A new perylene bisimide bola amphiphile: Synthesis, characterization, fluorescent properties and applications as a potential probe

Article abstract of DOI:10.1039/c3nj00116d

Fluorescently-tagged lipids are a powerful tool for investigating the dynamics of lipids in cell biological studies and in biophysical applications. Herein we report the synthesis and the characterization of a new bola amphiphile, PC12, based on a bisimidic perylene moiety: a central perylene unit is symmetrically linked to two aliphatic chains both ending with a quaternary ammonium group. Absorption and fluorescent properties of the newly synthesized compound were investigated in DMSO and in water as a function of concentration and temperature. Further, the entrapment efficiency, the fluorescence behavior in dimyristoyl-sn-glycero-phosphocholine (DMPC) liposomes, and cell uptake on human and murine glioblastoma cell lines were evaluated. When loaded in liposomes, PC12 follows the same destiny of liposomes themselves in the cell cultures: this is an interesting result because PC12 could be used both as an aspecific dye (free form) and as an organelle-specific lipid probe.

Full text of DOI:10.1039/c3nj00116d

NJC
View Article Online
View Journal | View Issue
PAPER
A new perylene bisimide bola amphiphile: synthesis,
characterization, fluorescent properties and
applications as a potential probe†
Cite this: NewJ.Chem., 2013,
37, 2166
Marco Franceschin,a Cecilia Bombelli,*b Silvia Borioni,a Giuseppina Bozzuto,c
Silvia Eleuteri,a Giovanna Mancini,b Agnese Molinaric and
Armandodoriano Biancoa
Fluorescently-tagged lipids are a powerful tool for investigating the dynamics of lipids in cell biological
studies and in biophysical applications. Herein we report the synthesis and the characterization of a new
bola amphiphile, PC12, based on a bisimidic perylene moiety: a central perylene unit is symmetrically linked
to two aliphatic chains both ending with a quaternary ammonium group. Absorption and fluorescent
properties of the newly synthesized compound were investigated in DMSO and in water as a function of
concentration and temperature. Further, the entrapment efficiency, the fluorescence behavior in dimyristoyl-
sn-glycero-phosphocholine (DMPC) liposomes, and cell uptake on human and murine glioblastoma cell lines
were evaluated. When loaded in liposomes, PC12 follows the same destiny of liposomes themselves in the
cell cultures: this is an interesting result because PC12 could be used both as an aspecific dye (free form)
and as an organelle-specific lipid probe.
Received (in Montpellier, France)
30th January 2013,
Accepted 22nd April 2013
DOI: 10.1039/c3nj00116d
www.rsc.org/njc
to the hydrophilic or to the hydrophobic region of the amphiphilic
molecules. Perylene bisimides are known as optimal fluorescent
Introduction
Fluorescently-tagged lipids are a powerful tool for investigating the
dynamics of lipids in cell biological studies and in biophysical
applications.1 Several fluorescent lipids have been proposed and
exploited since the early 1980s, some of which resemble their
natural counterpart (e.g. fluorescently labeled phosphatidylcholine,
phosphatidylethanolamine, lysophosphatidylethanolamine, and
fluorescently labeled fatty acid derivatives),2 whereas others simply
feature an amphiphilic molecular structure with no resemblance of
natural lipids (e.g. 3,30-diacylindocarbocyanine iodides3 laurdan,
6-dodecanoyldimethylaminonaphthalene,4 umbelliferone, 4-hepta-
decyl-7-hydroxycoumarin5). Depending on the lipid aspect to be
investigated, the fluorescent dye can be covalently coupled either
dyes because they feature excellent chemical, thermal and photo-
chemical stability and high fluorescence quantum yields.6,7 In fact,
they have been applied in different fields such as laser dyes,8
photovoltaic cells,9 and sensors.10 Moreover, the extended aromatic
system of the perylene dye has been exploited in the formation of
supramolecular systems with photophysical properties peculiar
with respect to single molecules.11 However, the study of supra-
molecular aggregates of perylene bisimide derivatives has been
restricted for many years to organic solvents; only recently, some
water soluble perylene bisimide amphiphiles have been developed,
belonging both to the class of conventional surfactants (where the
hydrophobic perylene moiety is located at one side of the molecule)
and to that of bola amphiphiles (where the perylene unit is the core
of the molecule, linked at both sides to hydrophilic residues). A
recent review of Wu¨rthner and coworkers provides an overview on
this topic.12 Amphiphiles with a perylene unit may be useful for
many applications in an aqueous environment such as (i) disper-
sion of poorly soluble functional carbon materials (SWCNTs and
graphenes), (ii) study of biomolecules (DNA and RNA) thanks to the
fluorescence and electron-acceptor properties of perylene dye;12b
(iii) development of anticancer drugs because of the ability of the
perylene unit to stabilize the G-quadruplex inhibiting telomerase
activity;12c–g (iv) development of fluorescent lipid probes for cell
a
`
Sapienza - Universita di Roma, Dip. Chimica, P.le A. Moro 5, Roma, Italy.
E-mail: marco.franceschin@uniroma1.it, siborioni@gmail.com,
silvia.eleuteri@uniroma1.it, agnese.molinari@iss.it,
armandodoriano.bianco@uniroma1.it
b CNR, Istituto di Metodologie Chimiche – Sezione Meccanismi di Reazione and
`
Dipartimento di Chimica, Sapienza-Universita di Roma, c/o dipartimento
`
di Chimica Sapienza-Universita di Roma, P.le A. Moro 5, Roma, Italy.
