Aqueous solution behaviour and membrane disruptive activity of pH-responsive PEGylated pseudo-peptides and their intracellular distribution.
ABSTRACT The effect of PEGylation on the aqueous solution properties and cell membrane disruptive activity of a pH-responsive pseudo-peptide, poly(l-lysine iso-phthalamide), has been investigated by dynamic light scattering, haemolysis and lactate dehydrogenase (LDH) assays. Intracellular trafficking of the polymers has been examined using confocal and fluorescence microscopy. With increasing degree of PEGylation, the modified polymers can form stabilised compact structures with reduced mean hydrodynamic diameters. Poly(l-lysine iso-phthalamide) with a low degree of PEGylation (17.4 wt%) retained pH-dependent solution behaviour and showed enhanced kinetic membrane disruptive activity compared to the parent polymer. It facilitated trafficking of endocytosed materials into the cytoplasm of HeLa cells. At levels of PEGylation in excess of 25.6 wt%, the modified polymers displayed a single particle size distribution unresponsive to pH, as well as a decrease in cell membrane lytic ability. The mechanism involved in membrane destabilisation was also investigated, and the potential applications of these modified polymers in drug delivery were discussed.
- Journal of The American College of Cardiology - J AMER COLL CARDIOL. 01/2011; 57(14).
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ABSTRACT: Pseudo-peptidic polymers have been synthesised by grafting L-valine (PV), L-leucine (PL) and L-phenylalanine (PP) onto the pendant carboxylic acid moieties of a pH-responsive polyamide, poly(L-lysine isophthalamide). The pH-responsive aqueous solution properties of PV-75, PL-75 and PP-75 with a stoichiometric degree of substitution of 75 mol% have been compared with those of the parent poly(L-lysine isophthalamide) using UV-visible and fluorescence spectroscopy. At low concentrations (≤0.1 mg mL−1), the grafted polymers displayed pH-dependent conformation. The pH at the onset of hydrophobic association (pHh) and the pH range over which association occurred varied significantly between the different amino acid grafts. The pHh values of PV-75, PL-75 and PP-75 at 0.025 mg mL−1 were 6.2, 7.0 and 7.2, respectively. Increasing concentration enhanced intermolecular aggregation. A bis-functional Cy5 derivative, incorporated within the backbones of poly(L-lysine isophthalamide) (polyCy5) and PP-75 (PDP-75), was demonstrated to act as a fluorescence reporter on the state of polymer conformation and aggregation. The intracellular trafficking of PDP-75, examined by confocal microscopy, indicates potential applications of the grafted polymers in drug delivery and medical imaging.Journal of Materials Chemistry 01/2009; 19(24). · 6.63 Impact Factor
- Journal of The American College of Cardiology - J AMER COLL CARDIOL. 01/2011; 57(14).
Aqueous solution behaviour and membrane disruptive activity of pH-responsive
PEGylated pseudo-peptides and their intracellular distribution
Rongjun Chena, Zhilian Yueb, Mark E. Ecclestonc, Nigel K.H. Slatera,*
aDepartment of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK
bInstitute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore
cVivamer Ltd, William Gates Building, JJ Thomson Avenue, Cambridge, CB3 0FD, UK
a r t i c l e i n f o
Received 20 June 2008
Accepted 28 July 2008
Available online 15 August 2008
a b s t r a c t
The effect of PEGylation on the aqueous solution properties and cell membrane disruptive activity of
a pH-responsive pseudo-peptide, poly(L-lysine iso-phthalamide), has been investigated by dynamic light
scattering, haemolysis and lactate dehydrogenase (LDH) assays. Intracellular trafficking of the polymers
has been examined using confocal and fluorescence microscopy. With increasing degree of PEGylation,
the modified polymers can form stabilised compact structures with reduced mean hydrodynamic
diameters. Poly(L-lysine iso-phthalamide) with a low degree of PEGylation (17.4 wt%) retained pH-
dependent solution behaviour and showed enhanced kinetic membrane disruptive activity compared to
the parent polymer. It facilitated trafficking of endocytosed materials into the cytoplasm of HeLa cells. At
levels of PEGylation in excess of 25.6 wt%, the modified polymers displayed a single particle size
distribution unresponsive to pH, as well as a decrease in cell membrane lytic ability. The mechanism
involved in membrane destabilisation was also investigated, and the potential applications of these
modified polymers in drug delivery were discussed.
