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Interaction of Polycationic Polymers with Supported Lipid Bilayers and Cells:
Nanoscale Hole Formation and Enhanced Membrane Permeability
Seungpyo Hong,|,#,† Pascale R. Leroueil,⊥,#,† Elizabeth K. Janus,§,# Jennifer L. Peters,#Mary-Margaret Kober,⊥,#
Mohammad T. Islam,#Bradford G. Orr,‡,X,# James R. Baker, Jr.,#and Mark M. Banaszak Holl‡,§,|,⊥,#,*
Programs in Applied Physics, Biophysics, and Macromolecular Science and Engineering, Departments of Chemistry and
Physics, and Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan,
Ann Arbor, Michigan 48109. Received March 27, 2006; Revised Manuscript Received April 5, 2006
Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid
bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM),
cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter
(FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran
(DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance
for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or
expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1-3µg/mL. By way
of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not
induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers
tested are not toxic to KB or Rat2 cells up to a 12 µg/mL concentration (XTT assay). In the concentration range
of 6-12 µg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase
(LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most
dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small
dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images
also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using
the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye
diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the
formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of
materials across the cell membrane.
INTRODUCTION
The water soluble synthetic polycationic polymers poly-L-
lysine (PLL), polyethylenimine (PEI), diethylaminoethyl-dextran
(DEAE-DEX), and polyamidoamine (PAMAM) dendrimers
have been employed in a variety of gene and drug delivery
system applications (1-3). The generally accepted mechanism
for internalization of these polymers is polycation-mediated
endocytosis, a three-step process composed of binding with
phospholipids and/or glycolipids in the membrane, internaliza-
tion into cells, and exit from the endosome (4-7). Having
particular interest in the first step of the process, several research
groups have used model systems including phosphatidyletha-
nolamine containing anionic vesicles to study the membrane/
particle interaction (8-10). Studies of model systems have
provided good evidence for membrane disruption caused by
cationic macromolecules such as PEI, PAMAM dendrimers, and
poly-(N-ethyl-4-vinylpyridinium bromide) as measured by
changes in fluorescence intensity of artificial vesicles containing
dyes such as fluorescein (FITC) after exposure to the polycations
(10-12). Several reports using living cell membranes have also
demonstrated increased membrane permeability as determined
by cytotoxicity (cytosolic enzyme leakage such as LDH) using
assay techniques (13,14). However, no clear connection has
been made between proposed models and in vitro studies.
We recently reported that positively charged PAMAM
dendrimers cause membrane disruption (dendroporation), al-
lowing the diffusion of molecules in and out of cells (15). This
in vitro result is in good agreement with the proposed hole
formation mechanism, as suggested by atomic force microscopy
(AFM) on a supported 1,2-dimyristoyl-sn-glycero-3-phospho-
choline (DMPC) model membrane (16-19). On the basis of
our previous results, we wanted to explore if nanoscale hole
formation could be caused by less exotic polycationic polymers
such as PEI, PLL, and DEAE-DEX which are substantially
cheaper and more commonly used materials than the PAMAM
dendrimers. The key hypotheses we address in this paper are
as follows:
(1) Commercially available polycationic polymers, PEI, PLL,
and DEAE-DEX cause nanoscale hole formation in supported
lipid bilayers.
(2) Commercially available polycationic polymers, PEI, PLL,
and DEAE-DEX cause substantial permeability of the cell
plasma membrane allowing enhanced enzyme and dye diffusion.
(3) Under the same conditions, neutral linear polymers (PEG
and PVA) do not cause nanoscale hole formation in supported
lipid bilayers nor do they generate comparable enhancements
in cell membrane permeability.
In this study, we employed AFM to observe the effect of
* To whom correspondence should be addressed. Phone: (734) 763-
2283. Fax: (734) 763-2307. E-mail: mbanasza@umich.edu.
‡Program in Applied Physics.
§Program in Biophysics.
|Program in Macromolecular Science and Engineering.
⊥Department of Chemistry.
XDepartment of Physics.
#Michigan Nanotechnology Institute for Medicine and Biological
Sciences.
†Both authors provided the same contribution for this work.
