Synthetic polyion-counterion transport systems in polymersomes and gels

Article abstract of DOI:10.1039/c1ob05835e

Transport across the membranes of polymersomes remains difficult in part due to the great thickness of the polymer bilayers. Here, we report that dynamic polyion-counterion transport systems are active in fluorogenic polymersomes composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PDMS-PMOXA). These results suggest that counterion-activated calf-thymus DNA can act as cation carrier that moves not only across lipid bilayer and bulk chloroform membranes but also across the "plastic" membranes of polymersomes. Compared to egg yolk phosophatidylcholine (EYPC) lipsosomes, activities and activator scope in PDMS-PMOXA polymersomes are clearly reduced. Embedded in agar gel matrices, fluorogenic PDMS-PMOXA polymersomes respond reliably to polyion-counterion transporters, with high contrast, high stability and preserved selectivity. Compared to standard EYPC liposomes, it cannot be said that PDMS-PMOXA polymersomes are better. However, they are different, and this difference could be interesting for the development of sensing devices.

Full text of DOI:10.1039/c1ob05835e

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PAPER
Synthetic polyion-counterion transport systems in polymersomes and gels†
Javier Montenegro,a Jo¨rg Braun,b Ozana Fischer-Onaca,b Wolfgang Meier*b and Stefan Matile*a
Received 26th May 2011, Accepted 11th July 2011
DOI: 10.1039/c1ob05835e
Transport across the membranes of polymersomes remains difficult in part due to the great
thickness of the polymer bilayers. Here, we report that dynamic polyion-counterion transport systems
are active in fluorogenic polymersomes composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline)
(PDMS-PMOXA). These results suggest that counterion-activated calf-thymus DNA can
act as cation carrier that moves not only across lipid bilayer and bulk chloroform membranes but also
across the “plastic” membranes of polymersomes. Compared to egg yolk phosophatidylcholine (EYPC)
lipsosomes, activities and activator scope in PDMS-PMOXA polymersomes are clearly reduced.
Embedded in agar gel matrices, fluorogenic PDMS-PMOXA polymersomes respond reliably to
polyion-counterion transporters, with high contrast, high stability and preserved selectivity. Compared
to standard EYPC liposomes, it cannot be said that PDMS-PMOXA polymersomes are better. However,
they are different, and this difference could be interesting for the development of sensing devices.
Introduction
Polymersomes have been introduced as liposome analogs that are
made from amphiphilic block copolymers with hydrophobic and
hydrophilic domains instead of phospholipids.1,2 Compared to
liposomes, these “plastic” vesicles have thicker, sturdier, yet less
organized, more deformable bilayer membranes. The self-assembly
of macromolecular amphiphiles into polymersomes is compatible
with a broad variety of possible sizes and functionalities. In part
for these reasons, polymersomes have received much attention
for possible applications as robust drug carriers, sensing devices,
or nanoreactors1,2 Whereas synthetic transport systems have
been studied extensively in lipid bilayer membranes,3 surprisingly
little is known about transport across the thicker membranes
of polymersomes.2 Taking advantage of unique possibility to
screen large activator collections without much synthetic effort,4
we here report that several dynamic polyion-counterion trans-
port systems4–8 could be identified as active in polymersomes
composed of poly(dimethylsiloxane)-b-poly(2-methyloxazoline)
(PDMS-PMOXA, Fig. 1).
Polyion-counterion transport systems are attractive because of
their significance in nature6–8 and their usefulness in differential4
and aptameric sensing systems.5 In these systems, amphiphilic
counterions are used to activate polyions such as the anionic DNA
and RNA7 or the cationic CPPs (cell-penetrating peptides)8 to
move across bulk or lipid bilayer membranes, enter cells, transduce
Fig. 1 (a) Preparation of fluorogenic polymersomes and (b) ion transport
with dynamic polyion-counterion complexes. Hydrophobic “tails” (e.g.,
cyclamen aldehyde T1) are covalently captured by hydrophilic cations
(e.g., trihydrazides G1H3) to give triple-tail amphiphiles (e.g., G1H3T1)
that can activate polyanions (e.g., ctDNA) as transporters in fluorogenic
liposomes or polymersomes (here the example for fluorescence recovery
in response to the export of trapped cationic quenchers (green) but not
anionic fluorophores (red) is shown).