? 2008 Elsevier Ltd. All rights reserved.
Efficient release of endocytosed drugs into the cytoplasm
represents a challenge for polymeric drug delivery. After entering
cells by passive or active endocytosis, macromolecular delivery
systems are commonly trafficked to lysosomes, where fragile drugs
(e.g. DNA and proteins) may be degraded by lysosomal enzymes
[1,2]. Even if the payload is not degraded, compartmentalisation
within endosomes or lysosomes can prevent drugs from reaching
their specific subcellular targets .
One approach to achieving cytoplasmic drug delivery is the
development of pH-responsive polymers with designed capability
to disrupt the endosomal membrane, thus avoiding non-productive
lysosomal trafficking . Anionic pH-responsive polymers are of
particular interest because they can mimic the pH-mediated
membrane lytic properties of fusogenic peptides that mediate viral
infection . These amphiphilic polymers, like viral peptides,
contain alternating hydrophobic segments and ionisable carboxyl
groups. Upon reduction of pH, they display a conformational
change from extended structures, dominated by electrostatic
repulsion, to collapsed structures, stabilised by hydrophobic
association , facilitating their interaction with and consequent
disruption of lipid bilayer membranes [7,8]. Hydrophobic associa-
tion, therefore, has been recognised as a determinant for control-
ling the conformational transition and membrane disruption [9,10].
A wide variety of pH-responsive polyanions have been developed,
ranging from non-biodegradable vinyl-based polymers  to
biodegradable poly(amino acid)s and pseudo-peptides [12–14].
Extensive research has been carried out on the pH-dependent
conformational transition and membrane destabilising effects of
vinyl-based polymers, including homopolymers and copolymers of
a-alkyl acrylic acids and alkyl acrylates [7–11,15,16]. These poly-
mers can be manipulated by controlling hydrophobic contents to
disrupt the lipid bilayer at endosomal pHs, whilst being essentially
non-lytic at physiological pH. This has prompted their potential
applications in the cytoplasmic delivery of drugs [17–20].
Additional requirements for an efficacious macromolecular
delivery system, including avoidance of both the reticuloendothe-
lial system and rapid renal clearance , restrict the use of non-
biodegradable vinyl-based polymers . These issues have been
addressed byPEGylation of a pH-responsive biodegradable pseudo-
system [13,14,23,24]. The pH-mediated aqueous solution behaviour
of the polymers with different degrees of PEGylation has been
investigated by conventional fluorescence characterisation at
concentrations up to 1.0 mg/ml using pyrene as a probe , and is
* Corresponding author. Tel.: þ44 1223 762953; fax: þ44 1223 334796.
E-mail address: email@example.com (N.K.H. Slater).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/$ – see front matter ? 2008 Elsevier Ltd. All rights reserved.
Biomaterials 29 (2008) 4333–4340
further substantiated in this paper at elevated polymer concentra-
tions by dynamic light scattering. pH-dependent cell membrane
lysis of the polymers with PEG grafting levels below 23.4 wt% has
been demonstrated using a haemolysis model and their in vitro
their kinetic properties and mechanisms of membrane disruption
are presented, together with the effect of levels of PEGylation above
the modified polymers and their potentials to facilitate release of
endocytosed materials into the cytoplasm are also assessed.
2. Materials and methods
iso-Phthaloyl chloride, potassium carbonate, N,N0-dicyclohexylcarbodiimide
poly(ethylene glycol) amine (mPEG-NH2, NH2(CH2CH2O)99CH3, Mn¼ 4400 Da),
fluorescein 5-isothiocyanate (FITC) and calcein were purchased from Sigma–Aldrich
(Dorset, UK). L-lysine methyl ester dihydrochloride was obtained from Fisher
from TCS Biosciences (Buckingham, UK). Nutrient mixture F-12 Ham medium and
RPMI-1640 medium were purchased from Invitrogen (Paisley, UK). Foetal bovine
serum (FBS), Dulbecco’s phosphate buffered saline (D-PBS), penicillin-streptomycin
solution (10,000 units/ml penicillin and 10 mg/ml streptomycin), L-glutamine solu-
tion (200 mM) and trypsin-EDTA solution (0.5 g/l porcine trypsin and 0.2 g/l
EDTA$4Na) were obtained from Sigma–Aldrich (Dorset, UK). CytoTox 96?non-
radioactive cytotoxicity assay kit was purchased from Promega (Southampton, UK).