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polymers upon supported DMPC lipid bilayers. Polymer
interactions with cells in vitro were investigated in terms of
morphological studies using confocal laser scanning microscopy
(CLSM) as well as toxicological studies using XTT, lactate
dehydrogenase (LDH), and luciferase (LUC) assays and flow
cytometry. AFM studies revealed that the presence of polyca-
tionic polymers induces nanoscale hole formation and/or the
expansion of preexisting defects in the supported DMPC
bilayers. The presence of polycationic polymers in the cell
medium made the cell membrane permeable to molecules such
as cytosolic enzymes (LDH and LUC), propidium iodide (PI),
and FITC. These results directly support the three hypotheses
described above and suggest an alternative model to polycation-
mediated endocytosis for the transport of materials across cell
membranes in the presence of polycationic polymers.
EXPERIMENTAL PROCEDURES
Materials and Measurement of Molar Masses. PLL, PLL-
FITC conjugate (PLL-FITC), PEI, and DEAE-DEX were
purchased from Sigma-Aldrich. G5 PAMAM dendrimers were
synthesized and then conjugated with FITC at the Michigan
Nanotechnology Institute for Medicine and Biological Sciences,
University of Michigan (15). DMPC lipids were provided by
Avanti Lipids, Alabaster, AL. Chemical structures of the
polycationic polymers used in this paper are illustrated in Figure
1. The molar mass moments and molar mass distribution of each
polymer sample was measured using gel permeation chroma-
tography (GPC). The number average molar mass (Mn) and
polydispersity index (PDI), a commonly used measure of the
breadth of the molar mass distribution defined as the ratio of
the weight and number average molar masses (Mw/Mn), of
individual samples are listed in Table 1. GPC experiments were
performed using an Alliance Waters 2690 separation module
(Waters Corp., Milford, MA) equipped with a Waters 2487 UV
absorbance detector (Waters Corp.), a Wyatt Dawn DSP laser
photometer (Wyatt Technology Corp., Santa Barbara, CA), an
Optilab DSP interferometric refractometer (Wyatt Technology
Corp.), and TosoHaas TSK-Gel Guard PHW 06762 (75×7.5
mm, 12 µm), G 2000 PW 05761 (300 ×7.5 mm, 10 µm), G
3000 PW 05762 (300 ×7.5 mm, 10 µm), and G 4000 PW (300
×7.5 mm, 17 µm) columns. Column temperatures were
maintained at 25 (0.2 °C with a Waters temperature control
module. Citric acid buffer (0.1 M) with 0.025% sodium azide
in water was used as a mobile phase. The pH of the mobile
phase was adjusted to 2.74 using NaOH, and the flow rate was
maintained at 1 mL/min. Sample concentration was approxi-
mately 2 mg/mL, and an injection volume of 100 µL was used
for all samples. Molar mass moments of the polymers
were determined using Astra software (version 4.7) (Wyatt
Technology Corp.).
Preparation and AFM Observation of Supported DMPC
Lipid Bilayers. A 0.67 mg/mL suspension of small, unilamellar
vesicles (SUVs) was prepared as previously reported (15,16,
20). Supported lipid bilayers were formed by depositing 80 µL
of liposome suspension on a 1 ×1cm
2piece of freshly cleaved
mica. After an incubation time of approximately 20 min, the
sample was gently rinsed with water to remove excess lipids
and placed in the AFM for imaging as described in previous
studies. All AFM measurements were performed in tapping
mode on a Nanoscope IIIa Multimode scanning probe micro-
scope from Digital Instruments (DI, Veeco Metrology Group,
Santa Barbara, CA). The AFM was equipped with a liquid cell
(DI) and a silicon nitride cantilever (DI model NPS, spring
constant 0.32 N/m, length 100 µm) operating at a drive
frequency of 6-9 kHz. After taking an initial image of the
bilayer, approximately 20 µL of polymer solution was injected
into the liquid cell. All solutions were prepared using high purity
water (Nerl Diagnostics, East Providence, RI). The temperature
inside the liquid cell was 28 °C and therefore above the gel to
liquid transition temperature of supported DMPC bilayers.
