Multimodal and multifunctional stealth polymer nanospheres for sustained drug delivery

Article abstract of DOI:10.1039/c2nj40016b

We report the preparation of fluorescent and magnetic PMMA nanospheres, and a corresponding PEGylated 'stealth' analogue prepared using a block copolymer. The nanospheres contain encapsulated magnetite nanoparticles and fluorescent BODIPY dyes, including a new such dye with pH-sensitive fluorescent emission. The new dye could potentially be used as an indicator of the immediate physiological environment. The nanospheres were non-toxic at up to 500 μg ml^-1 in PC12 cells. Lomerizine, a lipophilic calcium channel blocker, was also encapsulated in the nanospheres and displayed sustained, pH-dependent release characteristics. The nanospheres may be of use to release lomerizine and other water-insoluble drugs at central nervous system injury sites. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012.

Full text of DOI:10.1039/c2nj40016b

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New Journal of Chemistry
A journal for new directions in chemistry
www.rsc.org/njc
Volume 36 | Number 7 | July 2012 | Pages 1425–1528
ISSN 1144-0546
COVER ARTICLE
Iyer et al.
Multimodal and multifunctional stealth polymer nanospheres
for sustained drug delivery

Dynamic ArticVleiewLiAnrktiscle Online
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Cite this: New J. Chem., 2012, 36, 1457–1462
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PAPER
Multimodal and multifunctional stealth polymer nanospheres for
sustained drug deliveryw
Cameron W. Evans,ab Melissa J. Latter,a Diwei Ho,ab
Saquib Ahmed M. A. Peerzade,a Tristan D. Clemons,ab Melinda Fitzgerald,b
Sarah A. Dunlopb and K. Swaminathan Iyer*b
Received (in Montpellier, France) 9th January 2012, Accepted 24th March 2012
DOI: 10.1039/c2nj40016b
We report the preparation of fluorescent and magnetic PMMA nanospheres, and a corresponding
PEGylated ‘stealth’ analogue prepared using a block copolymer. The nanospheres contain
encapsulated magnetite nanoparticles and fluorescent BODIPY dyes, including a new such dye
with pH-sensitive fluorescent emission. The new dye could potentially be used as an indicator of
the immediate physiological environment. The nanospheres were non-toxic at up to 500 mg mlÀ1
in PC12 cells. Lomerizine, a lipophilic calcium channel blocker, was also encapsulated in the
nanospheres and displayed sustained, pH-dependent release characteristics. The nanospheres may
be of use to release lomerizine and other water-insoluble drugs at central nervous system
injury sites.
particles can be followed at the scales of systems and organs by
Introduction
MRI, or at the cellular level by fluorescence.3,9 Furthermore,
the physical encapsulation approach is suited to a variety
of molecular and nanoparticulate cargoes without needing
substantial modifications to the preparative procedure for
the carrier nanoparticles.