E-mail: cecilia.bombelli@uniroma1.it, giovanna.mancini@uniroma1.it
c
`
Istituto Superiore di Sanita, Department of Technology and Health, Roma, Italy.
E-mail: giuseppina.bozzuto@uniroma1.it, agnese.molinari@iss.it
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3nj00116d
c
2166 New J. Chem., 2013, 37, 2166--2173
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
biology and biophysical studies. A very recent paper reports13 the a BOC reactant at a 0.3 : 1 ratio with respect to the amine,19 in
design and obtainment of fluorescent amphiphilic probes where the dichloromethane at room temperature for 3 days, to obtain
bisimidic perylene residue and dendritic polyethylene glycol (PEG) the desired compound 3. The mono-protected product 3 was
constitute the hydrophobic and hydrophilic regions, respectively, efficiently purified by column chromatography. Even though due
arranged in two different main architectures, the head–tail and the to the use of a small amount of boc-anhydride a big percentage
core–shell dyes. The first one is the architecture of a ‘‘conventional of the unreacted amine remained in the reaction mixture, it was
amphiphile’’ with a hydrophilic head group (the dendritic PEG) and possible to recover by the same column chromatography the
a hydrophobic region (the perylene moiety), whereas in the core– purified starting compound, which could be recycled.
shell architecture the bisimidic perylene unit is linked in opposite
Once the asymmetrical compound 3 was synthesized, perylene
directions to symmetric dendritic PEGs, thus resulting in a bola diimide 4 was easily obtained by treatment of the brominated
type (or bipolar) amphiphile. The architecture of the fluorescent perylene anhydride 2 with an almost stoichiometric amount of 3,
amphiphiles has been proved to influence the cellular uptake of in DMA/dioxane under argon. No more than 10% excess of reactant
the probes, in particular, the bola amphiphile was internalized was used to avoid side reactions at the brominated positions, which
more efficiently by cells.
can occur at higher ratios. It is worth noting that, in principle,
Herein we report the synthesis of bola amphiphile PC12 (1) substitution of bromine atoms by the primary amine can actually
based on a bisimidic perylene moiety. A central perylene unit is occur even at a stoichiometric ratio. In our experience, this can be
symmetrically linked to two dodecyl chains both ending with a avoided by strictly controlling the temperature and the time of
quaternary ammonium group. The absorption and emission reaction, TLC was used to follow the appearance of any blue-green
features of the fluorescent bola lipid PC12 were investigated in spot, due to the formation of a new chromophore where a nitrogen
DMSO and in water as a function of concentration and temperature. atom from the primary amine is conjugated to the aromatic
Further, the entrapment efficiency in liposomes and cell uptake on system.17 If such a spot appears, the reaction must be stopped
glioblastoma cell lines were evaluated.
and the products can be purified by column chromatography.
The deprotection of pure compound 4 was conducted in
dichloromethane adding TFA dropwise to obtain pure compound 5.
Finally, to obtain a bola type molecule, the polar hydrophilic
heads were subjected to exhaustive methylation in order to
Results and discussion
Design rationale and synthesis of bola lipid PC12
Our objective was to synthesize a fluorescent amphiphilic probe obtain positively charged head groups. In particular, PC12 was
characterized by two hydrophobic tails each linked on one side obtained in quantitative yield by adding CH3I to perylene
with a polar hydrophilic head and on the other with the bisimide previously dissolved in dioxane.
fluorescent hydrophobic core.
Photophysical characterization of PC12
Perylene anhydride is an excellent candidate for creating the
desired fluorescent probe, since the insertion of side chains on
UV-visible absorption spectra. The absorption spectrum of
the major axis of perylene is a well studied procedure.14 Moreover 1 ꢁ 10ꢀ5 M PC12 in DMSO is reported in Fig. 1 (long dash). The
perylene bisimide dyes are known for their high molar absorption spectrum shows two main maxima in the UV/visible range, one
coefficients of 30 000–90 000 cmꢀ1 Mꢀ1 15
fluorescence quantum of high intensity at 528 nm and the other at 494 nm. Moreover,
,
yields in some cases close to unity16 and excellent photostability.17 a shoulder at higher energy (458 nm) can be detected. The observed
The first challenge of this approach is represented by the low pattern20 is due to the vibronic fine structure of the S0–S1 transition
solubility of perylene in common organic solvents. Based on our of the perylene moiety; in particular, the lower energy band,
previous experience,18 we bypassed this main problem using corresponding to the 0–0 transition, is more intense with respect
perylene derivatives modified on the bay-area by bromination to the higher energy band, corresponding to the 0–1 transition, and
(compound 2). In order to obtain a compound with the desired the shoulder can be assigned to the 0–2 transition. As expected, the
properties described above, we chose to insert as side chains on vibronic peaks are not well resolved as in other perylene derivatives
the dibromoperylene core a diamine with a long aliphatic chain: without substituents in the ‘‘bay’’ positions. Qualitatively, this broad-
1,12-diaminododecane. This compound has the right length to fit ening originates from the loss of planarity of the perylene ring due to
inside phospholipid bilayers and presents two amino groups the presence of a bromine atom in the bay area.21 However, little
which are suitable for the reaction with the anhydride function, changes in maxima absorption wavelengths are observed with
leaving a polar group at the opposite end of the chain.
