A novel bimodal lipidic contrast agent for cellular labelling and tumour MRI

Article abstract of DOI:10.1039/b910561a

We have synthesized a bimodal lipidie molecule bearing both fluorophore and contrast agent signatures on the same structure in order to create a robust bimodal liposome for both magnetic resonance imaging (MRI) and fluorescence microscopy utility. The dual-modality concept considered in the synthesis of this new paramagnetic and fluorescent lipid is valuable in that anatomical information (MRI) as well as very sensitive localization (ex vivo fluorescence microscopy) of signal, and therefore liposome biodistribution, is obtainable. Bimodal cationic and neutral PEGylated liposomes were formulated using this novel lipid probe and used to label cells in vitro and image human ovarian xenografts in vivo. Tumour signal enhancement was increased by over 6-fold post-administration of the neutral PEGylated liposomes, and was maintained at this level up to the 24 h end-point. Our results showed this lipid to be more effective and sensitive than the single signature paramagnetic lipid Gd·DOTA·DSA at cellular labelling and tumour MRI. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b910561a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A novel bimodal lipidic contrast agent for cellular labelling and tumour MRI†
Nazila Kamaly,a,b Tammy Kalber,b Gavin Kenny,b Jimmy Bell,b Michael Jorgensen*‡a and Andrew Miller*a
Received 29th May 2009, Accepted 29th September 2009
First published as an Advance Article on the web 5th November 2009
DOI: 10.1039/b910561a
We have synthesized a bimodal lipidic molecule bearing both fluorophore and contrast agent signatures
on the same structure in order to create a robust bimodal liposome for both magnetic resonance
imaging (MRI) and fluorescence microscopy utility. The dual-modality concept considered in the
synthesis of this new paramagnetic and fluorescent lipid is valuable in that anatomical information
(MRI) as well as very sensitive localization (ex vivo fluorescence microscopy) of signal, and therefore
liposome biodistribution, is obtainable. Bimodal cationic and neutral PEGylated liposomes were
formulated using this novel lipid probe and used to label cells in vitro and image human ovarian
xenografts in vivo. Tumour signal enhancement was increased by over 6-fold post-administration of the
neutral PEGylated liposomes, and was maintained at this level up to the 24 h end-point. Our results
showed this lipid to be more effective and sensitive than the single signature paramagnetic lipid
Gd·DOTA·DSA at cellular labelling and tumour MRI.
yl)amino]hexanoyl]-sn-glycero-3-phosphoethanolamine (DOPE-
Introduction
NBD).
Multimodal imaging agents have the potential to provide more
To our knowledge, only one type of bimodal lipidic molecule
than one signal from a biological sample and in this way
has been developed thus far that incorporates both a paramagnetic
enhance the visualization of biological processes. A number of
contrast agent and a fluorophore onto a single molecular frame-
multimodal probes with both paramagnetic and fluorescence
work. Li et al. have designed and synthesized a bimodal probe,
signatures have been developed for cellular imaging and use in
which has lipophilicity due to the attachment of a single alkyl
chain, that can incorporate into lipidic membranes.9 This bimodal
agent was used to label low-density lipoprotein (LDL) particles
for the in vivo detection of LDL receptors.
imaging of developmental biology, as MRI and optical techniques
are excellent complementary imaging methods.1–4 Cellular imaging
is an important area, which has facilitated the study of cell
behaviour in living animals; for example, the fate of stem cells has
been tracked and monitored in a serial manner over time.5 The
labelling of cells of interest is usually achieved through the use of
molecular probes or nanoparticles such as liposomes, which are
capable of effective cellular entry.6,7
Fluorescently tagged lipid probes have been used extensively
for the investigation of lipid trafficking and sorting in both
model and real cellular membranes; in particular, fluorescent
lipids have been used to highlight cellular processes such as
membrane fusion.8 In principle, there are two regions on lipids
where a fluorophore may be attached; this is achieved either
through covalent attachment of the fluorescent moiety to the
head group region or attachment to the lipid chain. The most
frequently utilized fluorescent lipids are fluorescent analogues of
dioleoylphosphatidylethanolamine (DOPE), which are DOPE-
Rhodamine and 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-
Previously, we have synthesized a number of paramagnetic
gadolinium-bearing lipids for liposomal cell labelling and vi-
sualization by MRI, with the additional incorporation of a
separate fluorescent lipid in the liposome formulation to obtain
bimodal liposomes.10,11 In order to create a more robust bimodal
imaging liposome for cell labelling and tumour imaging and to
avoid the use of two separate signalling lipids, our aim was
to synthesize the bimodal Gd lipid 6-Rhodamine-sulfonamide-
2-(amido-Gd(III) {1,4,7,10-tetraazacyclododec-1-yl}-acetic acid)
hexa-amido-N,N-dioctadecylacetamide, Gd·DOTA·Rhoda·DSA
1, which also incorporates a fluorescent moiety (Scheme 1). This
contrast agent is valuable for the bimodal imaging of tumours or
labelled cells, since in addition to MRI, fluorescence microscopy
provides further proof of particle location, with both signals co-
localized and arising from the same signaling probe. Utilizing
an orthogonally protected lysine linker, we have conjugated a
rhodamine moiety onto a DOTA-bearing C-18 dialkyl lipid
(Scheme 1) and complexed Gd into the molecule to obtain the
bimodal lipid Gd·DOTA·Rhoda·DSA 1. This lipid was then used
to label IGROV-1 human ovarian carcinoma cells and to image
xenograft tumours in mice.
aImperial College Genetic Therapies Centre, Department of Chemistry,
Flowers Building, Armstrong Road, Imperial College London, London, UK
SW7 2AZ. E-mail: a.miller@imperial.ac.uk; Fax: +44 (0)20 7594 5803;
Tel: +44 (0)20 7594 5869
bMetabolic and Molecular Imaging Group, Imaging Sciences Department,
MRC Clinical Sciences Centre, Imperial College London, Hammersmith
Hospital, London, UK W12 0HS
† Electronic supplementary information (ESI) available: NMR spectra for
compounds 4, 5, 7, 8 and 10; HPLC details and results for compounds 8
and 1. See DOI: 10.1039/b910561a
‡ Contact address for Dr M. Jorgensen: King’s College London, 8th Floor,
Capital House, 42 Weston Street, London SE1 3QD. Fax +44 (0)207 848
8122; Tel: +44 (0)20 7848 8125.
