Reduction-sensitive liposomes from a multifunctional lipid conjugate and natural phospholipids: Reduction and release kinetics and cellular uptake

Article abstract of DOI:10.1021/la201160y

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin m

Full text of DOI:10.1021/la201160y

ARTICLE
pubs.acs.org/Langmuir
Reduction-Sensitive Liposomes from a Multifunctional Lipid
Conjugate and Natural Phospholipids: Reduction and Release
Kinetics and Cellular Uptake
Bj€orn Goldenbogen,† Nicolai Brodersen,‡ Andrea Gramatica,† Martin Loew,† J€urgen Liebscher,‡
Andreas Herrmann,† Holger Egger,*,§ Bastian Budde,§ and Anna Arbuzova*,†
†Institute of Biology/Molecular Biophysics, Humboldt-University Berlin, Invalidenstrasse 42, 10115 Berlin, Germany,
‡Institute of Chemistry, Humboldt-University Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany,
§Bayer Technology Services GmbH, Process Technology, Building E41, 51368 Leverkusen, Germany
S Supporting Information
b
ABSTRACT: The development of targeted and triggerable
delivery systems is of high relevance for anticancer therapies.
We report here on reduction-sensitive liposomes composed of a
novel multifunctional lipidlike conjugate, containing a disulfide
bond and a biotin moiety, and natural phospholipids. The
incorporation of the disulfide conjugate into vesicles and the
kinetics of their reduction were studied using dansyl-labeled
conjugate 1 in using the dansyl fluorescence environmen-
tal sensitivity and the F€orster resonance energy transfer from
dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP),
dithiothreitol (DTT), L-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the
membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein
release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from
reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere
with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell
line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes
functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed,
implying the potential of using our system for active targeting and delivery.
’ INTRODUCTION
conjugates of poly(ethylene glycol) and phospholipids8,12ꢀ14 or
nucleoside-lipid based carriers,15 reduction-sensitive disulfide
copolymers,16ꢀ18 and redox-triggered liposomes built from a
quinone-lipid conjugate were reported.19,20 Disulfide-based con-
jugates are considered to be promising tools for designing
antitumor drug delivery systems.13,21ꢀ24 Disulfide bonds are
rather stable in normal healthy tissues and blood but can be reduced
in tumor tissues and inside cells after endocytosis.21,23,25,26 Indeed,
many tumor tissues were found to have higher GSH, thio-
redoxin and thioredoxin reductase concentrations than nor-
mal tissues.23,27,28
On the basis of our expertise on multifunctional nucleolipid
conjugates, pore-forming peptides, and membrane organization,29ꢀ32
we developed reduction-sensitive liposomes made from a mix-
ture of natural phospholipids and new multifunctional lipid-
anchored disulfide conjugate 1 or 2 (Scheme 1). The incorporation
Liposomes, for example, doxorubicin stealth liposomes, are
being used as vehicles to deliver drugs in anticancer therapies.1ꢀ6
They can encapsulate both water-soluble and hydrophobic drugs,
protect drugs from degradation, and allow the codelivery of
several drugs. In addition, liposomes accumulate in malignant
tissues because of the enhanced permeability and retention (EPR)
effect. Because of their surface properties, liposomes can be
functionalized with different tumor-specific ligands for tar-
geted delivery: liposomes functionalized with antibodies,
ligands, or affibodies were shown to accumulate in respective
tumors.3,7 The active and passive accumulation of nanocar-
riers in tumor tissues leads to a lower systemic toxicity of the
drugs.2,3,8,9
Although different carriers are already in clinical use, the
demand for more efficient delivery systems and a controlled triggered
release only at the site of destination is very high.10 Therefore,
different stimuli-sensitive systems were proposed. Giving only
a few examples, phospholipase-triggered drug release from
liposomal carriers,11 reduction-triggered delivery using disulfide
Received: March 29, 2011
Revised:
July 1, 2011
Published: August 05, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Conjugates 1 and 2 Described in the Experimental Section and Reduction Products 3, 7 and 3, 9,
Respectively
of the conjugates into lipid vesicles, the kinetics of the disulfide
bond reduction, and dye release from vesicles were investigated.
The nonspecific cellular uptake of the reduction-sensitive lipo-
somes and the release of the cargo in cells were tested using a
mouse macrophage cell line. The assembly of antibody-present-
ing reduction-sensitive liposomes was achieved using an anti-
HER2 biotin-conjugated antibody and a streptavidinꢀbiotin
block system, which is often used as a test building system
because of the high-affinity binding of the biotinꢀstreptavidin
complex and the versatility with respect to the functionalization
of polymerosomes.33,34 The uptake of the antibody-presenting
liposomes loaded with doxorubicin, a fluorescent anthracycline
antibiotic that is an anticancer drug with a strong antimitotic
effect, was tested using a breast cancer cell line overexpressing the
HER2 receptor.
’ EXPERIMENTAL SECTION
Materials. L-R-Phosphatidylcholine (PC, egg, chicken), 1,2-dio-
leoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-
glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)
(ammonium salt, Rh-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanola-
mine-N-(5-dimethylamino-1-naphthalenesulfonyl) (ammonium salt)
(Dansyl-PE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-
N-(cap biotinyl) (sodium salt, Biotinyl-PE) were purchased from Avanti
Polar Lipids, Inc. (Alabaster, AL) and used without further purification.
Tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), L-cy-
steine, reduced glutathione (GSH), streptavidin, antimouse IgG-perox-
idase, calcein, doxorubicin hydrochloride (DOX), and Sephadex G50
were obtained from Sigma-Aldrich (Taufkirchen, Germany). Triton
X-100 was from Roth (Carl Roth, Karlsruhe, Germany). Phosphate-
buffered saline (PBS), trypsin, L-glutamine, RPMI, DMEM cell culture
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mediums, fetal bovine serum (FBS), and penicillinꢀstreptomycin were
from PAA Laboratories GmbH (Co€lbe, Germany). Mouse antihuman
anti-p185HER2 biotin-conjugated antibody and unlabeled and FITC-
labeled streptavidin were from Invitrogen, Inc. (Karlsruhe, Germany).