Colloidal systems, including nanoparticles and liposomes,
have been extensively studied as potential drug carriers for
targeted or controlled release. The encapsulation of drugs in
nanoparticles offers one means for controlled or targeted
release, but the use of nanosystems could be increased if
tracking methods in the body were also incorporated to
visualise delivery. For this reason, there is considerable interest
in developing multifunctional nanoparticles, which combine
imaging and therapy in a single construct.1 Monitoring of the
nano-assembly will facilitate an indirect determination of the
site at which the therapy is administered.2
Nanoparticles and liposomes are known to be rapidly
cleared from tissues and blood by cells of the mononuclear
phagocytic system (MPS), particularly macrophages in the
liver and spleen10 (including hepatic Kupffer cells)11,12 and
circulating monocytes.13 Nanoparticle removal by the MPS
occurs by interaction of the hydrophobic surface of particles
with plasma proteins (opsonins), which are recognised by
specific receptors on macrophages, promoting the binding
and phagocytosis of these carriers.11,13 This is the primary
mechanism by which the organs of the reticuloendothelial
system (RES)—principally the liver, spleen and bone marrow—
recognise circulating nanoparticles.12
It is possible to incorporate more than one imaging tool in a
single nanoparticle system, producing a multifunctional particle
that supports several imaging modalities. One common combi-
nation of imaging modalities is magnetic resonance imaging (MRI)
and fluorescence microscopy, which combines the radiation-free,
whole-body, deep tissue imaging ability of MRI with the sensitivity
of fluorescence detection.3–7 Delivering drugs using this kind of
nanoparticle as the vehicle has several benefits; encapsulated drugs
are protected on their journey to the target tissue,8 and the resulting
Nanoparticle clearance by the RES is known to be affected
by size and surface characteristics.13,14 For example, nano-
particles with a hydrophobic surface are removed from circulation
more rapidly than those with hydrophilic and neutral surfaces.13
Previous studies demonstrate that the rate of opsonisation of
nanoparticles can be reduced by modifying the nanoparticle
surface with a hydrophilic, flexible, and non-ionic polymer.15
Examples include poly(ethylene glycol) (PEG), polysaccharides,
or poloxamers and poloxamines,10,11 which provide a steric
barrier on the particle surface that minimises opsonisation.12
Nanoparticles that have been modified in this way are typically
known as ‘‘stealth’’ particles, because they escape the surveillance
of the RES.12 Increased blood lifetimes of injected nanoparticles
a School of Chemistry and Biochemistry, The University of Western
Australia, 35 Stirling Hwy, Crawley WA 6009, Australia.
E-mail: swaminatha.iyer@uwa.edu.au; Fax: +61 8 6488 7330;
Tel: +61 8 6488 4470
b Experimental and Regenerative Neurosciences, School of Animal
Biology, University of Western Australia, 35 Stirling Hwy,
Crawley WA 6009, Australia
w Electronic supplementary information (ESI) available: TEM of
magnetite nanoparticles, magnetic characterisation by SQUID,
relaxometry plots. See DOI: 10.1039/c2nj40016b
c
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New J. Chem., 2012, 36, 1457–1462 1457

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have been related to the presence of PEG on their surfaces.11,14
Similarly, a PEG corona has been shown to impart a stealth
property to liposomes, suppressing recognition and uptake by
the RES and extending the circulation time of the particles in
the body.16 Importantly, high concentrations of PEG on the
surface alone do not lead to low uptake; the spatial configu-
ration and freedom of the PEG chains is also important.14 In
particular, block copolymers containing a PEG segment have
been shown to be very effective at preventing uptake.14
Herein, we report a stealth carrier for sustained drug release,
using PEG-modified PMMA nanoparticles that contain both
magnetite and a fluorescent probe. Using a novel, pH-sensitive
BODIPY dye we also show that the emission of the fluorophore
can be potentially used as an indicator of the immediate
physiological environment. We show that the distribution and
loading of magnetite nanoparticles inside these nanocarriers can
be regulated by the choice of solvents. Finally, we demonstrate
that these stealth nanocarriers can be used as a pH-responsive
release agent using lomerizine, a small-molecule drug that is
generally insoluble in aqueous systems.
Scheme 1 Preparation of BODIPY dyes 3 and 4: (i) dichloromethane,
triethylamine, boron trifluoride etherate; (ii) toluene, piperidine, acetic
acid, molecular sieves.
Results and discussion
The BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)
fluorophores are a popular choice for various sensing and
imaging applications. These dyes typically have small Stokes
shifts and narrow emission spectra,17 but most compounds of
this class are insoluble in water and emit in the green part of
the spectrum, limiting their biological use. On the other hand,
methods that increase the versatility of this dye class have been
recently reported,17–24 including strategies to impart water
solubility18–21 and modifications to shift the emission22–24
(for example, by incorporating chromophores to extend conjuga-
tion within the structure).