Results and discussion
The bimodal fluorescent and paramagnetic lipid Gd·DOTA·
Rhoda·DSA 1 was synthesized by initial coupling of the lysine
linker Na-Boc-Ne-CBz-L-lysine-N-hydroxysuccinimide ester 3
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Scheme 1 (i) Et3N, CH2Cl2, 40 ◦C, 5 h, 92%, (ii)TFA–CH2Cl2: 1 : 1 v/v, rt, 2.5 h, quantitative, (iii) Et3N, CH2Cl2, 45 ◦C, 12 h, 67% (iv) H2, Pd-C 10%,
1,4-cyclohexadiene, MeOH–H2O: 2 : 1 v/v, 60 ◦C, 3 h, 60%, (v) 1% Et3N, CH2Cl2, rt, 12 h, 95%, (vi) 6H2O·GdCl3, H2O, 90 ◦C, 12 h, quantitative.
with N¢N-distearylamidomethylamine (DSA) 2 in order to
obtain the orthogonally protected lipid 4 (Scheme 1). The
synthesis of lipid 2 has been previously described.10 A facile acidic
cleavage of the tert-butyloxycarbonyl (Boc) protecting group using
TFA yielded amine 5 in good yield and purity. A further amide
coupling was carried out with the mono-carboxylate activated
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-
N-hydroxysuccinimide ester (DOTA·NHS) 6 to obtain the
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carboxybenzyl (CBz) protected compound 7. Deprotection of the
CBz group was carried out with H2 gas and palladium on carbon,
with a major improvement in hydrogenolysis achieved by the
addition of 1,4 cyclohexadiene to the reaction mixture to obtain
DOTA lipid 8. With this precursor lipid in hand, a sulfonamide
coupling was achieved with Rhodamine Lissamine B sulfonyl
chloride 9 in basic conditions to produce the novel bimodal lipid
10 with excellent yield and purity. Finally, DOTA·Rhoda·DSA 10
was complexed with GdCl3 using a stoichiometric amount of the
chelating agent and heating the reaction in pure water (Scheme 1);
quantitative yields were obtained and the final compound was
characterized by ESI-MS and HPLC. The presence of free Gd3+
ions was investigated by using the xylenol orange colorimetric
assay, and no free Gd ions were detected. In addition, the
isotopic peaks of Gd were apparent in the mass spectrum of this
compound, confirming metal chelation with the DOTA head
group. The excitation and emission wavelengths measured for
Gd·DOTA·Rhoda·DSA 1 were found to be 543 and 568 nm,
respectively (MeOH).
2. Cationic bimodal Gd·DOTA·Rhoda·DSA liposomes
Liposomes were formulated with Gd·DOTA·Rhoda·DSA in ad-
dition to the cationic lipid N1-cholesteryloxycarbonyl-3,7-diaza-
1,9-diaminonane (CDAN) and the neutral helper lipid DOPE. Li-
posomes were formulated in water and their size measured by
photon correlation spectroscopy (PCS). Additionally, cryo-TEM
was used to visualize the cationic liposomes (Fig. 1).
1. T1 relaxation analysis of Gd·DOTA·Rhoda·DSA
Fig. 1 (a) Cationic liposomes of approximately 100 nm were formulated
using Gd·DOTA·Rhoda·DSA, CDAN and DOPE. (b) Cryo-TEM image
of liposomes (bar: 100 nm).
The T1 signal enhancement ability of lipid 1 in water was mea-
R
sured and compared with the clinical contrast agent Dotaremꢀ.
Aqueous solutions of Gd·DOTA·Rhoda·DSA were prepared at
the clinically relevant dose of 0.5 mM, and T1 measurements were
obtained using eqn (1):
3. Effect of lipid film hydration medium on T1 relaxation
)
Si = S0(1 - e(-x/T
)
(1)
1
As the novel bimodal lipid synthesized is a T1 reducing agent, we
were interested in assessing the effect of the lipid film hydration
medium on the relaxation efficiency of Gd·DOTA·Rhoda·DSA
1. An identical lipid mixture as above was used to prepare lipid
films, which were then hydrated with either doubly distilled water,
HEPES, PBS, non-supplemented media or supplemented media
(containing serum and antibiotics); the T1 of water was also
measured. These results demonstrated that the type of liquid used
to hydrate the lipid film, and in which the liposomes subsequently
assemble, does indeed affect T1 relaxation, with water being the
most suitable hydration medium, where 74% bulk T1 reduction
is achieved (Fig. 2). The other solutions reduced bulk solvent
T1 to 64% for HEPES, 50% for supplemented media, 26% for
non-supplemented media and 25% for PBS. Based on these
results cationic liposomes were prepared in water for relaxivity
measurements and cell labelling.
A standard T1 saturation recovery method (spin echo sequence)
was used to determine T1 values (according to eqn (1)), where x
is TR (time to repeat), and Si is the measured signal for a given
TR. The T1 measurement results obtained show the novel bimodal
lipid Gd·DOTA·Rhoda·DSA to be an effective T1 signal enhancer
capable of reducing the bulk water T1 by 95%, and to be 16%
R
more effective than Dotaremꢀ (Table 1). Interestingly, this data
showed that the T1 relaxation efficacy of Gd·DOTA·Rhoda·DSA
is two-fold greater than the measured T1 of the single signa-
ture compound Gd·DOTA·DSA shown previously.10 In solution,
Gd·DOTA·Rhoda·DSA is believed to form micelle-like structures,
leading to a slower “tumbling” motion of the paramagnetic head
group in water. This tumbling motion is faster for Gd·DOTA
R
(Dotaremꢀ) as it is a small molecular weight agent. We believe
that the lower T1 value obtained for Gd·DOTA·Rhoda·DSA is
perhaps due to this effect.
Table 1 T1 relaxation values for solutions of Gd·DOTA·Rhoda·DSA,
R
Dotaremꢀ, DOTA·Rhoda·DSA and pure water
Fig. 2 Phantom samples were prepared with Gd·DOTA·Rhoda·DSA
containing cationic liposomes hydrated in H2O, HEPES, supplemented
media (with antibiotics and serum), non-supplemented media or PBS. A
pure water sample (dark grey bar) was also included and T1 values
measured at ambient temperatures at 4.7 T.
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4. MRI visualization of labelled IGROV-1 cells
5. Fluorescence microscopy of labelled IGROV-1 cells
Based on the successful cell uptake results demonstrated through
the use of the cationic lipid CDAN and the neutral helper
lipid DOPE,10 these lipids were also utilised in our cell labelling
vector. A 5 mol% composition of Gd·DOTA·Rhoda·DSA 1 was
used in the liposomes for cell labelling experiments since this
ratio would ensure an adequate and non-excessive amount of
rhodamine fluorescence in the cells (for bimodal fluorescence
and MR imaging). IGROV-1 cells were labelled with the cationic
liposomes and the labelled cells had acquired a pink coloration
under normal light, and appeared as bright pellets with high signal
intensity when imaged by MRI (Fig. 3). The T1 measurement for
the labelled cells was measured to be 1122 21.38 ms compared
to 2825 70.85 ms for the unlabelled cells. The cells labelled
with Gd·DOTA·Rhoda·DSA appeared ~61% more enhanced
compared to control unlabelled cells. Inductively coupled plasma
mass spectrometry (ICP-MS) studies were carried out using cell
lysates post-8 h incubation with the cationic liposomes, to assess
the amount of Gd internalized into the cells more precisely. From
this, we determined that 22.84% of the administered Gd dose was
retained within the IGROV-1 cells. We found that post-incubation,
the cells contained a total mass of 132 ng of Gd, and assuming
a doubling of the IGROV-1 cell population post-seeding, we
estimate that each IGROV-1 cell contained 1.17 ¥ 107 atoms of
Gd.
