Constrained and UV-activatable cell-penetrating peptides for intracellular delivery of liposomes

Article abstract of DOI:10.1016/j.jconrel.2012.10.008

Herein we report on the development of a novel method of constraining a cell-penetrating peptide, which can be used to trigger transport of liposomes into cells upon in this case radiation with UV-light. A cell-penetrating peptide, which was modified on b

Full text of DOI:10.1016/j.jconrel.2012.10.008

Journal of Controlled Release 164 (2012) 87–94
Contents lists available at SciVerse ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Constrained and UV-activatable cell-penetrating peptides for intracellular delivery
of liposomes
Morten B. Hansen a, Ethlinn van Gaal b, Inge Minten a, Gert Storm b,c
,
Jan C.M. van Hest a, Dennis W.P.M. Löwik a,
⁎
a
Radboud University Nijmegen, Institute for Molecules and Materials, Department of Organic Chemistry, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, David de Wiedgebouw, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
MIRA Institute for Biomedical Technology & Technical Medicine, Faculty of Science & Technology, University of Twente, Building Zuidhorst, 7500 AE Enschede, The Netherlands
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report on the development of a novel method of constraining a cell-penetrating peptide, which
can be used to trigger transport of liposomes into cells upon in this case radiation with UV-light. A cell-
penetrating peptide, which was modified on both termini with an alkyl chain, was anchored to the liposomal
surface in a constrained and deactivated form. Since one of the two alkyl chains was connected to the peptide
via a UV-cleavable linker, disconnection of this alkyl chain upon irradiation led to the exposure of the
cell-penetrating peptide, and mediated the transport of the entire liposome particle into cells.
© 2012 Elsevier B.V. All rights reserved.
Received 20 April 2012
Accepted 9 October 2012
Available online 17 October 2012
Keywords:
Cell penetrating peptide
Liposomes
Peptide amphiphiles
Activatable cellular transduction
1. Introduction
pH, temperature and enzymatic triggers have been used to target drug
carrier systems specifically to diseased tissues, as the latter areas often
Over the past decades, numerous drug carriers have been developed
from a wide range of materials [1–12]. A common goal for most of these
carriers is to address some of the inherent problems associated with ad-
ministration of drugs, such as toxicity, instability and unfavorable
biodistribution. To deal with these issues, encapsulation of the drug in
a carrier is often the method of choice, as this serves to protect the
drug from the body environment and vice versa, and to shift the
biodistribution towards the target site hence the stability, safety and
targeting efficiency of the drug can be increased. In this respect, lipo-
somes have received a fair amount of attention as carriers of encapsulat-
ed drugs [13–16], because of their easy preparation (at lab scale), their
composition of natural and relatively inexpensive starting materials, as
well as approval of several liposome products by the FDA [17–19]. Of
particular interest are the so-called Stealth liposomes, which are coated
with poly(ethylene glycol) (PEG) to evade immune system interactions
and thereby achieve a long circulation time in the bloodstream [20–24].
However, while encapsulation provides a solution for unfavorable
biodistribution, toxicity and instability of drugs, encapsulated drugs
on the other hand have to escape from the interior of the carrier to
exert their activity. To gain control over this release, a plethora of trig-
gers has been employed such as reduction [25,26], ultrasound [27,28],
light [29–34], pH [6,35–40], temperature [41–43], magnetic [44] and
enzymatic activity [2,45–47] to mediate drug release. Of these triggers,
exhibit a decreased pH, elevated temperature or express specific en-
zymes [48]. The most precise control, however, is achieved when exter-
nal triggers such as ultrasound, magnetism and light are employed
since these can be applied to defined localized areas to induce drug re-
lease [49]. The disadvantage is that most of these externally applied
triggers are restricted to superficial tissues, though deep-seated tissue
may be reached with the aid of laparoscopy [49,50].
