Effect of chain length on cytotoxicity and endocytosis of cationic polymers

Article abstract of DOI:10.1021/ma102498g

We investigated the effect of chain length on the cytotoxicity and endocytosis of rhodamine B end-labeled cationic linear poly(2-(N,N- dimethylamino)ethyl methacrylate) (RhB-PDMAEMA) polymers [M_w = (1.1-4.8) × 10⁴ g/mol and M_w/M_n ～ 1.2], which were synthesized via atom transfer radical polymerization by using rhodamine B-based initiator. The 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and caspase-3/7 assays revealed that the cytotoxicity and cellular membrane disruption of HepG2 cells induced by PDMAEMA depend on both the polymer concentration and chain length, and the apoptosis depends on chain length at the concentration of 37.6 μg/mL. In the concentration range 10-110 μg/mL, PDMAEMA chains with different lengths are cytotoxic to HepG2 cells by different mechanisms. Namely, (1) for short PDMAEMA chains [M_w = (1.1-1.7) × 10⁴ g/mol], the cytotoxicity, membrane disruption, and apoptosis are very low, independent of the chain length; (2) in the medium range (1.7 × 10 ⁴ < M_w < 3.9 × 10⁴ g/mol), the cytotoxicity increases with the chain length and polymer concentration, mainly due to the cooperative effect of membrane disruption and apoptosis; and (3) for long chains [M_w = (3.9-4.8) × 10⁴ g/mol], they are very disruptive to the cellular membrane, pro-apoptotic, and able to enter the cytoplasm and nucleus faster than short chains, as revealed by the real-time confocal laser scanning microscopy images, and their much higher cytotoxicity is independent of the PDMAEMA chain length.

Full text of DOI:10.1021/ma102498g

ARTICLE
pubs.acs.org/Macromolecules
Effect of Chain Length on Cytotoxicity and Endocytosis of Cationic
Polymers
Jinge Cai,† Yanan Yue,† Deng Rui,† Yanfeng Zhang,‡ Shiyong Liu,‡ and Chi Wu*,†,§
†Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
‡CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, The University of Science and
Technology of China, Hefei, Anhui 230026, China
§The Hefei National Laboratory of Physical Science at Microscale, Department of Chemical Physics, The University of Science and
Technology of China, Hefei, Anhui 230026, China
ABSTRACT: We investigated the effect of chain length on the cytotoxicity and
endocytosis of rhodamine B end-labeled cationic linear poly(2-(N,N-dimethyla-
mino)ethyl methacrylate) (RhB-PDMAEMA) polymers [Mw = (1.1-4.8) ꢀ 104 g/
mol and Mw/Mn ∼ 1.2], which were synthesized via atom transfer radical
polymerization by using rhodamine B-based initiator. The 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH),
and caspase-3/7 assays revealed that the cytotoxicity and cellular membrane
disruption of HepG2 cells induced by PDMAEMA depend on both the polymer
concentration and chain length, and the apoptosis depends on chain length at the
concentration of 37.6 μg/mL. In the concentration range 10-110 μg/mL,
PDMAEMA chains with different lengths are cytotoxic to HepG2 cells by different
mechanisms. Namely, (1) for short PDMAEMA chains [Mw = (1.1-1.7) ꢀ 104 g/
mol], the cytotoxicity, membrane disruption, and apoptosis are very low, independent of the chain length; (2) in the medium range
(1.7 ꢀ 104 < Mw < 3.9 ꢀ 104 g/mol), the cytotoxicity increases with the chain length and polymer concentration, mainly due to the
cooperative effect of membrane disruption and apoptosis; and (3) for long chains [Mw = (3.9-4.8) ꢀ 104 g/mol], they are very
disruptive to the cellular membrane, pro-apoptotic, and able to enter the cytoplasm and nucleus faster than short chains, as revealed
by the real-time confocal laser scanning microscopy images, and their much higher cytotoxicity is independent of the PDMAEMA
chain length.
’ INTRODUCTION
escape, nuclear transport and entry, and final unpacking of DNA
inside the cell nucleus.1,12,23 The polymer/DNA polyplexes
could be blocked by any of these obstacles. Our current study
mainly focused on the effect of chain length on the endocytosis
and cytotoxicity of cationic polymers. Previous results indicated
that the chain length of a given cationic polymer has a significant
impact on the cytotoxicity and also on the gene transfection
efficiency.16,24-27 It is generally known that short chains are less
cytotoxic than long ones, but the detailed mechanism remains
elusive.