Cell Lines. The KB and Rat2 cell lines were purchased from
the American Type Tissue Collection (ATCC, Manassas, VA)
and grown continuously as a monolayer at 37 °C and 5% CO2
in RPMI 1640 medium (Mediatech, Herndon, VA) and Dul-
becco’s modified Eagle’s medium (DMEM, Gibco, Eggenstein,
Germany), respectively. The Rat2 cell line was transfected to
permanently express the LUC gene using PAMAM dendrimer-
mediated gene transfection as previously described (15,21,22).
The LUC expressing Rat2 cells are noted as Rat2pLUC. The
RPMI 1640 and DMEM media were supplemented with
penicillin (100 units/mL), streptomycin (100 µg/mL), and 10%
heat-inactivated fetal bovine calf serum (FBS) before use.
XTT, LUC, and LDH Assays and Flow Cytometry.
Cytotoxicity of the polycationic polymers was assessed with a
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)car-
bonyl]-2H-tetrazolium hydroxide (XTT) assay kit (Cell Prolif-
eration Kit II, Roche Molecular Biochemicals, Mannheim,
Germany). KB and Rat2 cell lines (at a concentration of 5 ×
104cells/well) were prepared as monolayers in 96 well plates,
followed by incubation with polymers in phosphate-buffered
saline (PBS) with Ca2+and Mg2+at 37 °C under 5% CO2for
4.5 h. Polymer solutions (supernatants) were removed and
followed by washing with PBS twice. Mitochondrial activities
in the cells were then measured using the assay kit.
The LDH and LUC activities in the cell supernatant after 3
h incubation were respectively analyzed using an LDH assay
kit (Promega Co., Madison, WI) and a chemiluminescence
assay(Promega Co., Madison, WI) as previously described (15).
The measured LDH and LUC activities were either recalculated
by percentage to the activities of cell lysates of intact cells (%
LDH released) or adjusted for the protein concentration of the
sample (relative light unit (RLU)/mg protein). Flow cytometry
was also performed according to our report (15). Fluorescence
signal intensities were measured by a Coulter EPICS/XL MCL
Beckman-Coulter flow cytometer and data were analyzed using
Expo32 software (Beckman-Coulter, Miami, FL).
CLSM Observation. A concentration of 2 ×104cells/mL
of Rat2 cells was seeded on MatTek glass bottom Petri dishes
(35 mm) and incubated at 37 °C under 5% CO2for 24 h. The
DMEM was removed and 2 mL of each PLL-FITC and
dendrimer-FITC conjugates in PBS (Ca2+,Mg
2+) solution was
added into the appropriate dish. The dishes were incubated with
added solutions at 37 °C under 5% CO2for 1 h. The conjugate-
Figure 1. Chemical structures of commercially available polycationic
polymers used in this study.
Effect of Polycations on Membranes
Bioconjugate Chem.,
Vol. 17, No. 3, 2006 729
containing solutions were removed, and the resulting cell
monolayer was washed with PBS. Cells were fixed with 2%
formaldehyde in the PBS (Ca2+,Mg
2+) at room temperature
for 10 min, followed by washing with PBS twice. Confocal and
differential interference contrast (DIC) images were taken on
an Olympus FV-500 confocal microscope using a 40×, 1.2 NA
oil immersion objective. For the confocal images, the 488 nm
line of an argon ion laser was used for excitation and the
emission was filtered at 505 nm.
RESULTS
Polycation-Induced Hole Formation on Aqueous Sup-
ported DMPC Lipid Bilayers. The polycation polymers used
in this study (PLL, PEI, and DEAE-DEX) were all shown to
disrupt DMPC-supported lipid bilayers. Several controls were
performed for the AFM study. Before the introduction (through
injection) of polymers into the solution containing the aqueous
supported lipid bilayers, the bilayers were imaged alone for 8
to 10 min. These preimaging steps were done to ensure that
the bilayers were stable and that preexisting defects that
appeared were not due to the act of imaging itself. A blank
injection of water (the solvent in which the polymers were
dissolved) was completed and resulted in no change to the
bilayer over the course of a normal imaging session (60 to 90
min). This shows that the injection itself is not responsible for
the formation of defects. Finally, injections of 1 µg/mL of poly-
(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA), two
neutral polymers, were performed and resulted in no change to
the supported bilayers over normal imaging time.
Images taken using AFM show the three polycationic
polymers (PEI, PLL, and DEAE-DEX) disrupt DMPC supported
lipid bilayers. Figures 2, 3, and 4 are representative data sets of
the polycationic polymers and DMPC lipid bilayer interactions.
