press Inc, Totowa, NJ, USA, 2007, pp. 427-434.
[17] N. Anderson, J. Borlak, Drug-induced phospholipidosis, FEBS Lett. 580 (2006)
5533-5540.
[18] M.J. Reasor, S. Kacew, Drug-induced phospholipidosis: are there functional
consequences?, Exp. Biol. Med. (Maywood). 226 (2001) 825-830.
[19] P. Nioi, I.D. Pardo, R.D. Snyder, Monitoring the accumulation of fluorescently
labeled phospholipids in cell cultures provides an accurate screen for drugs that
induce phospholipidosis, Drug Chem. Toxicol. 31 (2008) 515-528.
[20] C. Laboisse, A. Jarry, J.E. Branka, D. Merlin, C. Bou-Hanna, G. Vallette, Recent
aspects of the regulation of intestinal mucus secretion, Proc. Nutr. Soc. 55 (1996)
259-264.
[21] B. Maro, M.C. Marty, M. Bornens, In vivo and in vitro effects of the mitochondrial
uncoupler FCCP on microtubules, EMBO J. 1 (1982) 1347-1352.
[22] A.H. Guse, E. Roth, F. Emmrich, Intracellular Ca2+ pools in Jurkat T-lymphocytes,
Biochem. J. 291 ( Pt 2) (1993) 447-451.
21
[23] R.M. Martin, H. Leonhardt, M.C. Cardoso, DNA labeling in living cells, Cytom.
Part A. 67 (2005) 45-52.
[24] K.R. Gee, K.A. Brown, W.N. Chen, J. Bishop-Stewart, D. Gray, I. Johnson,
Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes, Cell
Calcium 27 (2000) 97-106.
[25] D. Floryk, J. Houstek, Tetramethyl rhodamine methyl ester (TMRM) is suitable for
cytofluorometric measurements of mitochondrial membrane potential in cells treated
with digitonin, Biosci. Rep. 19 (1999) 27-34.
[26] T. Zuliani, R. Duval, C. Jayat, S. Schnebert, P. Andre, M. Dumas, M.H. Ratinaud,
Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial
transmembrane potential and plasma membrane integrity: interest for cell death
investigations, Cytom. Part A. 54 (2003) 100-108.
[27] T. Nonoyama, R. Fukuda, Drug-induced phospholipidosis - pathological aspects and
its prediction, J. Toxicol. Pathol. 21 (2008) 9-24.
[28] P. Greenspan, E.P. Mayer, S.D. Fowler, Nile red: a selective fluorescent stain for
intracellular lipid droplets, J. Cell Biol. 100 (1985) 965-973.
[29] B.W. Halstead, C.M. Zwickl, R.E. Morgan, D.K. Monteith, C.E. Thomas, R.K.
Bowers, B.R. Berridge, A clinical flow cytometric biomarker strategy: validation of
peripheral leukocyte phospholipidosis using Nile red, J. Appl. Toxicol. 26 (2006)
169-177.
[30] Z. Xia, E.L. Appelkvist, J.W. DePierre, L. Nassberger, Tricyclic antidepressantinduced lipidosis in human peripheral monocytes in vitro, as well as in a monocytederived cell line, as monitored by spectrofluorimetry and flow cytometry after
staining with Nile red, Biochem. Pharmacol. 53 (1997) 1521-1532.
[31] G. Schmitz, G. Muller, Structure and function of lamellar bodies, lipid-protein
complexes involved in storage and secretion of cellular lipids, J. Lipid Res. 32 (1991)
1539-1570.
[32] J.P. Montenez, F. Van Bambeke, J. Piret, R. Brasseur, P.M. Tulkens, M.P. MingeotLeclercq, Interactions of macrolide antibiotics (Erythromycin A, roxithromycin,
erythromycylamine [Dirithromycin], and azithromycin) with phospholipids:
computer-aided conformational analysis and studies on acellular and cell culture
models, Toxicol. Appl. Pharmacol. 156 (1999) 129-140.