respect to pure perylene, as expected in the presence of electron-
On the other hand, the simultaneous presence of two withdrawing substituents in the ‘‘bay’’ positions. Moreover, the
equivalent groups on the same molecule represents the second maxima absorption wavelengths of the bands do not shift with
main challenge of the proposed synthetic route.
concentration and the ratio of vibronic bands is constant with
Thus our synthetic strategy (Scheme 1) begins with the concentration (see Fig. S1, ESI†). Thus, in DMSO, the overall features
protection with boc-anhydride of one of the two amino groups of the spectra suggest the absence of aggregation involving a p–p
of the aliphatic chain to be inserted.
interaction between the perylene rings.
The addition of classical amino protecting group BOC led to
In aqueous solution otherwise, PC12 shows the typical
an asymmetrical compound where the two amino groups absorption spectrum of aggregated perylene dyes (Fig. 1, solid
present a different reactivity. The reaction was conducted using line); in fact, the absorption intensities of all bands decrease,
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 2166--2173 2167

View Article Online
NJC
Paper
Scheme 1 Preparation of bola amphiphile PC12 (1).
and the 0–1 transition becomes more intense than the 0–0 one. 55 1C the relative intensity of the perylene absorption peaks does
The intensity reversal observed for the 0–0 and 0–1 bands not change (see Fig. 2b, ESI†).
indicates that in water the Franck–Condon factors favour the
Fluorescence spectra. The fluorescence emission spectrum
higher (0–1) excited state,22 suggesting the formation of H-type p–p of 6 ꢁ 10ꢀ6 M PC12 in DMSO is reported in Fig. 2a. It shows a
stacking aggregates. By heating the sample (1 ꢁ 10ꢀ5 M PC12) up to broad band with a maximum at 555 nm and a shoulder at
c
2168 New J. Chem., 2013, 37, 2166--2173
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
nature of the counterion, i.e. iodide, which is a well known
fluorescence quencher. The bromine atoms in the bay area, other-
wise, cannot be indicated as the main cause of the low quantum
yield; in fact, for different perylene bisimide derivatives, with the
same substituents on the nitrogen atoms, the presence of one27 or
two bromine atoms28 in the bay area decreases the quantum yield by
only 7% and 24%, respectively.
The fluorescence emission spectrum of 6 ꢁ 10ꢀ6 M PC12 in
water as a function of temperature (25–60 1C) is reported in
Fig. 2b. The emission spectrum shows, as in DMSO, a broad
band with a maximum at 562 nm and 595 nm. However, the
aggregation behaviour of PC12 in water is responsible for the
decrease in the fluorescence quantum yield with respect to
DMSO. In fact, with increasing temperature, the intensity of the
fluorescence emission increases, suggesting a temperature
driven disaggregation process. Note that the fluorescence emis-
sion of PC12 as a function of temperature in DMSO decreases
with increasing temperature, as expected for a fluorophore in
Fig. 1 Absorption spectra of 1 ꢁ 10ꢀ5 M PC12 in DMSO (long dash) and in
water (solid line).
595 nm, that is the mirror image of the absorption spectrum, as the absence of aggregation (Fig. 2a).
previously observed for other perylene derivatives.23 Emission
quantum yield for PC12 was evaluated in air equilibrated
Entrapment efficiency and fluorescence spectra of PC12 in
DMPC liposomes
DMSO medium with respect to Ru(bpy)3Cl2 (F = 0.028 in
H2O).24 Moreover, in order to have as a touchstone for quantum
yield a molecule belonging to the same class of PC12, we used also As a general rule, a solute unentrapped in liposomes remains in
another standard, not commercially available but described in the solution after the extrusion and can be removed from the liposome
literature, perylene-3,4,10-tetracarboxylic acid tetrapotassium salt. dispersion by filtration on an exclusion chromatography column.
The fluorescence of this standard is independent of oxygen and In some cases the unentrapped solute can self-assemble in solution
displays a very high quantum yield (F = 1.00 in 0.1 M K2CO3, with to give large aggregates; in this case, aggregates will be removed
respect to fluorescein).25 Relative emission quantum yield was in the extrusion procedure by the polycarbonate membrane.
found to be 0.072 with respect to Ru(bpy)3Cl2 and 0.039 with Therefore, the amount of unentrapped PC12 excluded by extrusion
respect to perylene-3,4,10-tetracarboxylic acid. In the field of and/or gel filtration is mainly dependent on its aggregation state
perylene derivatives, the quantum yield of PC12 is quite low. and water solubility. The amount in molar percentages of PC12 in
The reason for this low quantum yield can be ascribed mainly to DMPC liposome dispersions at each stage of the preparation has
the nature of the substituent at the imide group.14a The alkyl been calculated: 20% of PC12 is lost upon extrusion while the
chains on the nitrogen are not fixed in an orthogonal conforma- amount of PC12 remains constant upon gel filtration, suggesting
tion and are characterized by a high conformational freedom, the that all unentrapped PC12 is filtered off by the polycarbonate
vibronic motions (‘loose bolt effect’)26 are thus responsible for a membrane and it is probably present in the buffer assembled in
drop in the quantum yield of perylene. Another cause could be the large aggregates.