Although there is some variation, several repetitions were
completed for each polymer confirming that the degrees of
membrane/polymer interactions are qualitatively similar. Fol-
lowing the addition through injection of PLL into the AFM
liquid cell at a final concentration of ∼1 ug/mL (Figure 2),
new defects in the bilayer are formed. The depth of these defects
in the membrane is primarily in the range of 4.0-4.8 nm.
Addition of PEI onto the bilayer, however, results in mostly
the expansion of preexisting defects (Figure 3). These expanded
defects have measured 4.4 to 5.1 nm in depth which is consistent
with the removal of a full bilayer. Interestingly, the introduction
of DEAE-DEX to supported DMPC bilayers results in only 2-4
nm deep depressions.
Cytotoxicity of Polycation and Polycation-Induced En-
zyme Leakage. The cytotoxicity of the polymers was deter-
mined using an XTT assay (Figure 5). The relative cell viability
was calculated as:
The assay results show that the polymers are not cytotoxic
up to a 12 µg/mL concentration (>80% cell viability) for both
KB and Rat2 cells. Based on these data, the assays performed
to investigate the cytosolic enzyme release were carried out in
polymer concentrations of 6 and 12 µg/mL. Within this range,
the polymers are noncytotoxic, so the observed enzyme release
can be ascribed to an increase in cell membrane permeability
as opposed to general lysis due to cell death.
The effect of positively charged polymers on the cell
membranes was investigated in terms of cytosolic enzyme
release from the cells using LDH and LUC assay techniques.
Table 1. Physicochemical Properties of Polycations Used in This Study
polymer Mn(PDI)aorder of amines (deg) charge/monomer ratiobzeta potential (mV)cshape of chain
PLL 11210 (1.67) 1, 2 0.007519 17.59 (0.39 linear, flexible
PLL-FITC 23620 (1.06) 1, 2 0.007519 - linear, flexible
PEI 78220 (3.44) 1, 2, 3 0.02290 45.18 (16.84 branched, flexible
DEAE-DEX 18490 (32.90) 3 0.00218 30.44 (2.71 linear, intramolecular cross-linking
G5-NH226530 (1.02) 1, 3 0.00885 19.24 (16.63 sphere-like
PEG 16290 (1.22) N/A N/A -1.07 (1.10 linear, flexible
PVA 28490 (1.57) N/A N/A -0.68 (1.36 linear, inter-/intramolecular H bonding
aMeasured by GPC. bTheoretical number of charged groups per molecular weight of polymeric monomer. cMeasured by zeta potential/particle sizer
NICOMP 380 ZLS (PSS‚NICOMP, Santa Barbara, CA). The maximum count rate was 5 MHz and the measurements were carried out for 5 min. All the
polymer samples were at a concentration of 1 mg/mL, pH 7.4, and measurements for each polymer were repeated five times.
Figure 2. AFM images of supported DMPC lipid bilayers upon
exposure to poly-L-lysine (PLL). 20 µLof10µg/mL PLL injected
following image a, resulting in a final concentration of ∼1.0 µg/mL in
the AFM liquid cell. Total time between a and c is approximately 50
min. White arrows indicate formation of several new holes in the lipid
bilayers caused by PLL. Bar: 500 nm. Z-scale: 20 nm.
Figure 3. AFM images of supported DMPC lipid bilayers upon
exposure to poly(ethylenimine) (PEI). 20 µLof5µg/mL PEI injected
following image a, resulting in a final concentration of ∼0.5 µg/mL.
An additional 20 µLof10µg/mL was injected after b, resulting in a
final concentration of ∼1.5 µg/mL. Total time between a and c is
approximately 40 min. Note that there is no new hole formation but
instead the preexisting defects are expanded. One of the preexisting
defects and expansion of the defect is indicated by white arrows in
each image. Bar: 500 nm. Z-scale: 20 nm.
Figure 4. AFM images of supported DMPC lipid bilayers upon
exposure to diethylaminoethyl-dextran (DEAE-DEX). 50 µLof5µg/
mL DEAE-DEX injected following image a, resulting in a final
concentration of ∼1.3 µg/mL. Total time between a and c is ap-
proximately 90 min. Unlike that PLL and PEI create or expand defects
in the lipid bilayers, DEAE-DEX induces membrane thinning. The
newly formed defects are 2-4 nm deep instead of complete removal
of the lipid bilayers (∼4-5 nm deep). Bar: 500 nm. Z-scale: 20 nm.