aDepartment of Organic Chemistry, University of Geneva, Geneva, Switzer-
land. E-mail: stefan.matile@unige.ch; Fax: +41 22 379 3215; Tel: +41 22
379 6523; Web: www.unige.ch/sciences/chiorg/matile/
bDepartment of Chemistry, University of Basel, Basel, Switzerland;
Web: www.chemie.unibas.ch/~meier/index.html/
† Electronic supplementary information (ESI) available: Detailed experi-
mental procedures. See DOI: 10.1039/c1ob05835e
signals in vesicular sensing systems, and so on.5,6 In dynamic
polyion-counterion transport systems, the amphiphilic counterion
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activators are produced in situ by covalent capture of hydrophobic
molecules with reactive charged heads.4 This approach is attractive
for the generation of larger activator libraries to rapidly screen for
transport activity4–6 or cellular uptake7,8 and to generate patterns
for differential sensing applications.4,9
calf-thymus (ct) DNA as cation transporter in EYPC liposomes.4b
G1H1 and G1H6 were inactive because of insufficient and ex-
cessive hydrophobicity, respectively, the ammonium analogs were
consistently less active.4c With charge-inverted CPPs, carboxylate
amphiphiles were better than phosphates, and gemini amphiphiles
with two heads and two tails were best.4d
The hydrophobic tails T1–T10 are the survivors of an initially
much broader screen. Cyclamen aldehyde T1 and jasmine aldehyde
T2 are branched aromatic aldehydes, citronellals in racemic (T3)
and enantiopure form (T4, T5) are classics to study stereochem-
istry in transport and sensing.4a,b The collection is completed by
oleyl aldehyde T6 and the single carbon homologs heptanal T7,
octanal T8, nonanal T9, and decanal T10.4d
The peptidic headgroups G1H2, G1H3 and G1H4 were synthe-
sized following the reported procedures,4b all aldehyde tails were
commercially available or very easily prepared. The amphiphilic
activators were prepared in situ as described previously.4 For
example, incubation of two equivalents of cyclamen aldehyde T1
per hydrazide in G1H3 in DMSO for 1 h at 60 ◦C gave hydrazone
G1H3T1 (Fig. 1), incubation with octanal T8 gave G1H3T8, and
so on.
Addition of excess triton X-100 caused full fluorescence re-
covery, indicating that polymersomes are lysed as completely
as liposomes (Fig. 3, t = 200 s). As with liposomes, these
conditions could thus be used to calibrate transport experiments
in polymersomes to a fractional activity Y = 1.0 with excess triton
X-100.
Fluorescence recovery was not observed in response to the
addition of G1H3 to PDMS-PMOXA-LUVs … HPTS/DPX
polymersomes. Addition of either ctDNA or G1H3T8 alone did
not cause an increase in HPTS emission either (Fig. 3b, t < 0).
The inactivity of the isolated partners demonstrated that neither
polyanion nor countercation alone are capable to mediate the
export of either DPX or HPTS from PDMS-PMOXA-LUVs …
HPTS/DPX vesicles. However, the sequential addition of first
G1H3T8 and ctDNA next resulted in fluorescence recovery (Fig.