Fig. 2 Fluorescence emission spectra (lexc = 505 nm) at increasing temperature (25–60 1C) of 6 ꢁ 10ꢀ6 M PC12 (a) in DMSO; (b) in an aqueous solution.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 2166--2173 2169

View Article Online
NJC
Paper
Fig. 3 Fluorescence emission spectra (lexc = 505 nm) of 6 ꢁ 10ꢀ6 M PC12 (a)
entrapped in DMPC liposomes (solid line) and in PBS buffer, magnified (b) 10
times (dotted line) and (c) 100 times (dashed line).
Fig. 5 Intracellular localization of PC12 analyzed using laser scanning confocal
microscopy. (a, c, and e) Rat C6 and (b, d, and f) human LN229 glioblastoma cells
treated with PC12 in DMSO for 5 (a and b) and 27 h (c and d). (e) C6 and
(f) LN229 cells treated with PC12 entrapped in liposomes for 27 h.
Fig. 4 Fluorescence emission spectra (lexc = 505 nm) of 6 ꢁ 10ꢀ6 M PC12
compound as a fluorescent lipid probe. In particular, intracellular
localization of PC12 administered either in DMSO or entrapped in
liposomes was analyzed using laser scanning confocal microscopy
(LSCM) in both human (LN229) and murine (C6) glioblastoma
cells. As visualized in Fig. 5, after 5 (Fig. 5a and b) and 27 h
(Fig. 5c and d) of treatment with a DMSO solution of the probe,
PC12 stained preferentially cellular membranes of both C6
and LN229 cells. Plasma membranes and intracytoplasmic
organelles appeared strongly fluorescent, while no significant
signal was revealed inside the nuclei. The cellular distribution of
PC12 completely changed when it was entrapped in DMPC
liposomes. After 5 h of incubation it completely failed to stain
C6 and LN229 cells, as demonstrated by the lack of the fluorescent
signal in treated glioblastoma cultures (data not shown). In
contrast, after 27 h of incubation, strongly fluorescent liposomes
clusterized preferentially on the plasma membranes were revealed
on both C6 and LN229 cells, and a low intracytoplasmic signal
could be observed (Fig. 5e and f). Fluorescent clusters of
liposomes that seem to be localized in the cytoplasm of some
cells (Fig. 5e, arrows) are, actually, liposomes localized on the
entrapped in DMPC liposomes at increasing temperature.
The fluorescence emission spectrum of PC12 entrapped in
liposomes after gel filtration is shown in Fig. 3 (solid line). The
fluorescence intensity is very high with respect to PC12 at the same
concentration (6 ꢁ 10ꢀ6 M) in the buffer (Fig. 3, dotted and dashed
lines), suggesting that the perylene dye molecules are included in
the lipid bilayer in the monomeric form. Moreover, the whole
fluorescence spectrum is blue shifted, with the maximum at
543 nm (Fig. 3, solid line). Because the maximum is 562 nm in
water and 555 nm in DMSO, respectively, this blue shift suggests a
location of the perylene moiety in a less polar environment with
respect to water. The hypothesis of the inclusion of PC12 in the
lipid bilayer in the monomeric form is strengthened by the fact that
heating the PC12 liposome solution up to 60 1C (Fig. 4) does not
induce an increase in the intensity of fluorescence emission, rather,
a slight decrease of fluorescence emission is detected.
Intracellular distribution of PC12
Finally, the interactions of PC12 with cellular structures were plasma membrane and visualized by the optical sectioning
studied in order to evaluate the potential of the newly synthesized tangent to the cell surface. After the treatment with PC12
c
2170 New J. Chem., 2013, 37, 2166--2173
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
entrapped within liposomes, LN229 cells undergo morphologi-
1H and 13C NMR spectra were recorded using Bruker 300 and
cal changes, most likely due to the interaction of vesicles with Varian Mercury 300 instruments. J values are given in Hz.
the membrane rather than to the entrapped probe.
ESI-MS spectra were recorded on a Micromass Q-TOF MICRO
spectrometer.
Spectrophotometric experiments were carried out on a Varian
Cary 300 Bio using a cell of 1 cm path length.
Steady-state fluorescence emission spectra were carried out
Conclusion
A new dibromoperylene derivative, representing a fluorescently
labelled lipid analogue, was synthesized. The synthetic procedure on a HORIBA Jobin-Yvon Fluoromax 4 spectrofluorimeter, all
involved the bromination of the perylene bay-area to obtain a more the proper corrections for spectral sensitivity in the near
soluble compound that could be handled more easily, and the infrared region were applied in order to obtain true emission
insertion of a suitable alkyl chain to obtain molecules with the spectra.
proper size and the amphiphilic character required for the inter-
The fluorescence quantum yield (F) was estimated using
action with phospholipid bilayers. The absorption and the fluores- eqn (1) in DMSO by using as reference the integrated emission
cence features of the new compound were investigated in DMSO, in intensity of Ru(bpy)3Cl2 (F = 0.028 in H2O at RT) and of
water and upon its inclusion in DMPC liposomes. UV spectra of the perylene-3,4,10-tetracarboxylic acid tetrapotassium salt (F = 1.00
perylene derivative showed the classical opposite behaviour in in 0.1 M K2CO3, with respect to fluorescein):
organic solvent and in water due to its monomeric form in DMSO
2
IsampleAstdZsample
Ff0
and to the presence of aggregates in water. The emission spectra as
Ff ¼
2
IstdAsampleZstd
a function of temperature (25–60 1C) confirmed this picture.
