PH-sensitive PEG-based micelles for tumor targeting

Article abstract of DOI:10.3109/1061186X.2010.499466

A new acid sensitive nanocarrier based on lipid core micelles has been investigated for tumor targeted drug delivery. Sulfadimethoxine-PEG-phospholipid unimer (SD-PEG-DSPE) was designed to endow micelles with pH responsiveness in the physiopathologic rang

Full text of DOI:10.3109/1061186X.2010.499466

Journal of Drug Targeting, 2011, 19(4): 303–313
© 2011 Informa UK, Ltd.
ISSN 1061-186X print/ISSN 1029-2330 online
DOI: 10.3109/1061186X.2010.499466
ORIGINAL ARTICLE
pH-sensitive PEG-based micelles for tumor targeting
Stefano Salmaso, Sara Bersani, Marco Pirazzini, and Paolo Caliceti
Department of Pharmaceutical Sciences, University of Padova, Padova, Italy
Abstract
A new acid sensitive nanocarrier based on lipid core micelles has been investigated for tumor targeted drug delivery.
Sulfadimethoxine-PEG-phospholipid unimer (SD-PEG-DSPE) was designed to endow micelles with pH responsiveness
in the physiopathologic range. The unimer was synthesized according to a two-step procedure. Potentiometric
analysis showed that SD-PEG-DSPE has pK of 6.7. In water, the unimers assembled spontaneously in 20nm size
micelles with 60 μM critical micelle concentaration. The particle size was not affected by the pH in the 6.2–7.4 range.
The micelles loaded paclitaxel very efficiently and released the drug slowly regardless the incubation pH. Fluorescence
spectroscopy and cytofluorimetry carried out by MCF7 tumor cell incubation with labeled SD-PEG-DSPE micelles at
pH 7.4 and 6.2 showed that micelles associate with cells mostly at acidic pH with a time-dependent behavior. A cell
subpopulation took up the nanocarrier more efficiently at pH 6.2. Confocal microscopy confirmed that under these
conditions the systems are taken up by cells or fuse with cellular membrane. Cytotoxicity studies demonstrated that
the SD-PEG-DSPE micelles deliver more efficiently paclitaxel at pH 6.2 than at neutral pH confirming that the cell
internalization can be triggered by the external environmental conditions.
Keywords: pH-sensitive micelles, paclitaxel, nanocarrier, PEG-phospholipid colloidal systems
Introduction
properties often represent a hurdle to their systemic
administration and circulation in the blood stream. Poor
physicochemical features may provoke aggregation,
microvessel clogging and promote opsonization followed
by complement activation and removal by the RES (De
& Robinson, 2004; Makino et al. 2001). ese drawbacks
hamper their biopharmaceutical efficiency, application
reliability, and safety, which exclude their use as circu-
lating carriers for site-specific drug delivery (Verrecchia
et al., 1995; Owens 3rd & Peppas, 2006).
Amphiphilic materials that self assemble into
colloidal hydrophobic core/hydrophilic shell particles
have been successfully developed as drug carriers as
they can host poorly soluble and unstable drugs into the
core while drug release occurs by physical mechanism
which involve drug diffusion or particle disaggregation.
According to their physicochemical properties, these
systems can display long circulating properties and
disease site tropism thus enhancing the body exposure
to the drug. As an example, lipid core PEG nanocarriers
with diameter up to 300 nm have been investigated for
tumor targeting as they can accumulate into the tumor
In the last decades, nanoscience has been playing a
pivotal role in the investigation of novel biophysical
phenomena, processes, and materials at the nanome-
ter scale that find application in many fields of life and
health science. Nanotechnology advancements have
provided for a wide range of tools and devices with
unique features that can be properly exploited in thera-
peutic, diagnostic, and regenerative science. Accordingly,
physical or chemical combination of molecular modules
with different physicochemical and biological proper-
ties may afford nanocarriers with complex architectures
and “smart” behavior. Surface tailoring, for example, can
yield selective biological recognition and drug targeting
to specific tissues and avoid unwanted nanosystem inter-
face interactions with biological structures, namely cells
and proteins.
So far, a variety of nanospheres have been inves-
tigated and exploited for controlled release of drugs
with poor biopharmaceutical properties (Panyam &
Labhasetwar, 2003; Salmaso et al., 2009; Kumari et al.,
2010). Nevertheless, inadequate size, shape, and surface
Address for Correspondence: Stefano Salmaso, Department of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131
Padova, Italy. E-mail: stefano.salmaso@unipd.it
(Received 22 March 2010; revised 22 May 2010; accepted 24 May 2010)
303

304 Stefano Salmaso et al.
tissue by the enhanced permeability and retention
effect (Matsumura & Maeda, 1986; Yokoyama et al.,
1990; Lukyanov & Torchilin, 2004; Torchilin, 2007).
Nevertheless, due to the lack of cell recognition prop-
erties, plain micelles can distribute into the disease
site only by passive mechanisms that strongly limit the
selectivity of these formulations. Aimed at improving the
therapeutic performance of nanosystems, many efforts
have been dedicated to prepare smart carriers with cell
recognition moieties or environmentally sensitive mod-
ules that can target selectively tissues and cells, as well
as interact with them, undergo cell uptake, and release
the drug into specific compartments by physiopatho-
logical stimuli. Based on evidences that cancer tissues
have altered biological activity that affects temperature,
pH, enzyme composition, and redox activity, stimuli
sensitive colloidal drug delivery systems that exploit the
healthy tissue/disease tissue microenvironmental dif-
ferences have been designed for anticancer drug deliv-
ery (Tannock & Rotin, 1989; Gerweck & Seetharaman,
1996; Stefanadis et al., 2001). pH-sensitive nanoparticles
have interesting perspectives as anticancer drug deliv-
ery systems as they undergo increased cell associa-
tion under slightly acidic conditions, which mimic the
tumor interstitial conditions (Na et al., 2003). However,
since the pH environmental differences are very sharp,
nanosystems provided with adequate “molecular sen-
sors” having very precise responsiveness within narrow
environmental differences are required. A screening of
molecular sensors with pH sensitiveness that guaran-
tee for hydrophilic/hydrophobic switching suitable for
controlled drug release was first published by Bae and
co-workers (Kang & Bae, 2001, 2002). Sulfadimethoxine
(SD) has been associated to hardcore matrix–based
or hydrogels core–based nanoparticles to produce pH
responsive formulations. According to its pKa of 6.2, SD
can in fact undergo reversible pH-dependent protona-
tion that induces structural hydrophilic/hydrophobic
changes. erefore, the drug release rate from these
nanosystems is governed mostly by pH controlled core
shrinking (Na et al., 2004).