Up to now, most triggered-release carrier systems (including light-
triggered systems) work via a destabilization of the carrier causing re-
lease of the encapsulated drug in the extra-cellular space. This, howev-
er, may lead to poor cellular uptake of the released drugs in the case of
macromolecular and/or hydrophilic drugs, which do not easily cross the
cell membrane (e.g. oligonucleotides) [51,52]. Hence, cellular uptake of
the carrier before releasing its contents may be desirable. Regarding
uptake, this can be facilitated by functionalization of the carrier with
cell-penetrating peptides (CPPs), which is a class of peptides capable
of inducing cellular uptake of almost any kind of cargo to which they
have been attached [53–61]. However, modification of the liposome
surface with Tat-peptides leads to aspecific interaction and uptake in
non-target cells [62]. Moreover, its positive charge leads to the interac-
tion with blood components and enhances clearance via the reticuloen-
dothelial system [63], thereby compromising the stealth property that
is essential for prolonged circulation. In order to combine stealth behav-
ior and Tat-mediated cellular uptake, we designed a UV-activatable
cell-penetrating peptide which is inactivated by means of constraining
the peptide by ‘hiding’ it on the surface of a liposome. Reactivation of
⁎
Corresponding author.
E-mail address: d.lowik@science.ru.nl (D.W.P.M. Löwik).
0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jconrel.2012.10.008

88
M.B. Hansen et al. / Journal of Controlled Release 164 (2012) 87–94
the peptide can be accomplished by releasing the constrain via
UV-irradiation.
and the resin re-suspended in fresh DMF. DMAP (5 mg, 40.9 μmol)
and activated nitro-benzyl alcohol 4a (106 mg, 50.3 μmol) were
added and the suspension was shaken in the dark over night. Then
the resin was washed with DMF, CH2Cl2, i-PrOH, CH2Cl2 and Et2O
and suspended in the cleavage mixture (2 mL, trifluoroacetic acid/
water/triisopropyl-silane/thioanisole 90:5:2.5:2.5) for 18 h. The free
peptide was precipitated from Et2O, redissolved in water and lyophilized
yielding the crude peptide as a white powder. The crude peptide was pu-
rified by semi-preparative HPLC affording C12-UV-Tat 7a as a white
powder after lyophilization (20 mg, 27%). MALDI-TOF [M+H]+m/z:
2109.9 (calcd. 2108.2), fragment at 1778.4 and 1718.9 (the UV-
cleavable linker was cleaved by the MALDI laser).
2. Materials and methods
Breipohl resin was purchased from Novabiochem and Fmoc-L-amino
acids were from Bachem (Bubendorf, Switzerland) or Novabiochem
(EMD Chemicals, Gibbstown, USA). Lipids were from Avanti Polar
Lipids (Alabaster, USA) or Lipoid (Steinhausen, Switzerland). Atto655
was purchased from ATTO-TEC (Siegen, Germany). All other chemicals
were purchased from Baker, Fluka or Sigma Aldrich and used as re-
ceived. Mass spectra were recorded on a Bruker Biflex MALDI-TOF
(Bruker Daltronik, Bremen, Germany). Lyophilization was achieved
using an ilShin Freeze Dryer (ilShin, Ede, The Netherlands).
Hepes (99%) was obtained from Acros Organics BVBA (Geel,
Belgium). Phosphate-buffered saline (PBS) was purchased from
B. Braun (Melsungen AG, Melsungen); fetal bovine serum (FBS) was
from Integro, Zaandam, The Netherlands, Trypsin/EDTA, Plain DMEM
(Dulbecco's modification of Eagle's medium, with 3.7 g/L sodium bicar-
bonate, 1 g/LL-glucose, L-glutamine) and antibiotics/antimycotics
(penicillin, streptomycin sulfate, amphotericin B) were all from PAA
Laboratories (Pasching, Austria).
2.5. C16-UV-Tat (7b)
The title compound was prepared as described above for C12-
UV-cleavable anchor Tat 7a from Tat resin (333 mg, loading
0.59 mmol/g, 57.8 μmol peptide), activated benzyl alcohol 4b (186 mg,
317 μmol) and DMAP (5 mg, 40.9 μmol) affording C16-UV-Tat 7b as
a white powder after lyophilization (81 mg, 65%). MALDI-TOF
[M+H]+m/z: 2165.3 (calcd. 2164.3), fragment at 1787.7 and 1718.2
(the UV-cleavable linker was cleaved by the MALDI laser).
Human epithelial ovarian carcinoma (HeLa) cells were kindly
given by the Institute of Biomembranes (Utrecht University, The
Netherlands).
