Amphiphilic dynamic NDI and PDI probes: Imaging microdomains in giant unilamellar vesicles

Article abstract of DOI:10.1039/c2ob25119a

Dynamic amphiphiles provide access to transmembrane ion transport, differential sensing and cellular uptake. In this report, we introduce dynamic amphiphiles with fluorescent tails. Core-substituted naphthalenediimides (cNDIs) and perylenediimides (cPDIs) are tested. Whereas the latter suffer from poor partitioning, dynamic cNDI amphiphiles are found to be purifiable by RP-HPLC, to partition selectively into liquid-disordered (Ld) microdomains of mixed lipid bilayers and to activate DNA as transporters. Importantly, fluorescence properties, partitioning and activity can be modulated by changes in the structure of mixed amphiphiles. These results confirm the potential of dynamic fluorescent amphiphiles to selectively label extra- and intracellular membrane domains and visualize biological function.

Full text of DOI:10.1039/c2ob25119a

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PAPER
Amphiphilic dynamic NDI and PDI probes: imaging microdomains in giant
unilamellar vesicles†‡
David Alonso Doval, Andrea Fin, Miwa Takahashi-Umebayashi, Howard Riezman, Aurelien Roux,
Naomi Sakai and Stefan Matile*
Received 16th January 2012, Accepted 28th February 2012
DOI: 10.1039/c2ob25119a
Dynamic amphiphiles provide access to transmembrane ion transport, differential sensing and cellular
uptake. In this report, we introduce dynamic amphiphiles with fluorescent tails. Core-substituted
naphthalenediimides (cNDIs) and perylenediimides (cPDIs) are tested. Whereas the latter suffer from
poor partitioning, dynamic cNDI amphiphiles are found to be purifiable by RP-HPLC, to partition
selectively into liquid-disordered (Ld) microdomains of mixed lipid bilayers and to activate DNA as
transporters. Importantly, fluorescence properties, partitioning and activity can be modulated by changes
in the structure of mixed amphiphiles. These results confirm the potential of dynamic fluorescent
amphiphiles to selectively label extra- and intracellular membrane domains and visualize biological
function.
which are then recognized by principal component or hierarchi-
cal cluster analysis. Differential sensing with dynamic
Dynamic amphiphiles are amphiphiles that contain dynamic
Introduction
bonds or “bridges” to connect hydrophilic heads and lipophilic
tails.1–4 Although emphasis has so far been on hydrazone
bridges, there is much potential in introducing other dynamic
covalent bonds such as disulfides, boronic esters or oximes
to build chemoorthogonal bridges in dynamic amphiphiles.
Dynamic amphiphiles are of interest with regard to ion transport
in vesicles1–3 and applications to cellular uptake4 as well as bio-
sensing, aptamerosensing and differential sensing of analytes of
free choice.1 They have originally been introduced to expand
biosensing applications with synthetic transport systems to also
include lipophilic analytes such as cholesterol.2 However,
dynamic amphiphiles soon turned out to be most attractive for
differential sensing with synthetic transport systems. In brief,
hydrophobic analytes such as muscone 1 are covalently captured
in situ with reactive head groups like 2, that is simple glutamate
derivatives containing one guanidinium cation and two hydra-
zides each (Fig. 1a).1 The resulting dynamic amphiphile 3 with
two hydrazone bridges is then characterized as activator of DNA
transporters in fluorogenic vesicles (Fig. 1c, e). Different activi-
ties with different head groups are used to generate patterns
Fig. 1 Dynamic amphiphiles as fluorescent membrane probes (b), for
cellular uptake (c, d) transport (c, e), and sensing (a, c, e). Hydrophobic
“tails” (e.g., muscone 1) are covalently captured by hydrophilic cations
School of Chemistry and Biochemistry, University of Geneva, Geneva,
Switzerland. E-mail: stefan.matile@unige.ch; www.unige.ch/sciences/
tition into lipid bilayer membranes (b) and activate polyanions (e.g.,
chiorg/matile/; Fax: +41 22 379 5123; Tel: +41 22 379 6523
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
(e.g., dihydrazide 2) to give dynamic amphiphiles (e.g., 3) that can par-
DNA, c) for cellular uptake (d) or cation transport in fluorogenic vesicles
((e), shown here is the example of fluorescence recovery in response to
the export of trapped cationic quenchers (blue) but not anionic fluoro-
‡Electronic supplementary information (ESI) available: Detailed exper-
imental procedures. See DOI: 10.1039/c2ob25119a
phores (red)).