Although phospholipids-PEG pH responsive micelles
offernewinterestingopportunitiestotheanticancerdrug
delivery, there is still a lack of investigation about the
physicochemical requisites that dictate the biopharma-
ceutical behavior and benefits of these systems. In order
to advance the knowledge on the key parameters that
affect the drug delivery properties of pH-sensitive colloi-
dal systems, new phospholipid-PEG micelles decorated
with multiple copies of a pH responsive moiety have
been produced. e pH-sensitive molecule was selected
to modulate the surface properties of the nanocarrier
(namely hydrophilic/hydrophobic features) according
to narrow pH changes throughout the body districts. In
vitro studies were carried out using paclitaxel as anti-
cancer drug model to examine the physicochemical and
biopharmaceutical properties of these systems and their
effective potential as drug delivery systems.
Materials and methods
Materials
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-
[N-Hydroxysuccinimidyl (poly(ethylene glycol))-3400]
(NHS-PEG3.4 kDa-DSPE) and 1,2-distearoyl-sn-glycero-
3-phosphoethanolamine-N-[methoxy (poly(ethylene
glycol))5000] (mPEG5 kDa-DSPE) were purchased
from Laysan Bio Inc. (Arab, AL). Paclitaxel was
from LC Laboratories (Woburn, MA). 1,2-Dioleoyl-
sn-glycero-3-phosphoethanolamine-fluorescein
(fluorescein-DOPE), pyrene, MTT, cell culture media,
penicillin-streptomicin-amphotericin
B
solution,
trypsinsolution, fetalbovineserum(FBS)wereobtained
from Sigma (St. Louis, MO). SD, succinic anhydride and
1,2-diaminoethane (DAE) were purchased from Fluka
(Buchs, Switzerland). All analytical grade and deuter-
ated solvents were obtained from Aldrich (Ostheim,
Germany).
Sephadex G 25 superfine medium was provided by
GE Healthcare Bio-sciences AB (Uppsala, Sweden).
Human breast adenocarcinoma MCF7 cells were cul-
tured in RPMI-1640 medium supplemented with 10%
fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin,
100 μg/mL streptomycin, 0.25 μg/mL amphotericin B.
Cells were routinely treated with a 500 μg/mL trypsin
solution containing 2.5 mg/mL EDTA in Ca2+ and Mg2+
free 0.02 M phosphate buffer, 0.15 M NaCl, pH 7.4 and
transferred on a new flask.
Synthesis of SD-succinate
SD (2.0 g, 6.4 mmol) was dissolved in 45mL of anhydrous
acetone. e solution was added of succinic anhydride
(3.2 g, 32 mmol) and stirred overnight under anhydrous
conditions. e white precipitate was recovered by cen-
trifugation and washed three times with acetone. e
product was dried under vacuum and recovered at 62.6%
yield. in-layer chromatography of the product was
carried out on glass sheets coated with silica gel (Merck
F-254; Merck, Darmstadt, Germany). e plates were run
with CHCl3/methanol 12/1 volume ratio and detected by
254 nm UV light. SD-succinate (SD-succ) and SD showed
Rf of 0.85 and 0.89, respectively. e purity of the interme-
diate was evaluated by RP-HPLC using a Shimadzu SP-10
HPLC system equipped with a Phenomenex Luna C18
column (250 × 4.6 mm) eluted in a gradient mode, using
water-0.05% trifluoroacetic acid (TFA) (eluent A) and
acetonitrile-0.05% TFA (eluent B), from 15% to 50% eluent
B in 17 min. e UV detector was set at 268 nm. SD-succ
was found to have 98% purity. e product was analyzed
by 1H NMR spectroscopy using a Bruker Spectrospin 300,
300 MHz (Bruker Spectrospin, Billerica, MA).
1H NMR in DMSO-d6: δ 2.60 (t, J = 6.2 Hz, 2 H of
succinic acid -NH-CO-CH2-CH2-); δ 2.67 (t, J = 6.3 Hz,
2 H of succinic acid NH-CO-CH2-CH2-); δ 3.35 (s, 1 H of
aromatic anilinamide -NH-COO-CH2-); δ 3.75 and 3.79
(ds, 6 H, -OCH3 of pyrimidinic ring); δ 5.93 (s, 1 H, pyrim-
idinic ring); δ 7.88 (d, J = 9.2 Hz, 2 H, protons of aromatic
Journal of Drug Targeting

pH-sensitive PEG-based micelles for tumor targeting 305
anilinamide ring); δ 7.74 (d, J= 9.2, 2 H, protons of aro-
matic anilinamide ring); δ 10.40 (s, 1 H of -NHSO2-).
e final product (SD-succ) dissolved in methanol
(1 mg/mL) was analyzed by mass spectroscopy using
ring); δ 5.59 (s, 1 H, pyrimidinic ring); δ 7.66 (d, J = 8.6 Hz,
2H, protons of anilinamide ring); δ 7.58 (d, J = 8.8 Hz, 2 H,
protons of anilinamide ring); δ 8.08 (t, J= 5.5, 1 H, NH2-
CH2CH2-NHCO-); δ 10.16 (s, 1 H, -NHSO2-).
a
Mariner ESI-TOF system (PerSeptive Biosystems,
ESI-TOF mass spectroscopy showed m/z signal at
453.16 and at 227.09 m/z corresponding to the mono
and double charged ions, respectively [calculated for (M
+H)+: 453.16; calculated for (M +2H)2+: 227.08].