In order to assess the intracellular uptake of the cationic bimodal
liposomes, microscopy experiments were carried out using this
formulation. Fluorescence microscopy of the fixed IGROV-1
cells revealed uptake of these liposomes to be intracellular,
perinuclear, ubiquitous and dispersed throughout the cytoplasm
(Fig. 4). The successful in vitro cell labelling of IGROV-1 cells and
visualisation by both MRI and fluorescence microscopy revealed
Gd·DOTA·Rhoda·DSA 1 to be an effective bimodal cell labelling
vehicle and results were obtained that co-validated the presence
of the liposomes within cells by both MRI and fluorescence
microscopy.
6. In vivo tumour MRI with Gd·DOTA·Rhoda·DSA
Tumour imaging is valuable in the assessment of a range of
characteristics of solid tumours, whereby tumour characteris-
tics such as viable versus necrotic regions, vascularity, level of
aggressiveness, location and response to therapy can be both
visualised and quantitated with the use of appropriate probes.
A range of bimodal probes have been applied to the imaging
of tumours.12,13 Based on the successful tumour imaging results
using the first generation Gd·DOTA·DSA lipid,10 we were in-
terested in evaluating the use of the novel second generation
bimodal Gd·DOTA·Rhoda·DSA for tumour imaging of IGROV-
1 xenografts in vivo. Neutral bimodal PEGylated liposomes were
prepared using Gd·DOTA·Rhoda·DSA and characterized; r1 and
r2 relaxivities were found to be 3.627
0.183 and 12.935
0.384 mM-1 s-1, respectively. Relaxivity can be defined as the
capability of a contrast agent to enhance bulk solvent T1 or T2
relaxation rate per mM of Gd per second. r1 and r2 relaxivities for
lipid 1 in neutral PEGylated liposomes were found to be higher
than the single signature lipid Gd·DOTA·DSA by 2.8- and 2.5-
fold, respectively (see Table 2). This increase in relaxivity may
result due to the better proximity of the Gd·DOTA headgroup
with the surrounding bulk water due to the attached rhodamine
fluorophore, as it has been shown that charged aromatic probes
tend to locate at the polar surface of membranes, and due to
these charged groups the deep burial of these lipids within the
hydrocarbon region of the bilayer is restricted.14
An IGROV-1 human ovarian xenograft model was used to
test the tumour imaging efficacy of the novel bimodal lipid
Gd·DOTA·Rhoda·DSA. IGROV-1 cells lead to well vascularised
tumours, which become palpable within a few weeks. Once
tumours had grown large enough, baseline MR images of
Fig. 3 (A) T1-weighted MR images of IGROV-1 cells labelled with
Gd·DOTA·Rhoda·DSA liposomes. 1. Cationic Gd·DOTA·Rhoda·DSA
liposomes (1122
21.38 ms); 2. cationic DOTA·Rhoda·DSA control
liposomes (2254 59.01 ms); 3. unlabeled cells (2825 70.85 ms) and
4. cells plus PBS (2780 42.8 ms). (B) Corresponding photo.
Despite the fact that these cationic liposomes contain 25 mol%
less Gd lipid, their MRI cellular labelling efficiency is comparable
to the previously reported value of 68% cellular T1 enhancement
for the single signature Gd·DOTA·DSA lipid (used at 30 mol%).10
This level of signal enhancement demonstrates the effectiveness
of the novel bimodal lipid 1 for cell labelling and visualization by
MRI.
Fig. 4 (a) Bright field image of IGROV-1 cells incubated with cationic liposomes containing Gd·DOTA·Rhoda·DSA (bar represents 20 mm),
(b) corresponding fluorescent image of the labelled cells and (c) enlarged fluorescent imaging of two labelled IGROV-1 cells (¥400).
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Table 2 Comparison of Gd·DOTA·DSA and Gd·DOTA·Rhoda·DSA
conjugated and used as a headgroup, which is a fluorophore with
an extremely high extinction coefficient (up to 93 000 M-1 cm-1)
and forms a stable sulfonamide linkage with the primary amine
of 8. Relaxation properties of this lipid at reducing bulk water T1
were shown to be more effective than the clinical contrast agent
lipids at 4.7 T (19 ◦C ambient bore temperature).
T1/ms 0.5 mM
solution
r1/mM-1 s-1 r2/mM-1 s-1
Gd·DOTA·Rhoda·DSAa
Lipid
—
—
—
—
—
—
205.8 30.8
400.1 15.92
817.5 93.5
R
Dotaremꢀ when measured and compared at an equivalent Gd
atomic content. Both cationic and neutral PEGylated liposomes
were formulated using this lipid with ease, and cryo-TEM showed
the liposomes to have a uniform size and shape distribution,
despite initial concerns that the lipid head group may prove too
large for successful liposome formulation.
Gd·DOTA·DSAb
Lipid
R a
Dotaremꢀ
Gd·DOTA·Rhoda·DSA 3.627 0.183 12.935 0.384 —
Neutral liposome
Gd·DOTA·DSAc
0.926 0.325 5.096 1.8
—
The cationic formulation incorporating Gd·DOTA·
Neutral liposome
Rhoda·DSA for in vitro cell labelling was based on the
a As per Table 1. b See ref. 10. c See ref. 15.
R
commercial liposomal transfection agent Trojeneꢀ, which is
composed of a 1 : 1 mixture of CDAN and DOPE lipids, ensuring
maximal cell uptake. The incorporation of CDAN into the
formulations renders a positive surface charge to the liposome
membrane as CDAN has a cationic polyamino head group.33
Cellular uptake of liposomes is mediated by adsorption of these
nanoparticles onto the cell surface followed by endocytosis.34
Liposomes that contain the fusogenic lipid DOPE in particular,
have been shown to be capable of fusion to both the cellular and
endosomal membranes, with liposomal content released from the
cationic liposomes in cells by the action of anionic lipids initially
located in the cytoplasmic face of the endosome.33,35
For cell populations to be imaged by MRI, they must be
distinguishable from the natural background noise signal. The
markers that are to be used for their labelling should be able to
produce significant enhancement of either T1 (positive, bright) or
T2 (negative, dark) relaxation. Our in vitro MRI and fluorescence
microscopy results demonstrate effective cellular labelling, with
all cells brightly labelled with minimal toxicity (as confirmed by
Trypan blue staining, data not shown). Additionally, in order
to further assess the cell labelling efficiency of the developed
cationic liposomes, we are currently investigating the effects of
cell division and dilution of the labelled cells in vitro in a serial
manner.
We developed neutral particles by including the hydrophilic
polymer amphiphile DSPE-PEG(2000) into the liposomes incor-
porating Gd·DOTA·Rhoda·DSA, in addition to cholesterol and
DOPC lipids. In vivo imaging of xenograft IGROV-1 tumours
showed these liposomes to be trapped within the tumours and tu-
mour T1 signals to be enhanced by up to 65% at 14 h post-injection,
and persist up to the 24 h end point. We believe that the lipophilic-
ity of the rhodamine moiety maybe causative of the higher signal
enhancement at 14 h with the Gd·DOTA·Rhoda·DSA liposomes,
suggesting incorporation in cell membranes.