In this work, two such dyes were synthesised: the green-
fluorescent 1,3,5,7,8-pentamethyl BODIPY 325 and the new
1,3,7,8-tetramethyl-5-(4-dimethylaminostyryl) analogue 4. The
parent dye 3 was prepared in one pot by condensation of
pyrrole 1 and acid chloride 2 prior to coordination with boron
trifluoride-etherate (Scheme 1). Knoevenagel reaction of 3 in
the presence of the aldehyde afforded the new dye 4. Extending
the conjugation red-shifted the peak absorption wavelength
(lmax 591 nm for 4 in dichloromethane compared to lmax
499 nm for 3 in chloroform25), producing a dye that was purple
in dichloromethane, while also providing an acid-sensitive
group that opens the possibility for sensing behaviour (peak
emission wavelength lem E 500 nm protonated, 670 nm
deprotonated). This qualitative reversible molecular switching
of BODIPY 4 was also demonstrated in the presence of acid or
base (Fig. 1a) as previously seen for the structurally similar
meso-phenyl BODIPY dye.26
Fig. 1 (a) Fluorescence spectra of 4 (solid) and 4 + H+ (dashed)
recorded in CH2Cl2; both spectra have been normalised to give the
same maximum emission intensity. The photograph, under UV
illumination, shows the colour change observed upon the addition of
acid or base; (b) Nanosphere sample (containing BODIPY 3) dispersed
in water; (c) the same sample collected using a permanent magnet;
(d) Dynamic light scattering characterisation of nanoparticle size for
PMMA (solid) and PMMA-b-PEG (open) nanoparticles.
shown in Fig. 1b–c. Nanospheres contained multiple iron
oxide nanoparticles; the association of individual magnetite
nanoparticles into clusters has been used to increase the
transverse relaxivity of nanospheres of the same size while
maintaining the superparamagnetic characteristics.29 The nano-
spheres produced by this method were stable in aqueous dispersion,
even though the iron oxide and BODIPY constituents were not.
DLS of the nanospheres showed that the particle size was in the
range 100–300 nm, with an average size of approximately 170 nm
(Fig. 1d). SQUID data (Fig. S2 and S3) show that the nanospheres
maintained the superparamagnetic behaviour of the constituent
iron oxide nanoparticles, as indicated by the absence of hysteresis
at 300 K and the coincidence of the zero field-cooled and
field-cooled magnetisation curves. The FT-IR spectrum of
the prepared nanospheres strongly resembled that of PMMA
Iron oxide nanoparticles (with sizes 4–10 nm) were prepared
via the high temperature decomposition of Fe(acac)3 (Fig. S1).27,28
Polymer nanospheres were synthesised from either poly(methyl
methacrylate) (PMMA), to give unmodified nanospheres, or from
poly(methyl methacrylate)-block-poly(ethylene glycol) (PMMA-b-
PEG) to produce a PEGylated stealth analogue. By including
BODIPY dyes and iron oxide nanoparticles in the organic phase,
we prepared magnetic and fluorescent polymer nanospheres as
c
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(3000–2800 cmÀ1, C–H stretch; 1731 cmÀ1, CQO stretch),30
and suggested that the unmodified PMMA nanospheres were
not substantially PEGylated by the Pluronic surfactant used in
the preparation.
(PBS) to mimic physiological conditions (Fig. 3). Reverse-
phase HPLC (RP-HPLC) was used to separate the released
lomerizine, which was monitored by UV at 210 nm.43 The
drug loading was also measured by RP-HPLC and was found
to be 41 Æ 1% w/w. It has been reported that polymeric drug
delivery systems often release drug in an initial burst, followed
by extended period of slower release, perhaps due to the drug
being adsorbed on the particle surface or because of pores and
cracks in the polymer matrix.44 In this case, it would appear
that the low solubility of lomerizine results in the saturation of
the sink within a few hours. Thus, the release rate fell
considerably from this point onwards, but it is also likely that
a burst pattern was observed. The dependence of the release
rate on pH is likely due to the pH-dependent solubility of
lomerizine itself as PMMA is not a pH-responsive polymer, and
therefore drug release probably occurs simply by diffusion.