2.2. Polymer preparation and composition determination
The synthesis of poly(L-lysine iso-phthalamide) and its derivatives grafted with
mPEG-NH2, designated as PA-n (Fig. 1), has been reported previously [13,14,23].
Here, n refers to the stoichiometric molar percentage of mPEG-NH2 relative to
carboxylic acid used for grafting. The structures of these polymers were charac-
terised using a 500 MHz NMR spectrometer (Bruker Biospin GmbH, Germany). The
molecular weight of poly(L-lysine iso-phthalamide) (Mw¼ 24,000, Mn¼ 14,000,
polydispersity ¼ 1.7) was determined using an aqueous GPC system (Viscotek, UK)
. The actual degrees of PEGylationwere measured by1H-NMR spectroscopy, and
used to calculate the molecular weights of PEGylated polymers. The compositions of
PEGylated polymers are shown inTable 1. Fluorophore-labelled polymers (1.0 mol%)
were synthesised by coupling FITC–NH–(CH2)2–NH2, which was derivatised by the
reaction between FITC and ethylene diamine using dibutyltin dilaurate as a catalyst,
to the carboxylic acids of the polymers using standard DCC/DMAP coupling tech-
niques. They were purified by dialysis against deionised water and isolated by lyo-
philisation. A lowlevel of labelling was selected here to avoid significant modulation
of the polymer properties and fluorescence quenching.
2.3. Dynamic light scattering
The aggregation and conformation of the polymers in aqueous solution were
investigated using a PDDLS/Batch dynamic light scattering platform equipped with
a PD2000DLS dynamic light scattering detector (Precision Detectors, USA).
Poly(L-lysine iso-phthalamide) and PA-1.5 at various concentrations were prepared
in the buffers at specific pHs and allowed to equilibrate for 48 h. Stock solutions of
the polymers with higher degrees of PEGylation, such as PA-2.5, PA-5 and PA-6, were
prepared using a co-solvent system (methanol/water ¼ 1:1, v/v), followed by dial-
ysis against deionised water for 48 h and then serial dilutions to the required
concentrations with buffers at specific pHs. The samples were filtered through
0.45 mm pore size filters, and then the measurements were conducted in a 1.0-ml
quartz cuvette using a diode laser of 800 nm at a scattering angle of 90?.
2.4. Haemolysis assay
. NaCl aqueous solution (150 mM), 100 mM phosphate buffers (pH 5.0–7.4) and
100 mM citratebuffers (pH 4.0–5.0) were prepared tobe isosmotictothe inside of an
RBC. Stock solutions of poly(L-lysine iso-phthalamide) and its PEGylated derivatives
use.Sheep RBCswerewashed threetimeswith150 mMNaClandresuspendedin the
polymer solutions at required concentrations and pHs, to achieve a final RBC
alone (negative control), or in deionised water (positive control). The samples were
then incubated in a waterbath at 37?C for various periods up to 2 h, followed by
centrifugation at 4000 rpm for 4 min. The absorbance of the supernatants at 541 nm
and the percentages of erythrolysis were determined. Each test was performed in
2.5. Particle sizing of RBCs
A Coulter LS230 laser diffraction particle size analyser (Beckman Coulter, USA)
was used to measure the size distributions of erythrocytes. The sheep RBCs pre-
washed with 150 mM NaCl were incubated with PA-1.5 at the required concentra-
tion and pH in a waterbath at 37?C for 1 h according to the above procedure for
a haemolysis assay. Controls were prepared by resuspending RBCs in buffer alone
(negative control), or in deionised water (positive control). The suspension samples
were inverted several times before the measurements using a 750-nm diode laser.