3, t > 40 s). This finding demonstrated that polyion-counterion
transporters are active in polymersomes. The fluorescence in-
tensity just before lysis was taken as fractional activity Y and
plotted as a function of activator concentration. The resulting
dose response curves were subjected to Hill analysis to yield the
Results and discussion
To explore the activity of dynamic polyion-counterion complexes
in polymersomes, PDMS-PMOXA was selected as most promising
candidate. The self-assembly of these amphiphilic polymers into
large unilamellar vesicles (average size ª 100 nm) has been demon-
strated, their fluid, highly deformable but ultrastable membrane
appeared most promising for transport experiments (Fig. 1).10
Fluorogenic PDMS-PMOXA polymersomes were prepared
by swelling of a carefully dried PDMS-PMOXA film in water
containing not only buffers for pH (10 mM Tris, pH 7.4) and
osmotic stress (72 mM NaCl) but also an anionic fluorophore
(5 mM HPTS, 8-hydroxy-1,3,6-pyrenetrisulfonate) and a cationic
quencher (16.5 mM DPX, p-xylene-bispyridinium bromide).11
The obtained PDMS-PMOXA LUVs were extruded through a
polycarbonate membrane and external fluorescent probes were
removed by size exclusion chromatography over a sephadex
column. Fluorescence recovery in this assay can originate from
the export of cationic DPX, anionic HPTS or both from intact
vesicles, or from the destruction of the vesicles.4,6 Counterion-
activated DNA transporters have been shown to transport DPX
but not HPTS across bulk chloroform membranes, and to move
across intact lipid bilayer membranes.6b In the ideal case, export
of the DPX quencher could be observed as an increase in the
emission of the anionic fluorophore HPTS that is left behind
(lex = 413 nm, lem = 510 nm).
A focused collection of amphiphiles was used in this study
(Fig. 2). Peptidic mini-dendrons with guanidinium cations and two
(G1H2), three (G1H3) or four reactive hydrazides (G1H4) were
selected as head groups because they have been best to activate
Y
MAX, the maximal accessible activity under these conditions, the
EC50, the effective activator concentration needed to reach 50% of
Y
MAX, and the Hill coefficient n, which indicates the steepness of
the sigmoidal fitting (Fig. 4aꢀ, Tables S1, S2).11
Most dose-response curves could not be completed to full satu-
ration because of the onset of either precipitation or lysis at high
amphiphile concentrations. In fluorescence kinetics, precipitation
was visible by increasing noise from light scattering. Activity of the
amphiphiles alone was detectable by fluorescence recovery before
the addition of ctDNA. G1H3T1 for example, the amphiphile
obtained from cyclamen aldehyde T1, showed outstanding ability
to activate ctDNA in polymersomes (Fig. 3a, 4b ). However,
᭹
at high concentrations, G1H3T1 caused fluorescence recovery
already in the absence of ctDNA (Fig. 3d).
As far as the cationic head groups are concerned, significant ac-
tivity was only observed for trihydrazide G1H3. With dihydrazide
G1H2, most tested amphiphiles including, G1H2T8 and G1H2T9,
were active in polymersomes in the absence of ctDNA and did
Fig. 2 Reactive counterions G1H2, G1H3 and G1H4 composed of one
guanidinium cation (G1) and two to four hydrazides (H) for in situ reaction
with hydrophobic tails T1–T10 to yield amphiphilic hydrazones that can
activate DNA as transporter in fluorogenic polymersomes (see Fig. 1).
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Fig. 4 Dose response curves for ctDNA transporters in PDMS-PMOXA
polymersomes activated by a) G1H3T7 (᭛, from Fig. 3c), G1H3T8 (ꢀ,
from Fig. 3b) and G1H3T9 ( ). b) Same for G1H3T1 ( , from Fig. 3a),
᭹
᭺
G1H3T2 (᭜) and G1H3T6 (ꢁ).
for octanal T8. Compared to G1H3T8, the activation of ctDNA
transporters by the shortened n-alkyl homolog G1H3T7 revealed
a strongly reduced YMAX = 16.3 1.2% (Fig. 3c), whereas the
interesting EC50 ~ 10 mM was preserved (Fig. 4a᭛, Tables S1,
S2†). With the elongated homolog G1H3T9, YMAX remained
satisfactory, whereas the EC50 = 124.5 0.6 mM was very high (Fig.
4a , Tables S1, S2†). The resulting, quite remarkable selectivity
᭺
Fig. 3 Changes in fractional fluorescence intensity IF of HPTS (lex = 413
nm, lem = 510 nm) during addition of a) G1H3T1 (1.25 (ꢀ), 10 (+), 25
for G1H2T8 in the n-alkyl series with polymersomes was different
to the situation with liposomes, where rather similar activities were
obtained from pentanal up to dodecanal.