Extrapolating from the initial slope gives an indication that
the total loaded dose of lomerizine would be released after
approximately 5 h, 21 h, and 90 days for pH 5, 6, and 7.4
respectively under ideal sink conditions.
The assembly of the magnetite nanoparticles (either uniformly
dispersed throughout the nanosphere or selectively associated to
one side) could be controlled by changing the solvent mixture
employed in the organic phase. The selection of solvents affects
the dispersability of the iron oxide nanoparticles within the
solvated polymeric micelle, which determines the distribution
of these particles within the nanospheres (Fig. 2). The PMMA
nanospheres prepared using different solvent mixtures as shown
in Fig. 2 had longitudinal relaxivity r1 E 8 sÀ1 mMÀ1 and
transverse relaxivity r2 E 300 sÀ1 mMÀ1 as detailed in Fig. S4.
The high value of r2 is similar to other values reported in the
literature and confirms the suitability of PMMA nanospheres for
use as an MRI contrast agent.31
Using a similar technique, drug-loaded nanoparticles were
prepared by dissolving lomerizine in the organic phase.
Lomerizine is an L- and T-type voltage-gated calcium channel
blocker and selective cerebral vasodilator.32 It protects retinal
ganglion cells following optic nerve injury33–36 and displays
To ensure that the drug carrier was not innately toxic,
nanospheres (PMMA and PMMA-b-PEG) were incubated
with rat pheochromocytoma neural progenitor (PC12) cells
and the toxicity of the nanospheres after 24 h determined using
a Live/Dead assay (calcein AM and ethidium homodimer-1).
No reduction in cell viability (p > 0.05) was observed for
either type of particle at concentrations up to 500 mg mlÀ1
(Fig. 4a–b). At 1000 mg mlÀ1, however, PMMA-b-PEG particles
caused a reduction (p r 0.05) in cell viability, whereas PMMA
particles did not. Therefore, both types of nanospheres were non-
toxic at up to 500 mg mlÀ1 as assessed by cell viability relative to
control, and differences in measured viabilities are attributed
to statistical variation in the samples. Upon incubation for up
to 120 h, we did not observe any association with cells for
either the PMMA or PMMA-b-PEG particles as prepared.
To assess the effectiveness of the stealth surface, nano-
spheres were combined with branched polyethylenimine
(PEI) and incubated with PC12 cells. Polyethylenimine is a
synthetic polymer and a common transfection agent;45–47 the
positive charges on PEI assist in association with the plasma
neuroprotective effects in
a number of other injury
models.37–40 In in vivo studies, however, large and repeated
doses of lomerizine are used.33–36 Because injury to the central
nervous system results in an extracellular pH drop,41,42
a
pH-responsive, controlled-release drug delivery system will
likely be of therapeutic value, delivering greater doses of
lomerizine to the most acidic sites. In these kinds of injuries,
immune responses can include macrophage recruitment and
microglial activation;33–35 a stealth delivery mechanism may
be beneficial if delivery to these sites is desired.
For drug loading, the aqueous phase and subsequent washes
were buffered at pH 9 to prevent dissolution of lomerizine
during purification. Drug release data were subsequently
recorded at pH 5, 6, or 7.4 after magnetic separation of
nanoparticles and resuspension in phosphate buffered saline
Fig. 2 TEM images of particles prepared from the following hexane/
chloroform/acetone solvent systems: (a) PMMA 0 : 1 : 19 (scale bar =
50 nm); (b) PMMA 1 : 2 : 9 (scale bar = 50 nm); (c) PMMA-b-PEG
1 : 2 : 9 (scale bar = 50 nm). (d) Corresponding low magnification view
of the particles in (b) (scale bar = 100 nm), inset: elemental mapping
shows presence of carbon (red) and iron (green). (e) Lower magnification
view of particles in (c) (scale bar = 100 nm).