In line with the EPR effect, the formulated neutral liposomes are
believed to accumulate within the tumour tissues by extravasation
through the disrupted endothelium lining tumour vessels over
time. Their prolonged circulation is a direct result of the PEG
layer, as liposomes bearing a surface decorated with the neutral hy-
drophilic PEG polymer benefit from prolonged circulation times,
with half lives reported from 2–24 h in rodents, and as high as
45 h in humans.36 In addition, surface-grafted PEG liposomes
have reduced uptake by liver cells as the liposomes are not
effectively bound by plasma proteins.37 The utilization of neutral
lipids, in addition to the incorporation of between 5–10% molar
ratios of a PEGylated lipid in the liposome formulation, provides
tumour-bearing mice were obtained pre-injection, after which the
PEGylated bimodal liposomes were injected via the tail vein, and
the mice imaged at 2, 14 and 24 h post-administration intervals.
The measured T1 relaxation values from the tumour areas revealed
an excellent level of tumour signal enhancement of up to ~65%
at 14 h post-injection of the bimodal liposomes, and this signal
enhancement was maintained up to the 24 h end-point (Fig. 5a).
T1-weighted images of axial tumour slices were also obtained and
revealed the images taken at the 14 and 24 h time points to be
brighter than the baseline and 2 h post-injection images (Fig. 5b).
These results are even more encouraging than earlier findings,
where only a 10% signal enhancement was reported at 16 h
for the unimodal Gd·DOTA·DSA lipid, and similar T1-reducing
effects were observed only initially at 24 h post-administration
compared to the observed accelerated tumour accumulation of
Gd·DOTA·Rhoda·DSA at 14 h.15
After 24 h post-administration, the mice were sacrificed, and the
tumours excised, frozen and sectioned for histology. Fluorescence
microscopy of the sectioned tumour slices revealed a high level of
fluorescence signal in the tumour tissue (Fig. 5c,e). These results
validate the MRI data and show the ubiquitous presence of the
Gd·DOTA·Rhoda·DSA lipid in the tumour tissue. It is believed
that these neutral PEGylated liposomes accumulate in tumour
tissue due to the enhanced permeation and retention (EPR) effect.
This describes tumour endothelium vasculature, which, due to its
“leakiness”, allows the penetration of macromolecular particles
through to tumour interstitium.16–18
Cellular labelling to date has mainly involved the use of iron
oxide nanoparticles, as a strong negative signal enhancement
arises due to the superparamagnetic nature of these particles.19–25
Gadolinium-based probes have also been applied to the labelling
of various cell lines and their subsequent in vivo tracking achieved
using MRI.26–30 Cellular imaging is a growing field, for which
novel probes and labels are under continuous development. By
incorporating Gd lipids into the membranes of liposomes or by
encapsulating paramagnetic contrast agents in their aqueous core,
they can be rendered MRI visible and supramolecular systems
with a better control of size obtained.11,31,32 We have designed
and synthesized Gd·DOTA·Rhoda·DSA 1, a novel bimodal
paramagnetic and fluorescent lipid for liposome incorporation, in
vitro cell labelling and in vivo tumour MRI. This lipid incorporated
a fully saturated alkyl chain that bestows a good level of rigidity
and anchorage into the liposome bilayer, and in addition to
using the stable macrocyclic DOTA chelate, rhodamine was also
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Fig. 5 (a) Tumour T1 signal enhancement calculated as the percentage of T1 signal enhancement relative to the baseline (pre-injection) at 4.7 T. (b)
T1-weighted tumour images of Balb/c mice injected with PEGylated bimodal Gd·.DOTA·Rhoda·DSA liposomes (white arrow shows tumour location)
at baseline (pre-injection), 2, 14 and 24 h post-liposome administration. (c) and (d) Fluorescence microscopy images of sectioned tumour (7 mm thick)
post-Gd·DOTA·Rhoda·DSA liposomes injection. The images on the left are red fluorescent images and the images on the right are corresponding bright
field images (¥400, green bar represents 50 mm).
steric stabilization and protection from blood plasma proteins
such as opsonins, and leads to a reduced Kupffer cell uptake.
Cholesterol was incorporated into the formulation since this lipid
has been shown to increase the head group spacing in liposome
formulations and stabilize the resulting bilayer membranes.38
Here, cholesterol presence in the liposome formulation controls
the membrane permeability of both fluid and rigid bilayers by
inducing conformational ordering of the lipid chains. In addition,
cholesterol can reduce serum-induced aggregation as a direct result
of its neutral charge.39
The fluorescence pattern observed from the sectioned tu-
mour tissue post-liposome administration appears to be more
uniform and spread-out throughout the tumour tissue. This
may support the idea that the Gd·DOTA·Rhoda·DSA lipid is
able to incorporate within the cancer cell membranes. How-
ever, appropriate staining and confocal microscopy studies are
necessary to elucidate more information on the precise local-
ization of PEGylated bimodal liposomes formulated with the
bimodal lipid Gd·DOTA·Rhoda·DSA 1. Post mortem analysis
of mice injected with the PEGylated liposomes also revealed a
pink gastrointestinal tract, suggesting hepatobiliary excretion of
these nanoparticles. Future investigations will be carried out to
determine the organ biodistribution and clearance of our novel
in vivo bimodal liposomes. Bimodal liposomes formulated with
Gd·DOTA·Rhoda·DSA will also be used to label various other
cell types such as stem cells. Additionally, the developed cationic
liposomes will be utilised for concurrent nucleic acid delivery and
imaging to cells.
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Merck 0.040 to 0.063 mm, 230 to 400 mesh silica gel. Microscopy
experiments were conducted on a Nikon Eclipse E600 microscope.
MRI experiments were conducted on a 4.7 T Magnex magnet
(Oxford, UK) with a Varian Unity Inova console (Palo Alto, CA,
USA). All procedures on animals were conducted in accordance
with UK Home Office regulations and the Guidance for the
Operation of Animals (Scientific Procedures) Act (1986).
Conclusion
A
novel bimodal fluorescent and paramagnetic lipid,
Gd·DOTA·Rhoda·DSA was synthesized and utilized for
the in vitro cell labelling of IGROV-1 human ovarian cells, and
was shown to be an effective tumour T1 signal enhancer. These
findings were co-validated successfully in a bimodal manner
using both fluorescence microscopy of histological tissue and
real-time MRI of tumour-bearing mice. Our results confirm
the MRI advantages of the novel fluorescent and paramagnetic
lipid Gd·DOTA·Rhoda·DSA in comparison to the single
signature lipid Gd·DOTA·DSA, where in addition to indisputable
co-localization of fluorophore signal and MRI contrast agent
signature, a greater degree of relaxivity efficacy is also achieved.
This combination allows for an accurate assessment of tumour
signal enhancement using Gd contrast in real-time, in addition to
facilitating in vivo fluorescence imaging or histological analysis of
tumour tissue.
Synthesis of Gd lipid
6-Benzyloxycarbonylamino-2-(tert-butoxycarbonylamido) hexa-
amido-N,N-dioctadecylacetamide
4. Boc-Lys-(CBz)-succini-
mide ester (5.0 g, 10.471 mmol) and N,N-distearyl-
3
amidomethylamine (DSA), 2 (6.96 g, 12.041 mmol) were added
to an air evacuated flask and dissolved in anhydrous CH2Cl2
(200 mL). The solution was briefly stirred, followed by the slow
addition of Et3N (4.38 mL, 31.413 mmol, 3 eq.). The reaction was
stirred for a further 5 h at 40 ◦C until TLC indicated completion.