Previously, we have experimentally showed that the cytotoxi-
city increases with the length of a cationic polyethylenimine (PEI)
chain even though its structure contains no toxic moieties,28 which
leads us to hypothesize that its cytotoxicity must be physical;
namely, cationic charges on the polymer chain interact with
negatively charged membranes in both the extracellular and intra-
cellular spaces and complex with negatively charged protein chains
in the intracellular space to disrupt their pathways, resulting in the
apoptosis.19,27
In the gene therapy, exogenous nucleic acids are transferred to
targeted cells of patients to express a therapeutic level of defective
or deficient proteins.1-3 The first obstacle in such a gene delivery
is the anionic nature of the cellular membrane, preventing the
endocytosis of negatively charged DNA.1 Proper cationic agents
(vectors) are needed to (1) condense genetic materials into small
particles, (2) protect them against the degradation, and (3)
facilitate their cellular entry.4-6 Cationic polymers are one kind
of such nonviral vectors because of their low host immunogeni-
city, construction flexibility, and facile preparation.7-11 How-
ever, their transfection efficiency is still much lower than that of
viral carriers.12-15 Their cytotoxicity is another important issue
that has to be addressed before any applications in human gene
therapy.16-22 Therefore, how to simultaneously increase the
transfection efficiency and reduce the cytotoxicity has stood as a
very challenging and catch-22 problem in the gene therapy.
To deliver genes from a solution mixture of anionic DNA and
cationic polymer all the way through the cellular membrane and
cytoplasm into the cell nucleus, the complexes made of polymeric
vectors and DNA have to pass through a series of narrow gaps or
obstacles (not barriers because one can go over a barrier as long
as jumping higher than it), including endocytosis, endolysosomal
Received: November 3, 2010
Revised:
February 16, 2011
Published: March 07, 2011
r
2011 American Chemical Society
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Scheme 1. Synthetic Routes Employed for the Preparation of Rhodamine B End-Labeled Cationic PDMAEMA Polymers with
Different Desired Chain Lengths via Atom Transfer Radical Polymerization (ATRP)
To prove such a hypothesis, we synthesized a series of poly-
(2-(N,N-dimethylamino)ethyl methacrylate) (PDMAEMA)
chains with different lengths by using atom transfer radical
polymerization (ATRP) protocols. Starting from the rhodamine
B-based ATRP initiator, PDMAEMA chains were end-labeled
with a fluorophore to facilitate real-time cell imaging. Note that
PDMAEMA is not a very high efficient gene transfection vector
in comparison with other cationic polymers.18,29 The choice of
PDMAEMA is due to its well-documented synthesis and char-
acterization so that it becomes an excellent model for the
evaluation of relationships between the chain structures and
functions.4,18,30 Armed with this set of well-characterized poly-
mers, we studied the chain-length-dependent cytotoxicity, cell
membrane integrity, and apoptosis, respectively, by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), lactate dehydrogenase (LDH), and caspase-3/7 assays
in HepG2 cell line. We also investigated the effect of chain length
on the endocytosis kinetics by tracking the HepG2 cellular
internalization of the chains with a confocal laser scanning
Figure 1. 1H NMR spectrum recorded in CDCl3 for rhodamine
B-based ATRP initiator (2).
microscope (CLSM).
’ EXPERIMENTAL SECTION
distilled just prior to use and the others were used as received. 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
Hoechst 33342 were purchased from Invitrogen (Eugene, OR). Cyto-
Tox 96 nonradioactive cytotoxicity assay kit and Caspase-Glo 3/7 assay
kit were purchased from Promega (Madison, WI). Fetal bovine serum
(FBS), phosphate buffered saline (PBS), Dulbecco’s modified Eagle’s
medium (DMEM), and penicillin-streptomycin were purchased from
GIBCO (Grand Island, NY). HepG2 cells were grown at 37 °C, 5% CO2
in DMEM complete medium, supplemented with 10% FBS, penicillin,
and streptomycin (both at 100 units/mL).
PDMAEMA Synthesis. Synthetic routes employed for the pre-
paration of rhodamine B end-labeled cationic PDMAEMA polymers
(RhB-PDMAEMA) with different desired chain lengths are shown in
Scheme 1. First, we prepared 2-hydroxyethyl 2-bromoisobutyrate (1) as
follows. Into a round-bottom flask equipped with a magnetic stirring bar,
ethylene glycol (155.2 g, 2.5 mol) and Et3N (14.5 mL, 0.1 mol) were
charged. After cooling to 0 °C in an ice-water bath, 2-bromoisobutyryl
bromide (12.4 mL, 0.1 mol) was added dropwise over ∼30 min. The
Materials and Cell Lines. 2-(N,N-Dimethylamino)ethyl metha-
crylate (DMAEMA, 98%, from Acros, Morris Plains, NJ) was distilled at
reduced pressure just prior to use. Rhodamine B (RhB, 99%, from Acros,
Morris Plains, NJ) was used without further purification. Cuprous
bromide (CuBr, 98%), dimethyl sulfoxide (DMSO), 2-bromo-2-methyl-
propionyl bromide (98%), and N,N,N0,N00,N00-pentamethyldiethylene-
triamine (PMDETA, 99%) were purchased from Sigma-Aldrich
(Deutschland) and used as received. Triethylamine (Et3N) and ethylene
glycol were purchased from the China National Medicines Co. Ltd.
and distilled at reduced pressure just prior to use. 1-Ethyl-
3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC HCl,
3
99%, from GL Biochem Ltd., Shanghai, China) was used as received.