[33] J.K. Morelli, M. Buehrle, F. Pognan, L.R. Barone, W. Fieles, P.J. Ciaccio,
Validation of an in vitro screen for phospholipidosis using a high-content biology
platform, Cell Biol Toxicol. 22 (2006) 15-27.
22
[34] A.B. Cox, L.A. Rawlinson, A.W. Baird, V. Bzik, D.J. Brayden, In vitro interactions
between the oral absorption promoter, sodium caprate (C(10)) and S. typhimurium in
rat intestinal ileal mucosae, Pharm. Res. 25 (2008) 114-122.
[35] P.J. O'Brien, J.R. Haskins, In D. L. Taylor, J. R. Haskins and K. A. Giuliano (Eds.),
High content screening: A powerful approach to systems cell biology and drug
discovery, Humana Press Inc, Totowa, NJ, USA, 2007, pp. 415-425.
[36] S. Keely, A. Rullay, C. Wilson, A. Carmichael, S. Carrington, A. Corfield, D.M.
Haddleton, D.J. Brayden, In vitro and ex vivo intestinal tissue models to measure
mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers, Pharm.
Res. 22 (2005) 38-49.
[37] Y.Z. You, D.S. Manickam, Q.H. Zhou, D. Oupicky, Reducible poly(2dimethylaminoethyl methacrylate): synthesis, cytotoxicity, and gene delivery activity,
J. Control Release. 122 (2007) 217-225.
[38] R.A. Jones, M.H. Poniris, M.R. Wilson, pDMAEMA is internalised by endocytosis
but does not physically disrupt endosomes, J. Control Release. 96 (2004) 379-391.
[39] L. Hayflick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell
Res. 37 (1965) 614-636.
[40] Y. Kobayashi, R. Sakemura, A. Kumagai, E. Sumikawa, M. Fujii, D. Ayusawa,
Nuclear swelling occurs during premature senescence mediated by MAP kinases in
normal human fibroblasts, Biosci. Biotechnol. Biochem. 72 (2008) 1122-1125.
[41] D.H. Young, H. Kauss, Release of calcium from suspension-cultured glycine max
cells by chitosan, other polycations, and polyamines in relation to effects on
membrane permeability, Plant Physiol. 73 (1983) 698-702.
[42] S. van Cruchten, W. van den Broeck, Morphological and biochemical aspects of
apoptosis, oncosis and necrosis, Anat. Histol. Embryol. 31 (2002) 214-223.
[43] D.Y. Yu, Q.L. Zhao, Z.L. Wei, T. Nomura, I. Kashiwakura, T.V. Kagiya, T. Kondo,
Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by
sanazole, Apoptosis. 14 (2009) 655-664.
[44] E.J. Calabrese, L.A. Baldwin, A quantitatively-based methodology for the evaluation
of chemical hormesis, Hum. Ecol. Risk Assess. 3 (1997) 545-554.
[45] A.R. Stebbing, Interpreting 'dose-response' curves using homeodynamic data: with
an improved explanation for hormesis, Dose-Response 7 (2009) 221-233.
23
[46] Y. Z. You, D. S. Manickam, Q. H. Zhou, D. Oupicky, Reducible poly(2dimethylaminoethyl methacrylate): synthesis, cytotoxicity, and gene delivery activity,
J. Control. Release. 122 (2007) 217-225.
[47] E. Yancheva, D. Paneva, D. Danchev, L. Mespouille, P. Dubois, N. Manolova, I.
Rashkov, Polyelectrolyte complexes based on (quaternized) poly[(2dimethylamino)ethyl methacrylate]: behavior in contact with blood, Macromol.
Biosci. 7 (2007) 940-954.
[49] J. M. Layman, S. M. Ramirez, M. D. Green, T. E. Long, Influence of polycation
molecular weight on poly(2-dimethylaminoethyl methacrylate)-mediated DNA
delivery in vitro, Biomacromolecules. 10 (2009) 1244-1252.
[48] P. van de Wetering, J. Y. Cherng, H. Talsma, D, J. Crommelin, W. E. Hennink, 2(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents. J.
Control. Release 53 (1998) 145-153.
[50] Z. Bereznowski, In vivo assessment of methyl methacrylate metabolism and toxicity,
Int. J Biochem. Cell B. 27 (1995) 1311-1316.