2.6. C12 Tat (8a)
2.1. General peptide synthesis
Tat on Breipohl resin (300 mg, loading 0.59 mmol/g, 52.1 μmol
peptide) was suspended in DMF for 5 min, then DMF was removed
and the resin re-suspended in fresh DMF. Dodecanoic acid (75 mg,
337 μmol), DIPEA (120 μL, 1.25 mmol) and benzotriazole-1-yl-oxy-
tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP;
155 mg, 350 μmol) were added and the suspension was shaken over
night. Then the resin was washed with DMF, CH2Cl2, i-PrOH, CH2Cl2
and Et2O and suspended in cleavage mixture (2 mL, trifluoroacetic
acid/water/triisopropylsilane/thioanisole 90:5:2.5:2.5) for 18 h. The
free peptide was precipitated from Et2O, redissolved in water and
lyophilized affording a white powder. The crude peptide was purified
by semi-preparative HPLC affording C12-Tat 8a as a white powder
after lyophilization (10 mg, 10%). MALDI-TOF [M+H]+m/z: 1900.3
(calcd. 1900.2).
Peptides were synthesized on Breipohl resins [64,65] using a
Labortec640 peptide synthesizer (Labortec, Bubendorf, Switzerland)
and employing a standard Fmoc solid-phase peptide synthesis
(SPPS) protocol [66]. Briefly, the resin was swollen in DMF for
20 min prior to use. The first and subsequent Fmoc groups were re-
moved by washing the resin with piperidine in DMF (20%, v/v) and
then shaking for 25 min with another portion of piperidine in DMF.
The desired sequence of amino acids was coupled to the resin using
Fmoc-L-amino acids (3.0 eq), diisopropylcarbodiimide (DIPCDI, 3.3 eq)
and N-hydroxy benzotriazole (HOBt, 3.6 eq). Peptide couplings were
followed to completion using the Kaiser test [67]. After the final Fmoc re-
moval the resin was washed with DMF, CH2Cl2, i-PrOH, CH2Cl2, and Et2O
and air-dried for at least 2 h.
2.2. Synthesis of Tat
2.7. C16 Tat (8b)
The following sequence was synthesized by standard SPPS, as de-
scribed above: YGRKKRRQRRRGC. Tat on Wang resin (204 mg, load-
ing 0.59 mmol/g, 35.4 μmol peptide) was suspended in cleavage mixture
The title compound was prepared as described above for C12 Tat
8a from Tat on Breipohl resin (300 mg, loading 0.59 mmol/g,
52.1 μmol peptide), hexadecanoic acid (87 mg, 339 μmol), DIPEA
(120 μL, 1.25 mmol) and BOP (155 mg, 350 μmol) affording C16
Tat 8b as a white powder (20 mg, 19%). MALDI-TOF [M+H]+m/z:
1957.4 (calcd. 1956.2).
(2 mL,
trifluoroacetic
acid/water/triisopropyl-silane/thioanisole
90:5:2.5:2.5) for 18 h. The free peptide was precipitated from Et2O,
redissolved in water and lyophilized yielding the crude peptide as a
white powder. The crude peptide was purified by semi-preparative
HPLC affording Tat as a white powder after lyophilization (20 mg, 33%).
MALDI-TOF [M+H]+m/z: 1718.3 (calcd. 1718.0).
2.8. Optimization of irradiation time
2.3. Synthesis of Tat (R>A)
A solution of Atto655 (22.5 μg/mL) was prepared in HEPES buffer.
This corresponds to the expected concentration in the liposome prepara-
tions assuming a statistical encapsulation of the dye. This solution was ir-
radiated with a Bluepoint2 UV-lamp (Dr. Hönle, Münich, Germany)
placed directly above the quartz cuvette containing the solution for the
following time intervals: 0 s, 15 s, 30 s, 1 min, 2 min, 3 min, 4 min and
5 min. After each irradiation step a fluorescence spectrum was acquired
on a LS55 fluorescence spectrometer (PerkinElmer, Groningen, The
Netherlands) using the following settings: excitation at 600 nm, excita-
tion slit 5.0 nm, emission slit 7.0 nm, scan range 620–850 nm, and
scan speed 100 nm/min. Unless stated otherwise irradiation, when re-
quired, was performed for 2 min throughout this work.
The following sequence was synthesized by standard SPPS and
cleaved off the resin as described above: YGAKKARQRRAGC. Tat (R>A)
resin (140 mg, loading 0.59 mmol/g, 29.7 μmol peptide) afforded Tat
(R>A) as a white powder after HPLC purification (6 mg, 13%).
MALDI-TOF [M+H]+m/z: 1463.3 (calcd. 1462.8).