Framingham, MA). e positive ion was found to have
411.10 m/z [calculated for (M +H)+: 411.09].
Synthesis of SD-succ methyl ester (SD-succ-OCH3)
SD-succ (400 mg, 973 μmol) was dissolved in 25 mL
of methanol in presence of 500 μL of acetyl chloride
(9.7 mmol). After 30-min reaction, NaHCO3 (706 mg,
8.40 mmol) was added to quench the hydrogen chloride
released by the esterification reaction. e methanolic
solution was evaporated under reduced pressure. A total
recovery of the product was achieved.
1H NMR in DMSO-d6: δ2.60 (t, J = 6.2 Hz, 2 H of succinic
acid NH-CO-CH2-CH2-); δ 2.67 (t, J = 6.3 Hz, 2 H of suc-
cinic acid NH-CO-CH2-CH2-); δ 3.35 (s, 1 H of aromatic
anilinamide -NH-COO-CH2-); δ 3.58 (s, 3 H, -CO-OCH3);
δ 3.75 and 3.79 (ds, 6 H, -OCH3 of pyrimidinic ring); δ
5.93 (s, 1 H, pyrimidinic ring); δ 7.88 (d, J = 9.2, 2 H, pro-
tons of aromatic anilinamide ring); δ 7.74 (d, J = 9.2, 2H,
protons of aromatic anilinamide ring); δ 10.40 (s, 1 H of
-NHSO2-).
Synthesis of sulfadimetoxine-PEG-DSPE
(SD-PEG-DSPE)
SD-succ-ethyl-NH2 (125 mg, 277 μmol) was dissolved in
3 mL of anhydrous DMF and added of 38 μL of trieth-
ylamine (279 μmol) and 200 mg of commercial NHS-
PEG3.4 kDa-DSPE (46.1 μmol). e mixture was maintained
overnight under stirring and then the crude was recov-
ered by precipitation in cold anhydrous diethylether. e
precipitate was dissolved in 2 mL of dichloromethane,
centrifuged at 5000 rpm for 5 min and the organic solvent
was removed under vacuum. e dry film was rehydrated
by adding 5 mL of 5 mM NH4Cl, pH 8.0, and the conju-
gate was purified by size exclusion chromatography with
a column prepacked with Sephadex G 25 superfine resin
eluted with 5 mM NH4Cl, pH 8.0. Fractions positive to
both UV-Vis analysis at 268 nm and Iodine test (Sims &
Snape, 1980) were pooled together and lyophilized. e
recovery yield was 62%.
e PEG/SD molar ratio in the conjugate was assessed
by quantitative Iodine test for PEG and UV spectroscopic
analysis for SD (Sadagopa Ramanujam et al., 1980). e
SD/PEG molar ratio was found to be 0.79/1. e conjugate
purity was estimated by RP-HPLC that confirmed that
no traces of SD-succ-ethyl-NH2 were detectable. e
conjugate purity was 93%.
e chemical identity of the SD-succ-OCH3 was
confirmed by ESI-TOF mass spectrometry that showed
m/z signal of 425.14 [calculated for (M +H+): 425.12]. e
purity of the intermediate was evaluated by RP-HPLC as
reported above and was found to be 95.5%.
Synthesis of SD-succ-CONH(CH2)2NH2
(SD-succ-ethyl-NH2)
SD-succ-OCH3 (413 mg,971μmol)wasdissolvedin12 mL
of dimethyl formamide and the solution was added of
1.31 mL of DAE (19.6 mmol) and 0.15 mL of acetic acid
(973 μmol). e reaction was stirred for 6 h at 37°C under
anhydrous conditions. e mixture was poured dropwise
into cold anhydrous diethyl ether under stirring and the
precipitate was washed three times with diethyl ether
and desiccated under vacuum. e white powder was
dissolved in 10 mL of water and purified by solid phase
extraction with a SEP-PAK C18 cartridge from Millipore
(Milford, MA) eluted with 20 mL of water to remove unre-
acted 1,4-diaminoethane and then washed extensively
with methanol. e organic solvent was removed under
vacuum giving a final product yield of 120 mg (27.3%).
e derivative was analyzed by thin-layer chromatogra-
phy carried out on glass sheets coated with silica gel run
by methanol. e products were detected with 254 nm
UV light (SD-succ Rf = 0.85; SD-succ-ethyl-NH2 Rf = 0.29).
e purity of SD-succ-ethyl-NH2, assessed by RP-HPLC
as described above, was 84%.
1H NMR in CDCl3: δ 0.87 (m, 10 H, -CH2-CH3 of
phospholipid alkyl chains); δ 1.25 (m, ~52 H -(CH2)n- of
phospholipid alkyl chains); δ 1.58 (m, 4 H, -COOCH2-
CH2- of phospholipid alkyl chains); δ 2.26–3.31 (m, 4 H,
-COOCH2-CH2 of phospholipid alkyl chains); δ 3.64 (s,
309 H, -OCH2CH2O- of PEG); δ 3.86 and 3.89 (ds, 4.6 H,
-OCH3 of pyrimidinic ring of SD); δ 7.88 (d, J = 8.6 Hz, 2
H, protons of anilinamide ring); δ 7.80 (d, J = 8.6 Hz, 2 H,
protons of anilinamide ring).
pKa assessment of SD-PEG-DSPE
SD-PEG-DSPE(100.0mg,23.1μmol)wasdissolvedin20mL
of HCl 0.15M. e potentiometric analysis was carried
out by stepwise addition of 0.15M NaOH to the SD-PEG-
DSPE solution using a radiometer ABU93 Triburette titra-
tor (Radiometer Analytical, Villeurbanne Cedex, France)
equippedwiththreeautomaticregulatedburettes, adouble
internal voltmeter, and a temperature controller.