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Fig. 2 Fluorescent dynamic cNDI and cPDI amphiphiles introduced in
this study. All cNDIs are mixtures of 3,7- and 2,6-regioisomers in the
core and epimers in the alkyl tail (compare Fig. 4).
Scheme 1 Synthesis of red cNDI tail 8. (a) 1. Dibromoisocyanuric
acid; 2. EtI, EtOH, K2CO3, 35%; 3. NaOEt, 73%; (b) 1. KOH, iPrOH,
reflux, 15 h; 2. 11, AcOH/H2O 1 : 1, 160 °C, µW, 5 min; 3. ammonium
acetate, AcOH/H2O 1 : 1, pH 4, 160 °C, µW, 30 min, 15%; (c) 13/DCM
1 : 1, rt, 22 h, 72%; (d) Cu(OAc)2, TEA, DMAc, O2, 55 °C, 20 h, 62%.
8 and 14 are mixtures of 3,7- and 2,6-regioisomers and mixtures of
enantiomers, 4 isomers each in total.
amphiphiles has been exemplified with artificial noses for odor-
ants or perfumes as illustrative samples from the supermarket.1
For cellular uptake, dynamic amphiphiles are attractive
because they provide effortless access to significant libraries.4
With design principles largely obscure, screening will be essen-
tial to tackle significant current challenges such as the delivery
of siRNA5 or understanding and control over the mode of action
of cell-penetrating peptides.6 Preliminary results in this direction
are encouraging.4
In this report, a third general application of dynamic amphi-
philes is envisioned for the first time, i.e., fluorescent membrane
probes.7–9 Hydrophobic by nature, the poor delivery of dedicated
fluorescent membrane probes often hampers their usefulness in
practice.8 Dynamic probes have the potential that their properties
can be tailored on demand. This includes delivery not only to
the extracellular membrane, but also to intracellular locations
assisted by polyion transporters.1–4 Moreover, the release of
dynamic probes at membranes of interest is conceivable by fine
tuning the reactivity of the bridge, and targeting and positioning
could be modulated further in mixed multitail amphiphiles.
To test these ideas, we selected core-substituted naphthalene-
diimide (cNDI)10,11 and perylenediimide (cPDI)12,13 fluoro-
phores as examples (Fig. 2). Little explored as membrane
probes, these fluorophores were considered promising because
their long lifetime is interesting for single-molecule studies, their
ability to cover the full visible range without global structural
changes is ideal for FRET applications,10 and their compact
planar structure promised partitioning into more ordered mem-
brane domains.9,13 We report the design, synthesis and evalu-
ation of dynamic NDI and PDI amphiphiles, including mixed
systems. We show that they can be separated and purified by
RP-HPLC, activate DNA as transporters, and that their partition-
ing into liquid disordered (Ld) phase can be modulated in mixed
amphiphiles and used to label microdomains in GUVs.
Fig. 3 Covalent capture of aldehyde substrates as single-tail dynamic
amphiphiles.
then with ammonium acetate. Microwave-assisted imide
formation gave the mixed diimide 12 together with the sym-
metric side products.
Core substitution with iso-propylamine 13 converted the
yellow cNDI 12 into the red cNDI 14. In the final step, a Cu-
catalyzed C–N bond formation was employed to couple 14 and
boronic acid 15. Structure and homogeneity of the final product
8 was confirmed by NMR spectroscopy, mass spectrometry
(MS) and high-pressure liquid chromatography (HPLC).
The dynamic NDI amphiphile 4 with one single fluorescent
tail was obtained by incubation of aldehyde 8 with the cationic
monohydrazide 161 for 1 h in dry DMSO at 60 °C (Fig. 3).1–3
The originally envisioned aldehyde 17 could not be used for this
purpose because it was insoluble in DMSO and other applicable
solvents. The dynamic homologue 7 with a single PDI tail was
obtained by incubation of PDI aldehyde 18 under identical con-
ditions (Fig. 3). The synthesis of cPDI 18 with two ethoxy
groups in the core is described in the ESI.‡14
Results and discussion
The red, fluorescent tails 8 were accessible from naphthalene dia-
nhydride 9 in seven steps (Scheme 1).14 The conversion of sub-
strate 9 into the core-substituted naphthalene tetraester 10 was
accomplished in three steps following published procedures.10
From there, the esters were hydrolyzed with base and the
product reacted first with racemic 2-ethyl-1-hexylamine 11 and
One advantage with fluorescent tails was that formation and
stability of dynamic amphiphiles could be more easily followed
by reverse-phase (RP) HPLC.14 Single-tail amphiphiles 4 and 7
were identified and purified by this method, and their structure
was confirmed by electrospray ionization (ESI) MS. However,
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Fig. 4 Covalent capture of aldehyde substrates as double-tail dynamic amphiphiles. All four regioisomers of mixed amphiphile 6 are shown, they all
exist as pairs of epimers. Analogous regio- and stereoisomers for 5 and 20 are not shown.