2.6. Cell culture
Chinese Hamster Ovary (CHO) cells and human cervical cancer (HeLa) cells were
routinely grown in nutrient mixture F12 Ham medium and RPMI-1640 medium
respectively. The media were supplemented with 10% (v/v) FBS, 2 mM L-glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin. Both CHO and HeLa cells were
trypsinised using trypsin–EDTA and maintained in a humidified incubator with 5%
2.7. Lactate dehydrogenase (LDH) assay
The reduction of cell membrane integrity of CHO and HeLa cells following
exposure to the polymer solutions at pH 5.5 was determined by LDH assay . CHO
or HeLa cells (1 ?105cells/ml) were seeded into a 96-well round-bottomed plate
(100 ml/well)andincubatedat37?Cfor24 h.ThecellswerewashedwithD-PBSthree
times and then treated in quadruplicate with fresh serum-free medium containing
the polymers at required concentrations. Control wells contained the cells in the
absence of polymer. Each well was titrated to pH 5.5 using 0.1 M HCl and the cells
incubated at 37?C for various periods up to 60 min. The spent medium was then
replaced with fresh serum-free medium. The remaining viable cells were lysed by
freeze–thawmanipulation,andpelletedbycentrifugationat1000 rpmfor4 min.The
LDH released into the supernatants was quantified using a Cytotox 96?cytotoxicity
assay kit (Promega, UK). Background wells were prepared with serum-free medium
only. The absorbance of the resulting coloured formazan solutions at 490 nm was
measured using an EC 340 Bio Kinetics plate reader (Bio-Tek Instruments, USA) and
the relative cell viabilities were determined. It was confirmed that the absorbance
was directly proportional to the number of lysed cells (data not shown).
2.8. Laser scanning confocal microscopy
Two millilitres of HeLa cells (1–2 ? 105cells/ml) were seeded into a 35-mm
glass-bottom culture dish (MatTek, USA) and cultured for 24 h in an incubator at
37?C. The cells were washed three times with D-PBS and incubated for 30 min with
2 ml of serum and phenol red free medium containing the FITC-labelled polymer at
the required concentration. The dish was washed three times with D-PBS, replen-
ished with serum and phenol red free medium, and returned to the incubator for
another period up to 3.5 h. The images of the cells were taken within 15 min using
an SP1 compound laser scanning confocal microscope (Leica, Germany). In related
CC NH (CH2)4
R = OH or CH3(OCH2CH2)99NH
Fig.1. Repeat unit structure of poly(L-lysine iso-phthalamide) grafted with mPEG-NH2.
Composition measurements for poly(L-lysine iso-phthalamide) grafted with mPEG-
aStoichiometric molar ratio of mPEG-NH2relative to carboxylic acid used for
bDegree of substitution, defined as the number of PEG side chains per 100
carboxylic acid groups and determined by
cCalculated based on PEG (wt%) and the number average molecular weight Mnof
poly(L-lysine iso-phthalamide) determined by GPC.
1H-NMR in d6-DMSO at room
R. Chen et al. / Biomaterials 29 (2008) 4333–43404334
experiments, calcein (a membrane-impermeable fluorophore) was used as a tracer
molecule to monitor the uptake of the external medium by constitutive endocytosis
and the effect of polymers on internalised vesicles. HeLa cells were treated with 2 ml
of serum and phenol red free medium containing 2.0 mg/ml calcein in the presence
or absence of the polymer (pH values of the solutions were adjusted to 7.4). The
images were obtained within 15 min using a FluoView? FV300 inverted laser
scanning confocal microscope (Olympus, Japan). For detection of FITC and calcein
fluorescence, the excitation and emission wavelengths were 480 and 530 nm
2.9. Fluorescence microscopy
Fluorescence microscopy was employed to study subcellular trafficking of the
FITC-labelled polymers. HeLa cells (1–2 ? 105cells/ml) were treated with 2 ml of
FITC-labelled polymer solution in serum and phenol red free RPMI-1640 medium
according to the procedure for confocal microscopy. The cells were examined within
15 min on a Zeiss Axiophot microscope equipped with a UV bulb (OSRAM Short Arc
Mercury Lamps HBO, 103W/2) (Carl Zeiss, UK).
3. Results and discussion
3.1. Aqueous solution properties
The solution behaviour of PEGylated poly(L-lysine iso-phthal-
amide) was investigated by dynamic light scattering. The variations
in mean hydrodynamic diameter with concentration for poly-
(L-lysine iso-phthalamide), PA-1.5 and PA-5 at pH 7.4 are shown in
Fig. 2a. The mean hydrodynamic diameter of poly(L-lysine iso-
phthalamide) was almost constant (w880 nm) at a concentration
?3.0 mg/ml, but decreased rapidly to 388 nm at 5.0 mg/ml, fol-
lowed by a further gradual reduction to 285 nm at 20 mg/ml.