The reaction was stopped and the organic layer washed with
water (¥ 3, 50 mL) and the aqueous layer back extracted with
2 : 1 CH2Cl2 : MeOH (¥ 3, 50 mL). The combined organic layers
were subsequently washed with 7% citric acid (¥ 3, 50 mL) and
the aqueous layer extracted further with CH2Cl2 (¥ 3, 50 mL).
The final combined organic layers were collected and dried over
MgSO4, filtered and reduced in vacuo to yield a hygroscopic white
solid (9.05 g, 92%). Rf [CH2Cl2 : MeOH : H2O: 34.5 : 9 : 1 v/v] 0.56;
Experimental methods and materials
Materials and general procedures
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(pol-
yethylene glycol)-2000 (DSPE-PEG2000) were purchased from
Avanti Polar Lipids Inc. (Alabaster, AL, USA). The
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-
N-hydroxysuccinimide ester (DOTA·NHS ester) was purchased
from Macrocyclics (USA), and all other chemicals were of
analytical grade or the best grade available and purchased from
Sigma-Aldrich (UK).
n
max(nujol)/cm-1 3322.75, 1721.16, 1643.05, 1246.75, 1171.54;
dH(400 MHz; CDCl3) 7.34 (5H, m, J 4.4 Hz, aromatic), 5.08
meta
(2H, s, PhCH2-), 4.93 (1H, s, br, Boc-NHCH-), 4.03 (2H, d,
-NCOCH2NHCO-), 3.31 (2H, m, -CH2NHCOOBn), 3.16 (4H,
m, 2 ¥ CH2CH2NCO-), 1.84 (2H, s, br, (BocNH)CHCH2-), 1.52
(6H, m, 2 ¥ -CH2CH2NCO- and -CH2CH2NHCOOBn), 1.43
(9H, s, -OC(CH3)3), 1.32 (4H, 2 ¥ (CH3CH2-)) 1.26 (60H, s,
30 ¥ chain CH2’s), 0.89 (6H, t, 2 ¥ CH3CH2CH2-); dC(100 MHz;
CDCl3) 171.89 (-NHCOCH(NHBoc)), 167.01 (-NCOCH2NH-),
156.54 (PhCH2OCO), 155.59 (-OCOt-Bu), 136.63 (-C1 aromatic),
128.47 (-C3, C5 aromatic), 128.10 (-C4 aromatic), 128.01 (-
C2, C6 aromatic), 79.94 (-OC(CH3)3), 66.57 (PhCH2-), 54.31
(Boc-NHCH-), 46.26 (-CH2N-), 41.15 (-NCOCH2NHCO-),
40.55 (CBz-NHCH2-), 32.58 (-BocNHCHCH2-), 31.92 (-
BocNHCHCH2CH2CH2-), 29.71-29.36 (12 chain CH2’s), 28.72
(-CH2CH2N-), 28.33 (-OC(CH3)3), 27.61 (-CH2CH2CH2N-),
22.69 (CH3CH2-), 22.50 (BocNHCHCH2CH2-), 14.11 (CH3CH2-
). HPLC tR = 36.28 min, column C-4 peptide, gradient mix:
0.0 min [100% A], 15–25.0 min [100% B], 25.1–45.0 min [100% C],
45.1–55.0 min [100% A]; flow: 1 mL min-1; m/z HRMS (FAB+)
941.8031 (MH+, 100%, C57H104N4O6 requires 940.7956).
1H NMR spectra were recorded on a 400 MHz Bruker Advance
400 spectrometer. Chemical shifts are reported in parts per million
(ppm) downfield from TMS, using residual chloroform (7.27
ppm) as an internal standard. Data are reported as follows:
chemical shift, s = singlet, br = broad singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, coupling constants J
are given in hertz (Hz). 13C NMR spectra were recorded on
a 100 MHz Bruker Advance 400 spectrometer and chemical
shifts are reported in parts per million (ppm) downfield from
TMS, using the middle resonance of CDCl3 (77.0 ppm) as an
internal standard. Infrared (IR) spectra were recorded on a JASCO
FT/IR-620 infrared spectrophotometer; absorptions are recorded
in wavenumbers (nmax in cm-1). Analytical HPLC was conducted
on a Hitachi-LaChrom L-7150 pump system equipped with a
Polymer Laboratories PL-ELS 1000 evaporative light scattering
detector. HPLC gradient mixes assigned as follows: gradient mix
A = H2O–0.1%TFA; mix B = MeCN–0.1%TFA; mix C = MeOH,
all compounds obtained with purity of greater than 95%. Mass
spectra were performed using VG-070B, Joel SX-102 or Bruker
Esquire 3000 ESI instruments. Melting points were determined
on a Stuart Scientific SMP3 apparatus and are reported without
correction. Reactions with air sensitive material were carried out
by standard syringe techniques. CH2Cl2 was distilled over P2O5.
Thin layer chromatographic (TLC) analyses were performed on
Merck 0.2 mm aluminium-backed silica gel 60 F254 plates and
components werevisualized byillumination with UV, an Ultrospec
III (deuterium lamp at 300 nm) was used to visualize the UV
absorbance. Flash column chromatography was performed using
6-Benzyloxycarbonylamino-2-(amino) hexa-amido-N,N-dioc-
tadecylacetamide 5. The Boc-protected lipid 4 (288.0 mg,
0.306 mmol) was added to a round-bottom flask and to this
was added a 1 : 1 TFA : CH2Cl2 (20 mL, v/v) solution. The
reaction was stirred at room temperature for 2 h until TLC
indicated completion. The solvents were removed in vacuo
and the product freeze-dried from water to yield a white
hygroscopic solid (257.09 mg, quantitative, mp = 47–50 ◦C).
Rf [(CH2Cl2 : MeOH : H2O: 34.5 : 9 : 1) : CH2Cl2: 1 : 1 v/v] 0.39;
n
max(nujol)/cm-1 3848.26, 1676.8, 1203.36, 722.21; dH(400 MHz;
CDCl3) 7.99 (1H, s, br, -NH-) 7.33 (5H, m, J 6.4 Hz,
ortho
aromatic), 5.06 (2H, s, PhCH2-), 4.17 (2H, d, -NCOCH2-), 3.96
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(1H, d, NH2CH-), 3.25 (2H, m, -CH2NHCOOBn), 3.15 (4H,
m, 2 ¥ CH2CH2NCO-)), 1.89 (2H, s, br, NH2CHCH2-), 1.47
(8H, m, 2 ¥ (CH3CH2-) and -CH2CH2NCO-) 1.26 (60H, s, 30 ¥
chain CH2’s), 0.88 (6H, t, 2 ¥ CH3CH2-); dC(100 MHz; CDCl3)
169.13 (-NHCOCH-NH2), 167.53 (-NCOCH2NH-), 157.48
(PhCH2OCO), 136.56 (-C1 aromatic), 128.68 (-C3, C5 aromatic),
128.29 (-C4 aromatic), 128.05 (-C2, C6 aromatic), 67.08 (PhCH2-),
53.56 (NH2CH-), 47.57 (2 ¥ -CH2N-), 46.90 (-NCOCH2NHCO-),
41.30 (CBz-NHCH2-), 39.94 (-NCOCH2NHCO-), 32.08
(NH2CHCH2-), 30.76 (NH2CHCH2CH2CH2-), 29.87-29.52 (12
chain CH2’s), 29.18 (-CH2CH2N-), 28.72 (-CH2CH2CH2N-),
22.84 (CH3CH2-), 21.40 (NH2CHCH2CH2-), 14.25 (CH3CH2-).