Finally, the data collected for PC12 in liposomes suggest that
PC12 is embedded in the liposome bilayer where the dye molecules
are most probably in the monomeric form.
where F0f is the absolute quantum yield for the standard; Isample
and Istd are the integrated emission intensities of the sample and
of the standard, respectively; Asample and Astd are the absorbances
of the sample and of the standard that are equal at the used
excitation wavelength (484 nm for Ru(bpy)3Cl2 and 438 nm for
perylene-3,4,10-tetracarboxylic acid tetrapotassium salt), and Zsample
and Zstd are the respective refractive indices of the solvents (1.479 for
DMSO, 1.33 for 0.1 M K2CO3).
Laser Scanning Confocal Microscopy observations were per-
formed by using a Leica TCS SP2 laser scanning confocal micro-
scopy (Leica Microsystems, Mannheim, Germany).
In the treatment of both C6 and LN229 cells with PC12 (in the
free form), no apparent signs of cell suffering or death were
observed, suggesting that this new amphiphilic probe is not toxic
and could be used as ‘‘vital stain’’ in all biological experiments
where cells are not fixed. The different subcellular localization of
free and liposome-entrapped PC12 could provide the basis for its
different uses as a fluorescent lipid probe. In fact, free PC12 was
detectable both in the plasma membrane and intracytoplasmic
organelles, whereas PC12 loaded in liposomes accumulates only on
the plasma membrane of both C6 and LN229 cells. This difference
suggests that PC12 when loaded in liposomes follows the same
Synthesis
destiny of liposomes themselves in the cell cultures. Therefore, in Starting compound 2 (1,7-dibromoperylene-3,4:9,10-tetracarboxylic
the free form it could represent an aspecific stain for lipids of cell dianhydride) was prepared by reaction of 3,4,9,10-perylentetra-
membranes (plasma and organelle membranes), moreover, since carboxylic dianhydride with commercially available bromine,
no significant signal was revealed inside the nuclei, it can be as previously described.14b
particularly useful in multiple fluorescence experiments employing
Together with compound 2, we obtained also the corres-
other dyes for nucleus stains. In the liposome-entrapped form, ponding 1,6-derivative (about 10%). This minor isomer is not
since it follows the same destiny of liposome itself, it could be used separable from the 1,7-isomer at this stage; in the synthetic
to label specific cell subcompartments using liposomes specifically schemes only the main 1,7-isomer is reported.
formulated for targeting defined compartments.
12-(Amino-dodecyl)-carbamic acid tert-butyl ester (3). To a
solution of 505 mg (2.52 mmol) of 1,12-diaminododecane in
5 ml of CH2Cl2 and 5 ml of chloroform at 0 1C 168 mg
(0.77 mmol) of boc-anhydride in dichloromethane was added
dropwise. The reaction was stirred at room temperature for a
Experimental section
Materials and methods
All commercial reagents, exclusion gel Sephadex G-50, phos- week. The residue was taken up with ethyl acetate, washed with
phate buffer PBS tablets, RPE and HPLC grade solvents were brine (10 ml, 3ꢁ), dried over Na2SO4 and evaporated to obtain
purchased from Carlo Erba Reagenti, Fluka and Sigma-Aldrich an oil. The residual oil was subjected to column chromato-
and used without further purification. Dimyristoyl-sn-glycero- graphy using CHCl3/MeOH (90/10) to obtain 153 mg (66%) of
phosphocholine (DMPC) was purchased from Avanti Polar 12-(amino-dodecyl)-carbamic acid tert-butyl ester as a colour-
Lipids (Alabaster, AL). TLCs were run on Merck silica gel less oil. dH (300 MHz; CDCl3) 4.53 (1H, br s, NH-BOC), 3.08
60 F254 plates. Silica gel chromatography was performed by (2H, m, NBOC-CH2), 2.67 (2H, t, J 7, NH2AMMINIC-CH2), 1.43
using Merck silica gel 60 (0.063–0.200 mm). Perylene-3,4,10- (13H, br, CH2- b NAMMINIC, CH2- b NHBOC, (CH3)3-C), 1.31–1.18
tetracarboxylic acid tetrapotassium salt was prepared and (16H, br, -CH2- alkyl chain); dC (300 MHz, CDCl3) 142.5 (CQO),
purified as previously reported.25
d 42.5, 48.9, 34.0, 30.3, 29.8, 29.7, 29.5, 28.6, 27.1 (aliphatic C).