Similarly, the mean size of PA-1.5 was reduced considerably from
686 nm at 1.0 mg/ml to 202 nm at 5.0 mg/ml and remained
constant at higher concentrations. PA-5 exhibited markedly lower
mean hydrodynamic diameters throughout the concentration
range studied. Its size decreased gradually from 203 nm at 0.05
mg/ml to 41 nm at 1.0 mg/ml, followed by a marginal reduction to
30 nm at 10 mg/ml. This size reduction at sufficiently high polymer
concentrations is due to enhanced intermolecular interactions and
Hydrodynamic diameter (nm)
4.5 5.05.56.0 6.5 7.07.5
Hydrodynamic diameter (nm)
Fig. 2. Mean hydrodynamic diameters of poly(L-lysine iso-phthalamide) (-), PA-1.5
(:) and PA-5 (?) (a) as a function of concentration in 0.1 M PBS buffer at pH 7.4, and (b)
as a function of pH at 5.0 mg/ml in 0.1 M PBS buffer. Error bars represent standard
deviations of four samples.
Relative haemolysis (%)
Incubation time (min)
Fig. 4. Relative haemolysis of sheep RBCs as a function of incubation time in the
absence of polymer (:) and in the presence of poly(L-lysine iso-phthalamide) (;)
(1.0 mg/ml) and PA-1.5 (-) (1.0 mg/ml) at pH 5.0. Error bars represent standard
deviations of three samples.
% in class
Fig. 3. Hydrodynamic diameter and percentage of particle distributions as a function
of pH. The large distribution (?) and small distribution (B) of PA-1.5 and single
distribution of PA-5 (:) formed at 5.0 mg/ml in 0.1 M PBS buffer. Data points represent
the average of four samples.
R. Chen et al. / Biomaterials 29 (2008) 4333–43404335
micellisation of the polymers . The mean size and critical
concentration, where the mean size starts to decrease, were
reduced significantly with increasing degree of PEGylation. This
indicates that PEGylation can facilitate formation and stabilisation
of compact structures.
The effect of pH on polymer conformation and aggregation was
also investigated. As shown in Fig. 2b, the mean hydrodynamic
diameters of poly(L-lysine iso-phthalamide) and PA-1.5 at 5.0
mg/ml were strongly influenced by pH. The size of poly(L-lysine iso-
phthalamide) remained constant (w350 nm) until pH 6.0. A
significant reduction to 76 nm was noted with further acidification
to pH 5.0, after which a plateau was discovered. The size of PA-1.5
was constant at 196 nm at pH ? 7.0, followed by a gradual decrease
to 24 nm at pH 4.5. PA-1.5 displayed a higher pH, where the mean
size starts to decrease, compared to poly(L-lysine iso-phthalamide),
resulting from the influence of pH-induced hydrogen bonding
between the tethered PEG and protonated carboxylic acid groups
[23,25]. It is noteworthy that the size of the compact domains
formed by PA-5 (w35 nm) was independent of pH.
Diameter ( m)
Diameter ( m)
Fig. 5. Particle size distributions of sheep RBC suspensions in deionised water (:) and
0.1 M PBS buffer at pH 7.4 in the absence of polymer (-) and in the presence of PA-1.5
at 1.0 mg/ml (,), and at pH 5.0 in the absence of polymer (?) and in the presence of
PA-1.5 at 1.0 mg/ml (B). (a) Change of distributions across the entire range of diameter;
(b) enlarged portion of (a) containing only the diameter range 1.5–7.5 mm. RBC samples
were incubated in a 37?C water bath for 1 h.
4.5 5.0 5.56.06.57.0 7.5
Relative haemolysis (%)
Fig. 6. Relative haemolysis of sheep RBCs as a function of pH in the presence of poly-
(L-lysine iso-phthalamide) (-), PA-2.5 (?) and PA-6 (:) at 1.0 mg/ml. Error bars
represent standard deviations of three samples.