Highest activity YMAX = 75.3 3.2% could be reached with
the branched aromatic tails of cyclamen aldehyde (Fig. 3a,
(¥), 50 (᭛), 75 (ꢀ), 100 mM ( ), final concentrations, t ~ 0 s), b) G1H3T8
᭺
(0.05 ( ), 0.50 (ꢀ), 2.50 (+), 6.25 (¥), 12.50 (᭛), 25 (ꢀ), 45 mM ( )) or
᭹
᭺
c) G1H3T7 (0.82 ( ), 1.62 (ꢀ), 3.25 (+), 6.25 (ꢀ), 12.5 (ꢀ), 25 mM ( )),
᭹
᭺
ctDNA (1.25 mg ml-1 final concentration, t ~ 40 s) and triton X-100 (excess,
t ~ 200 s) to PDMS-PMOXA-LUVs … HPTS/DPX. d) Membrane activity
of amphiphiles without DNA.
4b ). The related jasmine aldehyde T2, an enone with excellent
᭹
activity in liposomes, gave poor activity in polymersomes (YMAX
=
22.6 4.5%, 4b᭜). Detectable YMAX = 19.7 2.1% found with
oleyl aldehyde T6 confirmed previous insights from liposomes that
central cis double bonds can activate long alkyl tails (Fig. 4bꢁ).9
Racemic citronellal T3, (-)-citronellal T4 and (+)-citronellal T5 all
gave detectable activities with G1H3 and ctDNA in polymersomes
with YMAX in the range from 6% to 37% and EC50’s from
40 mM to 80 mM (Table S2†). Uniqueness and reproducibility
of the data (EC50, YMAX, n) obtained for T1–T10 with G1H3 and
ctDNA in polymersomes were confirmed by principal component
analysis (PCA).4a,9 Except for the weakly active heptanal T7
and jasmine aldehyde T2, the obtained 3D score plots showed
surprisingly little overlap (Fig. 5). This result confirmed the
not show further increase in activity in the presence of ctDNA
(Fig. S1†). With the complementary tetrahydrazide G1H4, all
tested octopus amphiphiles were inactive in the presence and in
the absence of ctDNA (Fig. S2†). Most active in liposomes, the
inactivity of octopus amphiphiles in polymersomes presumably
originated from the disappearance of these more hydrophobic
amphiphiles in the thick “plastic” membranes. With liposomes,
a similar inactivation with increasing tail number was observed
previously for G1H6.4d
The screening of tails attached to G1H3 heads gave with YMAX
=
58.2 3.4% and EC50 = 14.4 0.2 mM outstanding activities
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Fig. 5 PCA score plot for the activity of odorants T1–T10 with G1H3 and
DNA transporters in polymersomes. Data points represent independent
experiments (Table S1†).11
Fig. 6 (a) Fluorescence image of an agar gel plate loaded with PDMS-P-
MOXA-LUVs … HPTS/DPX after the addition of 4 ml of 1 : 3 mixtures of
ctDNA (1 mg ml-1 Tris (10 mM, pH 7.4, 107 mM NaCl) and (2) G1H3T3,
(3) G1H3T1 and (4) G1H3T9 (DMSO; 0.6, 1,25, 2.5, 5.0, 10 mM, left
to right) or (1, 5) 4 ml of 50% aqueous Tween 20. b) ImageJ scan of row
3 in (a) (cyclamen amphiphile G1H3T1). c, d) Same for EYPC-LUVs …
HPTS/DPX, with 1.2% Triton X-100 in (1) and (5).