2.4. C12-UV-Tat (7a)
Tat on Breipohl resin (200 mg, loading 0.59 mmol/g, 34.7 μmol
peptide) was suspended in DMF for 5 min, then DMF was removed

M.B. Hansen et al. / Journal of Controlled Release 164 (2012) 87–94
89
2.9. Liposome preparation
4 °C protected from light. Samples were centrifuged for 5 min at
250 ×g at 4 °C. After removal of the supernatant, cells were washed
with ice-cold PBS (3×200 μL, each centrifugation step at 250×g for
5 min at 4 °C). Cells were resuspended in 200 μl icecold PBS and kept
on ice until time of analysis. Flow cytometric analysis was performed
on a FACSCantoII (Becton and Dickinson, Mountain View, CA, USA)
equipped with a 488 nm 20 mW Solid State diode laser and a 633 nm
20 mW HeNe laser. 10,000 cells were recorded per sample to determine
Atto655 signal (APC/Cy7 channel).
The liposomes were prepared via the reversed-phase evaporation
method [68] as follows: egg-derived hydrogenated phosphatidyl-
choline (62 mg, 81.4 μmol), 1,2-distearoyl-SN-glycero-3-phospho-
ethanolamine-N-[methoxy(polyethylene glycol)-2000] (12.5 mg,
4.5 μmol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[maleimido(polyethylene glycol)-2000] (13.3 mg, 4.5 μmol) and
cholesterol (17 mg, 44.0 μmol) were dissolved in CHCl3 (6.66 mL). A
solution of Atto655 (0.8 mL, 0.5 mg/mL) was added as well as 10 mM
HEPES buffer (4.54 mL, 10 mM HEPES, 1 mM EDTA, 0.8% NaCl, pH 7.5).
The resulting two-phase system was vortexed to form a blue emulsion.
The majority of the CHCl3 was removed in vacuo and the remaining re-
moved by flushing with N2. Vortex with a few glass beads was performed
to aid formation of a homogeneous colloidal suspension. Water (0.5 mL)
was added to compensate for loss during evaporation. The suspen-
sion was extruded through polycarbonate filters (3×600 nm and
6×200 nm) using a Lipex high-pressure extruder (Northern Lipids,
Vancouver, Canada) and the size of the resulting liposome suspen-
sion was evaluated by dynamic light scattering (Zetasizer Nano-S,
Malvern Instruments, Malvern, UK). A diameter of 194 nm and a poly-
dispersity index of 0.11 were found (average of three measurements).
2.13. Confocal laser scanning microscopy
The cellular uptake experiments were done with HeLa cells, which
were seeded one day before the experiment in 8-well microscopy
chambers (Nunc, Wiesbaden, Germany) at a density of 40,000 cells/
well. At the time of the experiment, cells had grown to approximate-
ly 50% confluence. The liposome samples (100 nmol phosphate
equivalents — typically around 10 μL) were diluted with Dulbecco's
Modified Eagle Medium supplemented with 10% fetal calf serum
(DMEM, 380 μL) and added to the cells, which were incubated for 2 h
at 37 °C. The cells were washed with DMEM (3×400 μL) and imaged
immediately. Confocal laser scanning microscopy was performed using
a Leica Microsystems TCS SP2 AOBS system (Mannheim, Germany). Ex-
citation of Atto655 was achieved with an argon laser [488 nm (52%),
514 nm (65%) and 561 nm (63%)] and the resulting emission was ac-
quired between 575 and 800 nm as an average of four scans.
2.10. Functionalization of liposomes and zeta-potential determination
The freshly prepared liposome suspensions were split in seven equal
aliquots. To each of six aliquots (one aliquot was saved as a control) was
added a solution in HEPES buffer (100 μL) of the peptide (1.0 eq. relative
to maleimido-groups) to be conjugated to the liposomes and a solution
of tris(2-carboxyethyl)phosphine (10 μL, 0.5 M) was added. The reac-
tions were shaken 1 h at rt, then 1 h at 40 °C and finally incubated at
4 °C over night. The liposome suspensions were diluted with HEPES buff-
er and ultracentrifuged for 40 min at 50,000 rpm and 4 °C (Sorvall
Discovery-100, Thermo Scientific, Breda, The Netherlands). The resulting
pellets were resuspended in HEPES buffer (500 μL). The zeta-potential of
the liposome preparations was measured on a Malvern Zetasizer Nano-Z
(Malvern Instruments, Malvern, UK) on liposome samples (50 μL) di-
luted into water (1.5 mL). The reported value is an average of three
measurements.