Fig. 3 Release of lomerizine over time at pH 5.0 (red triangles, solid
line), pH 6.0 (blue circles, dashed line) and pH 7.4 (black squares, dotted
line). All data points are averages of two lomerizine determinations Æ SE.
The inset shows a magnified scale for release at pH 7.4.
c
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Experimental
Materials
Boron trifluoride diethyl etherate, 4-(dimethylamino)benzal-
dehyde, 2,4-dimethylpyrrole, piperidine, Pluronic F-108, poly-
ethylenimine (Mn 1200, Mw 1300, 50% solution in water),
poly(methyl methacrylate) (Mw 120,000 g molÀ1), triethyl-
amine, and Tris were obtained from Aldrich, acetyl chloride and
triethylamine from Fluka, PMMA-b-PEG (Mn 40 000 g molÀ1
MMA, 11,500 g molÀ1 PEG, Mw/Mn 1.3) from Polymer Source,
Inc., and lomerizine dihydrochloride from LKT Laboratories.
Solvents were of analytical grade, except HPLC solvents which
were of HPLC grade and filtered (0.2 mm) before use. Hexane was
distilled before use, and CH2Cl2 was distilled according to
standard procedures.49 Milli-Q water (> 18 MO cm) was used
in all preparations. Cell culture materials were obtained from
Invitrogen unless otherwise stated: RPMI1640, horse serum,
fetal bovine serum, penicillin/streptomycin, L-glutamine, non-
essential amino acids, sodium pyruvate. Poly(L-lysine) hydro-
bromide was obtained from Sigma.
Fig. 4 PC12 cultures incubated with nanospheres. Error bars
indicate SE. (a) Viability of PC12 cells incubated for 24 h with PMMA
nanospheres (n = 7 per concentration, one-way ANOVA with
Bonferroni post hoc correction, p > 0.05). (b) Viability of PC12 cells
incubated for 24 h with PMMA-b-PEG nanospheres (n = 7 per
concentration, Mann-Whitney U, p r 0.05 indicated by *). (c) PMMA
nanospheres containing pentamethyl BODIPY 3 are associated with
cells in the presence of PEI (green = 3, PMMA nanospheres;
DIC overlay, 40 Â/1.25, scale bar = 20 mm). (d) The same is observed
for PMMA nanospheres containing BODIPY 4 (red = 4, PMMA
nanospheres, blue = Hoechst, nuclei; DIC overlay, 63 Â/1.40, scale
bar = 20 mm). (e) PMMA-b-PEG nanoparticles do not associate
with cells, even in the presence of PEI (red = 4, PMMA-b-PEG
nanospheres, blue = Hoechst, nuclei; DIC overlay, 63 Â/1.40, scale
bar = 20 mm).