compatibility of fluorogenic polymersomes with differential sens-
ing applications.4a,9
With activity of polyion-counterion transport in fluorgenic
polymersomes confirmed in solution, experiments with polymer-
somes in gels were envisioned next. Transport experiments with
vesicles in gels would be attractive for the construction of solid
or, more precisely, semi-wet devices.12 With fluorogenic EYPC
liposomes, transport and sensing experiments in gels have met with
little success so far. To elaborate on transport experiments with
polymersomes in gels, the polysaccharide agar-agar, a undisruptive
gel widely used from food industry to electrophoresis, cell culture
and so on, was selected.13
To gelate polymersomes, agar suspensions were boiling in buffer
(10 mM Tris, 107 NaCl, pH 7.4) and then allowed to cool
down. Fluorogenic PDMS-PMOXA-LUVs … HPTS/DPX vesicle
suspensions were added at 35 ◦C, just before gelation, and the
mixture was transferred to a Petri dish (10 cm diameter) and
allowed to cool down. With a 50 ml micropipette, 19 small holes
were punched in regular repeats into the obtained gel (Fig. 6a).
Interestingly, Triton X-100, most powerful in solution (Fig. 3),
was incapable to permeabilize PDMS-PMOXA polymersomes in
gels. For calibration, the detergent Tween 20 was used instead and
placed in rows 1 and 5 (Fig. 6a). Rows 2, 3 and 4 were loaded
with 4 ml of 1 : 3 mixtures of ctDNA with G1H3T3, G1H3T1 and
G1H3T9, respectively, at increasing concentrations from 625 mM
to 10 mM. Gratifyingly, the contrast of the fluorescent spots
obtained after 5 min of incubation at room temperature compared
to the background was sufficient to take the pictures without
removal of the gel from the plate.
Transport experiments with fluorogenic polymersomes in gels
were quantified with the Java-based image processing program
ImageJ (Fig. 6b). The values obtained for integrated fluorescent
areas were normalized against maximal fluorescent area covered
with excess surfactant (Fig. 6a, rows 1 and 5) and reported as
fractional activities Y. The resulting dose response curves with
polymersomes in gels (Fig. 7a and 8a) were compared to results
for the same polyion-counterion complexes with polymersomes in
solution (Fig. 7b and 8b), liposomes in gels (Fig. 6c, 7c and 8c)
and liposomes in solution (Fig. 7d and 8d).
Fig. 7 Dose response curves for G1H3T3 (᭜), G1H3T1 ( ) and G1H3T9
᭹
(
) with DNA and (a) polymersomes in gels (from Fig. 6a, 2–4), (b)
᭺
polymersomes in solution, (c) liposomes in gels (from Fig. 6c, 2–4), and
(d) liposomes in solution. The concentrations in a) and c) refer to the
concentrations of the 4 ml stock solutions added to the gel.
In general, the fluorescence responses of polymersomes and
liposomes in gels were remarkably different (Fig. 6a vs. 6c).
Transport experiments with fluorogenic polymersomes in gels gave
small and bright spots that translated into sharp peaks with single
maxima in ImageJ scans (Fig. 6a, 6b). Liposomes gave larger, more
diffuse spots, often with broad doublets in ImageJ scans (Fig. 6c,
6d). These distinct differences are consistent with the lower EC50s
found with EYPC LUVs compared to those with polymersomes,
both in solution (Fig. 7b vs. 7d).
Compared to transport experiments in solution, large diameters
of spots in gel assays should correspond to low EC50, whereas high
intensity and good contrast in gels should correspond to high
Y
MAX, perhaps supported by large Hill coefficients.
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gels. This conclusion is reached based on experimental evidence
secured with ctDNA as polyion, PDMS-PMOXA amphiphiles
for polymersomes, and internal DPX and HPTS as probes.
Dynamic hydrazone counterions are used as DNA activators
because covalent capture of hydrophobic aldehydes with cationic
hydrazides provides facile access to large libraries.
Compared to standard EYPC liposomes, it cannot be said that
PDMS-PMOXA polymersomes are better. They are different.
For instance, PDMS-PMOXA polymersomes gave higher EC50’s
and higher selectivity. These differences are in agreement with an
overall increased barrier to be overcome while moving across the
polymer membrane. Among the tested head groups, only peptide
dendrons with one guanidinium cation and three hydrazides (i.e.,
G1H3) were active. Best results were obtained after covalent
capture of cyclamen aldehyde and octanal, activity was detectable
for at least ten different hydrophobic aldehydes. Conjugates
of hydrophobic aldehydes with G1H2 were surfactants, and
conjugates with G1H4 were inactive.