with a retention time tR = 15.3 min (Fig. 5a). The split peak indi-
cated that 2,6- and 3,7-regioisomers could be separated. This
separation of regioisomers has already been accomplished with
similar samples.15
The dynamic amphiphile 5 with two NDI tails appeared at tR
= 14.1 min (Fig. 5a). Isolation of the sample and reinjection was
possible with negligible hydrolysis of the hydrazone bridges
(Fig. 5c). Traces of free aldehyde 8 appeared (<5%), whereas the
broad peaks of single tail intermediates 20 around tR ∼ 11 min
remained undetectable. The dynamic amphiphile 21 with two
alkyl tails was visible at 250 nm (Fig. 5a). The mixed amphi-
philes 6 appeared at tR = 15.9 min as a broad peak with a
shoulder. The product 6 was quite stable during isolation and
characterization. A dominant single peak was obtained for pure
material by ESI MS (Fig. 5d). Reinjection of purified material
gave mixed amphiphiles 6 as dominant product, confirming stab-
ility during purification, also under acidic conditions (Fig. 5b).
In HPLCs of purified samples, the broad peak for single-tail
degradation products around tR ∼ 10.2 min decreased with
decreasing acidity of the acid used during purification by HPLC
(i.e., 15% with 0.1% TFA, pKa 0.50; 8% with 0.1% formic acid,
pKa 3.75; 4% with 0.1% acetic acid, pKa 4.76). The amphiphiles
20 with single NDI tails appeared as main side products, confi-
rming that aryl hydrazone bridges are more stable than alkyl
hydrazones. Interestingly, the dynamic amphiphile 5 with two
NDI tails appeared as well as a minor side product. This finding
pointed toward the occurrence of tail scrambling during purifi-
cation and characterization.
To illustrate the beauty and power of the approach, we pre-
pared also the dynamic amphiphile 22 with three NDI tails by
incubating aldehyde 8 with trihydrazide 231 (Fig. 6). Eluted at tR
= 16.0 min, triple-tail amphiphile 22 could be isolated, reinjected
and subjected to ESI MS without significant tail loss. Mixed
triple-tail amphiphiles were not prepared because mixed double-
tail amphiphiles 6 were sufficient to outline the functional
benefits of tail mixing.
Fig. 5 PR-HPLC traces of (a) the crude reaction mixture obtained
from covalent capture of aldehydes 8 and 19 by dihydrazide 2, (b)
purified mixed amphiphile 6 and (c) purified di-NDI 5 with detection by
absorption at 540 nm (solid) and at 250 nm (dashed); (d) ESI MS of 6.
HPLC analysis was most interesting with double-tail amphi-
philes because mixed systems could be included. Incubation of
cNDI aldehyde 8 together with the cationic dihydrazide 21 as
well as octadecyl aldehyde 19 gave reasonably complex
RP-HPLC profiles (Fig. 4 and 5a). Best results were obtained on
a YMC-Pack OSD-A S-5 µm column (120A C18, 250 mm ×
10 mm) and a gradient elution moving from 4 : 6 to 7 : 1 THF–
The ability of dynamic amphiphiles 4, 6 and 7 to activate
DNA as cation transporter in lipid bilayer membranes was deter-
mined under established conditions.1–3 Fluorogenic large unila-
mellar vesicles (LUVs) were prepared by swelling dried egg
yolk phosphatidylcholine (EYPC) films in water containing
buffers, an anionic fluorophore (HPTS, 8-hydroxy-1,3,6-pyrene-
trisulfonate) and a cationic quencher (DPX, p-xylene-bispyridi-
nium bromide). The obtained EYPC LUVs ideally report the
export of DPX quenchers by counterion activated DNA as an
water with 0.1% TFAwithin 12 min at a flow rate of 3 ml min−1
.