Synthesis of Conjugates 1 and 2. Disulfide conjugates 1 and 2
were synthesized as described in detail elsewhere (in preparation). See
also Scheme 1 and a brief description of the synthesis below.
2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyl-N-(2-O-
(3-O-biotinyl-2-N-dansyl-serin-1-yl)-tetraethylenglykol-1-yl)-ethanam-
monium-tosylate (1). A solution of tosylate 6 (292 mg, 0.33 mmol) and
disulfide 5 (80 mg, 0.11 mmol) in CHCl3 (1 mL) was stirred under
argon in a sealed flask at 70 °C (2 bar) for 5 days. The solvent was
stripped off by a rotary evaporator, and the remainder was purified by
column chromatography (silica, 5:1 dichloromethane/MeOH, Rf = 0.2).
The product was obtained as a diastereomeric mixture in 50% yield (88
mg) as a yellow solid, mp 55ꢀ57 °C.
2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyl-N-(2-O-
biotinyl-tetraethylen-glykol-1-yl)-ethanammonium-tosylat (2). A sus-
pension of disulfide 5 (161 mg, 0.23 mmol) and biotin derivative 8 (332
mg, 0.58 mmol) in chloroform (1.7 mL) was put into a sealed flask under
argon and heated to 70 °C under stirring for 7 days. The pressure rose to
about 2 bar. The solvent was removed with a rotary evaporator, and the
remainder was purified by column chromatography (silica, 5:1 dichlor-
omethane/MeOH, Rf = 0.3) giving 115 mg (39% yield) of product 2 as a
yellowish solid, mp > 190 °C under decomposition.
Figure 1. Fluorescence emission spectra of vesicles composed of
7.4:92.6 conjugate 1/PC with or without 1 mol% Rh-PE (red-dotted
and green-dashed curves, respectively). The black curve reveals the
spectrum of the PC vesicles containing 1 mol% Rh-PE. The blue dashed-
dotted curve reveals the spectrum after the addition of 0.1% Triton
X-100 to the vesicles composed of 1:7.4:91.6 Rh-PE/conjugate 1/PC.
Excitation at 330 nm, 35 °C.
used to obtain 1.5 mL of 2 mM total lipid stocks in buffer. Lipid mixtures
were rehydrated with calcein (70 mM calcein, 10 mM HEPES, 1 mM
EDTA, pH 7.4) or KCl buffer (100 mM KCl, 10 mM HEPES, pH 7.4) at
70 °C. The suspension was mixed vigorously and subjected to five
freezeꢀthaw cycles with thawing in a water bath at 70 °C and 11
extrusions through a 100 nm pore size polycarbonate filter (Nucleopore
GmbH, T€ubingen, Germany) using a Mini-Extruder with a heating block
(Avanti Polar Lipids, Alabaster, AL) at approximately 70 °C.37 Thin
layer chromatography was used to prove that conjugates 1 and 2 could
be reduced (data not shown). Vesicles with entrapped calcein were
separated from free calcein by gel filtration using spin Sephadex G50
columns (1 mL polypropylene column, Qiagen, Hilden, Germany) at
(100ꢀ300)g for 3 min. Sephadex G50 was presoaked in the isoosmotic
KCl buffer with 10 mM HEPES and 1 mM EDTA at pH 7.4. Reduction-
sensitive liposomes were stable and retained entrapped calcein for at
least 2 weeks.
Fluorescence Spectroscopy. An Aminco Bowman series 2
fluorometer with a temperature-controlled cell and continuous stirring
was used. Measurements were made in 4 mL cuvettes using 1 mL
samples containing a 10 to 30 μM lipid concentration. Dansyl was
excited at 330 nm, and emission was measured at 520 nm. Rhodamine
was excited at 560 nm, and emission was measured at 590 nm. F€orster
resonance energy transfer from dansyl to rhodamine was measured with
excitation at 330 nm and emission at 590 nm. Calcein was excited at
492 nm, and emission was measured at 520 nm. Time-dependent release
curves were normalized using eq 1 as described elsewhere
2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethy-
lethanamine (5). PTAD (114 mg, 0.66 mmol) was added to a
solution of 3-mercapto-1,2-bis-O-(octadecyl)-propane (3)35 (400 mg,
0.66 mmol) in a mixture of dry CH2Cl2 (14 mL) and dry MeCN
(5.4 mL). The resulting red solution was stirred under argon at room
temperature for 30 min and then heated to 50ꢀ55 °C. When the mixture
had become colorless (after about 2 h), 2-dimethylaminoethanethiol
hydrochloride 4 (243 mg, 1.72 mmol) was added. After being stirred at
room temperature for 20 h, the solvents were removed with a rotary
evaporator and the remainder was purified by column chromatography
(silica, 12:1 CH2Cl2/MeOH, Rf = 0.6), providing 322 mg (68% yield) of
product 5 as a colorless solid.
Tosyl-biotinyl-tetraethylene Glycol (8). A suspension of (+)-biotin
(1.00 g, 4.1 mmol), O-tosyltetraethylene glycol36 (1.04 g, 4.1 mmol),
DCCI (1.01 g, 4.9 mmol), and DMAP (70 mg, 0.57 mmol) in CH2Cl2
(30 mL) was stirred at room temperature for 96 h. The solvent was
removed with a rotary evaporator, and the remainder was purified by
column chromatography (silica, 10:1 CH2Cl2/MeOH, Rf = 0.3), giving
1.03 g (43% yield) of product 8 as a colorless solid.
Conjugates 1 and 2 have molecular weights of 1611 and 1291 g/mol,
respectively.