[OD492nm]sample/[OD492nm]control ×100
730
Bioconjugate Chem.,
Vol. 17, No. 3, 2006 Hong et al.
As the polymer concentration increases, the amounts of both
LDH and LUC released increased as illustrated in Figures 6
and 7. Induced membrane permeability as measured by LDH
release shows a degree of cell type (KB and Rat2) dependence
as shown in Figure 6. In both cell types (Figure 6), however,
PEI induced more LDH leakage as compared to the other
polycationic polymers. This is not surprising because PEI
possesses a much greater charge/monomer ratio (see Table 1)
as compared to the other polymers. G5-NH2and PLL cause
similar amount of LDH release and the least LDH was released
from the cells after exposure to DEAE-DEX among the
polycationic polymers, particularly in the case of the Rat2 cells.
PEG and PVA did not cause enzyme leakage. LUC release from
Rat2pLUC exhibits a trend similar to the LDH release from
cells seen as discussed above (Figure 7).
Binding and Internalization of PLL-FITC. PLL-FITC
conjugate was used for direct observation by CLSM. This
experiment was carried out to compare the internalization
behavior of common commercial polycationic polymers to that
of dendrimeric G5-NH2. Figure 8 shows that PLL-FITC is
internalized into the Rat2 cells resulting in strong fluorescence
of FITC from inside the cells. In particular, the intracellular
location of the internalized conjugates appears to exclude the
nucleus. This internalization behavior is particularly interesting
because penetration of the polymers into the nucleus should be
avoided for nontoxic gene delivery as it may alter the DNA
sequence. G5-Ac-FITC, which remains neutral in water due
to the conversion of all the surface primary amines groups to
acetamide groups, is not internalized as shown in Figure 8e,
allowing the conjugate to be used as a negative control (15).
The DIC image is provided to show that an appropriate cell
density was used (Figure 8f).
Diffusion of Small Molecules through the Permeabilized
Membranes. It is known that PI and fluorescein diacetate (FDA)
can be employed as indicators of diffusion-in and out, respec-
tively (15,23). PI is readily internalized into cells with disrupted
membranes but is excluded from cells with intact membranes.
In contrast, FDA is able to traverse intact membranes and is
then converted to fluorescein (FITC) by endogenous esterase
in cells. The resulting FITC is unable to travel across intact
membranes but is able to traverse permeabilized membrane.
Therefore, it is assumed that PI fluorescence should increase
and FITC fluorescence should decrease with increasing mem-
brane permeability. Figure 9a shows the increase in PI signal
intensity induced following the incubation of KB cells with the
polycationic polymers. The greatest PI intensity increase is seen
using PEI, followed by G5, DEAE-DEX, and PLL. Charge
neutral PVA and PEG do not induce PI internalization.
On the other hand, FITC fluorescence intensities are decreased
after exposure to the polycationic polymers in a concentration-
dependent manner (Figure 9b). The largest amount of intensity
Figure 5. Cell viability determined by XTT assay of (a) KB and (b)
Rat2 cells after incubation with PLL, PEI, DEAE-DEX, and G5-NH2
PAMAM at 37 °C for 4.5 h. Note that all the polymers are not cytotoxic
up to a concentration of 12 µg/mL.
Figure 6. Dose-dependent LDH release from (a) KB and (b) Rat2
cell lines incubated with PLL, PEI, DEAE-DEX, G5-NH2PAMAM,
PEG, and PVA at 37 °C for 3 h. All the polycationic polymers induce
LDH leakage but the neutral polymers PEG and PVA do not cause
any significant leakage.
Figure 7. Dose-dependent luciferase (LUC) release from Rat2pLUC
cell line incubated with PLL, PEI, DEAE-DEX, G5-NH2PAMAM,
PEG, and PVA at 37 °C for 3 h. Before the incubation, Rat2 cells
were transfected by PAMAM dedrimer-mediated cell transfection to
express LUC in their cytosols. As seen in the LDH assay data in Figure
6, all the polycationic polymers used in this study also cause LUC
leakage but the neutral polymers do not.