Under these conditions, cNDI 8, the starting material, appeared
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ctDNA transporters in EYPC LUVs. The intensity before lysis
(Fig. 7a, t = 200 s) was taken as fractional activity Y and plotted
as a function of activator concentration at constant DNA concen-
tration (Fig. 7b). Hill analysis gave YMAX, the maximal accessi-
ble activity under these conditions, the EC50, the effective
activator concentration needed to reach 50% of YMAX, and the
Hill coefficient n, which indicates the steepness of the sigmoidal
fitting (Table 1).
All activators functioned with a very satisfactory EC50
∼ 8 µM. With single-tail NDI amphiphile 4, the highest EC50
coincided with highest YMAX = 72% as well as highest n = 17
(Table 1, entry 1). This exceptional cooperativity indicated that
at least 17 amphiphiles 4 are needed to activate DNA, and that
the resulting active polyion-counterion complexes are thermody-
namically unstable.17 A poor YMAX = 16% suggested that single-
tail PDI amphiphiles 7 and/or their stable complexes with
polyions prefer to precipitate rather than partition into the EYPC
membrane (Table 1, entry 3). Decreasing EC50 = 7.2 µM and
n = 5.7 were in agreement with this interpretation. An improve-
ment to EC50 = 6.2 µM and n = 3.1 without significant losses in
YMAX = 66% was possible with the mixed double-tail NDI
amphiphile 6 (Table 1, entry 2). This attractive behavior
suggested that the alkyl tail in amphiphile 6 improves the stab-
ility of the active complexes without losses in their partitioning
into EYPC membranes.
Fig. 6 Covalent capture of aldehyde substrates as triple-tail dynamic
amphiphiles.
With fluorescent tails as intrinsic probes, the partitioning of
dynamic NDI and PDI amphiphiles could be determined directly.
Because their fluorescence is quenched in water, partitioning in
lipid bilayers was detectable as fluorescence recovery at 575 nm
(Fig. 8a). Partitioning into 1,2-dioleoyl-sn-glycero-3-phospho-
choline (DOPC) LUVs in liquid disordered (Ld) phase was
tested first. The obtained response to increasing DOPC concen-
trations was fitted to
Fig. 7 (a) Changes in fractional fluorescence intensity IF of HPTS (λex
= 413 nm, λem = 510 nm) during addition of increasing concentrations
of mixed amphiphile 6 (t ∼ 0 s), ctDNA (1.25 µg ml−1 final concen-
tration, t ∼ 40 s) and excess triton X-100 (t ∼ 200 s) to EYP-
C-LUVs⊇HPTS/DPX. (b) Dose response curves for ctDNA activated by
dynamic counterions 4 (●), 6 (○) and 7 (◇) with fit to the Hill
equation.
increase in the emission of the ratiometric fluorophore HPTS. In
reality, fluorescence recovery in this assay can originate from the
export of cationic DPX, anionic HPTS or both from intact ves-
icles, or from the destruction of the vesicles. However, internal
trapping experiments have shown that counterion activation
enables DNA to move across intact lipid bilayers, and DPX but
not HPTS transport across bulk chloroform membranes has been
confirmed experimentally.16
The addition of either amphiphile 4, 6 or 7 (Fig. 7a, t < 40 s)
or calf thymus (ct) DNA to the vesicles did not cause a signifi-
cant fluorescence response. However, both together, 4, 6 or 7
and ctDNA, caused fluorescence recovery (Fig. 7a, t > 40 s).
This finding indicated that amphiphiles 4, 6 and 7 activate
I ¼ IMIN þ ðIMAX ꢀ IMINÞ=ð1 þ ½H2Oꢁ=Kx ½lipidꢁÞ
ð1Þ
where I is the fluorescence response, IMIN = I without lipids,
IMAX = I at saturation, [H2O] the concentration of water (55.3
M), and Kx the partition coefficient.18 With Kx ∼ 15 000, both
NDI amphiphiles 4 and 6 partitioned well into DOPC LUVs
(Table 1, entries 1 and 2). Fluorescence recovery of PDI amphi-
phile 7 was not detectable above scattering, a finding that was
consistent with the poor YMAX = 16% for DNA activation
(Table 1, entry 3).