Preparation of Liposomes. Conjugate 1 or 2 was dissolved in 5:1
chloroform/methanol to 5ꢀ10 mM. Lipid mixtures were prepared in
5:1 chloroform/methanol, and a thin lipid film was formed in round-
bottomed tubes by a rotary evaporator. Reduction-sensitive liposomes
were produced from conjugate 1 or 2 (5ꢀ20 mol%), L-R-phosphati-
dylcholine (PC, 20ꢀ30 mol%), and 1,2-dioleoyl-sn-glycero-3-phos-
phoethanolamine (DOPE, 60ꢀ65 mol%). Rh-PE (1 mol%) was
added to lipid mixtures as an acceptor in studying the incorporation
and reduction kinetics when stated. Lipid mixtures used were: 7.4:92.6
conjugate 1/PC with and without 1 mol% Rh-PE; 10:30:60, 18:27:55,
15:22:63, and 10:25:65 conjugate 1/PC/DOPE or conjugate 2/PC/
DOPE; and 20:80 conjugate 2/PC. Control-reduction-insensitive lipo-
somes were made from 40:60 PC/DOPE, 1:99 Rh-PE/PC, 1:99 dansyl-
PE/PC, and 1:40:60 biotinyl-PE/PC/DOPE. Note that the amount of
conjugate 1 in the vesicle samples was estimated by measuring the
absorption of dansyl at 330 nm and was 7.4 mol% for the experiments
shown in Figure 1. Lipid films were redissolved in cyclohexane with 5%
(v/v) ethanol, frozen, and lyophilized overnight. Lipids (3 μmol) were
FðtÞ ꢀ Fo
ð1Þ
Release ðtÞ ¼
Fmax ꢀ Fo
where Release (t) is the normalized release, F(t) is a raw data fluorescence
curve, Fo is the fluorescence intensity before the addition of a reducing agent,
and Fmax is the fluorescence intensity after the complete release of calcein
upon the disruption of liposomes by Trition X-100.29,38 Time was set to
0 s at the point of addition of a reducing agent. Light scattering at 90° was
measured at 550 nm on a Jobin Yvon Fluoromax-4 fluorometer (Horiba
Jobin Yvon, NJ, USA) and at 600 nm on an Aminco Bowman series 2 to
obtain the data shown in Figures S4 and S5 (left), respectively.
Uptake and Release Experiments in a Macrophage Cell
Line. Mouse macrophage cell line J774A.1 was used to study the
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nonspecific uptake of calcein-loaded vesicles. Cells were grown to 70%
confluency in 12 mL cell flasks in the presence of RPMI, 10% FBS, 5%
penicillin-streptomycin, and 2 mM L-glutamine. Then cells were gently
scraped off, plated in 35 mm glass-bottomed dishes (MaTek Corp.,
USA; 1:4 dilution), and incubated at 37 °C in 5% CO2 for 24 h. Cells
were washed six times with PBS with Ca2+ and Mg2+, incubated with
25 μL of reduction-sensitive or -insensitive liposomes, 15:22:63 conjugate
2/DOPE/PC, and 37:63 DOPE/PC, and reincubated in RPMI medium
at 37 °C with 5% CO2.
Fluorescence Microscopy. The fluorescence microscopy images
were taken with an Olympus IX-81 inverted epifluorescence microscope
in 1 mL of PBS with Ca2+ and Mg2+ to avoid cell detachment during
the observation. Before observation on the microscope, the cells were
washed six times with PBS with Ca2+ and Mg2+. Note that cells were
always incubated in a medium without phenol red to avoid the back-
ground fluorescence due to phenol red.
Fluorescence-Activated Cell Sorting (FACS). FACScan
(Becton Dickson Bioscience, USA) was used. Macrophages were treated
with 25 μM reduction-sensitive or -insensitive liposomes (15:22:63
conjugate 2/DOPE/PC or 37:63 DOPE/PC, respectively) for 1 h.
Subsequently, cells were washed six times with PBS without Ca2+ and
Mg2+ and scraped off. A 200 μL sample from the cell culture were taken,
treated with 1 μL of propidium iodide (PI, 1 mg/mL) to stain dead cells,
and used for FACS. The data obtained were analyzed with FlowJo (Tree
Star, Inc.) flow cytometry data analysis software. Only living cells, the
population of cells not stained with PI, was analyzed for their green
fluorescence intensity, corresponding to the uptake of loaded liposomes
and the release of calcein.
cycles with thawing in a water bath at 70 °C and lyophilized at 0.2 mbar
overnight. Doxorubicin (0.1 mL, 0.5 mg/mL) and after 5 min another
portion of doxorubicin (0.4 mL, 0.5 mg/mL) were added to the
lyophilized sample. A final concentration of doxorubicin of ∼3 mM
entrapped in liposomes was obtained. Note that we used doxorubicin
only for the detection of release, and the entrapped concentration was
significantly lower than that necessary for therapeutic treatment (e.g.,
∼200 mM). The obtained suspensions were then extruded 11 times
through a 100 nm pore size polycarbonate filter using a Mini-Extruder
with a heating block at approximately 70 °C. Vesicles with entrapped
doxorubicin were separated from the free dye by gel filtration using spin
Sephadex G50 columns (e.g., a 1 mL polypropylene column) for 3 min
at (100ꢀ300)g. Sephadex G50 was soaked in the physiological buffer.
Subsequently, 25 μL of streptavidin (1 mg/mL) and 50 μL (50 μg/mL) of
anti-p185HER2 biotin-conjugated antibody were added, and the mixture
was incubated at 4 °C for 1 h before being applied to cells. Cells were
washed four times with PBS with Ca2+ and Mg2+. Thenantibody-presenting
liposomes were added to the cells in PBS with Ca2+ and Mg2+, and cells were
incubated at 4 °C for 1 h to minimize unspecific uptake due to energy-
dependent endocytosis. The cells were then washed twice with PBS with
Ca2+ and Mg2+, fresh medium (DMEM) was added, and the cells were
incubated at 37 °C in 5% CO2 for 1 h. Before observation, the cells were
washed four times with PBS with Ca2+ and Mg2+. The fluorescence
microscopy images, taken with inverted epifluorescence microscope
Zeiss Axiovert 100, were recorded in 1 mL of PBS with Ca2+ and Mg2+
and 50 mM HEPES to avoid cell detachment during the observation.