Effect of Polycations on Membranes
Bioconjugate Chem.,
Vol. 17, No. 3, 2006 731
drop is observed in the PEI case, although DEAE-DEX, PLL,
and G5 also show a decrease in FITC fluorescence intensity.
As expected, PVA and PEG do not induce FITC escape.
DISCUSSION
Evidence for Hole Formation by Polycationic Polymers
PEI, PLL, and DEAE-DEX. We previously reported nanoscale
hole formation on both supported lipid bilayers and cellular
membranes induced by positively charged PAMAM dendrimers
(15-19). In this study we test the possibility of extending the
proposed mechanism of dendrimer/membrane interactions, i.e.,
hole formation by sphere-like PAMAM dendrimers, to the more
common and inexpensive polycationic polymers PEI, PLL, and
DEAE-DEX. Polycationic polymer-induced disruption of vari-
ous supported phospholipid bilayers including dioleoyl phos-
phatidyl choline (DOPC), dioleoyl phosphatidyl serine (DOPS),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA),
and their mixtures has been reported using various methods.
These techniques include quartz crystal microbalance with
dissipation, leakage assay, 31P NMR, and electrophoretic mobil-
ity (9,10,24). Furthermore, polycationic polymer-induced
permeabilization of living cell membranes has also been
observed in vitro using enzyme assays and flow cytometry (13,
15). In this paper, we correlate two separate types of studies
(model membrane and in vitro studies) by performing simul-
taneous experiments at similar conditions to provide a better
understanding of polymer/membrane interactions.
There are two basic types of disruption seen in AFM
images: membrane hole formation and membrane thinning.
‘Membrane hole formation’ is the complete removal of the lipid
bilayer from the mica surface, resulting in defects of 4 to 5 nm
in depth. The apparent discrepancy between the expected depth
for a removal of a full bilayer, 5 nm, and the depths
experimentally measured in this study can be explained by the
varying degrees of polymer adsorption to the surface of the mica.
‘Membrane thinning’, which could result from the reorientation
of lipids or the removal of a layer of lipid from the lipid bilayer,
yields depressions ranging between approximately 2 and 3 nm
(25-28).
Although the effect of each of the polymers used in the study
varies in degree, each of these commercial polycationic polymers
induce disruption of the bilayers as shown in the AFM images
(Figures 2-4). This study is consistent in terms of observing
bilayer disruption but differs in both the type of disruption and
concentration dependence as compared with the previous studies
which examined the interaction of positively charged dendrimers
Figure 8. Confocal microscopy images of Rat2 cells incubated with (a) 6 µg/mL PLL-FITC and (b) 12 µg/mL PLL-FITC conjugates. (c) A
zoomed-out image of b. Rat2 cells incubated with (d) 6 µg/mL G5-NH2-FITC and (e) 12 µg/mL G5-Ac-FITC conjugates. (f) Differential interference
contrast (DIC) image. Image f illustrates that there are a sufficient number of cells at the focal plane although nothing can be seen in fluorescence
image e. Note that the green fluorescence from either PLL-FITC or G5-NH2-FITC does not occur from within cell nuclei which are indicated by
several white arrows. The location of the nuclei and the exclusion of the polycationic polymers were confirmed in previously published work using
DAPI staining of the nucleus (15).
Figure 9. Fluorescence intensity of (a) propidium iodide (PI) and (b)
fluorescein (FITC) from KB cells measured by flow cytometer. Note
that fluorescence intensity of PI should increase while that of FITC
should decrease with increase of membrane permeability. All the
polycationic polymers cause an increase of PI fluorescence intensities
and a decrease of FITC fluorescence. However, the neutral polymers
do not cause such changes.
732
Bioconjugate Chem.,
Vol. 17, No. 3, 2006 Hong et al.
with a DMPC-supported lipid bilayers (16). Our in vitro study
shows enzyme (LDH with MW 135-140 kDa and ∼4.3 nm in
radius and LUC with MW 61 kDa and ∼2.7 nm in radius)
leakage out of the cells, polymer internalization into the cells,
and diffusion in and out of small molecular probes (PI and FITC
respectively) through the living cell membranes (Figures 6-9)
which are also consistent with our previous report using cationic
PAMAM dendrimers (15). Therefore, it is clear that PEI, PLL,
and DEAE-DEX also cause the formation of defects in both
supported lipid bilayers and cell membranes.