1530 45 60
Relative viability (%)
1530 45 60
Relative viability (%)
Fig. 7. Time-dependent relative viabilities of (a) CHO and (b) HeLa cells treated with
poly(L-lysine iso-phthalamide) (blank) and PA-5 (striped) at 1.0 mg/ml determined by
LDH assay, pH adjusted to 5.5 with HCl. Error bars represent standard deviations of
R. Chen et al. / Biomaterials 29 (2008) 4333–43404336
Further insight into the conformation and aggregation of the
PEGylated polymers was obtained by analysing the effect of pH on
their size distributions in aqueous solution. Fig. 3 depicts the
changes of hydrodynamic diameters and corresponding percent-
ages of different size distributions as a function of pH for PA-1.5 and
PA-5 at 5.0 mg/ml. At pH ? 7.0, PA-1.5 had a single particle size
distribution (w200 nm). At pH 6.5, the particles were divided into
two narrowly distributed populations, 44% of which was a small
particle population of 12 nm and the rest was a large one of
200 nm. As the polymer solution was further acidified, the
percentage of the small particle population increased significantly
to 87% at pH 5.0, with a concomitant decrease of the large particle
population to 13%. It has been argued by Croucher et al. [26,27] that
such small assemblies represent individual micelles, while the large
ones indicate loose aggregates of micelles analogous to the
secondary aggregates of micelles known to be formed from non-
ionic surfactants in water. Cao et al.  proposed that, in the case
of polysaccharides, large species resulted from reduced stabilisa-
tion of small ones due to neutralisation of carboxylate groups. The
pH-mediated reduction of particle size and change of particle size
distribution of PA-1.5 can be ascribed to the increased hydrophobic
association of the polymer resulting from neutralisation and
hydrogen bonding between PEG and carboxylic acid groups, which
has been noted by Yue et al.  by fluorescence characterisation
using pyrene as a probe. This may stabilise the hydrophobic
domains formed by PA-1.5, leading to the formation of an
increasing amount of the small particle population. By contrast, as
seen in Fig. 3, PA-5 displayed a single size distribution and its mean
size (w35 nm) did not change with pH within the range examined.
Relative viability (%)
20001000 7005002001007050 10
Relative viability (%)
Concentration ( g/ml)
Concentration ( g/ml)
Fig. 8. Concentration-dependent relative viabilities of (a) CHO and (b) HeLa cells
treated with poly(L-lysine iso-phthalamide) (blank) and PA-5 (striped) for 1 h deter-
mined by LDH assay, pH adjusted to 5.5 with HCl. Error bars represent standard
deviations of four samples.
Fig. 9. Confocal microscopy images of HeLa cells showing the subcellular distribution of FITC fluorescence. The cells were incubated for 30 min in medium containing (a) 2.0 mg/ml
FITC, (b,c) 1.0 mg/ml FITC-labelled PA-1.5, and (d) 1.0 mg/ml FITC-labelled poly(L-lysine iso-phthalamide). Image (b) was acquired at 30 min after uptake (?2.4 zoom), while images
(a), (c) and (d) at 3.5 h after uptake with a Leica SP1 confocal unit (?63 magnification).
R. Chen et al. / Biomaterials 29 (2008) 4333–4340 4337
This suggests that PA-5 adopted a stable compact micellar struc-
ture, and its hydrophilic corona prevented formation of loose
micelle aggregates. Therefore, the effect of PEGylation is two-fold,
promoting hydrophobic association of polymer backbones while
stabilising the polymer aggregates.
3.2. Cell membrane disruption
The effect of grafting poly(L-lysine iso-phthalamide) with less
than 23.4 wt% hydrophilic mPEG on the pH- and concentration-
dependent disruption of bilayer membranes has been shown
previously using a haemolysis model with the erythrocyte
membrane serving as a model for the endosomal membrane
. Fig. 4 shows the kinetic haemolytic activity of poly(L-lysine
iso-phthalamide) and PA-1.5 (17.4 wt% mPEG) at 1.0 mg/ml
measured at pH 5.0. In the absence of polymer, no haemolytic
activity was detected within 120 min. The presence of poly(L-lysine
iso-phthalamide) caused avery lowlevel of haemolysis of 9.8% after
120 min of incubation. PA-1.5 displayed haemolytic activity of 11.4%
after 15 min, followed by a sharp increase in the degree of hae-
molysis to a maximum of 51.7% after 60 min. Endocytosed macro-
molecules have been reported to be trafficked from early
endosomes to lysosomes within several hours . The kinetic
haemolytic activity of PA-1.5, therefore, matched the time
requirement for destabilisation of the endosomal membrane before
the vesicular evolution from endsomes to lysosomes.