’ RESULTS
Enzyme-Linked Immunosorbent Assay (ELISA). Biotin-con-
jugated mouse antihuman p185HER-2 (50 μL, 50 μg/mL) was coupled
to streptavidin (100 μg/mL) by incubation in PBS at room temperature.
The conjugate was then mixed with the reduction-sensitive liposomes
(60 min of incubation at room temperature, followed by 19 h of
incubation at 4 °C), and the resulting construct was size-separated on
a gel filtration column (Sephacryl S200 HR). Elution was carried out
with PBS, and the eluate fractions were subjected to ELISA and particle
count analysis. Briefly, fractions were adsorbed to a high-binding 96-well
ELISA plate (Greiner Bio-One GmbH, Frickenhausen, Germany)
blocked with skim milk powder and incubated with antimouse IgG-
peroxidase 1:1000 in Tris-buffered saline for 1 h. After a washing step
(three times with Tris-buffered saline), the remaining peroxidase was
detected by chromogenic detection (24.3 mM citric acid, 51.4 mM
disodium phosphate, 0.04% (w/v) o-phenylene diamine, and 0.012%
(v/v) hydrogen peroxide) and measurement in a UV/vis spectrophot-
ometer (492 nm). In parallel, the particle size and count of the fractions
were determined by dynamic light scattering using a Malvern Zetasizer
3000 HSA at 633 nm, 90°, and 25 °C.
Antibody-Mediated Liposomes’ Uptake and Release. BT-
474 breast cancer cells were grown to 70% confluency in 12 mL cell
flasks in DMEM with 10% FBS, 5% penicillin-streptomycin, and 2 mM
L-glutamine. Then they were detached using 0.05% trypsin/EDTA and
replated a day before the uptake experiment as described above for
macrophages.
To assemble anti-p185HER2-presenting reduction-sensitive and -
insensitive vesicles loaded with doxorubicin, 150 μL of vesicles, pre-
tested for the stability and triggered release, and 25 μL of streptavidin
were mixed in 1 mL PBS without Ca2+ and Mg2+ and incubated at room
temperature for 30 min. For the preparation of the liposomes, the
following lipid molar ratios were used: 20:20:60 conjugate 1/PC/DOPE
(RS-LUVs) and 0.1:40:60 Biotinyl-PE/PC/DOPE (RI-LUVs). The
samples were frozen (ꢀ80 °C) overnight and lyophilized with a freeze
dryer. Doxorubicin water solution (1 mL, 0.5 mg/ml) was added to
the lipid mixtures. The suspension was sonified (50% interval, 20%
intensity) for 10 min and subsequently subjected to five freezeꢀthaw
Synthesis of Multifunctional Conjugates. Disulfide conju-
gates 1 and 2 were synthesized as described in detail elsewhere.
Briefly, Scheme 1 shows the synthesis steps used to obtain
amphiphilic conjugates 1 and 2 starting from lipophilic ami-
noethyl disulfide 5 and tetraethylene glycol-biotin tosylates 6 and
8 as alkylating reagents establishing a tetraalkylammonium
moiety (details in the Experimental Section). Disulfide conju-
gates 1 and 2 each have a hydrophobic part made of two
octadecyl chains, allowing incorporation into lipid bilayers. This
hydrophobic part is connected via a disulfide to a polar head-
group containing a positively charged ammonium moiety. In
addition, the polar headgroup was functionalized with biotin via a
tetraethylene glycol linker and fluorescent dye dansyl for con-
jugate 1. Reductive cleavage of the disulfide moiety of 1 or 2 will
produce lipophilic thioglyceroldiether 3 and hydrophilic thiol 7
bearing the charged ammonium moiety, dansyl, and biotin or 9
bearing the charged ammonium moiety and biotin.
Incorporation of Conjugate 1 into Liposomes. First, using
dansyl-labeled conjugate 1 we proved that disulfide conjugate 1 is
incorporated into the membrane of the vesicles. Dansyl is an
environmentally sensitive fluorophore that has a higher quantum
yield in hydrophobic environments (e.g., when a dansyl-labeled
lipid analogue is incorporated into a lipid membrane).39 The
quantum yield will decrease if dansyl-labeled headgroup 7 is
transferred to an aqueous solution upon cleavage of the disulfide
bond. In addition, because of the overlap of dansyl emission and
rhodamine excitation spectra, F€orster resonance energy transfer
(FRET) from dansyl to closely located rhodamine molecules is
expected. The F€orster radius of the dansyl-rhodamine FRET pair
is about 4.3 nm.39,40
Phosphatidylcholine vesicles containing the FRET pair, con-
jugate 1, and Rh-PE; only a donor, conjugate 1; or only an
acceptor, Rh-PE, were prepared in parallel. The corresponding
emission spectra by the excitation of dansyl at 330 nm of the
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Figure 3. Normalized release of calcein from a 30 μM lipid concentra-
tion of 18:27:55 conjugate 2/PC/DOPE liposomes triggered by the
addition of 1, 0.5, and 0.25 mM DTT at time 0 s and 40 °C (green, black,
and red curves, respectively). The addition of reducing agent to 40:60
PC/DOPE liposomes did not induce release (cyan curve). After the
release reached a plateau phase, Triton X-100 was added, leading to the
complete dequenching of calcein.