1H NMR in DMSO-d6: δ 2.42 (t, J = 6.8, 2 H of succinic
acid -CH2-CH2-); δ 2.59 (t, J = 6.9, 2 H of succinic acid
-CH2-CH2-); δ 2.84 (t, J = 6.3, 2 H, diaminoethane -CH2-
CH2-NH2); δ 3.27 (m, J = 6.2 Hz, 2 H, diaminoethane -CH2-
CH2-NH2); δ3.62 and δ3.65 (ds, 6 H, -OCH3 of pyrimidinic
Micelles preparation
Micelles were prepared by film rehydration as reported
by Zhang and co-workers (2003). SD-PEG-DSPE (10 mg)
was dissolved in 10 mL of chloroform. e organic solu-
tion was removed under vacuum by rotary evaporation.
© 2011 Informa UK, Ltd.

306 Stefano Salmaso et al.
e dry thin layer was rehydrated with 5mL of 0.02 M
phosphate buffer, 0.15 M NaCl, pH 7.4.
Plain micelles were prepared as reported above using
mPEG5 kDa-DSPE.
For biological studies, SD-PEG-DSPE micelles were
prepared as reported above using 1% fluorescein-DOPE/
SD-PEG-DSPE molar ratio. e dry thin layer was rehy-
drated with RPMI-1640 at a final 1 mg/mL SD-PEG-DSPE
concentration.
split in 2 mL samples. One of the samples was adjusted to
pH 6.2 by 1 M HCl addition. e two samples were placed
into a Spectra/Por regenerated cellulose 3.6kDa cut-off
dialysis membrane (Spectrum Laboratories Inc., Rancho
Dominguez, CA) and dialyzed against 45 mL of 20 mM
phosphate 0.15 M NaCl at the same pH and at 37°C. At
scheduled intervals the receiving buffer volume was
replaced with fresh buffer. Paclitaxel was extracted from
the removed volume with 5 mL of dichloromethane for
four times. e organic solvent was removed under vac-
uum and the residue redissolved in 300 μL of methanol
and analyzed by RP-HPLC as reported above for pacli-
taxel estimation. e extraction efficiency of the method
was validated to be 97.4%
Determination of micellar hydrodynamic diameter
and size distribution
Micelles size and size distribution were measured by
dynamic light scattering analysis. Micelles at a concen-
tration of 2 mg/mL in 0.02 M phosphate buffer, 0.15 M
NaCl were used at pH 7.4, and the sample solutions were
also adjusted to pH 6.5 and 6.2 by 1M HCl addition.
e micelles were filtered with a 0.45 μm cut-off mem-
brane and analyzed using a Zetasizer Nano S instrument
(Malvern Instruments, Malvern, UK) by detecting the
backscatter of the beam at an angle of 173°.
Micelle biocompatibility
MCF7 cells were seeded in a 96-well plates (5000 cells/well)
and grown for 24 h in complete RPMI-1640 medium.
Afterwards, the medium was replaced with RPMI-1640 at
pH 6.2 and 7.4 containing increasing concentrations of
either SD-PEG-DSPE or mPEG-DSPE and the cells were
incubated at 37°C for 48 h. Each well was added of 20
μL of 5 mg/mL MTT in 20 mM phosphate buffer, 0.15 M
NaCl, pH 7.4, and the plates were incubated for further
4 h at 37°C. e medium was then carefully discharged
from each well and replaced with 200 μL of DMSO. e
plates were gently shaken overnight in order to dissolve
the formazan crystals and absorbance was measured
by a Bio-Tek Instruments microplate reader (Highland,
VT) at 570 nm and at 620 nm as reference. e cell viabil-
ity was expressed as percentage of the 570 nm/620 nm
absorbance ratio (Hansen et al., 1989).
Critical micelle concentration
Critical micelle concentration (CMC) was estimated
according to the method reported in the literature (Kwon
et al., 1993). Briefly, aliquots of 50 μL of a 10 mg/mL
pyrene solution in methanol were spiked in tubes and the
organic solvent was removed under vacuum. e tubes
were added of different volumes of 2 mg/mL of a SD-PEG-
DSPE solution in 20 mM phosphate buffer, 0.15 M NaCl
at 7.4, and buffer was added to yield 1 mL final volume
in order to have final concentrations of unimers rang-
ing from 0 mg/mL to 2 mg/mL. e tubes were shacked
at 37°C overnight and then centrifuged at 10,000 rpm for
5 min. e solution was analyzed by fluorescence (λex
339 nm; λem 390 nm). e CMC value was referred to the
concentration of the unimer at which a sharp increase in
fluorescence was observed. For comparison, the CMC of
commercial mPEG5 kDa-DSPE was investigated.
Cell uptake study by fluorimetric analysis of lysates
MCF7 cells were seeded in 6-well tissue culture plates
(1.5×106 cells/well) and grown for 24h in complete
RPMI-1640 medium. e cells were washed with RPMI-
1640 and 2mL of 1% fluorescein-DOPE-labeled SD-PEG-
DSPE or mPEG-DSPE micelles in 20mM phosphate
buffer, 0.15M NaCl at pH 7.4 or pH 6.2 (1mg/mL) was
added. e plates were incubated at 37°C. At scheduled
intervals, the medium was removed, the samples were
washed three times with 20mM phosphate buffer, 0.15M
NaCl at the same pH and detached by ice contact treat-
ment and pipetting (Salmaso et al., 2009). e cells were
recovered by centrifugation, lysed by addition of 1mL of
0.1% triton in water and the lysates were analyzed by fluo-
rescence spectroscopy (λex 494nm, λem 520nm). e rela-
tive fluorescence was normalized to the cell content that
was calculated according to the protein concentration of
lysated cells by BCA analysis (Pierce Biotechnology, Inc.,
Rockford, IL).
Paclitaxel-loaded micelle preparation
SD-PEG-DSPE (20.4 mg) was dissolved in 3 mL of
chloroform and the organic solution was added of 1mL
of methanol containing 2.6 mg of paclitaxel. e solvents
wereremovedbyrotaryevaporationandtheresultingthin
layer was rehydrated in 20 mM phosphate buffer, 0.15 M
NaCl, pH 7.4 to a final SD-PEG-DSPE concentration of
5 mg/mL. e colloidal dispersion was filtered through a
0.22 μm pore size membrane and the paclitaxel content
in the solution was determined by RP-HPLC using a
Phenomenex Luna C18 column isocratically eluted with
acetonitrile/H2O 55/45 vol/vol containing 0.05% TFA.
e UV detector was set at 227 nm and the peak area was
referred to a standard curve.