Dichloromethane (CH2Cl2), 4-(dimethylamino)pyridine (DMAP), iso-
propanol (IPA), n-hexane, methanol, tetrahydrofuran (THF), petro-
leum ether (30-60 °C), anhydrous sodium sulfate, hydrochloric acid,
sodium bicarbonate, and sodium chloride were all purchased from the
China National Medicines Co. Ltd. CH2Cl2 was dried over CaH2 and
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freeze-pump-thaw cycles, sealed under vacuum. After stirring at room
temperature for 10 h, the reaction mixture was exposed to air, diluted
with THF, and passed through a silica gel column to remove the copper
catalysts. After removing the solvents, the residues were dissolved in
THF and precipitated into an excess of petroleum ether (30-60 °C).
The above dissolution-precipitation cycle was repeated for three times.
After drying in a vacuum oven overnight at room temperature, RhB-
PDMAEMA sample was obtained. GPC analysis gave an Mw of 1.10 ꢀ
104 and an Mw/Mn of 1.26. Following similar protocols, other RhB-
PDMAEMA samples with different molar masses were also synthesized
by varying the amount of ATRP initiator 2, and the structural parameters
are listed in Table 1.
Sample Characterization. All nuclear magnetic resonance spec-
troscopy (NMR) spectra were recorded on a Bruker AV300 NMR
spectrometer (resonance frequency of 300 MHz for 1H) operated in the
Fourier transform mode. CDCl3 was used as the solvents. The gel
permeation chromatography (GPC) equipped with a Waters 1515
pump and a Waters 2414 differential refractive index detector (set at
35 °C) was used to determine the molar mass and molar mass
distributions. It used a series of three linear Styragel columns HT2,
HT4, and HT5 at an oven temperature of 40 °C. The eluent was THF
and the flow rate was 1.0 mL/min. A series of narrowly distributed
polystyrene (PS) standards were employed for calibration.
Figure 2. GPC elution profiles recorded for seven rhodamine B-labeled
PDMAEMA samples (see Table 1 for average molar masses and
polydispersities).
reaction was complete after stirring for another 3 h at room temperature.
The mixture was quenched with 1 L of H2O and extracted with CH2Cl2
(100 mL ꢀ 3). The combined organic phase was further extracted with
deionized water (100 mL ꢀ 3). After drying over anhydrous Na2SO4,
CH2Cl2 was removed on a rotary evaporator. The residues were purified
by distillation (85 °C, 30 mTorr) to yield a viscous, clear, and colorless
liquid (15.6 g, 75%). 1H NMR (300 MHz, CDCl3): δ (TMS, ppm): 4.41
(t, 2H, J = 3.5 Hz), 3.87 (t, 2H, J = 3.3 Hz), 3.21 (s, 1H), 1.95 (s, 6H).
13C NMR (75 MHz, CDCl3): δ (TMS, ppm): 171.80, 67.16, 60.31,
55.72, 30.49.31
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Bromide (MTT) Cell Viability Assay. HepG2 cells were seeded in a
96-well plate at an initial density of 6000 cells/well in 100 μL of the
DMEM complete medium. After 24 h, the cells were treated with
polymers at varying concentrations. The treated cells were incubated in a
humidified environment with 5% CO2 at 37 °C for 48 h. The MTT
reagent (in 20 μL of PBS, 5 mg/mL) was then added to each well. The
cells were further incubated for 4 h at 37 °C. The medium in each well
was then removed and replaced by 150 μL of DMSO. The plate was
gently agitated for 15 min before the absorbance (A) at 490 nm was
recorded by a microplate reader (Bio-Rad). The cell viability is calcul-
ated as A490,treated/A490,control ꢀ 100%, where A490,treated and A490,control
are the absorbance values of the cells cultured with and without cationic
PDMAEMA, respectively. Each experiment was done in quadruple. The
data were shown as the mean value plus a standard deviation ((SD).
It should be noted that when the result from the MTT assay shows
low cell viability, there are two possibilities. Namely, the addition of
polymer chains inhibits the cellular growth, leading to a low cellular
growing rate. In this case, the treated cells will morphologically similar as
those in the control group so that the MTT result will not be able to
reflect the cytotoxicity. On the other hand, if the low cell viability is due
to the cell death, the MTT results do indicate the cytotoxicity because we
can see some changes in their morphology. In our study, some of the
cells were detached from the plate and began to lyse, revealed by
confocal images. Therefore, a combination of the MTT assay and
confocal observation should be used.