Figure 2. Time traces of the dansyl fluorescence of 7.4:92.6 conjugate
1/PC vesicles (red curve, excitation at 330 nm, emission at 520 nm) and
the FRET-mediated rhodamine fluorescence of 7.4:91.6:1 conjugate 1/
PC/Rh-PE vesicles (black curve, excitation at 330 nm, emission at
590 nm) at 37 °C. TCEP (16 mM) was added at about 80 s. Note that
these are raw data, which are not corrected for dilution due to TCEP
addition, causing a sharp drop in fluorescence directly at the time of
addition.
conjugates 1 were detached from the membrane, leading to the
decrease in fluorescence due to the lower quantum yield of dansyl
in aqueous solution. The black curve represents the time trace of
the rhodamine fluorescence intensity upon excitation of dansyl at
330 nm (FRET). After about 80 s, a reducing agent was added,
cleaving the disulfide bridges and detaching headgroups 7 of
conjugates 1 off the membrane interface, hence leading to a
decrease in FRET.
Although disulfide conjugate 1 was incorporated into the
membrane of liposomes (Figure 1) and could be reduced by
TCEP (Figure 2), no release of entrapped molecules from PC/
conjugate 1(2) liposomes could be induced by the addition of a
reducing agent. Therefore, we have added unsaturated phospha-
tidylethanolamine, DOPE, to the lipid mixture to provoke the
liposome instability upon reduction of conjugate 1 or 2. By using
less than 70 mol% but at least 55 mol% DOPE, stable liposomes
were formed and reduction-induced release was studied.
Triggered Calcein Release. Calcein, a membrane-imperme-
able fluorescent dye, was encapsulated into liposomes at a high
self-quenching concentration (70 mM) as a mimic of a water-
soluble drug, allowing detection of the release kinetics due to the
increase in fluorescence upon the dequenching of calcein due to
dilution.38 Figure 3 shows the triggered release of the dye from
18:27:55 conjugate 2/PC/DOPE liposomes by DTT at 40 °C.
The curves were normalized as described in the Experimental
Section with the maximum fluorescence intensity measured after
the addition of surfactant Triton X-100 to destroy the liposomes
and hence to obtain the complete release of calcein. The release
started after a lag time, denoting the time required before the
onset of a rapid increase in the fluorescence signal (Figure 3).
The lag time was decreasing with increasing concentration of the
reducing agent (e.g., approximately 200, 120, and 50 s for 0.25,
0.5, and 1 mM DTT at 40 °C, respectively). The lag time also
decreased with increasing temperature and the relative fraction of
reducible conjugate 2 in the membrane (Supporting Information
Figure S2). The rate of release, defined as the steepest slope of
the fluorescence signal increase following the lag time, was
increasing with increasing temperature, the relative fraction of
reducible conjugate 2 in the membrane (Figure S2), and the
vesicles containing conjugate 1 with and without the rhodamine-
labeled phospholipid are shown in Figure 1 (red dotted and
green dashed curves, respectively). When both fluorophores
were incorporated, we observed a strong decrease in the dansyl
fluorescence and an increase in the rhodamine fluorescence (red
dotted curve) because of the strong energy transfer from dansyl
on conjugate 1 to rhodamine on the phospholipid, proving the
incorporation of conjugate 1 into the phospholipid membrane of
the vesicles.
For comparison, the black curve in Figure 1 shows the
spectrum of Rh-PE in vesicles prepared without conjugate 1.
The blue dashed-dotted curve shows the spectrum of the same
sample as shown by the red dotted curve (containing both
dansyl-labeled conjugate 1 and Rh-PE) but after the addition
of Triton X-100. The addition of the detergent led to the
destruction of liposomes and an increase in the distance between
donor (dansyl) and acceptor (rhodamine) and therefore a decrease
in energy transfer. High dansyl fluorescence in the vesicles without
the acceptor (green dashed curve) indicates that conjugate 1 is
incorporated into a lipid membrane. Together these results prove
that dansyl on conjugate 1 was located at the membrane interface
(high dansyl fluorescence) and that conjugate 1 was incorporated
into the same vesicles as rhodamine-labeled phospholipids.
Cryoelectron microscopy and dynamic light scattering mea-
surements revealed that the presence of the conjugate in the lipid
membrane did not change the morphology of the vesicles:
reduction-sensitive liposomes prepared by extrusion through
100 nm pore size filters had diameters of 100ꢀ150 nm
(Supporting Information Figure S1 and below).
Disulfide Reduction Kinetics. To assess the cleavage of the
disulfide bonds, both the environmental sensitivity of dansyl
fluorescence and the FRET between dansyl and rhodamine were
used to measure the reduction kinetics (Figure 2).
The red curve shows the time trace of dansyl fluorescence
changes of the probe containing 7.4:92.6 conjugate 1/PC vesicles.
Upon addition of the reducing agent (16 mM tris(2-carbox-
yethyl)phosphine, TCEP) at about 80 s, dansyl headgroups 7 of
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Figure 4. Normalized release of calcein from 30 μM 10:25:65 conjugate
2/PC/DOPE liposomes triggered by the addition at time 0 s of 30 mM
L-cysteine and from 20 μM 15:22:63 conjugate 2/PC/DOPE triggered
by the addition at time 0 s of 4 mM glutathione (GSH, 1.3 mg added to a
1 mL sample) shown as solid and dotted curves, respectively, at 37 °C.
The addition of 30 mM L-cysteine to 40:60 PC/DOPE liposomes did
not induce release (dashed curve).
Figure 5. Comparison of the cleavage (dansyl fluorescence decrease,
red curves, right ordinate) and normalized release kinetics (calcein
fluorescence dequenching, black curves, left ordinate) upon addition of
33 and 9 mM TCEP at time 0 s to 80 μM 15:22:63 conjugate 1/PC/
DOPE vesicles (solid and dotted curves, respectively). The black dotted
line shows negligible calcein release from the liposomes without the
addition of a reducing agent. The upper red dashed-dotted-dotted line
shows a negative control: 9 mM TCEP was added to 50 μM 20:80
dansyl-PE/PC vesicles in degassed buffer. The lower red dashed line
shows a decrease in the dansyl fluorescence of conjugate 1 (80 μM
liposomes) due to oxidation by air when the buffer was not degassed.
concentration of the reducing agent as can be expected from
simple kinetic considerations.