[51] S. Maher, T. W. Leonard,J. Jacobsen, D. J. Brayden, Safety and efficacy of sodium
caprate in promoting oral drug absorption: from in vitro to the clinic. Adv. Drug
Delivery Rev. 61 (2009):1427-1449.
24
Table 1
Dye cocktails for HCA assay.
Dye
Hoechst 33342
Fluo 4-AM
TOTO®-3 iodide
(642/660)
TMRM
Volume
[Stock]
32 µl
40 µl
1 mM, in dH20
1 mM, in DMSO
40 µl
8 µl
1 mM, in DMSO
100 µM in dH20, from
100 mM stock (DMSO)
TMRM: tetramethyl rhodamine methyl ester; TOTO®-3: TOTO®-3
iodide (642/660); total volume=10 ml.
Table 2
Positive controls and dyes for HCA assay.
Control/Dye
Target
[Conc]
Ref
FCCP (mitochondrial
uncoupler)
Ionomycin (calcium
ionophore)
Triton X-100 (membranepreturbing detergent)
Hoechst 33342*
Fluo 4-AM*
TMRM*
TOTO®-3*
Decrease MMP
10 µM
[21]
Increase [Ca2+]i
20 µM
[22]
Decrease PMP
0.2 %
[11]
Nuclear DNA
[Ca2+]i
MMP
PMP
0.8 µM
1 µM
20 nM
1 µM
[23]
[24]
[25]
[26]
FCCP: carbonylcyanide-p-trifluoromethyphenylhydrazone; MMP: mitochondrial membrane
potential; [Ca 2+]i: intracellular calcium; PMP: plasma membrane permeability. * Dye.
25
Table 3
Settings for In Cell® 1000 Workstation analysis for U937 cells. Bracketed numbers
indicate units used for Caco-2.
Feature
Source
Segmentation
Nuclei
Wave 1
(NA, NI, CN)
Wave 2
([Ca2+]i)
Wave 4
(PMP)
Wave 3
(MMP)
Top-hat
Cells
Reference 1
Reference 2
Min. Area
Sensitivity
Collar
30 (50) µm2
10 (100) %
-
Collar
-
-
1 (5) µm
Pseudo Nuclei
Pseudo - Cells
-
-
-
-
-
-
Glossary. Feature: the parameter that the analysis package is analysing; Source: the wavelength
channel used for the feature; Segmentation: how the analysis package analyses the data, e.g.
based on nucleus, cytoplasm or whole cell; ‘Top-hat’ is the method used to identify nuclei,
‘Collar’ is the method used to identify cell cytoplasm and ‘Pseudo’ is the method used to identify
other dyes within specific areas of the cell; Min.(Minimum) area: area of the smallest cell to be
tested; Sensitivity: sensitivity for picking cells from background noise; Collar (size): radius
around nucleus for analysis.
Table 4
Settings for In Cell® 1000 Workstation analysis for phospholipidosis in cells.
Feature
Source
Segmentation
Min. area
Sensitivity
Nuclei
Cells
Wave 1
Wave 2
Top-hat
Collar
40 µm2
100 %
Collar
5 µm
26
Table 5
Summary of pDMAEMA effects on cells. Concentrations (µg/ml) of pDMAEMA that
produce significant changes from controls (P < 0.05 compared to 100 % controls
containing no drug at all time points).
A. U937
Time
(Hours)
24
NA
NI
[Ca2+]i
PMP
MMP
CN
↑100
↑50
↓50
25
↑0.01-1
48
↑100
↑50
↓100
25
72
↑100
↑50
↓100
50
↑0.01-0.1,
↓50
↑0.01-0.1,
↓50
↓50
NA
NI
[Ca2+]i
PMP
MMP
CN
↓100
↓100
↑250
↑100
↑250
↑250
250
250
↓10
↓500
↓0.1
↑100
↑100
250
↓500
↓5000
↑10,
↓1000
↓0.1
B. Caco-2
Time
(Hours)
24
48
72
↑0.1-1
>1000
Upward arrows indicate increase compared to control, downward arrows indicate decrease
compared to controls. NA: nuclear area; NI: nuclear intensity; [Ca2+]i: intracellular calcium;
PMP: plasma membrane permeability; MMP: mitochondrial membrane potential; CN: cell
number.