Partitioning of NDI amphiphile into the solid-ordered (So)
phase of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
LUVs and the liquid-ordered (Lo) phase of DPPC/cholesterol
Table 1 Transport and partitioning dataa
Cpdb
YMAX (%)c
nd
EC50 (µM)e
Kx DOPCf
Kx DPPCg
Kx DPPC/CLh
Kx DOPC/CLi
4
6
7
71.6 2.4
66.0 3.0
16.3 0.9
17.1 1.2
3.1 0.4
5.7 1.6
10.3 0.1
6.2 0.3
7.2 0.4
(1.6 0.5) 104
(1.3 0.5) 104
—
(7.8 4.5) 103
(2.2 0.9) 103
—
(2.2 1.4) 103
(4.1 1.1) 103
—
(6.1 2.6) 103
(8.8 3.8) 103
—
a From HPTS/DPX assay (YMAX, n, EC50, Fig. 7) and fluorescence recovery of NDI tails in membranes (Fig. 8). b Compounds (Fig. 2). c Maximal
activity accessible relative to full vesicles destruction. From Hill analysis of dose response curves (Fig. 7b). d Hill coefficient. e Effective concentration,
f–i
concentration needed to reach 50% of YMAX
.
Partition coefficient Kx in LUVs of different composition (Fig. 8); DOPC, 1,2-dioleoyl-sn-glycero-3-
phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; CL, cholesterol.
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Fig. 8 (a) Increase in fluorescence emission intensity IF of mixed NDI
amphiphile 6 (λex = 540 nm) in the presence of increasing concentrations
of DOPC LUVs. (b) Dose response curve for data in (a), showing emis-
sion I at 575 nm as a function of DOPC concentration with curve fit to
eqn (1).
(CL) LUVs was clearly less favorable and more difficult to
detect above scattering. With all due caution, it can be said that
single-tail amphiphile 4 preferred the So phase (Kx = 7800) over
the Lo phase (Kx = 2200). This trend was reversed with addition
of an octadecyl tail in mixed double-tail NDI amphiphile 6,
which showed some preference for the Lo phase (Kx = 4100)
over So phase (Kx = 2200). Insensitivity in DOPC/CL LUV con-
trols suggested that the observed trends are valid. The addition
of a saturated alkyl tail was thus sufficient to increase the parti-
tioning into the Lo phase but clearly insufficient to “push” the
mixed amphiphile 6 from the Ld phase (Kx = 13 000) into the
Lo phase (Kx = 4100).
Partitioning of fluorescent NDI and PDI probes into mixed
LUVs with different microdomains was not determined because
fluorescence spectroscopy cannot differentiate between the
microdomains and the results would be as for microdomain-free
vesicles and thus meaningless. To more qualitatively probe for
selective partitioning within mixed membranes, confocal
imaging of giant unilamellar vesicles (GUVs) is the method of
choice (Fig. 9).7,19 The larger GUVs are required for detection
by fluorescence microscopy. One of the most reliable compo-
sitions of GUVs to study microdomains is SM–DOPC–CL
56 : 24 : 20.7i Under these optimized conditions, it is possible to
quite frequently observe GUVs that show separation into Lo and
Ld phases at room temperature, a situation that is perfect for
unambiguous data analysis. Egg sphingomyelin (SM) and CL
form the microdomains in the Lo phase, whereas DOPC segre-
gates into the separate microdomains in the Ld phase. Compared
to standard experiments in LUVs described above, DPPC is thus
replaced by SM to form, together with cholesterol, the Lo micro-
domains in the GUVs.
In heterogeneous GUVs composed of SM–DOPC–CL
56 : 24 : 20, Lo phases can be labeled with 0.05 mol% naphtho-
pyrene, a fluorescent probe that emits blue light. The fluorescent
probe BODIPY FL C5-HPC can be used alternatively to selec-
tively label the Ld domains. The partitioning of the new NDI
probes 4–6 in mixed GUVs was explored under these standard
conditions. For this purpose, GUVs composed of SM/DOPC/CL
56 : 24 : 20 were prepared in the presence of 0.1–0.5 mol% of an
NDI probe and 0.05 mol% naphthopyrene. Confocal microscopy
z-scanning was recorded simultaneously for red emission from
NDI probes (λex 543 nm) and blue emission from naphthopyrene
Fig. 9 Fluorescent images of GUVs composed of SM/DOPC/CL
56 : 24 : 20 with 0.1–0.5 mol% of (a, b) 4, (c) 5 and (d, e) 6 (red emis-
sion, λex 543 nm) and 0.05 mol% naphthopyrene in the Lo phase (blue
emission, λex 405 nm). Single plane images of the equator region show
simultaneously recorded red and blue emission separately (c–e, left) and
merged (c–e, right). Complete GUVs were reconstructed from z-scans in
0.8 µm increments (a, b and e, far right). The diameters of all shown
GUVs were around ∼10 μm, their dispersity covered roughly one order
of magnitude.
in the Lo phase (λex 405 nm). Single plane images obtained at
the equatorial region of the GUVs revealed little overlap of the
blue and the red fluorescence (Fig. 9c–e, left side). Merging of
the individual z-stacks fully confirmed the impression that the
blue and red fluorescence do not overlap (Fig. 9c–e, right side).