Figure 4 shows the reduction-triggered release of calcein upon
the addition of 30 mM L-cysteine (solid curve) and 1.3 mg of
glutathione (GSH, dotted curve) to liposomes, respectively, at
37 °C. The addition of physiological amounts of L-cysteine or
GSH41 to the conjugate 2/PC/DOPE liposomes led to the
significant release of entrapped calcein. The rate of release induced
upon the addition of 4 mM glutathione was slower and the final
fraction of dye released was lower than the rate and release level
induced upon the addition of 30 mM L-cystein. We assume that
this is due to the higher rate of GSH oxidation lowering the
efficient concentration of the reducing agent. Spontaneous
leakage without the addition of a reducing agent from the vesicles
was negligible. (For a typical spontaneous release curve, see the
black dashed line in Figure 5.) These results prove that water-
soluble molecules can be entrapped in the liposomes and that the
reduction of the disulfide bond of conjugate 2 using physiological
reducing agents induces the release of these molecules. In agreement
with previously published results,13 the release of calcein from
PC/DOPE-liposomes lacking the disulfide conjugates was neg-
ligible, regardless of whether one of the reducing agents was added
(Figure 3, cyan curve; Figure 4, dashed curve).
The reduction kinetics was fast, hence most of the probe
accessible for reduction was reduced during the slow release
phase, indicating that a certain amount of the cleaved conjugate
molecules is necessary to perturb the membrane of the vesicles to
allow release due to the formation of transient defects and/or
structural rearrangement. The upper dashed-dotted-dotted red
line shows the result of a negative control: the addition of TCEP
to vesicles containing dansyl-labeled phospholipid (Dansyl-PE)
in degassed buffer did not change the dansyl fluorescence. Note
that dansyl is sensitive to oxidation, leading to a decrease in
fluorescence; therefore, when working with dansyl-labeled
conjugates, buffers should be degassed and saturated with N2.
The dashed red curve below shows the approximately linear
decrease in dansyl fluorescence with time when the buffer was
not degassed.
Reduction of the conjugate disulfide bond led to the separa-
tion of the hydrophilic headgroup from the membrane surface
and to the aggregation of conjugate 1, PC, and DOPE-
containing liposomes at higher lipid concentrations. Aggregation
of the liposomes was observed with dynamic light scattering (not
shown). Preliminary results on the pH-sensitivity of the disulfide
conjugate, PC, DOPE-containing liposomes are discussed in the
Supporting Information
Comparison of Reduction and Release Kinetics. To com-
pare the reduction and release kinetics, liposomes from 15:22:63
conjugate 1/PC/DOPE with and without entrapped calcein
(70 mM) were prepared in parallel.
Red solid and dotted curves in Figure 5 represent the reduc-
tion-mediated decrease of dansyl fluorescence upon addition of
33 or 9 mM TCEP, respectively. Black solid and dotted curves in
Figure 5 represent the increase in calcein fluorescence due to the
reduction-triggered release of the dye from the liposomes upon
the addition of 33 or 9 mM TCEP, respectively. Without the
addition of a reducing agent, the release of calcein from the
liposomes was negligible (black dashed line). In agreement with
the results described above (Figures 2ꢀ4), in these experiments
conducted in parallel we also observed that cleavage started
immediately upon addition of the reducing agent, whereas a lag
phase was observed before the onset of the fast calcein release.
Unspecific Uptake of Liposomes by Macrophages and
Release of Calcein. Macrophages were used to study cargo
release from the reduction-sensitive liposomes in cells because
they have a high unspecific uptake efficiency and a high reducing
capability.16 First, we verified that the vesicles were stable in the
cell culture medium with 0ꢀ20% fetal bovine serum, and the
reduction-triggered release of calcein was preserved in the
medium after the addition of TCEP (data not shown). Then
the cells were incubated with 30 μM reduction-sensitive
or-insensitive vesicles in RPMI. After 10 min and 1 or 2 h of
incubation with vesicles, the cells were washed six times with PBS
with Ca2+ and Mg2+. The cells incubated for 10 min had only a
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Figure 6. (a, b) Fluorescence microscopy images of macrophage cells (J774A.1) treated with calcein-containing reduction-sensitive and reduction-
insensitive liposomes at 37 °C for 2 h, respectively. (c, d) Cell count versus calcein fluorescence histograms from the FACS data analysis of macrophages
incubated with reduction-sensitive and reduction-insensitive liposomes, respectively, at 37 °C for 1 h. The white bars correspond to 25 μm.
low level of fluorescence inside the cells, mainly localized to the
regions close to plasma membrane and small compartments,
presumably endosomes, in agreement with previous studies on
the uptake of reduction-sensitive polymerosomes.16 After 10 min
of incubation, almost no difference between cells treated with
reduction-sensitive and -insensitive liposomes was observed
(Figure S3). After 1 or 2 h, however, the fluorescence intensity
inside the cells increased remarkably because of the dequenching
of calcein released from liposomes. Cells treated with reduction-
sensitive vesicles showed higher and more homogeneously
spread fluorescence compared to that of the cells treated with
reduction-insensitive vesicles as shown in Figure 6a,b, respec-
tively. To quantify the release of calcein from the liposomes taken
up by macrophages, we used fluorescence-activated cell sorting
(FACS). Figure 6c,d shows the corresponding cell count versus
fluorescence histograms for macrophages treated with reduction-
sensitive and -insensitive liposomes, respectively, for 1 h. A
comparison of the two histograms reveals that cells incubated
with reduction-sensitive liposomes showed about a 10-fold
higher fluorescence than cells incubated with reduction-insensi-
tive liposomes demonstrating that more calcein was released
inside cells treated with vesicles containing conjugate 2.