Cytofluorimetric analysis
MCF7 cells were seeded in
a 12-well plate at a
Paclitaxel release studies
4 mL Paclitaxel-loaded micelles in 20 mM phosphate buf-
fer, 0.15 M NaCl pH 7.4 (SD-PEG-DSPE 5 mg/mL) were
concentration of 5×105 cells/well and grown for
48h. e medium was discharged and the cells were
incubated with 0.5mL/well of medium at pH 7.4 or
Journal of Drug Targeting

pH-sensitive PEG-based micelles for tumor targeting 307
6.2 containing 1mg/mL of 1% fluorescein-DOPE-labeled
SD-PEG-DSPE or mPEG-DSPE micelles. e plates were
incubatedfor4hat37°Candthenthemediumwasremoved,
thecellswashedtwicewith20mMphosphatebuffer,0.15M
NaClatthesamepHofincubationanddetachedbyicecold
contact treatment and pipetting. e cells were collected
by centrifugation at 1000rpm for 5min and resuspended in
1% paraformaldehyde in 20mM phosphate buffer, 0.15M
NaCl, pH 7.4. e samples were analyzed by cytofluo-
rimetry with a FACSCalibur Flow Cytometer (Becton
Dickinson, San Jose, CA). e experiment was performed
in triplicate.
for further 48 h. Cell viability was assessed by the MTT
test as reported above.
Results
Synthesis and chemical characterization of SD-PEG-
DSPE
e preparation of SD-PEG-DSPE was carried out
accordingtoatwo-stepprocedure:(1)synthesisofSD-succ-
CONH(CH2)2NH2 (SD-succ-ethyl-NH2); (2) conjugation
of SD-succ-ethyl-NH2 to commercially available NHS-
PEG3.4 kDa-DSPE.
Scheme 1 describes the synthesis of SD-succ-ethyl-
NH2. is product was obtained by the SD aniline group
amidation by reaction with succinic anhydride, carboxyl
group activation to methyl ester and finally reaction
with an excess of 1,2-diaminoethane that allowed for
the introduction of an amino group in the SD structure.
RP-HPLC, 1H NMR, and ESI-TOF analysis demonstrated
that the SD succinylation was quantitative yielding a
high purity acid intermediate. Also, the subsequent
esterification with methanol was found to be a very effi-
cient reaction. Unreacted acetyl chloride was quenched
by NaHCO3. e molecule amination was finally carried
out by treatment of the methyl ester activated derivative
with 20-fold molar excess of 1,2-diaminoethane. Such
an excess was necessary in order to avoid unwanted
cross-linking reactions. Acetic acid was added to the
reaction mixture in order to preserve the SD-succ
integrity as it can easily undergo hydrolysis (data not
shown). As diethyl ether extraction did not allow for
complete elimination of unreacted 1,2-diaminoethane,
an additional purification step was efficiently carried
out by solid phase extraction. e identity of SD-succ-
ethyl-NH2 and its purity degree were confirmed by
Confocal microscopy
After the initial passage in tissue culture flasks, MCF7
cells were seeded in 4-well tissue culture detachable
BD-Falcon CultureSlides chambers at a concentration of
2 × 105 cells per well and grown in RPMI-1640 with 10%
FBS. After 24 h the chambers were washed twice with
RPMI and then incubated at 37°C with 0.5 mL of 1 mg/mL
1% fluorescein-DOPE-labeled SD-PEG-DSPE or mPEG-
DSPE micelles in RPMI at pH 7.4 or 6.2. After 2 h, the
medium was removed, the samples were washed three
times with 20 mM phosphate buffer, 0.15 M NaCl at the
same pH of incubation and the cells were fixed by 15 min
contact with 0.5 mL of 1% paraformaldehyde in 20 mM
phosphate buffer, 0.15 M NaCl, pH 7.4. Individual cov-
erslips were mounted with glass slides using Vectashield
mounting medium with DAPI for nuclei staining (Vector
Laboratories, Burlingame, CA). ecellswereanalyzedby
confocal scanning laser microscope Leica TCS SP5 (Leica
Microsystems CMS, Mannheim, Germany) equipped
with a Leica HCX PL APO 63X/1.40 oil immersion objec-
tive. e blue fluorescence for DAPI was detected with
351 nm laser illumination and green fluorescence for
fluorescein-DOPE with 488 nm laser illumination.
1
chromatographic and spectrometric analysis. H NMR
and ESI-TOF spectra confirmed the product chemi-
cal identity. RP-HPLC chromatogram and the ESI-TOF
spectra showed that the reaction yielded a product with
high purity degree.
Scheme 2 describes the synthesis of SD-PEG-DSPE
that was carried out by reaction of commercial available
NHS-PEG3.4 kDa-DSPE with a 20-fold molar excess of
SD-succ-NH2. Large amount of SD-succ-ethyl-NH2 was
used to maximize the product yield. e crude product
precipitation in diethyl ether and rehydratation in water
In vitro selective drug delivery
MCF7 cells were seeded at a concentration of 5 × 103
cells/well on a 96-well plate in RPMI-1640 supple-
mented with 10% FBS and grown for 24 h. Afterwards,
the medium was replaced with medium containing
1 mg/mL of 0–50 nM paclitaxel-loaded SD-PEG-DSPE
or mPEG-DSPE micelles at pH 7.4 or 6.2. e cells were
incubated for 12 h and then the medium was replaced
with fresh complete medium and the cells were grown
O
O
O
H
N
NH2
OCH3
OH
HN
HN
HN
NH2
O
O
O
O
Succinic
anhydride
O
O
acetyl chloride
methanol
O
S
S
S
S
NH
DAE
O
NH
O
NH
O
O
NH
N
N
N
N
H3CO
N
OCH3
N
H3CO
OCH3
N
OCH3
H3CO
H3CO
OCH3
N
Scheme 1. Synthesis of SD-succ-ethyl-NH2.