Full images of complete GUVs were constructed from serial con-
focal optical sections at 0.8 µm intervals (Fig. 9a, 9b and 9e, far
right). The most revealing smaller GUVs with two to three large
domains were observed most frequently (Fig. 9b–d). Smaller
GUVs with several domains were seen less frequently (Fig. 9e).
Multiple domains in a single, usually larger GUV were only
found occasionally (Fig. 9a).
Coexisting Ld and Lo phases were observed for all GUVs,
and the blue and red fluorescence never overlapped strongly.
Knowing that naphthopyrene partitions into the Lo phase com-
posed of SM and CL, this finding demonstrated that in hetero-
geneous SM–DOPC–CL GUVs, the new NDI probes partition
into the Ld phase composed of DOPC. Overlapping emission
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with green-fluorescent BODIPY FL C5-HPC control probes for
Ld domains (λex 488 nm) corroborated the validity of this
interpretation (Fig. S8‡). Uniform partitioning of dynamic NDI
amphiphiles into Ld phases in GUVs was consistent with the
partition coefficients determined in LUVs (Table 1).
Acknowledgements
We thank D. Jeannerat, A. Pinto and S. Grass for NMR measure-
ments, the Sciences Mass Spectrometry (SMS) platform for
mass spectrometry services, and the University of Geneva, the
European Research Council (ERC Advanced Investigator), the
National Centre of Competence in Research (NCCR) Chemical
Biology and the Swiss NSF for financial support.
Quantitative determination of differences in partitioning by
fluorescence imaging in GUVs is not as straightforward as by
fluorescence spectroscopy in LUVs. However, separate single
plane images of the equator region indicated that NDIs 4 and 5
with more pronounced preferences for Ld phases left not a shade
of red emission in the area occupied by the blue fluorescent Lo
phase (Fig. 9c). The increasing acceptance of mixed NDI amphi-
philes 6 in Lo phases could be guessed from the appearance of
red fluorescence within blue domains (Table 1, entry 2;
Fig. 9d, e). However, these qualitative differences between the
NDI probes in GUVs are relatively minor and much less evident
than in quantitative measurements in LUVs. Controls confirmed
that dynamic triple-tail amphiphile 22 suffers increasingly from
intramolecular fluorescence quenching but still partitions into the
Ld phase (Fig. S8‡). The partitioning of dynamic PDI amphi-
philes was too poor for meaningful fluorescence imaging in
GUVs.
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Conclusions
This is the first report on dynamic amphiphiles with fluorescent
tails. NDIs and PDIs were selected as representative fluoro-
phores, mainly for convenience but also because of their poten-
tial as photostable FRET systems in Lo phases. The synthesis of
NDIs and PDIs with one aldehyde was as straightforward as
expected, solubilizing groups had to be added and refined. With
fluorescent tails, covalent capture by hydrazide head groups
could be easily followed by RP-HPLC. Dynamic NDI amphi-
philes could be purified, isolated and characterized without
excessive decomposition. Dynamic NDI amphiphiles activated
DNA as transporters in lipid bilayers, and activity could be
modulated by the composition of the of mixed systems.
Dynamic PDI amphiphiles were nearly inactive due to poor par-
titioning. This finding confirmed that dynamic NDI (but not
PDI) amphiphiles could be used to visualize cellular uptake path-
ways and label intracellular membranes. For example, one can
imagine active complexes with DNA being taken up by endocy-
tosis, amphiphilic NDI hydrazones being hydrolyzed in the more
acidic endosomes and hydrophobic NDI probes being released
to report on the nature of endosomal membranes.
Qualitative imaging in mixed GUVs revealed that dynamic
NDI amphiphiles partition into the Ld phase, independent of
their structure. However, quantitative partitioning studies in
LUVs revealed that partitioning in mixed membranes can be
modulated by the composition of mixed amphiphiles. Taken
together, these results fully confirm the potential of dynamic flu-
orescent amphiphiles to selectively label extra- and intracellular
membrane domains, and that the selectivity can be modulated
with the structure of mixed amphiphiles. With cNDIs and par-
ticularly cPDIs not fully convincing, we currently focus on
dynamic fluorescent amphiphiles that operate with environ-
mental-sensitive, conceptually innovative fluorophores rather
than by partitioning.
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