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 2166--2173 2171

View Article Online
NJC
Paper
N,N0-Bis[(amino-dodecyl)-12-carbamic acid tert-butyl ester]- flask by evaporation of CHCl3 solution containing the proper
1,7-dibromoperylene-3,4:9,10-tetracarboxylic diimide (4). To amount of DMPC and PC12 to obtain the desired percentage
257.6 mg of compound 2 (0.47 mmol) and 309 mg of compound mixture (DMPC/PC12 500/1). To the obtained film stored in a
3 (1.03 mmol) anhydrous dioxane (2.5 ml) and anhydrous DMA desiccator overnight under reduced pressure 10 ml of PBS
(2.5 ml) were added. The reaction mixture was then refluxed, buffer solution was added (Aldrich, 10ꢀ2 M pH 7.4) in order
stirred under argon for 6 h. Successively, ice and brine were to obtain a final concentration of 2.5 ꢁ 10ꢀ2 M in DMPC and
added and the solution was stored at 4 1C for a night. Then the 5 ꢁ 10ꢀ5 M in PC12. The dispersion was vortex-mixed and
solution was filtered on Hirsh imbute, washed with water. After then freeze-thawed six times from liquid nitrogen to 313 K.
drying the precipitate in an oven, 450 mg of compound 4 Dispersion was then extruded (10 times) through a 100 nm
was obtained (85% yield). dH (300 MHz; CF3COOD) 9.81 (2H, polycarbonate membrane (Whatman Nuclepore). The extrusion
d, J 8, ar.), 9.16 (2H, s, ar.), 8.94 (2H, d, J 8, ar.), 4.44 (4H, t, procedure was carried out at 307 K, well above the transition
J 8, Nimidic-CH2), 3.35 (4H, t, J 8, NHBOC-CH2), 1.93 (4H, br, temperature of DMPC (297.2 K), using a 10 ml extruder (Lipex
NHBOC-CH2-CH2), 1.72 (18H, br, (CH3)3-C), 1.60–1.37 (40H, br Biomembranes, Vancouver, Canada).
m, aliphatic C); dC (300 MHz, CF3COOD): 165.7 (CQO), 165.7
(CQO), 139.6 (ar.), 134.5 (ar.), 134.3 (ar.), 131.5 (ar.), 129.3 (ar.),
129.1 (ar.), 127.0 (ar.), 122.3 (ar.), 121.8 (ar.), 121.5 (ar.), 100.3
Determination of entrapment efficiency
((CH3)3-C), 42.1, 41.7, 29.3, 29.2, 29.0, 28.6, 27.7, 27.1, 26.8, 25.8 The unentrapped PC12 in extruded DMPC liposomes was
(aliphatic C). MS (ESI) m/z: 1137 (M + Na)+.
separated from the liposomes on a Sephadex G-50s gel
N,N0-Bis(12-aminododecane) 1,7-dibromoperylene-3,4:9,10- column, equilibrated in PBS buffer solution. PC12 concen-
tetracarboxylic diimide (5). 372.1 mg of compound 4 was tration in liposome preparations before and after extrusion
dissolved in dichloromethane (12 ml) and TFA (0.5 ml) was and before and after Sephadex filtration was determined by
added dropwise. The reaction mixture was stirred for 12 hours, measuring the absorbance maximum (at 521 nm) of 3 ml of
at 0 1C. Then, the reaction mixture was poured into ice and PC12/liposomes before extrusion, after extrusion, and after
neutralized carefully with NH3 (solution at 10%) until pH 8. The filtration; liposomes in the samples were disrupted by complete
residue was extracted with dichloromethane or chloroform, evaporation of water and dissolution of the residue in absolute
washed with brine (5 ml, 3ꢁ), dried over Na2SO4 and evaporated ethanol. The percentage of entrapped drug was calculated
to obtain 171.8 mg of the desired product 5 (57.0% yield). dH using the equation:30
(300 MHz; CF3COOD): 9.84 (2H, d, J 8, ar.), 9.18 (2H, s, ar.), 8.96
(2H, d, J 8, ar.), 4.47 (4H, t, br, Nimidic-CH2), 3.37 (4H, br,
NH2AMMINIC-CH2), 1.95 (8H, br, NH2AMMINIC-CH-CH2 and Nimidic
%PC12 = 100 ꢁ (MPaC12 Mlbip)/(MPbC12 Mlaip
)
-
CH-CH2), 1.60–1.30 (32H, br, aliphatic C); dC (300 MHz, CF3COOD): where MaPC12 and MPbC12 are the perylene derivative concen-
165.3 (CQO), 139.6 (ar.), 137.4 (ar.), 134.6 (ar.), 134.4 (ar.), 131.6 tration in liposome dispersion, respectively, before and after
(ar.), 129.3 (ar.), 129.1 (ar.), 127.1 (ar.), 122.4 (ar.), 121.9 (ar.), extrusion and before and after gel filtration and Malip and Mlbip
47.9, 29.3, 29.2, 29.1, 28.7, 27.7, 27.2, 26.8, 25.9 (aliphatic C). are the total lipid concentration before and after extrusion and
MS (ESI) m/z: 915 (M+); 937 (M + Na)+.
before and after gel filtration, respectively.
N,N0-Bis(12-trimethylammonium-dodecane) 1,7-dibromoper-
ylene-3,4:9,10-tetracarboxylic diimide iodide (1). To 84.7 mg of
compound 5 (0.09 mmol) 3 ml of anhydrous 1,4-dioxane was
added. The reaction mixture was stirred at 120 1C to favour the
complete dissolution of compound 5. Then the reaction mixture
was cooled down, CH3I (1 ml) was added and the reaction was
heated to 65 1C for 30 minutes. The solid was separated by
filtration to obtain 113.9 mg of the desired compound (yield
98%). dH (300 MHz; DMSO-d6): 9.35 (2H, d, J 8, ar.), 8.60 (2H, s,
ar.), 8.56 (2H, d, J 8, ar.), 4.03 (4H, t, J 7, Nimidic-CH2), 3.27 (4H, br,
NH2AMMINIC-CH2), 3.03 (18H, s, NH2AMMINIC-CH3), 1.66 (8H, br,
NH2AMMINIC-CH-CH2 and Nimidic-CH-CH2), 1.40–1.20 (32H, br,
aliphatic C); dC (300 MHz, DMSO-d6): 163.0 (CQO), 162.4
(CQO), 137.3 (ar.), 132.7 (ar.), 132.4 (ar.), 130.3 (ar.), 129.2
(ar.), 129.0 (ar.), 127.0 (ar.), 123.6 (ar.), 123.3 (ar.), 120.9 (ar.),
66.1, 52.9, 39.5, 22.7–22.6 (aliphatic C). MS (ESI) m/z: 500 (M2+).