2.14. Dynamic light scattering of liposomes in medium
The liposome samples (100 nmol phosphate equivalents — typically
around 5 μL) were diluted with DMEM, HEPES (200 μL) or HEPES
supplemented with bovine serum albumin (200 μL, 4 mg/mL) and mea-
sured by dynamic light scattering. The DMEM and HEPES supplemented
with bovine serum albumin solutions were also measured.
3. Results and discussion
As CPP, we used a Tat peptide derived from HIV [70,71], and im-
posed the UV-activatable property on the Tat peptide by incorporat-
ing it into a loop anchored to the surface of a PEG-ylated liposome
via two alkyl-chains of which one contained a UV-cleavable linker
(Fig. 1). While being in this loop, the surface PEG-chains shield the
CPP from cells and thus enable stealth behavior and oppose interac-
tion with target cells and thereby cellular uptake of the liposome.
However, once in vicinity of target cells, irradiation can be applied
to cleave the anchor, followed by loop-opening and exposure of Tat,
2.11. Phosphate concentration determination
The phosphate concentration was determined according to Rouser's
method [69]. Briefly, a standard dilution series of NaH2PO4 was set up
in triplicate (0–160 μL of a 0.5 mM NaH2PO4 solution) and treated the
same way as the samples (triplicate). The samples were dried at 180 °C,
then perchloric acid (0.3 mL, 70%) was added to each, and the samples
were incubated at 180 °C. Water (1.0 mL), a solution of (NH4)6Mo7O24
(0.5 mL, 1.2% w/v) and ascorbic acid (0.5 mL, 5% w/v) were added to
each sample and these were incubated at 100 °C for 5 min. The absor-
bance at 797 nm was measured after cooling with cold water. The phos-
phate concentration could then be determined by means of the standard
dilution series.
UV
2.12. Analysis of cellular uptake by flow cytometry
O
O
O
O
O
O
NH
O
NO2
O
HeLa cells were grown in DMEM supplemented with antibiotics/
antimycotics and 10% heat-inactivated FBS. Cells were maintained at
37 °C in a 5% CO2 humidified air atmosphere and split twice weekly.
Cells were confirmed to be free from mycoplasm by periodical testing
with a MycoAlert® Mycoplasma Detection Kit (Lonza, Verviers, Belgium).
Cells were harvested by trypsin/EDTA treatment, counted and di-
luted in cold medium to a concentration of 106 cells/mL. Liposome
samples were diluted to 1 mM or 0.5 mM in PBS and 100 μL liposome
sample was pipetted in triplicate into a 96-well plate. 100 μL cell sus-
pension was added to the liposome suspensions and incubated 1 h at
P
O
O
O
O
S
45
NH
N
O
O
Fig. 1. Schematic overview of the UV-activatable Tat-liposome delivery system.
A
cell-penetrating peptide, Tat, is incorporated in a PEG-loop and anchored to the surface
of a liposome with two lipid anchors, of which one can be cleaved by UV-light. Upon irra-
diation, the UV-cleavable anchor is cleaved leading to opening of the loop and exposure of
the Tat-peptide. This enables uptake of the liposome as a result of the Tat-peptide being
able to interact with cells. Dark gray box = Tat-peptide, light gray = UV-cleavable linker,
black = PEG, yellow = fatty acids and blue = phosphate head groups.

90
M.B. Hansen et al. / Journal of Controlled Release 164 (2012) 87–94
which can now bind to cells and mediate the cellular internalization
of the entire liposome particle.
BOP as a coupling reagent. Completion of the coupling reaction was con-
firmed with a Kaiser-test and the peptides were cleaved off their resins
and treated as described above.
Hence, to incorporate Tat in a surface-anchored loop on a liposome
two alkyl anchors are required — a C-terminal and an N-terminal one.
C-terminal anchoring was achieved via conjugation of a C-terminal cys-
teine thiol (introduced in Tat for this purpose) to a maleimide-PEG2000
functionalized distearoyl phosphatidylethanolamine, which was intro-
duced into the liposomes during their preparation (see Materials and
methods). In this regard, liposomes containing 10 mol% PEG of which
5 mol% were terminated with a maleimide group were found to be
optimal for Tat-functionalization [56,72]. N-terminal anchoring was
achieved by coupling either a UV-cleavable or non-cleavable anchor to
the N-terminus of the Tat-peptide to afford a UV-activatable and
non-activatable Tat-peptide, respectively (see below). In addition, two
alkyl-chains with different lengths (C12 or C16) were used to prepare
the UV-cleavable (C12-UV-Tat and C16-UV-Tat) and non-cleavable an-
chors (C12-Tat and C16-Tat) to provide insight into the influence of the
alkyl chain-length on anchoring into the liposome.