BODIPY synthesis
BODIPY 3 was prepared according to literature procedure
and characterisation data agrees with that reported for
1H NMR spectrum.25 The 13C NMR spectrum was not
previously reported. 13C NMR (100 MHz, CDCl3, 25 1C):
d = 153.56, 141.41, 140.99, 132.04, 121.20, 17.27, 16.33,
14.39 ppm. The synthesis of dye 4 was adapted from the
Knoevenagel method previously reported.26 BODIPY 3 (127.4 mg,
0.486 mmol) was combined with 4-(dimethylamino)benz
aldehyde (81.0 mg, 0.543 mmol) in toluene (10 mL) in the
presence of acetic acid (0.4 mL), piperidine (0.38 mL) and
molecular sieves. The reaction mixture was heated to reflux for
3 h, purified by column chromatography (silica) eluting with
dichloromethane/hexane (7 : 3), affording the desired product
BODIPY 4 (33.7 mg, 20% based on recovery of 14 mg of
starting BODIPY 3). 1H NMR (400 MHz, CDCl3, 25 1C): d =
7.47 (m, 3H), 7.19 (d, J(H,H) = 16 Hz, 1H), 6.68 (m, 3H), 6.04
(s, 1H), 3.01 (s, 6H), 2.60 (s, 3H), 2.55 (s, 3H), 2.46 (s, 3H),
2.42 ppm (s, 3H); 13C NMR (100 MHz, CDCl3, 25 1C): d =
153.28, 151.47, 150.92, 140.96, 138.87, 138.33, 137.05, 133.55,
131.90, 129.05, 124.78, 120.55, 117.73, 117.75, 114.44, 112.02,
40.24, 29.69, 17.63, 17.20, 16.28, 14.46 ppm; UV/Vis (CH2Cl2):
membrane of cells and may promote endocytosis.48 When PEI
was combined with particles, adhesion of PMMA particles to
the cell membrane (Fig. 4c–d) was observed, but particles did
not appear to be internalised, probably because PEI was not
covalently bound to the nanospheres. On the other hand,
stealth PMMA-b-PEG nanospheres did not associate with
cells, even when PEI was added (Fig. 4e). This suggests that
the block copolymer particles sterically hinder the electrostatic
attraction of PEI, and that they may be useful as a stealth
delivery agent.
Conclusions
We report a method to synthesise nanoparticles containing
non-water-soluble dyes and iron oxide nanoparticles that
can then be used in aqueous biological experiments. This
method is suited to a variety of lipophilic dyes, and we have
demonstrated the incorporation of two BODIPYs in the
nanospheres without modifying the synthesis procedure. The
fluorescent emission of 4 is pH-dependent, with acidification
causing a blue-shift in the maximum emission wavelength. The
neuroprotective drug lomerizine was also encapsulated in
these nanoparticles and exhibited a pH-dependent release
profile. The nanoparticles are non-toxic to PC12 cells at
concentrations up to 500 mg mlÀ1 and the PEGylated particles
did not associate with PC12 cells, even in the presence of the
transfection agent PEI. This strategy enables multimodal
tracking and delivery of drugs, potentially improving other-
wise poor bioavailability of a neuroprotective agent.
l
max(e) = 591 nm (38300); HR-ESMS calculated for
C23H26BF2N3 392.2224; found 393.2299 [M+H]+.
Iron oxide nanoparticle synthesis
Magnetite nanoparticles were prepared by decomposition of
Fe(acac)3 in benzyl ether, as previously described.27,28
Preparation of nanospheres
PMMA (75 mg, Mw 120,000 g molÀ1) and iron oxide nano-
particles (5 mg) were dissolved in chloroform and the solvent
evaporated in vacuo to help solvate the polymer. BODIPY 3 or
4 (5 mg), lomerizine (20 mg) and hexane/chloroform/acetone
(6.0 mL, typically 0.5 : 1 : 4.5) were added and all components
were dissolved. This mixture was added dropwise with vigorous
stirring to an aqueous solution of Pluronic F-108 (1.25 mg mLÀ1),
c
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buffered with 10 mM Tris at pH 9.0. The mixture was then
homogenised with a probe-type ultrasonicator for 1 min at low
power and stirred overnight under a slow flow of N2 to
evaporate the solvents. The resulting suspension was centri-
fuged at 3000 g for 45 min and the supernatant was passed
through a magnetic separation column (Miltenyi Biotec). The
collected nanospheres were washed from the column and
isolated by centrifugation at 16 000 g for 30 min. PEGylated
nanospheres were prepared in the same way, using PMMA-b-
PEG in place of PMMA. Samples were prepared for electron
microscopy by air-drying a drop of aqueous dispersed nano-
spheres on a carbon-coated copper grid; TEM was performed
on a JEOL 2100 operated at 120 kV. Magnetometry was
performed on a Quantum Design 7 T MPMS instrument,
and DLS size and zeta measurements were carried out using a
Malvern ZetaSizer Nano.
horse serum (10%), fetal bovine serum (5%), penicillin/
streptomycin (50 U mLÀ1, 50 mg mLÀ1), L-glutamine (2 mM),
non-essential amino acids (1%), and sodium pyruvate (1 mM).