Uptake of Antibody-Presenting Doxorubicin-Loaded Lip-
osomes by BT-474 Cells. To demonstrate the potential of the
active targeting, we used biotinꢀstreptavidin-specific binding to
cover liposomes with an antibody against the extracellular part of the
p185HER2 receptor. This receptor is known to be overexpressed
in about 20ꢀ30% of breast, ovarian, prostate, and gastric cancer
patients, and this high expression correlates with more aggressive
tumors and a poor prognosis.7,42,43 The p185HER2 receptor also
represents an attractive target for antibody-based therapy because it
is stably and homogeneously overexpressed in the tumor tissues,44
whereas in normal tissues p185HER2 is expressed only at low
levels.45 HER2-targeted immunoliposomes have been shown
to deliver anticancer drugs specifically to p185HER2-positive
BT-474 cells.46,47
The streptavidinꢀbiotin building system was applied to allow
variable targeting. Biotin was covalently attached to the head-
group of conjugates 1 and 2 (Scheme 1), and streptavidin was
used as a specific linker to attach the biotinylated anti-HER2
antibody to the liposomes containing conjugate 1 or 2. Binding
of streptavidin to the liposomal carriers was detected by three
independent methods: by the increase in fluorescein-labeled
streptavidin fluorescence upon attachment to biotin on the
surface of the carriers (data not shown), by the increase in light
scattering due to the aggregation of carriers via streptavidin
molecules (Supporting Information Figures S4 and S5, left), and
by an enzyme-linked immunosorbent assay (ELISA, Figure 7).
The average liposome size was constant (110ꢀ150 nm) in the
eluent volume range from 0.375 to 0.8, whereas the ELISA signal
first peaked and then diminished over the same elution period
(black squares, Figure 7, right) in good congruence with the
particle (liposome) count trend (red circles, Figure 7, right).
Thus, the ELISA signal corresponds to antibody-presenting lipo-
somes because the free antibodies were separated by chromatog-
raphy as described in the Experimental Section. It can therefore
be concluded that a major portion of the detected liposomes have
first bound streptavidin and then biotinylated antibody; there-
fore, the antibody-presenting reduction-sensitive liposomes, shown
as a cartoon on the left side of Figure 7, were obtained.
BT-474 breast cancer cells were incubated in DMEM with
antibody-presenting reduction-sensitive or -insensitive vesicles
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Figure 7. ( Right) Elution profile of reduction-sensitive the liposome/streptavidin/anti-p185HER2 complex determined by ELISA (black squares) and
dynamic light scattering (red circles). Lines were drawn to guide the eye. (Left) Liposomal carrier built of phospholipids (bilayer, gray) and the
multifunctional lipid conjugate (red, blue, and black), filled with small water-soluble molecules (green dots). Streptavidin tetramer (light blue) serving as
a linker between biotin (red) covalently attached to the headgroup of the conjugate and the biotinylated anti-HER2 antibody (red, black).
Figure 8. (a) Fluorescence microscopy and (b) differential interference contrast images of BT-474 cells treated with doxorubicin-loaded anti-
p185HER2 antibody-presenting reduction-sensitive liposomes at 37 °C for 1 h. White bars correspond to 25 μm. For a comparison of images of cells
treated with antibody-presenting reduction-sensitive and -insensitive liposomes and reduction-sensitive liposomes without antibody, see Supporting
Information Figure S6.
and vesicles without antibody, prepared as described in the
Experimental Section, at 37 °C for 1 h and then washed four
times with PBS before imaging. Figure 8 shows typical fluores-
cence and differential contrast microscopy images of BT-474
treated with antibody-presenting reduction-sensitive liposomes
(Supporting Information Figure S6a,b). The partial release of
doxorubicin inside the cells was observed. Cells incubated with
antibody-presenting reduction-insensitive liposomes showed
uptake, but doxorubicin fluorescence was merely concentrated
in small granularlike structures (Supporting Information Figure
S6c,d). Cells incubated with reduction-sensitive liposomes with-
out antibody showed nonspecific uptake, which was lower than
the specific uptake, and more release than for reduction-insensi-
tive liposomes (Supporting Information Figure S6e,f). Untreated
cells showed no fluorescence signal (not shown). Preliminary
MTT cell viability tests revealed no toxicity of liposomes contain-
ing disulfide conjugates (25ꢀ30 μM of total lipid, not shown).
Together with the results described above on streptavidin and
antibody binding to the liposomes containing conjugate 2, these
results illustrate the possibility of the targeted delivery of a drug
encapsulated by reduction-sensitive liposomes.
Scheme 2. Schematic Representation of the Multifunctional
Conjugate 1
’ DISCUSSION
Multifunctional Conjugates. We have developed a multi-
functional amphiphilic biotin-tagged fluorescently labeled disul-
fide conjugate. Scheme 2 summarizes different functionalities of
the conjugate: (1) hydrophobic chains offering stable incorpora-
tion into lipid membranes; (2) a disulfide bridge (yellow)
allowing the cleavage of the hydrophilic headgroup and therefore
destabilization of the liposomes and triggered release; (3) a
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fluorescent moiety (dansyl, blue) offering the possibility of the
detection of the incorporation and the reduction kinetics; and
(4) a recognition moiety (biotin, red).
primes the membranes for instabilities, caused by sufficient
reduction of disulfide conjugate 1 or 2. The reduction of conjugate
1 or 2 disulfide bond leads to a separation of the hydrophilic
headgroup (thiol 7 or 9, Scheme 1) from the membrane surface.