HPLC tR = 27.98 min, column C-4 peptide, gradient mix: 0.0 min
[100% A], 15–25.0 min [100% B], 25.1–45.0 min [100% C],
45.1–55.0 min [100% A]; flow: 1 mL min-1; m/z HRMS (FAB+)
841.7529 (MH+, 100%, C52H96N4O4 requires 840.7432.
from cyclohexane to yield a white fluffy powder (67.0 mg, 85%).
Rf [CH2Cl2 : MeOH : H2O: 34.5 : 9 : 1] 0.12;
n
max(nujol)/cm-1
2360.44, 1698.98, 1650.28, 1558.20, 1540.85; dH (400 MHz;
MeOD : CDCl3 3 : 1) 4.67 (1H, s, -CH-), 4.42 (-NCOCH2-),
4.04 (2H, d, (-HNCOCH2N-), 3.55 (4H, m, -CH2N-), 3.30-2.39
(16H, m, 8 ¥ macrocycle CH2’s), 2.33 (6H, m, HOOCCH2N-),
1.88-1.40 (8H, m, br, -CHCH2-, 2 ¥ -CH2CH2N-, NH2CH2CH2-),
1.25 (60H, s, chain CH2’s), 0.89 (6H, t, 2 ¥ CH3); dC(100 MHz;
MeOD : CDCl3
3 : 1)
175.90-175.34
(COOH),
172.95
(-CHNHCO-), 168.36 (-NCOCH2-), 59.01 (HOOCCH2N-),
52.34 (-NHCOCH2N-), 51.50 (-CH-), 50.01 (macrocyle CH2’s),
49.10 (-NCOCH2NHCO-), 47.14 (-CH2N-),41.74 (NH2CH2-),
32.93 (-CHCH2), 30.65-30.34 (chain CH2’s), 23.61 (CH3CH2-),
14.44 (CH3CH2-). HPLC tR = 36.20 min, column C-4 peptide,
gradient mix: 0.0 min [100% A], 15–25.0 min [100% B], 25.1–
45.0 min [100% C], 45.1–55.00 min [100% A]; flow: 1 mL min-1;
m/z HRMS (EI) 1093.8951 (MH+, 100%, C60H116N8O9 requires
1092.8865).
6-Benzyloxycarbonylamino-2-(amido-{1,4,7,10-tetraazacyclo-
dodec-1-yl}-acetic acid) hexa-amido-N,N-dioctadecylacetamide, 7.
Amine 5 (84.44 mg, 0.100 mmol) and DOTA-NHS ester 6
(100 mg, 0.120 mmol) were added to an air evaporated flask
fitted with a reflux condenser. Anhydrous CH2Cl2 (20 mL) was
added and the reaction stirred briefly at room temperature. To
this was added Et3N (41.81 mL, 0.300 mmol, 3 eq.) and the
reaction stirred at 45 ◦C under reflux for an overnight period. The
solvent was evaporated in vacuo to yield a white hygroscopic solid
(64.98 mg, 53%). Rf [CH2Cl2 : MeOH : H2O: 34.5 : 9 : 1 v/v] 0.26;
6-Rhodamine-sulfonamide-2-(amido-{1,4,7,10-tetraazacyclod-
odec-1-yl}-acetic acid) hexa-amido-N,N-dioctadecylacetamide 10.
Compound 8 (143.1 mg, 0.131 mmol) and Lissamine Rhodamine
B sulfonyl chloride 9 (75.63 mg, 0.131 mmol) were added to
an air evacuated flask to which was added anhydrous CH2Cl2
(100 mL). Then Et3N (1% of solvent, v/v) was added slowly
and the reaction stirred under an atmosphere of N2 at room
temperature for 12 h. The solvent was removed in vacuo and the
crude product purified by flash column chromatography (eluted
with (CH2Cl2 : MeOH : H2O: 34.5 : 9 : 1) : CH2Cl2: 9 : 1 v/v). The
solvents were removed in vacuo and the product was freeze-
dried from cyclohexane to yield a fluffy purple solid (175.6 mg,
82%, decomposition = 255–260 ◦C). Rf [CH2Cl2 : MeOH: H2O:
34.5 : 9 : 1 v/v] 0.31; nmax(nujol)/cm-1 3853.08, 3748.94, 3629.37,
2854.13, 2360.44, 2339.23, 1992.11, 1868.68, 1747.19, 1716.34,
1683.55, 1590.99, 1419.35, 1274.72, 1180.22, 1072.23, 1043.30,
719.32, 669.18; dH(400 MHz; MeOD : CDCl3 : AcOD: 3 : 1 : 1)
n
max(nujol)/cm-1 3741.23, 3681.44, 3621.66, 2360.44, 2333.45,
1953.54, 1835.9, 1743.33, 1691.27, 1646.91, 1513.85, 1481.06;
dH(400 MHz; CDCl3) 7.32 (5H, m, J 4.0 Hz, aromatic),
meta
5.07 (2H, m, PhCH2-), 4.77-2.37 (33H, m, -CH-, -NCOCH2NH-,
3 ¥ HOOCCH2N-, -NHCOCH2N-, 2 ¥ -CH2N-, CBzNHCH2-,
8 ¥ -macrocycle CH2’s), 2.02 (2H, m, br, -CHCH2-), 1.91-1.38
(10H, m, 2 ¥ -CH2CH2N-, CBzNHCH2CH2- and 2 ¥ CH3CH2-),
1.26 (60H, s, chain CH2’s), 0.89 (6H, t, 2 ¥ CH3); dC(100 MHz;
MeOD: CDCl3 1 : 3) 178.61 (COOH), 175.08 (-HNCOCH-),
167.83 (-CHNHCOCH2N-), 162.03 (-CH2NCOCH2-) 157.27
(PhCH2OCO-), 136.48 (-C1 aromatic), 128.29 (-C3, C5 aromatic),
127.84 (-C4 aromatic), 127.71 (-C2, C6 aromatic), 66.39 (PhCH2),
58.67 (HOOCCH2N-), 52.94 (-NHCOCH2N-), 51.42 (-CH-),
50.97-50.08 (macrocyle CH2’s), 49.10 (-NCOCH2NHCO-), 47.14
(-CH2N-), 46.36 (CBzNHCH2-), 40.94 (CBzNHCH2CH2-), 31.71
(-CHCH2), 29.49-26.67 (chain CH2’s), 22.45 (CH3CH2-), 13.74
(CH3CH2-). HPLC; tR = 29.33 min, column C-4 peptide, gradient
mix: 0.0 min [100% A], 15–25.0 min [100% B], 25.1–45.0 min [100%
C], 45.1–55.00 min [100% A]; flow: 1 mL min-1; m/z (EI) 1227.80
(MH+, 100%, C68H122N8O11 requires 1226.9233).