© 2011 Informa UK, Ltd.

308 Stefano Salmaso et al.
O
O
O
NH
O
O
NH2
HN
S
8
O
8
+
O
P
O
O
HO
O
O
O
NH
O
N
O
N
O
NH
O
77
N
H3CO
OCH3
O
O
DMF
TEA
OCH3
H3CO
N
O
O
N
8
HN
S
O
O
8
O
O
P
HO
O
O
O
O
O
NH
NH
NH
O
O
NH
77
O
Scheme 2. Synthesis of SD-PEG-DSPE.
allowed for micelle assembling and their fractionation
by size exclusion chromatography. Gel chromatography
was carried out under slightly basic conditions (pH 8.0)
that avoided the possible micelle association into mac-
roaggregates. e RP-HPLC analysis showed that the
purification process allowed for complete elimination of
the low molecular weight unconjugated SD-succ-ethyl-
NH2. e chemical identity of the purified conjugate was
14
12
10
8
6
1
confirmed by H NMR spectroscopy. Furthermore, the
4
1H NMR analysis showed that the reaction yielded 77%
NHS-PEG-DSPE conjugation with SD, which is in good
agreement with the results obtained by UV-Vis analysis.
e NHS-PEG-DSPE derivatization was very high in
consideration that the starting material was 91% NHS-
activated PEG-DSPE (provider information). According
to the analytical results the final product contained 23%
of inactivated PEG-DSPE (PEG-DSPE/SD-PEG-DSPE
molar percentage).
2
0
0
0,5
1
1,5
2
NaOH 0.15 M (mL)
Figure 1. Potentiometric titration of SD-PEG-DSPE dissolved in HCl
solution.
e data summarized in Table 1 show that neither the
micelle size nor the size distribution were affected by the
pH of the colloidal dispersion.
e CMC was found to be 60 μM, which is slightly
higher as compared to the values reported in the litera-
ture for PEG-phospholipid with similar molecular weight
(Lukyanov et al., 2002).
Physicochemical characterization of SD-PEG-DSPE
Potentiometric analysis was undertaken to investigate
acid/base properties of the new SD derivatized phos-
pholipid (Öhman & Sjöberg, 1996). Figure 1 reports the
potentiometric profile. e bioconjugate was found to
possess a pKa of 6.7 0.1, which is fairly close to the value
reported in literature for SD (Kang & Bae, 2002).
SD-PEG-DSPE biocompatibility
Biocompatibility studies were carried out by MCF7 cell
incubation with a wide range of SD-PEG-DSPE concen-
trations in order to examine the biological effect of the
phospholipids derivative either below or above its CMC
value. Furthermore, the study was performed at pH 7.4
and 6.2 to evaluate the material impact under differ-
ent physiopathological conditions. As control, mPEG-
Physical characterization of micelles
e dynamic light scattering profile reported in Figure 2
shows that SD-PEG-DSPE assembles into micelles with
~20 nm mean diameter and narrow size distribution
(PDI 0.35–0.619). Micelle size was in fair agreement with
data reported in the literature for micelles composed of
PEG-phospholipid with similar PEG molecular weight.
Journal of Drug Targeting

pH-sensitive PEG-based micelles for tumor targeting 309
A
25
20
15
10
5
100
80
60
40
20
0
1
10
100
Size (nm)
1000
10000
0
50
100
150
200
250
300
Figure 2. SD-PEG-DSPE-based micelle size analysis in 0.02M
phosphate buffer, 0.15M NaCl, pH 7.4.
SD-PEG-DSPE (µM)
B
100
80
60
40
20
0
Table 1. Micelle size (mean diameter SD, nm) at pH 7.4, 6.5 and 6.2.
pH conditions
Size (nm)
19.96 11.80
19.93 13.66
19.05 14.98
7.4
6.5
6.2
DSPE biocompatibility was investigated under the same
experimental conditions.
e cell viability profiles described in Figure 3 show
that neither SD-PEG-DSPE (panel A) nor mPEG-DSPE
(panel B) elicited cytotoxicity, regardless the unimer
concentration or the incubation pH.
0
50
100
150
200
250
300
mPEG-DSPE (µM)
Figure 3. SD-PEG-DSPE (A) and mPEG-DSPE (B) intrinsic cytotoxicity
at pH 6.2 (○) and pH 7.4 (▪) after 48h contact time.
micelles at pH 7.4 (2.1%), the MFI (178) of the former was
significantly high.
Cell uptake studies
eassociationofmicelle/cellatpH7.4and6.2wasinves-
tigatedbyMCF7cellincubationwithfluorescein-labeled
SD-PEG-DSPE. Fluorescein-labeled mPEG-DSPE was
used as control. e fluorescence values obtained from
MCF7 cells lysates are depicted in Figure 4.
e results reported in Figure 4A and B show that
the fluorescein-labeled mPEG-DSPE micelles were only
slightly taken up by the cells either at pH 7.4 or 6.2. Also
the cell uptake of fluorescein-labeled SD-PEG-DSPE
micelles was negligible when incubation was carried out
at pH 7.4. On the contrary, at pH 6.2, fluorescein-labeled
SD-PEG-DSPE-based micelles were significantly taken
up by the cells according to a time-dependent behavior.
e steady cell uptake was reached in about 5 h. At this
time point the cell-associated fluorescence obtained
with the SD-PEG-DSPE derivative was 4.8 times higher
as compared to that obtained with mPEG-DSPE.
Flow cytometry studies (FACS) partially confirmed the
data obtained by fluorescence analysis. e dot plot pro-
files reported in Figure 5 show that 97.8% and 97.3% of
MCF7 cells incubated with mPEG-DSPE micelles at pH
7.4 and 6.2, respectively, have a mean fluorescence inten-
sity (MFI) lower than 70 (19.3 at pH 7.4 and 9.5 at pH 6.2).