Then, rhodamine B-based ATRP initiator (2) was synthesized by
charging rhodamine B (4.8 g, 0.010 mol), EDC HCl (2.9 g, 0.015 mol),
3
2-hydroxyethyl 2-bromoisobutyrate (1) (3.2 g, 0.015 mol), and dry
CH2Cl2 (40 mL) into a round-bottom flask equipped with a magnetic
stirring bar. After cooling to 0 °C in an ice-water bath, DMAP (1.8 g,
15 μmol) was added. The reaction was conducted at room temperature
for 12 h. The reaction mixture was then sequentially extracted with 0.1 M
HCl (50 mL ꢀ 3), aqueous saturated NaHCO3 (50 mL ꢀ 3), and
aqueous saturated NaCl solution (50 mL ꢀ 3). After drying over anhydrous
Na2SO4, CH2Cl2 was removed on a rotary evaporator. The crude product
was purified by column chromatography (first THF/n-hexane = 4/1, v/v;
then THF/methanol = 10:1, v/v), yielding a purple powder (4.2 g, 65%). 1H
NMR (CDCl3, see Figure 1 for peak assignments), δ(TMS,ppm):8.26(1H,
i, aromatic protons), 7.81 (1H, g, aromatic protons), 7.71 (1H, h, aromatic
protons), 7.26 (1H, f, aromatic protons), 7.03 (2H, e, e0, aromatic protons),
6.89 (2H, c, c0, aromatic protons), 6.79 (2H, d, d0, aromatic protons), 4.28
(4H, j, k, -CH2CH2-), 3.62 (8H, b, b0, CH3CH2-), 1.68 (6H, l, -CH3),
and 1.30 (12H, a, a0, CH3CH2-).
We finally synthesized a set of narrowly distributed RhB-PDMAEMA
samples (3) by ATRP. In a typical example, DMAEMA (1.258 g, 8.0
mmol), rhodamine B-based initiator (2) (0.255 g, 0.4 mmol), PMDETA
(69.3 mg, 0.4 mmol), and dry IPA (3.0 mL) were charged into a Schlenk
tube. The mixture was degassed by two freeze-pump-thaw cycles.
CuBr (57.4 mg, 0.4 mmol) was then introduced into the Schlenk tube
under the protection of N2 flow. The tube was further degassed via three
Table 1. Structural Parameters of Seven Rhodamine B End-Labeled PDMAEMA Samples
RhB-PDMAEMAa
DMAEMA monomer (mmol)
initiator 2 (mmol)
Mw (g/mol)b
Mw/Mn
b
sample a
sample b
sample c
sample d
sample e
sample f
sample g
8.0
8.0
8.0
8.0
8.0
8.0
8.0
0.044
0.056
0.08
0.10
0.20
0.30
0.40
4.79 ꢀ 104
3.90 ꢀ 104
3.11 ꢀ 104
2.65 ꢀ 104
1.73 ꢀ 104
1.48 ꢀ 104
1.10 ꢀ 104
1.25
1.20
1.20
1.19
1.23
1.24
1.26
a All rhodamine B end-labeled PDMAEMA samples were synthesized at a feed ratio of [initiator 2]:[CuBr]:[PMDETA] = 1:1:1 in IPA (25 °C).
b Determined by GPC analysis using THF as the eluent.
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Figure 3. Concentration dependence of HepG2 cell viability of RhB-
PDMAEMA with different chain lengths, where MTT assay is used.
Figure 5. Concentration dependence of LDH release from HepG2 cells
treated with RhB-PDMAEMA of different chain lengths.
polymers at a final concentration of 37.6 μg/mL. The treated cells were
incubated in a humidified environment with 5% CO2 at 37 °C for 4 h.
Remove 50 μL of the cell culture medium from each well of 96-well
plate. Add 50 μL of the Caspase-Glo 3/7 reagent to each well of the 96-
well plate. The plate was gently shaken at room temperature for 2 h
before 90 μL of the mix contents of each well was moved to a white-
walled 96-well plate. Using a GloMax 96 microplate luminometer, we
determined the luminescence from each well. The activity of caspase-3/
7 is expressed as RLUtreated/RLUcontrol, where RLUtreated and RLUcontrol
are the relative luminescence unit (RLU) of the cells cultured with and
without cationic PDMAEMA, respectively. Each experiment was done in
quadruple. The data were shown as the mean value plus a standard
deviation ((SD).
Confocal Laser Scanning Microscopy. A total of 96 000 cells
were seeded in a μ-Dish35 mm,high (ibidi GmbH, Germany). After 24 h,
the cellular nucleus was stained with Hoechst for 5 min and then washed
once with PBS. After the cell culture medium was aspirated, RhB-
PDMAEMA in serum-free DMEM was applied at a fixed final concen-
tration (37.6 μg/mL). Live cell imaging was performed for 6 h using a
Nikon C1si CLSM equipped with a standard fluorescence detector
(Nikon, Japan) and an INU stage-top incubator (Tokai Hit, Japan).
Image sequences were captured at an ∼5 min interval. RhB-PDMAEMA
and Hoechst were excited at 543 and 408 nm, respectively. The
corresponding emissions were detected at 605/75 and 480/25 nm,
respectively. The mean RhB-PDMAEMA fluorescence intensities inside
the cytoplasm and nucleus are normalized by that in the extracellular
space. All the images were analyzed using the Nikon EZ-C1 software.
Figure 4. Chain length dependence of half-maximal inhibitory concen-
tration (IC50) of RhB-PDMAEMA in HepG2 cells.