=
=
8.71 (1H, s, SO2C CH-), 8.11 (1H, d, SO2CCH CH-), 7.73
(1H, s, -NH-), 7.33 (1H, d, xanthenyl, aromatic), 7.17 (1H,
d, xanthenyl), 6.97 (1H, d, xanthenyl, aromatic), 6.95 (1H, d,
xanthenyl, aromatic), 6.89 (1H, s, br, xanthenyl), 6.87 (1H, s, br,
xanthenyl), 4.56 (1H, s, -CH-), 4.18 (2H, d, (-HNCOCH2N-),
3.67 (16H, 2 ¥ -NCH2CH3, 3 ¥ HOOCCH2N-, -NHCOCH2N-,
2 ¥ -CH2N-, SO2NHCH2-), 3.35-3.12 (16H, m, 8 ¥ macrocycle
CH2’s), 2.92 (6H, m, HOOCCH2N-), 2.39–1.40 (4H, m, br, 2 ¥
-CH2CH2N-), 1.59 (4H, 2 ¥ -N+CH2CH3), 1.33 (78H, m, chain
CH2’s and -CHCH2CH2-, 2 ¥ CH3CH2N-), 2 ¥ CH3CH2N+,
2 ¥ CH3CH2CH2-), 0.89 (6H, t, 2 ¥ CH3); dC(100 MHz;
MeOD : CDCl3 : AcOD: 3 : 1 : 1) 174.74 (COOH), 155.68 (-COC-),
144.54 (-CN(CH2CH3)2), 132.62 (xanthenyl), 130.09 (aro-
matic), 127.31 (aromatic), 125.72 (aromatic), 113.56 (xan-
6-Amino-2-(amido-{1,4,7,10-tetraazacyclododec-1-yl}-acetic
acid) hexa-amido-N,N-dioctadecylacetamide 8. The CBz-
protected lipid 7 (100 mg, 0.0724 mmol) was added to a two
necked round-bottom flask equipped with a reflux condenser and
stirrer bar. To this was added a 2 : 1 MeOH : H2O solution (40 mL,
v/v) followed by the addition of 1,4-cyclohexadiene (212.3 mL,
2.27 mmol). The reaction was stirred and 10% Pd-C (10 mg)
was added, whilst ensuring the catalyst was fully dispersed in
solution. The air was displaced with H2 gas and the reaction
stirred at 60 ◦C under reflux and a stream of H2 gas for 12 h. The
reaction was stopped and the solution filtered through Celite.
The solvents were removed in vacuo and the product lyophilized
=
thenyl, aromatic), 113.56 (xanthenyl), 96.69 (-CH COC-), 50.01
(macrocyle CH2’s), 47.73 (-NCOCH2NHCO-), 46.64 (-CH2N-),
32.81 (NH2CH2-), 30.22 (-CHCH2), 30.55 (chain CH2’s),
23.50 (CH3CH2-), 14.49 (CH3CH2-), 12.85 (-NCH2CH3) 9.19
(-N+CH2CH3). HPLC tR = 36.20 min, column C-4 peptide,
gradient mix: 0.0 min [100% A], 15–25.0 min [100% B], 25.1–
45.0 min [100% C], 45.1–55.00 min [100% A]; flow: 1 mL min-1;
m/z HRMS (EI) 1634.2624 (MH+, 100%, C87H144N10O15S2 requires
1633.0254).
208 | Org. Biomol. Chem., 2010, 8, 201–211
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and in vivo liposome formulations were prepared and diluted in
water in order to obtain five different gadolinium concentrations
(in vitro liposomes: 0.340–0.042 [Gd] mM, in vivo liposomes:
1.577–0.197 [Gd] mM) in 200 mL of distilled water, and the molar
relaxivities r1 and r2 (mM-1 s-1), which define the slope of the linear
regression generated from a graph of 1/T1 or 1/T2 vs. the atomic
concentration of Gd, were determined. T1 values were obtained
using saturation recovery experiments performed with a standard
spin-echo sequence and a 2 mm single slice acquisition (TR = 50,
100, 200, 300, 500, 700, 1200, 3000, 5000, 7000 ms, TE = 11.19 ms),
number of signal averages: 10, FOV: 100 ¥ 50 mm2, collected into
a matrix of 256 ¥ 128. For T2 measurements, the following TE
values were used: 11.19, 30, 60 and 100 ms, and the TR was set to
3000 ms.
6-Rhodamine-sulfonamide-2-(amido-Gd(III)-{1,4,7,10-tetraaza-
cyclododec-1-yl}-acetic acid) hexa-amido-N,N-dioctadecyl-
acetamide 1. The fluorescent lipid 10 (20.02 mg, 0.0539 mmol)
was added to a round-bottom flask and stirred in pure distilled
water (80 mL). To this was added a stoichiometric amount of
GdCl3·6H2O (88 mg, 0.0539 mmol) and the reaction stirred
at 90 ◦C for 12 h. The water was freeze-dried to yield a dark
purple solid (93 mg, 96%, decomposition. = 327–330 ◦C). Rf
[CH2Cl2 : MeOH : H2O: 34.5 : 9 : 1 v/v] 0.20; nmax(nujol)/cm-1:
3748.94, 3673.73, 2854.13, 2339.23, 1920.75, 1868.68, 1772.26,
1683.55, 1 573.63, 1419.35, 1396.21, 1274.72, 1178.29, 1072.23;
HPLC tR = 34.19 min, column C-4 peptide, gradient mix:
0.0 min [100% A], 15–25.0 min [100% B], 25.1–45.0 min [100%
C], 45.1–55.00 min [100% A]; flow 1: mL min-1. lex; 545 nm
and lem; 568 nm (MeOH). m/z MS (EI) 1809.50 (MNa+, 100%,
C87H141GdN10O15S2 requires 1787.926).
Cell labelling (microscopy) with Gd·DOTA·Rhoda·DSA liposomes
Cationic liposomes were formulated in water as per
the liposome preparation method described above using
Gd·DOTA·Rhoda·DSA/CDAN/DOPE: 5/50/45 mol% (total
lipid concentration of 1.2 mg mL-1). The size of the liposomes was
assessed using PCS and was found to be on average ~200 nm. At
24 h prior to cell labelling experiments, adherent IGROV-1 human
ovarian carcinoma cells (1.25 ¥ 105 in a 6-welled plate) were grown
on cover slips in Dulbecco’s-Modified Eagle culture medium
(DMEM) supplemented with 10% fetal calf serum (FCS) and
Xylenol orange test
The presence of free gadolinium ions in solutions containing
the Gd-incorporated compound was determined using Xylenol
orange as described previously.10
Liposome preparations
All lipids were stored as stock solutions in anhydrous organic
solvents (CHCl3, MeOH or a mixture of both), at -20 C under
◦
◦
1% penicillin–streptomycin (Sigma-Aldrich) in a wet (37 C) 5%
argon. Liposomes were made with a range of defined molar
ratios of individual lipids to give a predetermined total lipid
concentration of 1.2 mg mL-1 for in vitro and 15 mg mL-1 in for in
vivo experiments. The in vitro cell labelling formulation consisted
of: Gd·DOTA·Rhoda·DSA/CDAN/DOPE: 5/50/45 mol% (hy-
drated in water) and the in vivo tumour imaging formulation con-
sisted of Gd·DOTA·Rhoda·DSA/DOPC/Cholesterol/DSPE-
PEG2000: 30/33/30/7 mol% (hydrated in HEPES (20 mM, NaCl
135 mM, pH 6.5)). Appropriate volumes of each lipid stock
were placed in a round-bottom flask (typically 5 mL) containing
distilled CHCl3 (500 mL) and stirred to ensure thorough mixing
of the lipids. The solvent was slowly removed in vacuo to ensure
production of an even lipid film. The film was re-hydrated with
buffer at a defined volume. The resulting solution was sonicated
for 30 min (at 30 ◦C) in order to form liposomes of appropriate size.