Roughly reproducing the selectivity in solution, activity of
fluorogenic PDMS-PMOXA polymersomes in gels was also best
for G1H3 with cyclamen aldehyde, followed by nonanal and
octanal. For these experiments, a simple solid support device for
pattern printing was developed using agar gelation of fluorogenic
vesicles solutions. With high EC50s and variable YMAX, polymer-
somes in gels gave bright fluorescent spots with high contrast
and small diameter. The low EC50s and high YMAX of liposomes
produced diffuse spots with large diameter and low contrast in gels.
Compared to liposomes, polymersomes in gels also showed better
matching with the selectivity sequences observed in solution.
These results do not only provide experimental evidence for the
activity of polyion-counterion transporters in fluorogenic poly-
mersomes. They also demonstrate compatibility with differential
sensing in solution. The compared to liposomes superior perfor-
mance of polymersomes in gels is of particular interest for the
development of semi-wet differential sensing devices.12 Attractive
future directions include the screening of membranes formed by
other di- or tri-block copolymers,1,2 charge inversion experiments
to study counterion-activated cell-penetrating peptides (CPPs) in
polymersomes,4c or the study of different gelators to improve
activity, selectivity, contrast and stability.12a,14
Fig. 8 Dose response curves for G1H3T8 (ꢀ), G1H3T9 ( ), and
᭺
G1H3T10 (᭛) with DNA and (a) polymersomes in gels, (b) polymersomes
in solution, (c) liposomes in gels, and (d) liposomes in solution. The
concentrations in a) and c) refer to the concentrations of the 4 ml stock
solutions added to the gel.
In the series G1H3T1 > G1H3T9 > G1H3T3, characterized
by different YMAX, high contrast with fluorogenic polymersomes
in gels coincided with high YMAX in solution (Fig. 6a, 7a, 7b).
Cyclamen amphiphile G1H3T1 with highest YMAX in solution
emerged as most powerful activator in gels (Fig. 7a, 7b, ).
᭹
Citronellal amphiphile G1H3T3 with poor YMAX in solution
performed also poorly in gels (Fig. 7a, 7b, ᭜). Whereas the
differences in YMAX were decisive for the outcome with fluorogenic
polymersomes in gels, small differences in EC50 appeared almost
irrelevant in this series (Fig. 7a, 7b).
The origin of differences in maximal activity YMAX is often
unclear. YMAX refers to the percentage of vesicles involved in
transport at saturation with transporters, the occurrence of YMAX
< 100% has been associated with transporter aggregation and
precipitation from the media at high concentration, with hindrance
of intervesicular transfer by irreversible partitioning, and more.
Presumably due to their increased fragility, the results with
liposomes in gels in the series G1H3T1 > G1H3T9 > G1H3T3
were clearly less satisfactory (Fig. 6c, 7c).
The series with alkyl homologs G1H3T8 > G1H3T9 ~
G1H3T10 is characterized by similar YMAX but more different
EC50 for both polymersomes and liposomes in solution (Fig.
8b, 8d). In this situation, the outstanding EC50 of G1H3T8 with
polymersomes in solution was at least partially preserved in gels
(Fig. 8a, 8b, ꢀ). However, the clear trend G1H3T8 > G1H3T9 >
G1H3T10 for liposomes in solution was lost in gels (Fig. 8c, 8d). In
this series, trends in gels were overall less significant and contained
more complex relationships between YMAX and EC50 (Fig. 8a, 8c).
Hill coefficients seemed to generally decrease in gels. This effect
is likely to originate from the exposure of the vesicles to transporter
gradients. These reduced Hill coefficients are thus unrelated to
transport mechanisms, the larger concentration range accessible
can be viewed as a potential advantage for sensing applications.
Acknowledgements
We thank the University of Geneva, the University of Basel, the
European Research Council (ERC Advanced Investigator), the
National Centre of Competence in Research (NCCR) Chemical
Biology, the NCCR Nanosciences and the Swiss NSF for financial
support. W.M. is thankful to P. Vajkozcy.
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