3.3. Coupling of peptides to liposomes
Liposomes were prepared from egg-derived phosphatidylcholine
and 10 mol% PEG2000-distearoyl phosphatidylethanolamine of which
half of the PEG chains was terminated with maleimide and half with a
methoxy group [56]. The Tat peptides with UV-cleavable and non-
cleavable anchors were coupled to maleimide-functionalized liposomes
via their C-terminal cysteine residue (Fig. 2A). In addition, Tat and Tat
with three Arg→Ala mutations (Tat R>A) both without alkyl anchors
were included as a positive and negative control respectively. Coupling
of the peptides to the liposomes was evaluated by measuring the
zeta-potential of the liposomes (Fig. 2B).
Unfunctionalized liposomes have a negative zeta-potential in water
because of the presence of negatively charged phosphate head groups.
However, after coupling of the positively charged Tat peptides to the
liposomes all, except the Tat mutant (Tat R>A), had a positive zeta-
potential, which indicated that the couplings were successful. The Tat
mutant, on the other hand, yielded liposomes, which still had a negative
zeta-potential although less negative than unfunctionalized liposomes.
This might be due to the smaller positive net charge on the mutant Tat,
though a less efficient coupling cannot be ruled out.
3.1. Synthesis of the UV-cleavable anchor and its coupling to Tat
Two UV-cleavable anchors with different alkyl lengths (C12 and C16
)
were synthesized from a common and commercially available starting
material, 4-(bromomethyl)-3-nitrobenzoic acid 1 (Scheme 1).
First, the bromide was substituted with a hydroxy group under
basic, aqueous conditions affording 4-(hydroxymethyl)-3-nitrobenzoic
acid 2 in excellent yield [73]. Next, two different alkyl anchors were
introduced by coupling either dodecylamine or hexadecylamine
to the carboxyl group using BOP (benzotriazole-1-yl-oxy-tris-
[dimethylamino]-phosphonium hexafluorophosphate) as a coupling
reagent providing anchor 3a and 3b in good yields. Subsequently, the
two anchors 3a and 3b were activated with 4-nitrophenyl chloroformate
for coupling to the Tat peptide (Scheme 2).
The Tat peptide was prepared via solid phase peptide synthesis on
a Wang-resin using standard Fmoc chemistry [66]. After the final
Fmoc removal with 20% piperidine in DMF, the activated anchors 4a
and 4b were coupled to the free N-terminus using DMAP as a catalyst.
A negative Kaiser-test [67] confirmed that the coupling was complete.
Global deprotection and cleavage of the peptides 6a and 6b from the
solid support were achieved by suspension of the immobilized pep-
tides in trifluoroacetic acid. After removal of the resin by filtration,
the crude peptides were precipitated in diethyl ether, air-dried, ly-
ophilized and purified by HPLC affording the UV-anchor-Tat peptides
7a and 7b.
3.4. Cell-adhesion and uptake of Tat-liposomes
The cellular uptake of all Tat-liposome conjugates were investigated
via flow cytometry analysis using an encapsulated Atto655 dye as fluo-
rescent marker (Fig. 3). In this regard, irradiation experiments (supple-
mentary data) showed that a 2 min irradiation period was a good
optimum for sufficient cleavage of the UV-cleavable anchor without
bleaching the Atto655 dye too much; hence a 2 min irradiation period
was used whenever UV-mediated cleavage was required.
No unspecific adhesion was observed for unfunctionalized lipo-
somes (negative control), whereas substantial and dose-dependent ad-
herence was observed for Tat-modified liposomes (lip-Tat). Lip-Tat
(R>A) showed limited cellular uptake (Fig. 3). Importantly, cellular up-
take of lip-Tat-UV-C16 was marginal before irradiation. However, after
irradiation cell-adhesion increased more than 10-fold (more than
15-fold, when corrected for photo-bleaching) to the same high level
as observed for lip-Tat (positive control). The same trend was evident
in the cellular uptake of the liposomes as demonstrated by confocal mi-
croscopy (Fig. 4). This clearly showed cellular uptake of lip-Tat but bare-
ly any uptake of lip-Tat (R>A), and unfunctionalized liposomes were
undetectable. It should be noted, that the decreased adhesion of
Tat-liposomes observed after irradiation is merely a result of photo-
bleaching and does not reflect an actual decrease in adhesion. Together,
this implies that the cell-adhesion and uptake observed for the series of
Tat-liposomes is indeed mediated by functional Tat-peptides on the
liposomes.