In the case of SD-PEG-DSPE micelles at pH 7.4, 97.9%
of cells had an MFI of 34.0 while after incubation at pH
6.2, 94.0% of cells displayed an MFI of 32. Despite these
results indicate that the number of cells involved in the
SD-PEG-DSPE micelles uptake at pH 6.2 was only slightly
higher (6%) than that incubated with mPEG-DSPE (2.2
and 2.7 at pH 7.4 and 6.2, respectively) and SD-PEG-DSPE
e confocal analysis performed with MCF7 cells
incubated with fluorescently labeled micelles at two pH
conditions, 7.4 and 6.2, confirmed the results obtained
by flow cytometry. e images reported in Figure 6 show
that diffused feeble cell fluorescence was obtained at both
incubation pH. Nevertheless, at pH 6.2 few cells displayed
high fluorescence (Figure 6, panel A). Interestingly, the
fluorescein-labeled mPEG-DSPE micelles used as con-
trol displayed undetectable fluorescence at both pHs and
no cell subpopulation was observed.
Paclitaxel loading and release study
e preparation of paclitaxel-loaded SD-PEG-DSPE
micelles resulted in 2.5% drug incorporation (paclitaxel/
SD-PEG-DSPE, w/w), which is similar to the payload
obtained with mPEG-DSPE micelles reported in the
literature (Gao et al., 2002, 2003). e drug release from
the micelles was investigated at pH 7.4 and 6.2, which
mimic the blood and the tumor interstitial compartment
microenvironment, respectively. In order to guarantee
sink conditions throughout the study, the releasing buffer
was frequently replaced with fresh medium.
e profiles depicted in Figure 7 show that paclitaxel
is released from the micelles with similar behavior at pH
7.4 and 6.2. About 5% of the loaded drug was released
in 12 h and about 50% in 200 h. Preliminary studies
demonstrated that paclitaxel in a micelle-free solution
freely diffused through the dialysis membrane as 100%
of the incubated drug was dialyzed in 10 h. erefore,
© 2011 Informa UK, Ltd.

310 Stefano Salmaso et al.
the slow paclitaxel appearance in the releasing medium
could not be attributed to drug/dialysis membrane
interaction phenomena.
CellculturestudiescarriedoutbyMCF7cellincubation
with paclitaxel-loaded SD-PEG-DSPE micelles at pH 6.2
and 7.4 were undertaken to evaluate the effect of the SD
decoration on the drug delivery properties of the colloidal
formulation. e cell culture protocol was adapted to
emphasize the pH-sensitive micelle contribution to the
drug uptake. As compared to other protocols reported in
the literature, a short exposition to drug-loaded carrier
could provide for a better discrimination between the
two pH conditions (Michalakis et al., 2005; Nonaka et al.,
2006). As a control, studies were performed under the
same conditions using paclitaxel-loaded mPEG-DSPE
micelles. e results reported in Figure 8 show that the
former have higher toxic effect than the latter. ese
results are in fair agreement with the higher cell interac-
tion properties of SD-PEG-DSPE micelles as compared
to the mPEG-DSPE formulations. However, though the
fluorescence studies showed that the SD-PEG-DSPE
micellesaretakenupbycellsaccordingtoapH-dependent
mechanism, the cytotoxicity studies showed only slight
differences in the drug delivery performance between
pH 7.4 and 6.2. At acidic condition, paclitaxel-loaded
SD-PEG-DSPE micelles have EC50 of 2 nM which is very
close to the values obtained with paclitaxel in solution,
while at pH 7.4 the EC50 value was 5 nM (Figure 8, panel
A). Control micelles displayed higher EC50 values: 10 and
15 nM at pH 7.4 and 6.2, respectively (Figure 8, panel B).
A
1
0,8
0,6
0,4
0,2
0
30
80
120
180
240
300
Contant time (min)
B
1
0,8
0,6
0,4
0,2
0
Discussion
Poly(ethylene glycol) phospholipids were conjugated
with SD to obtain pH-sensitive amphiphilic derivatives
(SD-PEG-DSPE) that can reversibly switch from the
ionized to the deionized form in a narrow pH range.
According to the SD pKa, the phospholipids derivatives
may undergo hydrophilic/hydrophobic changes within
the physiopathological microenvironmental conditions.
erefore, SD-PEG-DSPE micelles can be triggered into
the tumor site, where the environment is mildly acidic
as compared to blood, providing for site selective drug
delivery (Vaupel et al., 1989).
30
80
120
180
240
300
Contant time (min)
Figure 4. Time course fluorescence profiles of lysated MCF7 cells
after cell incubation with fluorescein-DOPE-labeled SD-PEG-
DSPE (A) and mPEG-DSPE (B) micelles. e cells were incubated
at pH 6.2 (dotted columns) and 7.4 (dashed columns).
A
1000
800
600
400
200
0
B
1000
800
600
400
200
0
C
1000
800
600
400
200
0
2.2%
2.7%
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Fluoresecence intensity
Fluoresecence intensity
Fluoresecence intensity
F
1000
800
600
400
200
0
E
1000
800
600
400
200
0
D
1000
800
600
400
200
0
2.1%
6%
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Fluoresecence intensity
Fluoresecence intensity
Fluoresecence intensity
Figure 5. FACS analysis of MCF7 cells incubated without carrier at pH 7.4 and 6.2 (panels A and D, respectively). Cells were treated with
fluorescein-labeled mPEG-DSPE (panel B and C) and SD-PEG-PE micelles (panel E and F) at pH 7.4 (panel B and E) and 6.2 (panel C and F).