Relationship between Polymer Properties and Membrane
Permeabilization. As modern biotechnology tends toward the
adaptation of biocompatible polymers for advanced biomedical
applications, a fundamental understanding of these polymers,
particularly in physiological conditions, should be obtained. In
this paper, we try to provide a better understanding of the
relationship between physical properties of polymers and
biological membranes. GPC measurement enables us to rank
hydrodynamic radii of the polycationic polymers in the follow-
ing order: DEAE-DEX >PEI >PLL >G5 PAMAM.
According to our AFM and in vitro studies, however, the size
of the polymers does not seem to markedly affect their ability
to induce hole formation in the membranes. Instead, it was found
that the degree of membrane permeability is strongly dependent
on the number of formal charges on the polymer chain. As
shown in Table 1, PEI has the greatest density of charged groups
on its single polymeric chain among the polymers used in this
study. Not surprisingly, a significantly high amount of LDH
and LUC was released in both cell lines after incubation with
PEI. Furthermore, changes in fluorescence in PI and FITC as a
result of interactions between PEI and cells were greater than
changes induced by the other polycationic polymers. This
indicates that charge interactions play a key role in inducing
changes of membrane permeability, i.e., nanoscale hole forma-
tion. Our data on PLL, DEAE-DEX, and G5-NH2, however,
suggests that electrostatic interactions are not the only factor
contributing to the effectiveness of polymer-induced nanopo-
ration. Although PLL has an order of magnitude higher charge/
monomer ratio than DEAE-DEX and G5-NH2, the difference
among those polymers in terms of enzyme leakage, PI inter-
nalization, and FITC escape is not remarkable. One possible
explanation is that efficacy of hole formation is dependent on
the architecture of polymers. That is, sphere-like (PAMAM),
branched (PEI), or ring-containing (DEAE-DEX) molecular
structures are likely more effective than linear polymers (PLL)
at increasing membrane permeability. Mecke et al. also sug-
gested that the macromolecular architecture is an important
factor in the polymer/membrane interactions (16).
Taken as whole, it is found that the common, commercial
polycationic polymers PEI, PLL, and DEAE-DEX result in the
formation of defects on supported lipid bilayers, enzyme leakage
out of cells, polymer internalization into cells, and diffusion in
and out of small molecular probes (PI and FITC) through cell
membranes. Our results directly support our hypotheses: (1)
commercially available polycationic polymers, PEI, PLL, and
DEAE-DEX cause nanoscale hole formation in supported lipid
bilayers, (2) commercially available polycationic polymers, PEI,
PLL, and DEAE-DEX cause substantial permeability of the cell
plasma membrane leading to enzyme and dye diffusion, and
(3) charge-neutral linear polymers do not induce hole formation
in supported lipid bilayers or induce a significant change in
membrane permeability. The proposed hypotheses are also
consistent with our previous study using PAMAM dendrimers
(15). An interesting addition to hypothesis 1 was raised by the
DEAE-DEX experiments showing membrane thinning. Poly-
cationic polymers appear capable of both nanoscale hole
formation and membrane thinning events. Our data suggest that
these polycationic polymer agents may be internalized into cells
through the nanoscale holes generated in the membranes and
that this is also a route of internalization for other materials.
In summary, we directly observe that polycationic polymers
induce hole formation in supported lipid bilayers. Our in vitro
results showing that polycationic polymers permeabilize living
cell membranes allowing for the diffusion of molecules in and
out, are consistent with a hole-formation mechanism (15,16).
Once the concentration is above the nontoxic range (up to ∼12
µg/mL), the polymers cause substantial membrane damage
resulting in cell death. From this point of view, these polyca-
tionic polymers are not ideal as biocompatible drug or gene
delivery agents. Instead, charge-neutral polymers that do not
cause nanoscale hole formation and cell membrane permeabi-
lization would likely serve as a better choice when paired with
a targeting moiety (29-31).
ACKNOWLEDGMENT
This project has been funded with federal funds from the
National Cancer Institute, National Institutes of Health, under
contract no. N01-CO-27173.
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