It should also be noted that the Trypan blue can also be used to study
the cytotoxicity, but it is not able to rapidly process a large number of
cells as the MTT assay with a microplate reader. Counting a small
number of cells in the trypan blue exclusion may lead to a relatively large
error in the results. Therefore, the Trypan blue is not suitable for the
study of the cytotoxicity of a large amount of samples. In addition, the λex
and λem of PI are 490 and 635 nm, respectively, so that the observation of
PI staining will be largely affected by our rhodamine-labeled PDMAE-
MA that also has a strong absorption at 490 nm and fluoresce in red. This
also explains why we used the MTT assay instead of other probes.
Lactate Dehydrogenase (LDH) Membrane Integrity Assay.
HepG2 cells were seeded in a 96-well plate at an initial density of 6000
cells/well in 100 μL of the DMEM complete medium. After 24 h, the
cells were treated with polymers at different chosen concentrations. The
treated cells were incubated in a humidified environment with 5% CO2
at 37 °C for 4 h. To determine the maximum LDH release, the 10ꢀ lysis
solution was added to the control group 2 h prior to the use of the
CytoTox 96 nonradioactive cytotoxicity assay kit. In each measurement,
50 μL of the cell culture solution from each well of the plate was carefully
aspirated and mixed with 50 μL of the reconstituted substrate mix in a
new 96-well plate. After 30 min incubation at room temperature, 50 μL
of stop solution was added to each well. The absorbance (A) at 490 nm
was recorded by a microplate reader (Bio-Rad). The LDH release,
defined as (A490,treated - A490,control)/(A490,maximum - A490,control) ꢀ
100%, represents the membrane disruption, where A490,treated and A490,
control are the absorbance values of the cells cultured in the presence and
absence of cationic PDMAEMA, and A490,maximum is the absorbance
value of cells in the maximum LDH release control group. Each
experiment was conducted in triple. The data were shown as the mean
value plus a standard deviation ((SD).
’ RESULTS AND DISCUSSION
Figure 3 shows that (1) at CPDMAEMA = 9.4 μg/mL, ∼60% of
the HepG2 cells are survived, irrespective of the chain length; (2)
when CPDMAEMA = 113 μg/mL, only ∼20% of the cells are
survived, also independent of the chain length; and (3) when 9.4 <
CPDMAEMA < 113 μg/mL, the cytotoxicity increases with the
chain length. On the other hand, Figure 4 reveals that in the range
Mw < 1.7 ꢀ 104 g/mol, the cytotoxicity of PDMAEMA is low and
nearly independent of the chain length, whereas in the range
Mw > 3.9 ꢀ 104 g/mol, the cytotoxicity is rather high but also
independent of the chain length. Therefore, for shorter chains
(Mw < 1.7 ꢀ 104 g/mol) and lower concentration (CPDMAEMA
<
∼10 μg/mL), PDMAEMA is much less cytotoxic; but for longer
chains (Mw > 3.9 ꢀ 104 g/mol) at higher concentrations
(CPDMAEMA > ∼110 μg/mL), PDMAEMA is fairly cytotoxic,
also independent of the chain length. Using the LDH assay, we
like to find whether short and long chains are cytotoxic to HepG2
cells in a different way.
Caspase-3/7 Activity Assay. HepG2 cells were seeded in a 96-
well plate at an initial density of 20 000 cells/well in 100 μL of the
DMEM complete medium. After 24 h, the cells were treated with
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Figure 6. Chain length dependence of RhB-PDMAEMA concentration
at which LDH release from HepG2 cells reaches 50%.
Figure 8. Chain length dependence of HepG2 cell death rate (top) and
LDH release (bottom) of RhB-PDMAEMA in the middle concentra-
tion range.
Figure 7. Chain length dependence of HepG2 cell death rate (top)
and LDH release (bottom) at low and high RhB-PDMAEMA con-
centrations.
Figure 9. Chain length dependence of caspase-3/7 activity of RhB-
PDMAEMA at a concentration of 37.6 μg/mL.
Figure 5 shows that when CPDMAEMA = 9.4 μg/mL, PDMAE-
MA induces a low level release of LDH, independent of the chain
length; while in the range 10 < CPDMAEMA < 110 μg/mL, the
release level of LDH increases with the chain length, revealing
that longer PDMAEMA chains are more disruptive to the cellular
membrane than shorter ones. To further illustrate this point,
Figure 6 summarizes the chain length dependence of PDMAE-
MA concentration at which the release of LDH from HepG2 cells
reaches 50%, reflecting the ability of different cationic PDMAE-
MA chains in the destabilization and disruption of the cell
membrane. It is clear that the disruption of the cell membrane
by shorter chains requires a much higher concentration. In the
molar mass range of (1-5) ꢀ 104 g/mol, the cellular membrane
disruption potential of the PDMAEMA chain linearly increases
with its length. Figures 3-6 show that both the cytotoxicity and
membrane disruption ability of PDMAEMA to HepG2 cells are
dependent not only on the chain length but also on the polymer
concentration. Therefore, we have to separate theses complicate
convolutions in a more clear and understandable way.
the chain length when CPDMAEMA g 113 μg/mL. In the middle
concentration range of 10-110 μg/mL, HepG2 cell death rate
and membrane disruption much depend on the chain length, as
shown in Figure 8.