Very likely, the lipid anchor remaining in the membrane adopts
an inverted conelike structure similar to that of unsaturated
phosphatidylethanolamines (e.g., DOPE).52 It is known that this
lipid shape interferes with the stability of the bilayer structure: at
high concentrations of cone-shaped lipids, the integrity and low
permeability of membranes are lost; even nonlamellar phases
might be formed as reported for membranes of phosphatidy-
lethanolamine at physiological temperatures and pH.53,54 In
agreement with previous studies (e.g., reviewed in ref 55),
reduction was accompanied by the subsequent collision-limited
aggregation of the liposomes and release could be triggered by
lowering the pH to 4.5. Very likely, the separation of the
hydrophilic headgroup facilitates the aggregation of liposomes
by close contact. The potential to form nonlamellar phases might
also trigger fusion with adjacent membranes, which might allow
the escape of the content of liposomes onto the cytosol of cells.56
A similar mechanism of triggered release has been suggested by
several groups for different liposomal systems.13,19,57 For exam-
ple, Thompson and co-workers reported a cryoelectron micro-
scopy and 31P NMR study of the structural changes of the acid-
triggered liposomes with decreasing pH, first showing the
aggregation of the liposomes and then the formation of small
condensed, presumably hexagonal, structures.57
In agreement with previous reports, the reduction kinetics is
first order in the concentration of both the reducing agent and the
substrate,58 whereas the release kinetics is more complex having a
delay, a slow, and a fast release phase. Although further quantitative
analysis of the release kinetics is necessary, our results, in particular
on the lag time, indicate that a certain amount of lipophilic
reduction product thioglyceroldiether 3 in the membrane was
necessary to produce defects or reorganization allowing the
release of entrapped molecules. Although further optimization
of the reduction-sensitive liposomes (e.g., in lipid composition
for enhanced stability and delivery of hydrophobic and hydro-
philic substances to specific cellular organelles) is necessary, our
results give the first proof of principle allowing the development
of a targeted triggered delivery system based on the multifunc-
tional lipophilic disulfide conjugates and natural phospholipids.
To the best of our knowledge, our conjugate 1 is the first to
combine four different functionalities in one molecule. However,
because the reducible conjugates are considered to be promising
for triggered delivery, several other reduction-sensitive lipophilic
conjugates and even more reduction sensitive polymers forming
nanocarriers were synthesized and studied by different groups (e.
g., refs 15, 23, 24, and 48 and references therein). Disulfide
conjugates of drugs and carrier molecules are also under intensive
investigation.49,50 M€uller et al.50 reported the suppression of
human lymphocyte proliferation upon incubation with a lipo-
philic disulfide prodrug presumably due to the liberation of the
drug as a result of the disulfide bond cleavage. The lipophilic
disulfide prodrugs incubated with serum in a cell-free medium
were stable, also in agreement with our observations.
Reduction-Sensitive Liposomal Carriers. We provide here
the proof of principle that disulfide conjugates 1 and 2 enable
the formation of liposomes that (i) can be filled with cargo,
(ii) recognize a biological target, and (iii) release the cargo upon
triggering with physiological reducing agents. The release of
calcein from liposomes containing conjugate 1 or 2 was induced
using different reducing agents: final concentrations of DTT as a
more potent reducing agent of 0.5ꢀ2 mM, of TCEP as a less
potent reducing agent of 3ꢀ30 mM, and of L-cysteine and GSH
at physiologically relevant concentrations of 30 and 4 mM,
respectively, were used.
Furthermore, we show that more calcein is released from
reduction-sensitive than reduction-insensitive liposomes upon
uptake into macrophages and that the specific targeting of our
reduction-sensitive liposomes to breast cancer cells using bioti-
nylated anti-HER2 antibody is possible. Hubbell and co-workers16
suggested that the internalization of the polymerosomes was an
early and late endosome-mediated process. In agreement with
this work, after 10 min of incubation both reduction-sensitive and
-insensitive liposomes were taken up to similar extent and calcein
was mostly localized inside the endosomes, giving a weak
fluorescence signal. After 1 and 2 h, we observed a significantly
higher increase in calcein fluorescence inside cells incubated with
reduction-sensitive than with reduction-insensitive liposomes.
This shows the accessibility of the disulfide conjugates to reduction
by intracellular reducing substances leading to the efficient
release of calcein and the dequenching of the fluorescence signal.
Functionalization of the liposomes using the streptavidinꢀ
biotin building system and the biotinylated anti-HER2 antibody
allowed the efficient delivery of doxorubicin into the breast cell
line known to overexpress the HER2 receptor. We used doxor-
ubicin here as a dye, entrapped at 3 mM, giving a final concentration
lower than IC50 (IC50 ≈ 10 μM51). Furthermore, the cells
were imaged after 1 h of incubation, whereas the toxic effects
would first be expected after 24ꢀ48 h of incubation. For therapeutic
applications, doxorubicin must be entrapped in carriers at a high
concentration (e.g., 200 mM), giving after release a local con-
centration higher than IC50. The entrapment of other
drugs, subsequent uptake, release, and viability of the cells, and
the toxicity of the carriers should be tested. Further development
of the controlled targeted drug delivery system for applications
(e.g., in cancer therapy) is necessary and promising.
’ ASSOCIATED CONTENT
S
Supporting Information. Cryoelectron microscopy im-
b
age of liposomes; dependence of the lag phase on temperature;
preliminary results on the pH sensitivity of the liposomes; images
of macrophages after 10 min of incubation with liposomes; aggrega-
tion of liposomes by streptavidin; and fluorescence images of
BT-474 cells incubated with DOX-loaded liposomes. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: holger.egger@bayer.com; arbuzova@cms.hu-berlin.de.
’ ACKNOWLEDGMENT
Mechanism of Release and Comparison of Reduction and
Release Kinetics. We surmise that although the high mol% of
DOPE in the carrier does not disrupt the bilayer structure it
The J774A.1 mouse macrophage cell line was a generous gift
from Prof. Dr. T. F. Meyer, MPI for Infection Biology, Berlin,
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Germany. Human breast cancer cell line BT-474 was a generous
gift of Prof. Dr. H. Lage, Charitꢀe, Berlin, Germany. This work was
supported by Bayer Technology Services GmbH.
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