Liposomes were sized by photon light scattering using a Coulter
Delta N4 PCS plus 440SX photon correlation spectrometer (PCS).
In each experiment the liposome suspensions (20 mL) were diluted
with PBS to a final volume of 200 mL. The mean average diameter
and the polydispersity index of the vesicles were determined at
25 ◦C from the result of at least three reliable readings. The cationic
in vitro liposomes were measured to be 222.27 11.2 nm with a
polydispersity index (PI) of 0.20. The PEGylated neutral in vivo
liposomes were 89.24 8.2 nm with a polydispersity index of 0.48.
CO2/90% air atmosphere. Cells were grown until 80% confluent.
The media was then removed and the cells washed with PBS. The
bimodal liposomes were then added to the cells (150 mL of cationic
liposomes, total lipid concentration of 1.2 mg mL-1) and allowed to
incubate for 5 h. After liposome uptake, the media was removed
and the cells were washed with PBS (¥ 2), heparin (¥ 1 (20 mg
mL-1), in order to remove unbound liposomes from cell surface),
PBS (¥ 2), paraformaldehyde (PFA, ¥ 1 (20 min at 37 ◦C)), PBS (¥
2), glycine (¥ 1 (20 mM, 20 min at 37 ◦C)), and finally PBS (¥ 2).
The slides were removed from the wells and fixed using Vectashield
mounting medium (Vector Laboratories, CA, USA). Microscopy
images were obtained on an Olympus 251 microscope.
MRI visualization of IGROV-1 cells labelled with
Gd·DOTA·Rhoda·DSA liposomes
24 h prior to liposome uptake, adherent IGROV-1 cells were
grown in DMEM with 10% FCS and 1% penicillin–streptomycin
(Sigma-Aldrich), in a 25 cm3 culture flask (5 ¥ 105 cells per well,
5 mL of growth medium) and a wet (37 ◦C) 5% CO2/90% air
atmosphere. The cells were grown until 80% confluent, and the
media was then removed and replaced with fresh media. The
cationic liposomes and controls (utilizing the non-Gd precursor
lipid DOTA·Rhoda·DSA 10 instead) were then added to the flask,
swirled to ensure even dispersion, and incubated in a wet (37 ◦C)
5% CO2/90% air atmosphere for 8 h (150 mL for each, with
Gd·DOTA·Rhoda·DSA liposomes containing a total of 578 ng
of Gd per flask). After liposome uptake, the cells were washed
with PBS (2 ¥ 2 mL) and then treated with trypsin-EDTA (500 mL
trypsin, 1mM EDTA) for 2 min at 37 ◦C. DMEM (complete,
3 mL) was added to neutralise the trypsin. The cells were washed
with PBS and then centrifuged in PBS buffer (1 mL), the solution
MRI analysis of Gd·DOTA·Rhoda·DSA 1
For T1 analysis, Gd·DOTA·Rhoda·DSA 1, DOTA·Rhoda·DSA
R
10 and Dotaremꢀ were added to water to give a final concen-
tration of 0.5 mM. The solutions (200 mL) were transferred to
eppendorf tubes, placed in a quadrature 1H coil and T1 relaxation
values measured on a 4.7 T Varian MR scanner at 19 ◦C ambient
bore temperature. For relaxivity measurements, both the in vitro
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Org. Biomol. Chem., 2010, 8, 201–211 | 209
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was decanted and the cells reconstituted in PBS by vortexing and
spun (1500 rpm for 5 min) into pellets in eppendorf tubes filled
with 200 mL PBS. The tubes were imaged whilst placed in a plastic
sample holder, and T1 values were measured on a 4.7 T Varian
Histology experiments
Following MRI, the animals were sacrificed and the tumours were
excised. The tumours were frozen on liquid nitrogen, embedded in
OCT (VWR) embedding compound, 7 mm thick sections cut and
mounted on microscopy slides.
◦
MR scanner at 19 C ambient bore temperature. T1 values were
obtained from saturation recovery experiments performed with a
standard spin-echo sequence and a 10 mm single slice acquisition
in the sagittal plane (TR = 50, 100, ms, TE = 15 ms), 4 signal
averages, FOV: 100 ¥ 50 mm2, collected into a matrix of 256 ¥ 128.
Post-MRI assessment, the cell pellets were vortexed and diluted up
to 5 mL with 2% nitric acid to give a total dilution of 1 in 25, and
the concentration of Gd determined using a Varian VISTA PRO
ICP-MS instrument. All data were corrected for these dilutions,
and a blank 2% nitric acid sample was also analyzed.
MRI data analysis
A region of interest (ROI) was drawn around the samples
containing the contrast agents or around the whole tumour in
each MR image using Image J software (U.S. National Institutes
of Health, Maryland, USA). Mean signal intensities of the ROIs
at different TR values were transferred to Graphpad Prism (San
Diego, California, USA) and T1 values calculated. Mean T1 values
for each sample or tumour were derived and a two-tailed unpaired
t-test assuming equal variances performed at each time point
to determine significant difference, with a 5% level of statistical
significance. The percentage change in T1 from pre-treatment was
also calculated for each tumour-bearing animal.
Effect of hydration solution on T1 relaxation
Cationic liposomes (as above) used to label cells were also prepared
to a total concentration of 1.2 mg mL-1 using various hydration
solutions (doubly distilled water, PBS, HEPES, supplemented
or non-supplemented DMEM) as described in the Liposome
Preparations section. For each liposome, 200 mL was placed in
an eppendorf tube and the samples were placed in a plastic
sample holder. T1 measurements at 4.7 T and 19 ◦C ambient
bore temperature were used. Saturation recovery experiments were
performed with a standard spin-echo sequence and a 10 mm single
slice acquisition in the sagittal plane (TR = 50, 100 ms, TE =
15 ms), 4 signal averages, FOV; 100 ¥ 50 mm2, collected into a
matrix of 256 ¥ 128.
Acknowledgements
Funding for Nazila Kamaly was provided by the GTC and
MRC. We thank Dr Tillmann Pape (Imperial College London) for
cryo-TEM imaging and the Natural History Museum (London)
for ICP-MS measurements.
Notes and references
Cryo-TEM of in vivo neutral PEGylated bimodal liposomes
1 M. M. Hu¨ber, A. B. Staubli, K. Kustedjo, M. H. B. Gray, J. Shih, S. E.
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