For short chains (Mw < 1.7 ꢀ 104 g/mol), the cytotoxicity and
membrane disruption of HepG2 cells are less influenced by their
lengths. In the middle range (1.7 ꢀ 104 < Mw < 3.9 ꢀ 104 g/mol),
both the cytotoxicity and membrane disruption increase with the
chain length. For longer chains (Mw > 3.9 ꢀ 104 g/mol), the
longer the chain, the more disruptive it appears to be, but the
cytotoxicity nearly remains a constant. Presumably, longer chains
can interact with negatively charged proteins and membranes in
the intracellular space more effectively than short ones due to a
relatively less entropic loss from the pure thermodynamic point
of view.
To find the contribution of apoptosis to cytotoxicity induced
by PDMAEMAs with different chain length in the middle
concentration range of 10-110 μg/mL, caspase-3/7 activity
assay was performed. Caspase-3/7 activity occurs as proteins
begin to degrade in the milieu and disappears after cell death.32
Figure 9 shows that in the range Mw < 1.7 ꢀ 104 g/mol the
caspase-3/7 activation of treated sample is only 1.5-fold higher
than that of untreated control, indicating that apoptosis rate of
PDMAEMA is very low. Moreover, no significant difference
between these three PDMAEMA chains was observed, revealing
Figure 7 shows the chain length dependence of HepG2 cell
death rate and the LDH release (membrane disruption) at a low
and a high concentration (CPDMAEMA = 9.4 and 113 μg/mL).
RhB-PDMAEMA is less cytotoxic and nearly not disruptive to
the cellular membrane at CPDMAEMA e 9.4 μg/mL regardless of
its chain length. On the other hand, it becomes highly cytotoxic
and its ability to disrupt the cell membrane slightly increases with
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Figure 10. Interpenetration of RhB-PDMAEMA chains (Mw = 1.1 ꢀ 104 g/mol) into HepG2 cells and subsequent intracellular trafficking, where time
(t) is counted after addition of RhB-PDMAEMA, CPDMAEMA = 37.6 μg/mL in serum-free DMEM, and RhB-PDMAEMA chains were visualized by
excitation with a 543 nm laser light.
Figure 11. Cellular uptake kinetics of RhB-PDMAEMA (Mw = 1.1 ꢀ
104 g/mol) on one typical HepG2 cell in terms of normalized fluores-
Figure 12. Time dependence of HepG2 cellular uptake of RhB-
PDMAEMA with different lengths, where 17, 24, and 26 cells are
analyzed after adding RhB-PDMAEMA with different chain lengths.
cence intensity inside nucleus and cytoplasm, respectively, where time
(t) is counted after addition of RhB-PDMAEMA at a final concentration
of 37.6 μg/mL in serum-free DMEM.
total of 6 h after the cells were treated with RhB-PDMAEMA
(Mw = 1.1 ꢀ 104 g/mol and CPDMAEMA = 37.6 μg/mL). Figure 10
that the apoptosis rate is independent of the chain length. In the
range 1.7 ꢀ 104 g/mol < Mw < 3.9 ꢀ 104 g/mol, the apoptosis rate
shows that as expected the initial uptake times are not identical
increases with the chain length. In the range Mw > 3.9 ꢀ 104 g/mol,
the apoptosis rate is rather high and almost independent of the chain
length.
for different HepG2 cells. However, ∼6 h after adding PDMAE-
MA, both the fluorescence intensities inside the cytoplasm and
nucleus reach their maximum values, indicating that the uptake of
PDMAEMA ceases after ∼6 h for most of the HepG2 cells. To
further elucidate the cellular uptake process, we tracked one
single cell after the treatment of RhB-PDMAEMA (Mw = 1.1 ꢀ
104 g/mol and CPDMAEMA = 37.6 μg/mL). Figure 11 shows that
after 120 min RhB-PDMAEMA chains form some clumps on the
cellular membrane. At 140 min postaddition, the number of the
clumps on the membrane increases, and at the same time, both
the cytoplasm and the nucleus become faintly fluorescent with
some discrete and easily defined patches inside the nucleus. The
fluorescence intensity inside the cell, especially for that inside the
nucleus, further increases with time. Finally, the overall fluores-
cence intensity inside the cytoplasm and nucleus approach their
maxima after 180 min. Figure 11 also shows the time-dependent
fluorescence intensity within the cytoplasm and cell nucleus,
revealing the cellular uptake kinetics. The simultaneous increases
of the fluorescence intensities inside the cytoplasm and nucleus
Therefore, according to Figures 8 and 9, we can conclude that
at the concentration of 37.6 μg/mL (1) for short chains (Mw <
1.7 ꢀ 104 g/mol), the cytotoxicity, membrane disruption and
apoptosis rate of HepG2 cells are less influenced by their lengths;
(2) in the middle range 1.7 ꢀ 104 g/mol < Mw < 3.9 ꢀ 104 g/mol,
the cytotoxicity, membrane disruption, and apoptosis rate in-
crease with the chain length, indicating that the cytotoxicity of
PDMAEMA in this range might be due to the cooperative effect
of apoptosis and the destabilization of the cellular membrane;
and (3) for longer chains (Mw > 3.9 ꢀ 104 g/mol), the longer the
chain, the more disruptive it appears to be, whereas the apoptosis
rate is not significantly altered, even slightly decreases, so that the
cytotoxicity remains a constant, almost independent of the chain
length.