Cell culture
Human (LN229) and murine glioblastoma (C6) lines (kindly
provided by Dr S. Ciafre’, University of Rome ‘‘Tor Vergata’’,
Italy) were grown as a monolayer in DMEM medium supple-
mented with 1% non-essential amino acids, 1% L-glutamine,
100 IU mlꢀ1 penicillin, 100 IU mlꢀ1 streptomycin, and 10%
fetal bovine serum at 37 1C in a 5% CO2 humidified atmo-
sphere in air.
Laser scanning confocal microscopy
Glioblastoma cells were analyzed using laser scanning confocal
microscopy (LSCM) in order to investigate the intracellular
distribution of 1 administered to cells in DMSO or delivered
by liposomes. Cells, grown on 12 mm glass coverslips, were
inoculated with 1 in DMSO or in liposomes, the final concen-
tration of the dye was, in both cases, 8 mM. After 5 and 27 h of
incubation at 37 1C, cells were fixed in 3.7% paraformaldehyde
in PBS, for 10 min at room temperature.
Liposome preparation
The aqueous dispersion of DMPC/PC12 liposomes was prepared
according to the procedure described by Hope et al.29 Briefly, a
film of lipid was prepared on the inside wall of a round-bottom
c
2172 New J. Chem., 2013, 37, 2166--2173
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
S. Borioni, L. Mayol, G. Ortaggi, A. Bianco, J. Amato and
M. Varra, Bioconjugate Chem., 2011, 22, 1309–1319;
(c) L. Rossetti, M. Franceschin, S. Schirripa, A. Bianco,
G. Ortaggi and M. Savino, Bioorg. Med. Chem. Lett., 2005,
15, 413–420; (d) M. Franceschin, Eur. J. Org. Chem., 2009,
2225–2238; (e) E. Micheli, D. D’Ambrosio, M. Franceschin
and M. Savino, Mini–Rev. Med. Chem., 2009, 9, 1622–1632;
( f ) V. Casagrande, E. Salvati, A. Alvino, A. Bianco,
A. Ciammaichella, C. D’Angelo, L. Ginnari-Satriani,
A. M. Serrilli, S. Iachettini, C. Leonetti, S. Neidle, G. Ortaggi,
M. Porru, A. Rizzo, M. Franceschin and A. Biroccio, J. Med.
Chem., 2011, 54, 1140–1156; (g) D. D’Ambrosio, P. Reichenbach,
E. Micheli, A. Alvino, M. Franceschin, M. Savino and J. Linger,
Biochimie, 2012, 94, 854–863.
Acknowledgements
This work was supported by the Italian Ministry of Education,
University and Research (MIUR) Program for Research of
National Interest (PRIN 2009) and Sapienza Universita di Roma.
`
Notes and References
1 O. Meier, V. Oberle and D. Hoekstra, Chem. Phys. Lipids,
2002, 116, 3–18.
2 (a) D. K. Struck and R. E. Pagano, J. Biol. Chem., 1980, 255,
5405–5410; (b) Z. Derzko and K. Jacobson, Biochemistry,
1980, 19, 6050–6057; (c) S. Spiegel, S. Kassis, M. Wilchek
and P. H. Fishman, J. Cell Biol., 1984, 99, 1575–1581.
3 R. D. Klausner and D. E. Wolf, Biochemistry, 1980, 19, 6199–6203.
4 T. Parasassi, G. De Stasio, A. D’Ubaldo and E. Gratton,
Biophys. J., 1990, 57, 1179–1186.
13 H. Liu, Y. Wang, C. Liu, H. Li, B. Gao, L. Zhang, F. Bo, Q. Bai
and B. Xinwu, J. Mater. Chem., 2012, 22, 6176–6181.
14 (a) F. Wu¨rthner, Chem. Commun., 2004, 1564–1579;
(b) M. Franceschin, A. Alvino, G. Ortaggi and A. Bianco,
Tetrahedron Lett., 2004, 45, 9015–9020.
15 (a) C. Kohl, T. Weil, J. Qu and K. Mullen, Chem.–Eur. J.,
2004, 10, 5297–5310; (b) H. Langhals, R. Ismael and
O. Yu¨ru¨k, Tetrahedron, 2000, 56, 5435–5441.
5 N. J. Zuidan and Y. Barenholz, Biochim. Biophys. Acta, 1997,
1329, 211–222.
6 (a) S. Demmig and H. Langhals, Chem. Ber., 1988, 121–225;
(b) W. Herbst and K. Hunger, Industrial Organic Pigments,
ed. Wiley-VCH, Weinheim, 2nd completely revised edn,
1997; (c) H. Zollinger, Color Chemistry, VCH, Weinheim,
3rd edn, 2003; (d) F. Wu¨rthner, Chem. Commun., 2004,
1564–1579.