27
Table 6
IC50 values for pDMAEMA on Caco-2 and U937 cells after exposure to pDMAEMA.
A: 72 hours treatment, B: Time course. Notation as for Table 5.
A. pDMAEMA IC50 values after 72 hours
Parameter
Caco-2
U937
NA
NI
[Ca2+]i
MMP
PMP
CN
>5000
250
100
140
550
100
>1000
>1000
100
50
45
>1000
B. pDMAEMA IC50 values over time
Time Hours
Caco-2
24
250
48
150
72
100
Values given in µg/ml
A
U937
25
25
50
B
28
Fig. 1.
Caco-2
U937
29
% Intracellular Ca % Nuclear Intensity % Nuclear Area
% Mem Permeability % Mito Potential
% Cell Number
120
***
NA
100
**
***
80
60
250
200
NI
150
***
***
100
50
0
600
***
[Ca2+]i
400
*
200
0
200
150
***
MMP
**
**
100
**
50
0
500
400
***
***
***
***
PMP
300
200
***
***
100
0
300
CN
***
200
100
***
**
***
***
***
1
10
10
0
10
00
10
00
0
0.1
1
10
10
0
10
00
10
00
0.0 0
01
0.0
1
0.1
0.0
01
0.0
1
0
[pDMAEMA] (g/ml)
Fig. 2.
30
A
B
C
D
Fig. 3.
31
% Fluorescence
140
A
***
120
***
100
**
**
Ery
TTX
50
10
1
Control
80
[pDMAEMA] (g/ml)
180
B
***
% Fluorescence
160
140
*
120
100
Ery
TTX
100
50
10
Control
80
[pDMAEMA] (g/ml)
Fig. 4.
32
A
C
B
D
Fig. 5.
33
A
B
C
D
Fig. 6.
Figure Legends
Fig. 1. Representative fused images of the HCA assay. A: U937, B: Caco-2. Cells were grown
for 24 hours and then exposed to 20µM ionomycin for 30 min. Blue/green and red cells indicate
healthy cells with active MMPs. Green cells indicate cells with increased [Ca2+]i. Blue cells
indicated cells with no MMP and an increased PMP. Horizontal bars = 50 µm.
Fig. 2. Concentration-response curves of effects of pDMAEMA on Caco-2 and U937
cells after 72 hours. Readings were compared to 100 % negative controls. * P < 0.05, **
34
P < 0.01, *** P < 0.001 compared to controls containing no drug. Notation as for Table 5
legend.
Fig. 3. Representative fused images of the phospholipidosis HCA assay. A: U937 cells,
B: U937 cells treated with erythromycin (250 µg/ml), C: Caco-2 cells, D: Caco-2 cells
treated with erythromycin (250 µg/ml). Exposure time was 24 hours. Nuclei were
stained red by Hoechst 33342. The phospholipids were stained green by Nile red.
Horizontal bars = 10 µm.
Fig. 4. Effect of pDMAEMA on phospholipidosis. A: U937, B: Caco-2. ■ = 24 hours,
▲ = 48 hours, □ = 72 hours. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to 100 %
controls containing no drug at all time points. Ery = erythromycin, TTX = Triton X-100.
Ery: 250 µg/ml, TTX: 0.01 %.
Fig. 5. Representative images from 3 independent experiments of H & E staining of
Ussing chamber-mounted rat ileal mucosae. A: Untreated, B: 1 mg/ml pDMAEMA, C:
10 mg/ml pDMAEMA, D: 0.1 % Triton X-100. Horizontal bars = 50 µm.
Fig. 6. Representative images from 3 independent experiments H & E staining of Ussing
chamber-mounted rat colonic mucosae. A: Untreated, B: 1 mg/ml pDMAEMA, C: 10
mg/ml pDMAEMA, D: 0.1 % Triton X-100. Horizontal bars = 50 µm.
35
36