Journal of Drug Targeting

pH-sensitive PEG-based micelles for tumor targeting 311
A
100
A
B
80
60
40
20
0
0
5
10
15
20
25
30
Figure 6. Confocal microscopic examination of MCF7 treated
with SD-PEG-DSPE micelles 1% DOPE-fluorescein at pH 6.2
(A) and 7.4 (B). Cell images were acquired in blue channel for
nuclei detection after labeling with DAPI and green channel for
fluorescein-labeled micelle detection.
paclitaxel (nM)
B
100
80
60
40
20
0
60
50
40
30
20
10
0
0
5
10
15
20
25
30
paclitaxel (nM)
0
50
100
150
200
Figure 8. Cytotoxicity profiles obtained by MCF7 cell incubation
with paclitaxel-loaded SD-PEG-DSPE (A) and mPEG-DSPE (B)
micelles at pH 6.2 (○) and 7.4 (▪).
time (h)
Figure 7. Release kinetic profile of paclitaxel from SD-PEG-DSPE
micelles at pH 6.2 (▪) and 7.4 (○).
morphological changes, namely bending of the poly-
mer chains, disassembling of the unimers or micelles
aggregation. e CMC value was slightly higher as com-
pared to that obtained with mPEG-DSPE reported in the
literature, probably because the presence of the negative
charges of SD on the micelle surface promotes PEG
chain repulsion. Nevertheless, the CMC was low enough
to bestow high thermodynamic stability on micelles,
preventing their de-aggregation upon dilution. Micelle
stability is a requisite in the development of physically
assembled drug delivery systems as the association
integrity must be maintained in the bloodstream while
structural alterations guarantee site selective drug release
(Lukyanov et al., 2002).
Cell culture studies carried out by quantitative analysis
of cells incubated with SD-PEG-DSPE fluorescently
labeled with fluorescein-DOPE showed that the micelles
interact with cells. However, cell toxicity studies showed
that the SD-PEG-DSPE interaction did not hamper the
cell biological activity either in the unimer or in the
micellar form, underlining the high biocompatibility of
this material. Cell/SD-PEG-DSPE micelle interaction
was found to take place according to a pH-dependent
behavior. e amount of a lipid core–loaded fluorescence
probe taken up by the cells incubated with SD-PEG-
DSPE micelles at pH 6.2 was significantly higher than
that obtained at pH 7.4 and with mPEG-DSPE micelles.
Furthermore, the time-dependent cell uptake observed
at pH 6.2 with the pH-sensitive micelles seems to
SD-PEG-DSPE unimers were synthesized according
to a simple, reproducible, and efficient protocol, as dem-
onstrated by the spectrometric and chromatographic
analysis of intermediates and final product. e use
of proper reaction conditions resulted in high product
yield and purity degree. In the first step, the bicarbon-
ate addition to the carboxylated SD activation reaction
avoided degradation phenomena due to hydrochloric
acid released by thionyl chloride, while the use of a
large excess of 1,2-diaminoethane for the SD amination
prevented the product dimerization. In the second step,
excess of SD-succ-ethyl-NH2 was used to achieve high
reaction yield with NHS-PEG-DSPE and then the unre-
acted reagent was eliminated by gel chromatography.
e potentiometric analysis showed that the SD-PEG-
DSPE unimers have pKa of 6.7, which is slightly higher as
compared to SD pKa reported in the literature. is dif-
ference may be ascribed to the presence of hydrated and
hindered PEG chains that can alter the SD microenviron-
ment and protonation.
e SD-PEG-DSPE unimers were found to assemble
in aqueous medium to produce micelles with ~20 nm
diameter, which is the typical size of these nanosystems
(Lukyanov et al., 2002). e micelle dimension was not
significantly affected by the pH conditions indicating
that under the experimental conditions the hydrophobic
character of SD at acidic pH does not induce significant
© 2011 Informa UK, Ltd.

312 Stefano Salmaso et al.
indicate that the internalization process undergoes cell
saturation. ese results were in apparent contradiction
with the flow cytofluorimetry and confocal microscopy
data, which showed that the micelles could be taken up
by all cells regardless the incubation pH. Nevertheless,
both cytofluorimetry and confocal microscopy analysis
clearly indicated that the higher cell uptake of SD-PEG-
DSPE micelles at pH 6.2 as compared to SD-PEG-DSPE
micelles at pH 7.4 or to mPEG-DSPE micelles could be
ascribed to a cell subpopulation, about 6% of the incu-
bated cells, which was significantly active in micelle
payload internalization under acidic conditions. is
unexpected behavior may be due to specific cell cycle
phase associated uptake. Additionally, the confocal
images showed that no fluorescence was associated to
the nucleus while it was located in the membrane and
cytosolic compartment.
e feasibility to use SD-PEG-DSPE micelles for drug
delivery was verified with paclitaxel, a poorly soluble
and unstable anticancer drug. Paclitaxel was efficiently
incorporated into the lipid core of micelles resulting in
dramatic solubility increase, from 0.3 μg/mL (Choa et al.,
2004) to 125 μg/mL. e slow drug release observed in
vitro highlights the high stability of paclitaxel-loaded
system, which is in agreement with the high micelle sta-
bility shown by the CMC value. Actually, the high stabil-
ity of the micelles at pH 7.4 is pre-requisite to avoid the
systemic drug release that may be responsible for unspe-
cific toxicity. On the other hand, the slow drug release
observed in buffer at pH 6.2 suggests that micelles are
stable under acidic conditions too, but the drug may be
released upon tumor cell interaction that is promoted by
the lower pH in this tissue. However, it should be noted
that, although the drug release has been evaluated in
buffer which simulates physiological conditions, a dif-
ferent behavior is expected in blood or the tumor tissue.
erefore additional investigations will be undertaken to
examine the micelle behavior either in ex vivo matrices
or in vivo.
a cell subpopulation as observed by flow cytometry and
confocal analysis.
Conclusions
e data described in this paper show that pH-
sensitive micelles can be obtained by conjugating SD
to amphiphilic molecules which spontaneously self
assemble into colloidal structures. e resulting materi-
als possess the requisites for pharmaceutical applica-
tion, namely biocompatibility, high drug payload, and
stability in the body compartment. Furthermore, the SD
decoration endows micelles that can interact with cells to
provide for selective drug delivery into tumor site.
However, although the results show the potential
applicability of these formulations for tumor targeting
opening interesting perspectives in the development of
these systems for chemotherapy, it was shown that the
attachment of one SD moiety is not per se sufficient to
guarantee extensive drug release into the tumor cells.
Declaration of interest
e authors report no conflicts of interest.
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