It has been noted that the overall RBC numbers were not
reduced significantly in the solutions of PA-1.5 (1.0 mg/ml) at both
pH 7.4 and 5.0, as determined by a haemocytometer . The
percentage of lysed RBCs by PA-1.5 at pH 5.0 relative to the total
RBC number in buffer alone was w8%, inconsistent with the hae-
molysis data (w54%). The mechanism of haemolysis was further
investigated by examining the size distributions of RBC particles
using static light scattering following incubation for 1 h at pH 7.4
and pH 5.0 in the absence and presence of PA-1.5 at 1.0 mg/ml. As
shown in Fig. 5a, RBCs were broken into debris following complete
membrane disruption in deionised water, with particle sizes
<0.1 mm in diameter. As expected, as RBCs were incubated in PBS
buffer in the absence of polymer, only a slight change in particle
size at pH 5.0 was observed compared to pH 7.4. The diameters fell
into the range of 1.5–7.5 mm, without occurrence of small particles
with sizes <0.1 mm in diameter, indicating that no RBC was lysed
into debris. Interestingly, the diameters of RBCs also remained
unaffected in the presence of PA-1.5 at pH 5.0 compared to pH 7.4.
These results suggest that the presence of PA-1.5 at pH 5.0 could
facilitate pore formation or make the cell membrane more
permeable so that haemoglobin can pass through without breaking
the erythrocyte membrane.
The membrane lytic activity of the polymers at levels of PEGy-
lation higher than 25.6 wt% was also investigated. Fig. 6 displays
the ability of poly(L-lysine iso-phthalamide), PA-2.5 (25.6 wt%
mPEG) and PA-6 (42.2 wt% mPEG) to haemolyse the erythrocyte
membrane at 1.0 mg/ml as a function of pH. PA-2.5 and PA-6
showed the maximum degrees of haemolysis of 15.0 and 22.1%
respectively, much lower than PA-1.5 with a lower degree of
grafting (17.4 wt% mPEG) . The effect of poly(L-lysine iso-
phthalamide) and PA-5 (40.9 wt% mPEG) on the extracellular
membrane integrity of CHO and HeLa cells was determined at pH
Fig. 10. Fluorescence microscopy images of HeLa cells treated for 30 min with (a,b) 1.0 mg/ml FITC-labelled PA-1.5 and (c) 1.0 mg/ml FITC-labelled poly(L-lysine iso-phthalamide).
Image (a) was acquired at 30 min after uptake, while images (b) and (c) at 3.5 h after uptake with a Zeiss Axiophot microscope (?40 magnification).
R. Chen et al. / Biomaterials 29 (2008) 4333–4340 4338
5.5 (as another model for the late endosomal membrane) by
measuring lactate dehydrogenase (a stable cytosolic enzyme)
activity in the extracellular medium. As shown in Figs. 7 and 8, the
two polymers influenced the membrane integrity of CHO and HeLa
cells, expressed conventionally as relative viability, in both time-
and concentration-dependent manners in the concentration range
of 10–2000 mg/ml over the periods up to 60 min. Only a slight
decrease in cell viability was observed in the presence of PA-5
compared to the unmodified polymer. The membrane lytic activity
of the two polymers was noticeably lower than that of PA-1.5 .
These observations indicate that over grafting poly(L-lysine iso-
phthalamide) with mPEG (?25.6 wt%) can cause a marked reduc-
tion in membrane lytic capability, probably as a result of micelli-
sation of the polymers at these levels of PEGylation .
3.3. Subcellular localisation
The potential applications of the PEGylated polymers, in
particular PA-1.5, to release endocytosed materials into the cyto-
plasm were assessed. Fig. 9a shows the subcellular distribution of
free FITC in HeLa cells, which is visualised as homogenous green
fluorescence throughout the cells following incubation for 3.5 h
after 30 min of uptake. When HeLa cells were treated for 30 min
with 1.0 mg/ml FITC-labelled PA-1.5 containing equivalent fluo-
rophore, a large number of punctate structures were evident in the
cells following 30 min of further incubation (Fig. 9b), indicating
FITC must have been associated with the polymer carrier. This
suggests that the fluorescently labelled polymer was internalised
by endocytosis and transported through early endosomes before
moving on to other vesicular compartments, instead of by diffusion
for free FITC. Following prolonged incubation for 3.5 h, a significant
amount of diffuse staining, indicative of escape of FITC-labelled
PA-1.5 into the cytoplasm, was observed together with some
residual punctate staining within intact endocytic vesicles (Fig. 9c).