3.2. Synthesis of Tat peptides with non-cleavable anchors
Tat peptides with non-cleavable C12 and C16 anchors were synthesized
and included in this study as negative controls, as these peptides were
expected to form loops, which cannot be opened by UV-irradiation. Tat
peptides with C12 and C16 anchors were synthesized by coupling lauric
acid and palmitic acid, respectively, to the free N-terminus of Tat using
Labeling of the cell membrane with CellMask showed some
co-localization with the liposomal dye (Atto655) suggesting that
some of the liposomes were adhered to the cell membrane and not
internalized. However, a large part of the liposomes were found in-
side the cells and separate from the membrane label, serving as a
proof that these liposomes are indeed taken up by the cells.
Br
O
OH
O
Na2CO3
BOP, DIPEA, H2NCnH2n+1
CH2Cl2, rt, 21 h
H2O-acetone (1:1)
reflux, 3 h
HO
HO
NO2
NO2
2 (98%)
1
A strong cell-adhesion before and after irradiation was also, but unex-
pectedly, observed for lip-Tat-C12 and lip-Tat-C16 as well as lip-Tat-
UV-C12 showed strong cell-adhesion before irradiation, suggesting that
shielding of the Tat-peptide was incomplete in these cases. However, in-
spection of the corresponding confocal images revealed the presence of
large aggregates on the surfaces of the cells in the case of lip-Tat-C12
and lip-Tat-C16 and modest aggregation in the case of lip-Tat-UV-C12.
In all these cases, the cells also seemed to be less viable (as discerned by
O
O
Cl
O
NO2
O
OH
O
O
O
NO2
Pyridine, Anhyd.
C, 5 h
NH
NH
n
n
NO2
NO2
3a, n=11 (90%)
3b, n=15 (92%)
4a, n = 11 (81%)
4b, n = 15 (78%)
Scheme 1. Synthesis of the UV-cleavable anchors.

M.B. Hansen et al. / Journal of Controlled Release 164 (2012) 87–94
91
O
Tat-Cys
NO2
Tat-Cys
NO2
O
O
O
Tat-Cys
4a or 4b, DMAP
90% TFA (aq.)
rt, 18 h
DMF, rt, 14 h
5
O
NH
O
NH
6a, n = 11
6b, n = 15
7a, n = 11 (27%)
7b, n = 15 (65%)
1) C11H23-COOH or C15H31-COOH,
BOP, DIPEA, DMF or CH2Cl2, 20 h, rt
Tat-Cys
O
Tat-Cys
5
2) 90% TFA (aq), rt, 18 h
8a, n = 10 (10%)
8b, n = 14 (19%)
Scheme 2. Coupling of UV-cleavable and non-cleavable anchors to Tat (Tat = YGRKKRRQRRRG).
the transmission images) than cells treated with the other Tat-liposome
conjugates. This implies that the observed cell-adhesion should not be
considered as a simple adhesion of the Tat-liposomes to the cells but
rather be regarded as the propensity to aggregate caused by incomplete
loop insertion of the N-alkyl chain into the liposome membrane, leading
to sedimentation of liposomes on cells rather than specific adherence
and uptake. Interestingly, this propensity seemed to decrease going
from lip-C12-Tat to lip-C16-Tat to UV-lip-C12-Tat suggesting that the
hydrophobic driving force for spontaneous insertion of the N-alkyl anchor
into the liposome membrane increases along this series. In the case of
lip-Tat-UV-C12, a substantial part of the Tat peptide was effectively
shielded in the loop conformation, as this system exhibited an increased
cell-adhesion after irradiation (even without taking photo-bleaching
into account). The improved shielding observed for lip-Tat-UV-C12 im-
plies that the aromatic ring in the UV-cleavable anchors provides a con-
siderable additional hydrophobic driving force for loop-formation.
Indeed, the lip-Tat-UV-C16, containing both the aromatic ring and the
longer alkyl-chain showed the most efficient anchoring as can be in-
ferred from the superior irradiation-triggered increase in cellular
uptake upon irradiation.