Further, we investigated the effect of chain length on HepG2
cellular uptake kinetics. The live cell imaging was recorded for a
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the cytotoxicity, cellular membrane disruption, and apoptosis of
HepG2 cells by using a set of narrowly distributed linear cationic
rhodamine B end-labeled poly(2-(N,N-dimethylamino)ethyl
methacrylate) (RhB-PDMAEMA) chains with different molar
masses [Mw = (1.1-4.8) ꢀ 104 g/mol]. This systematic study
shows that at CPDMAEMA < ∼10 μg/mL and CPDMAEMA > ∼110
μg/mL, regardless of the chain length, PDMAEMA is less and
highly cytotoxic to HepG2 cells, respectively; while in the
medium concentration range of 10-110 μg/mL, the cytotoxicity
of PDMAEMA to HepG2 cells increases with the chain length
but through different mechanisms. Namely, for short chains
(Mw < 1.7 ꢀ 104 g/mol), the cytotoxicity, the membrane
disruption ability and apoptosis rate are very low and indepen-
dent of the chain length; in the medium range of Mw = (1.7-3.9) ꢀ
104 g/mol, the cytotoxicity is due to the cooperative effect of
apoptosis and the destabilization of the cellular membrane;
whereas for long chains (Mw > 3.9 ꢀ 104 g/mol), the membrane
disruption and apoptosis rate are rather high, and the pro-
nounced cytotoxicity is also independent of the chain length.
Presumably, long cationic chains are more toxic in the intracel-
lular space due to their more effective complexation with
negatively charged proteins. The study of the uptake kinetics
also shows that long chains can penetrate HepG2 cellular and
nuclear membranes more quickly than their short counterparts.
Figure 13. Effect of chain length on HepG2 cellular uptake of RhB-
PDMAEMA, where t is the duration period after adding RhB-PDMAE-
MA, and 17, 24, and 26 cells are analyzed for different chain lengths.
indicate that short RhB-PDMAEMA chains can go all the way
through the cytoplasm and the nucleus membranes to the
nucleus. Moreover, Figure 11 reveals that HepG2 cellular uptake
of short RhB-PDMAEMA chains only starts at ∼120 min after
the addition in serum-free DMEM and ends at ∼190 min;
namely, the whole uptake process occurs within ∼3 h. Using
such a procedure, we tracked the cellular uptake of 17 cells to
obtain distributions and statistics.
Further, we repeated the same kinetic study for longer
PDMAEMA chains (Mw = 2.6 ꢀ 104 and 4.8 ꢀ 104 g/mol,
CPDMAEMA = 37.6 μg/mL) to investigate the effect of chain
length on HepG2 cellular uptake. According to the time at which
the cellular uptake starts, we analyzed the time-dependent
cellular uptake, as shown in Figure 12. For the longest chains,
nearly 20% HepG2 cells start to uptake PDMAEMA chains
within 0.5 h; for the chains in the middle length range, the cellular
uptake starts after 0.5 h and reaches 12% during the first hour;
while for the shortest chains, the cellular uptake occurs only after
1 h. Therefore, long chains are more effective in penetrating
HepG2 cellular and nuclear membranes. According to how long
the cellular uptake lasts, cells are separated into three groups, as
shown in Figure 13.
’ AUTHOR INFORMATION
Corresponding Author
*The Hong Kong address should be used for all correspondence.
’ ACKNOWLEDGMENT
The financial support of the National Natural Scientific
Foundation of China (NNSFC) Projects (50773077 and
20934005) and the Hong Kong Special Administration Region
Earmarked (RGC) Projects (CUHK4037/07P, 2160331;
CUHK4046/08P, 2160365; CUHK4039/08P, 2160361; and
CUHK4042/09P, 2160396) is gratefully acknowledged.
For the longest chains studied, nearly 30% of the cells take
0.5-1.0 h to finish their cellular uptake process; while for the
shortest chains, the cellular uptake lasts for more than 1 h. Although
the duration time of HepG2 cellular uptake of PDMAEMA chains
falls into a wide range of 40-180 min, varying from one cell to
another, we can still see that the uptake of longer chains is much
faster than that of their short counterparts. This is consistent with
the effect of chain length on the cellular membrane disruption.
Nevertheless, it is worth noting that HepG2 cellular uptake of either
the DNA/PDMAEMA polyplexes or free PDMAEMA chains is
much faster than the gene transfection (>12 h); i.e., most of the gene
expression occurs afterward.
Finally, we like to emphasize that the purpose of the current
study is focused on the effect of polymer chain length on the
cytotoxicity not on all the intracellular biochemical markers and
other proapoptotic proteins. The detailed pathways of polymer
chains with different lengths in the intracellular space have been
planned for our future study.