A related phenomenon has been noted that release of calcein from
vesicles mediated by poly(a-propylacrylic acid) and poly(a-ethyl-
acrylic acid) was increased progressively with time, reaching the
maximum at about 4 h after uptake . Following incubation for
3.5 h after30 min of uptake of 1.0 mg/ml FITC-labelled poly(L-lysine
iso-phthalamide), only a limited amount of diffuse staining
appeared in the cytoplasm, showing that most of the polymer was
still trapped within intracellular vesicles (see Fig. 9d). These
confocal results were substantiated by the fluorescence micro-
scopic study of the FITC-labelled polymers, as shown in Fig.10. The
white arrows indicate the fused vesicles formed prior to membrane
disruption, which has been noted by Jones et al.  in a study of
Fig. 11. Confocal microscopy images of HeLa cells showing the subcellular distribution of calcein fluorescence. (a) Cells without treatment of polymer or calcein. (b) Cells treated
with 2.0 mg/ml calcein. (c) Cells treated with 2.0 mg/ml calcein and 1.0 mg/ml poly(L-lysine iso-phthalamide). (d) Cells treated with 2.0 mg/ml calcein and 1.0 mg/ml PA-1.5. Images
were acquired with an Olympus FluoView? FV300 confocal unit (?60 magnification) at 3.5 h after 30 min of uptake.
R. Chen et al. / Biomaterials 29 (2008) 4333–4340 4339
poly(a-ethylacrylic acid)-mediated release of calcein. These results
show that PA-1.5 was more efficient at destabilising the endoso/
lysomal membrane and releasing endocytosed materials, consis-
tent with the haemolysis results .
The release of non-conjugated calcein, a membrane-imperme-
able fluorophore, was investigated to further evaluate the influence
of the polymers on internalised vesicles. As shown in Fig. 11a, no
fluorescence was detected in HeLa cells without treatment of
polymer or calcein. When the cells were incubated in the culture
medium containing calcein alone, fluorescent intracellular vesicles
appeared as small bright punctate spots in Fig.11b, consistent with
constitutive endocytosis of the external medium. As poly(L-lysine
iso-phthalamide) (Fig. 11c) and PA-1.5 (Fig. 11d) were added in the
culture mediumat a concentration of 1.0 mg/ml, some diffuse green
fluorescence was noted spreading over the cells, indicative of the
release of calcein into the cytoplasm. PA-1.5 demonstrated
enhancedcapability tofacilitate thisreleasecomparedtothe parent
polymer. A number of bright vesicles still existed in most areas
within the cells. The larger spots indicate fusion of these vesicles,
which is possibly a precursor to calcein release upon membrane
destabilisation. This effect appears similar to poly(a-alkylacrylic
acid)s-induced fusion of calcein-containing endosomes  and
phosphatidylcholine vesicles at endosomal pHs .
The conformation and aggregation of poly(L-lysine iso-phtha-
lamide) can be tailored by the degree of PEGylation. At low levels of
grafting (e.g. PA-1.5, 17.4 wt% mPEG), the hydrodynamic diameter
and particle size distribution of the modified polymer in aqueous
solution were strongly influenced by pH and concentration, whilst
higher levels (e.g. PA-5, 40.9 wt% mPEG) led to pH-independent
size and single distribution. PA-1.5 demonstrated improved pH-
responsive membrane disruptive activity compared to poly(L-lysine
iso-phthalamide), reaching a maximum degree of haemolysis
(51.7%) at pH 5.0 after 60 min. Over grafting poly(L-lysine iso-
phthalamide) with mPEG caused a marked reduction in membrane
lytic activity. The cytoplasmic localisation of FITC-labelled PA-1.5
and effective release of non-conjugated calcein into the cytoplasm
by PA-1.5 indicates the potential applications of the pH-responsive
PEGylated polymer for drug delivery.
Rongjun Chen is sponsored by the Gates Cambridge Trust (Gates
Scholarship) and the Universities UK (Overseas Research Students
Awards). The authors are also grateful for the financial support of
the Cambridge–MIT Institute for this project, and assistance
provided by Dr Feng Li and Ms Xiaowen Dai in confocal microscopy.
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