For confocal microscopy, cells were grown on poly(L-lysine)-
coated coverslips, coated by incubation for at least 1 h with
poly(L-lysine) hydrobromide (10 mg mLÀ1). Nanospheres were
added at a concentration of 10 mg mLÀ1 and PEI was added at a
final dilution of 1 mg mLÀ1 when used. For viability assess-
ments, cells were plated at a density of 2 Â 105 mLÀ1 in 96-well
plates coated with poly(L-lysine) 24 h prior to experiments. Cells
were incubated with nanospheres dispersed in complete media
for 24 h, and viability was determined using Live/Dead assay
(Invitrogen); briefly, cells were incubated with calcein AM
(1 mM) and EthD-1 (2–3 mM) in PBS for 30 min, and then
fluorescence was quantified using a spectrophotometer (BMG
FluoStar Optima) or cells were counted in four fields of view
per well at 20 Â magnification (Olympus IX-71) with assess-
ment of approximately 1000 cells per replicate.
Relaxometry
We thank Mr B. Padman and the Centre for Microscopy,
Characterisation and Analysis at UWA for assistance with
electron microscopy, and Dr A. Reeder (UWA) for mass
spectrometry analysis. Funding by the Australian Research
Council, National Health & Medical Research Council of
Australia and the Sir Harry Secombe Trust is graciously
acknowledged. MJL appreciates the support of the University
of Western Australia (Research Development Award).
Relaxivity data were measured (Bruker minispec mq) at
1.41 T. A Carl-Purcell-Meiboom-Gill (CPMG) spin echo
sequence was used to measure T2. The echo spacing was 1 ms
(2000 echoes). An inversion recovery (IR) sequence was used to
measure T1 using 10 inversion times (TI) logarithmically spaced
between 10 and 10 000 ms. Nanosphere samples were suspended
in water, and data were recorded at 27 1C. The iron content of
the samples was determined by ICP-AES after acid digestion.
Determination of lomerizine release
Notes and references
Release experiments were performed in pre-warmed phos-
phate buffered saline (PBS) at various pH levels (pH 5.0, 6.0
and 7.4). Nanospheres (10 mg) were dispersed in PBS (10 mL)
and maintained at 37.0 Æ 0.1 1C. The sinks were sampled in
duplicate over 10 h; aliquots of 150 mL were transferred to
filter tubes (Millipore, Amicon Ultra-0.5, 50 kDa cutoff),
centrifuged at 17 000 g for 5 min, and analysed by RP-HPLC.
No fresh PBS media was introduced into the sinks. Lomerizine
concentrations were calculated from a standard curve and
were reported as mean values Æ SE. The determination of
lomerizine by RP-HPLC was adapted from Waki and Ando.43
The measurements were run on a Waters 2695 separations
module coupled with Waters 2489 UV/Vis detector. A C18
column (150 Â 4.60 mm, 5 mm, 25 Æ 5 1C) was used with
isocratic elution using a 69 : 31 mixture of acetonitrile and
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0.1% w/w potassium phosphate buffer (pH 6) at 10.0 mL minÀ1
,
monitoring the eluent at 210 nm. Each sample was run for 13 min
and the integrated area of the largest peak between the
retention time 9–10 min was used for the calculation of
lomerizine concentration. The limit of detection for lomerizine
in water at 210 nm was 0.1 mg LÀ1. The loading of lomerizine
in nanospheres (1.69 mg) was determined by adding methanol
(1 mL) and sonicating in an ultrasonic cleaning bath (10 min).
Samples were left for 1 h, centrifuged to remove nanoparticles
(17 000 g, 10 min), and analysed by RP-HPLC as above.
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