Fig. 2. Coupling of the Tat-peptides to liposomes. A) The Tat-peptides were coupled via
a C-terminal cysteine residue and a liposome-PEG-maleimide group. B) Because of the
positive charge of the Tat-peptides the coupling can be assessed as an increase in the
zeta-potential of the liposomes.
Fig. 3. Cellular uptake of Tat-liposomes. Flow cytometry study of the celluar uptake of the
different Tat-liposomes before and after irradiation. Two different concentrations of lipo-
somes were used – 0.5 and 1 mM (phosphate equivalents) – except for UV C16 for which
only 1 mM was measured.

92
M.B. Hansen et al. / Journal of Controlled Release 164 (2012) 87–94
Fig. 4. Cellular uptake and adhesion. The cellular uptake and adhesion of the different Tat-liposomes was studied by confocal laser scanning microscopy before (top) and after irradiation
(bottom). The liposomes were visualized by means of the encapsulated dye, Atto655 (red color). Transmission-fluorescence overlay images of cells treated with lip-Tat-C12 (top, lower
right corner) and lip-Tat-UV-C16 (bottom, lower right corner) are included as examples (overlay images of all experiments can be found in the Supplementary data).
used in the experiments is rich in bovine serum albumin (BSA), it
was speculated that this might play an important role in the aggrega-
tion process. In serum, the function of BSA is to bind fatty acids
(to solubilize these), so it is reasonable to expect that BSA can bind
to N-alkyl chains that failed to insert into the liposome membrane.
Since BSA has six fatty acid binding sites [74] cross-linking and aggrega-
tion of liposomes with non-inserted N-alkyl chains would be possible.
To test this hypothesis, lip-Tat-C12 were diluted into a HEPES buffer
containing BSA at the same concentration as in the medium. Immedi-
ately, the solution turned turbid and DLS showed the presence of big
particles (Fig. 5) strongly suggesting that BSA is profoundly involved
in the aggregation observed in confocal microscopy possibly by binding
to the non-inserted N-alkyl chains.
3.5. Dynamic light scattering — aggregation of liposomes
To shed light on the aggregation behavior of the Tat-liposomes
their sizes were monitored by dynamic light scattering (DLS) in buffer
and in cell medium. In HEPES buffer all liposomes had the expected
mean size of approximately 175 nm, though lip-Tat-UV-C16 and lip-
Tat-C16 showed a somewhat broader size distribution (PDIb0.25;
Fig. 5).
However, when the same DLS measurements were conducted in
cell medium, lip-Tat-C12- and lip-Tat-UV-C12 liposomes increased
markedly in size thus serving as a strong indication that the medium
is responsible for the aggregation observed in the confocal microsco-
py experiments. For lip-Tat-C16 the already broad size distribution
makes it difficult to draw firm conclusions about their aggregation be-
havior, though it seems that the size of these liposomes does not
increase as much as observed for lip-Tat-C12. Because the medium
Lip-Tat-UV-C16, on the other hand did not increase in size at all nei-
ther in HEPES buffer nor in medium indicating that the loop-formation
is complete in case of lip-Tat-UV-C16.

M.B. Hansen et al. / Journal of Controlled Release 164 (2012) 87–94
93
Fig. 5. Aggregration of Tat liposomes. Aggregation of lip-Tat-C12, lip-Tat-UV-C12-Tat, lip-Tat-C12 and lip-Tat-UV-C16 was monitored by dynamic light scattering in cell culture medium
and in HEPES buffer.
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4. Conclusions
Herein, we have described the synthesis of a UV-cleavable lipid-
anchor and demonstrated how this anchor can be used to constrain
a cell-penetrating peptide (CPP) onto a liposomal surface. Further,
we have shown that when the CPP was engaged in this loop forma-
tion no cellular adhesion or uptake did take place. However, upon irra-
diation opening of the loop and exposure of the CPP induced cellular
adhesion and uptake. The next step will be to explore other triggers to
release the constrained CPP such as enzymatic cleavage of the lipid
anchor.
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Acknowledgments
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Louis van Bloois is acknowledged for assistance during liposome
preparation. Prof. Joop van Zoelen, Inez Meijer and Walter van Rotterdam
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Appendix A. Supplementary data
Experimental details for the synthesis of the UV-cleavable anchors
including characterization data. Characterization data of all the pep-
tides. Additional confocal microscopy images. Supplementary data to
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