’ REFERENCES
(1) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug
Discovery 2005, 4, 581–593.
(2) Emery, D. W. Clin. Appl. Immunol. Rev. 2004, 4, 411–422.
(3) Verma, I. M.; Somia, N. Nature 1997, 389, 239–242.
(4) Georgiou, T. K.; Vamvakaki, M.; Patrickios, C. S.; Yamasaki,
E. N.; Phylactou, L. A. Biomacromolecules 2004, 5, 2221–2229.
(5) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res.
2000, 17, 113–126.
(6) Bloomfield, V. A. Biopolymers 1997, 44, 269–282.
(7) Han, S.; Mahato, R. I.; Sung, Y. K.; Kim, S. W. Mol. Ther. 2000,
2, 302–317.
(8) Kabanov, A. V. Pharm. Sci. Technol. Today 1999, 2, 365–372.
(9) Yue, Y.; Jin, F.; Deng, R.; Cai, J.; Chen, Y.; Lin, M.; Kung, H.; Wu,
C. J. Controlled Release 201110.1016/j.jconrel.2010.10.028.
(10) Itaka, K.; Yamauchi, K.; Harada, A.; Nakamura, K.; Kawaguchi, H.;
Kataoka, K. Biomaterials 2003, 24, 4495–4506.
(11) von Harpe, A.; Petersen, H.; Li, Y. X.; Kissel, T. J. Controlled
Release 2000, 69, 309–322.
(12) Morille, M.; Passirani, C.; Vonarbourg, A.; Clavreul, A.; Benoit,
J. P. Biomaterials 2008, 29, 3477–3496.
(13) Schaffert, D.; Wagner, E. Gene Ther. 2008, 15, 1131–1138.
(14) Read, M. L.; Logan, A.; Seymour, L. W. Adv Genet. 2005,
53, 19–46.
’ CONCLUSIONS
Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT), lactate dehydrogenase (LDH), and
caspase-3/7 assays, we have studied the effect of chain length on
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ARTICLE
(15) Wightman, L.; Kircheis, R.; Rossler, V.; Carotta, S.; Ruzicka, R.;
Kursa, M.; Wagner, E. J. Gene Med. 2001, 3, 362–372.
(16) vandeWetering, P.; Cherng, J. Y.; Talsma, H.; Hennink, W. E. J.
Controlled Release 1997, 49, 59–69.
(17) Morimoto, K.; Nishikawa, M.; Kawakami, S.; Nakano, T.;
Hattori, Y.; Fumoto, S.; Yamashita, F.; Hashida, M. Mol. Ther. 2003,
7, 254–261.
(18) Layman, J. M.; Ramirez, S. M.; Green, M. D.; Long, T. E.
Biomacromolecules 2009, 10, 1244–1252.
(19) Parhamifar, L.; Larsen, A. K.; Hunter, A. C.; Andresen, T. L.;
Moghimi, S. M. Soft Matter 2010, 6, 4001–4009.
(20) Fischer, D.; Bieber, T.; Li, Y. X.; Elsasser, H. P.; Kissel, T.
Pharm. Res. 1999, 16, 1273–1279.
(21) Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. J. Controlled
Release 2006, 114, 100–109.
(22) Breunig, M.; Lungwitz, U.; Liebl, R.; Goepferich, A. Proc. Natl.
Acad. Sci. U.S.A. 2007, 104, 14454–14459.
(23) Jackson, D. A.; Juranek, S.; Lipps, H. J. Mol. Ther. 2006,
14, 613–626.
(24) Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Biomed. Mater. Res.
1999, 45, 268–275.
(25) Breunig, M.; Lungwitz, U.; Liebl, R.; Fontanari, C.; Klar, J.;
Kurtz, A.; Blunk, T.; Goepferich, A. J. Gene. Med. 2005, 7, 1287–1298.
(26) Georgiou, T. K.; Phylactou, L. A.; Patrickios, C. S. Biomacro-
molecules 2006, 7, 3505–3512.
(27) Moghimi, S. M.; Symonds, P.; Murray, J. C.; Hunter, A. C.;
Debska, G.; Szewczyk, A. Mol. Ther. 2005, 11, 990–995.
(28) Deng, R.; Yue, Y.; Jin, F.; Chen, Y. C.; Kung, H. F.; Lin,
M. C. M.; Wu, C. J. Controlled Release 2009, 140, 40–46.
(29) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl Acad. Sci. U.S.A.
1995, 92, 7297–7301.
(30) Cherng, J. Y.; vandeWetering, P.; Talsma, H.; Crommelin,
D. J. A.; Hennink, W. E. Pharm. Res. 1996, 13, 1038–1042.
(31) White, M. A.; Johnson, J. A.; Koberstein, J. T.; Turro, N. J. J. Am.
Chem. Soc. 2006, 128, 11356–11357.
(32) Huang, R.; Southall, N.; Cho, M. H.; Xia, M.; Inglese, J.; Austin,
C. P. Chem. Res. Toxicol. 2008, 21, 659–667.
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