7 (a) W. Herbst and K. Hunger, Industrial Organic Pigments, ed.
Wiley-VCH, Weinheim, 2nd completely revised edn, 1997;
(b) G. Seybold and G. Wagenblast, Dyes Pigm., 1989, 11, 303–317.
8 (a) M. Sadrai and G. R. Bird, Opt. Commun., 1984, 51–62;
(b) W. E. Ford and P. V. Kamat, J. Phys. Chem., 1987, 91,
6373–6380.
16 G. Seybold and G. Wagenblast, Dyes Pigm., 1989, 11, 303–317.
¨
17 A. Rademacher, S. Markle and H. Langhals, Chem. Ber.,
1982, 115, 2927–2934.
18 A. Alvino, M. Franceschin, C. Cefaro, S. Borioni, G. Ortaggi
and A. Bianco, Tetrahedron, 2007, 63, 7858–7865.
19 F. Ji, L. Zhu, X. Ma, Q. Wang and H. Tian, Tetrahedron Lett.,
2009, 50, 597–600.
20 R. Gvishi, R. Reisfeld and Z. Burshtein, Chem. Phys. Lett.,
1993, 213, 338–344.
9 (a) C. W. Tang, Appl. Phys. Lett., 1986, 48, 183–185;
21 M. Sadrai, W. Hadel, R. R. Saws, S. Husain, K. Krogh-
Jespemn, J. D. Westbrook and G. R. Bird, J. Phys. Chem.,
1992, 96, 7988–7996.
22 (a) S. Yagai, T. Seki, T. Karatsu, A. Kitamura and
F. Wu¨rthner, Angew. Chem., Int. Ed., 2008, 47, 3367–3371;
(b) J. Wang, A. Kulago, W. R. Browne and B. L. Feringa,
J. Am. Chem. Soc., 2010, 132, 4191–4196.
¨
(b) L. Schmidt-Mende, A. Fechtenkotter, K. Mu¨llen,
E. Moons, R. H. Friend and J. D. MacKenzie, Science, 2001,
293, 1119–1122; (c) J. L. Li, F. Dierschke, J. S. Wu,
A. C. Grimsdale and K. Mullen, J. Mater. Chem., 2006, 16,
96–100; (d) Y. Liu, C. Yang, Y. Li, Y. Li, S. Wang, J. Zhuang,
H. Liu, N. Wang, X. He, Y. Li and D. Zhu, Macromolecules,
2005, 38, 716–721; (e) Y. Liu, N. Wang, Y. Li, H. Liu, Y. Li,
J. Xiao, X. Xu, C. Huang, S. Cui and D. Zhu, Macromolecules,
2005, 38, 4880–4887.
¨
23 M. A. Abdalla, J. Bayer, J. O. Radler and K. laus Mu¨llen,
Angew. Chem., Int. Ed., 2004, 43, 3967–3970.
24 K. Nakamaru, Bull. Chem. Soc. Jpn., 1982, 55, 2697–2705.
25 H. Langhals, J. Karolin and L. B.-Å. Johansson, J. Chem. Soc.,
Faraday Trans., 1998, 94, 2919–2922.
10 (a) X. He, H. Liu, Y. Li, S. Wang, N. Wang, J. Xiao, X. Xu and
D. Zhu, Adv. Mater., 2005, 17, 2811–2815; (b) B. Wang and
C. Yu, Angew. Chem., Int. Ed., 2010, 49, 1485–1488; (c) Y. Che,
X. Yang, G. Liu, C. Yu, H. Ji, J. Zuo, J. Zhao and L. Zang, J. Am.
Chem. Soc., 2010, 132, 5743–5750; (d) Y. Che, A. Datar,
X. Yang, T. Naddo, J. Zhao and L. Zang, J. Am. Chem. Soc.,
2007, 129, 6354–6355; (e) Y. Huang, L. Fu, W. Zou, F. Zhang
and Z. Wei, J. Phys. Chem. C, 2011, 115, 10399–10404.
11 F. Wu¨rthner, T. E. Kaiser and C. R. Saha-Mu¨ller, Angew.
Chem., Int. Ed., 2011, 50, 3376–3410.
¨
26 A. Rademacher, S. Markle and H. Langhals, Chem. Ber.,
1982, 115, 2927–2934.
27 H. Langhals and S. Kirner, Eur. J. Org. Chem., 2000, 365–380.
28 S. Dey, A. Efimov and H. Lemmetyinen, Eur. J. Org. Chem.,
2011, 5955–5958.
29 M. J. Hope, R. Nayar, L. D. Mayer and P. R. Cullis, in
Liposome Technology, ed. G. Gregoriadis, Boca Raton, FL,
2nd edn, 1992, vol. 1, pp. 123–139.
¨
12 (a) D. Gorl, X. Zhang and F. Wu¨rthner, Angew. Chem., Int.
30 S. H. Hwang, Y. Maitani, X. R. Qi, K. Takayama and T. Nagai,
Int. J. Pharm., 1999, 179, 85–95.
Ed., 2012, 51, 6328–6348; (b) M. Franceschin, N. Borbone,
G. Oliviero, V. Casagrande, M. Scuotto, T. Coppola,
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 2166--2173 2173