Author's personal copy

Journal of Colloid and Interface Science 348 (2010) 129–136

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

www.elsevier.com/locate/jcis

Micelle-like nanoparticles of block copolymer poly(ethylene oxide)-block-

poly(methacrylic acid) incorporating fluorescently substituted metallacarboranes
designed as HIV protease inhibitor interaction probes
Mariusz Uchman a, Petr Cígler b, Bohumír Grüner c, Karel Procházka a, Pavel Matějíček a,*
a
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 2030, 128 40 Prague 2, Czech Republic
b
Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and Biochemistry, ASCR, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic
c
Institute of Inorganic Chemistry, ASCR, v.v.i., Area of Research Institutes 1001, 25068 Husinec-Řež, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: We prepared nanoparticles differing in morphology from double-hydrophilic block copolymer poly(eth-
Received 18 March 2010 ylene oxide)-block-poly(methacrylic acid), PEO–PMA, and two types of fluorescein–[3-cobalt(III) bis(1,2-
Accepted 15 April 2010 dicarbollide)] conjugates, GB176 and GB179, in alkaline buffer. GB176 molecule consists of fluorescein
Available online 18 April 2010
attached to the metallacarborane anion. In GB179 molecule, the fluorescein moiety connects two metal-
lacarborane anions. The self-assembly is based on the unusual interaction of metallacarborane clusters
Keywords: with PEO blocks which form insoluble micellar cores. The GB176 containing nanoparticles are loose
AFM
and irregular, while the GB179 ones are rigid and spherical. The structure of nanoparticles depends to
Dicarbollide anions
Drug delivery
some extent on a procedure of preparation. The micelles were studied by static and dynamic light scat-
Fluorometry tering, fluorometry and atomic force microscopy. Since the metallacarborane conjugates act as potent
Light scattering inhibitors of HIV protease, the presented system is important from the point of view of drug delivery.
Micelles Ó 2010 Elsevier Inc. All rights reserved.
Nanoparticles
Metallacarboranes
Polymeric composites

Introduction complex [16]. This is of importance because PEO is frequently used

Metallacarboranes belong to promising species in medicine
[1,2]. Some years ago we started a systematic research on the phys-
ico-chemical behavior of [3-cobalt(III) bis(1,2-dicarbollide)] anion
in aqueous solutions. This was promoted by the discovery of a
strong inhibition activity of several metallacarborane conjugates
against HIV protease [3–5]. The inhibition activity is however
strongly influenced by the complex behavior of metallacarboranes
in aqueous solutions [5] and the key problem is to prepare well-de-
fined and stable dispersions of the boron containing compounds in
water. From this point of view, the detailed knowledge of the
metallacarborane self-assembly is a challenging task [6–10]. Our
research covers studies of parent metallacarboranes [11] and
(metalla)carborane conjugates [12–14] in water to their interac-
tion with surfactants [13], cyclodextrins [14,15], phospholipid
membranes [14] and with high-molar-mass hydrophilic polymers
[16]. It was recently published that [3-cobalt(III) bis(1,2-dicarbol-
lide)] interacts with high-molar-mass poly(ethylene oxide), PEO,
in aqueous solutions forming an insoluble but stimuli-responsive
* Corresponding author. Fax: +420 224919752.
E-mail address: matej@lynette.natur.cuni.cz (P. Matějíček).
in medical applications [17–19].
In this paper, we investigate how to make use of this interaction
for increasing the solubility of metallacarboranes in aqueous sys-
tems. We are focused on a physico-chemical behavior of boron
cluster containing systems in water and their biological evaluation
should be a task for a separate research. We used two types of fluo-
rescein–[3-cobalt(III) bis(1,2-dicarbollide)] conjugates, GB176 (one
metallacarborane cluster bound to fluorescein molecule) and
GB179 (two metallacarborane clusters bound to fluorescein mole-
cule; structures shown in Fig. 1), as model systems. The conjugates
exhibit the HIV protease inhibition activity [20], their solubility in
aqueous solutions is restricted and they form large aggregates in
water, stability of which depends on pH due to the presence of
fluorescein part. Details on their synthesis and behavior in aqueous
solutions are described in Ref. [14].
Here we should point out several features of the probes, which
are important when we want to put our study in a broader perspec-
tive. The probes represent single (GB176) and double (GB179) clus-
ter compounds designed as HIV protease inhibitors [3–5], where the
latter seem to be more promising in view of pharmacological
applications [3–5,20]. Recent data suggest that the chemical
structure of the ‘‘nonboron” linker, such as bulkiness, charge, and
hydrophobicity can significantly influence both inhibition activity

0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcis.2010.04.037
 Author's personal copy
130 M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136
Fig. 1. Chemical structure of GB176 and GB179.
and mechanism [3–5]. As the linker, we chose the fluorescein moiety
primarily due to its photoluminiscence [14]. Interestingly, its intro-
duction to the structural patterns responsible for the inhibition of
HIV protease did not deteriorate the activity substantially [20]. Be-
sides the number of boron clusters in GB176 and GB179, we should
keep in mind that also different ability to be ionized could determine
the association and micellization behavior as demonstrated later.
Namely, GB179 cannot lose two protons from the fluorescein part
but GB176 can do in the borate buffer. This ‘‘drawback” of fluores-
cein will be considered in designing of fluorescent probes based on
boron clusters in our future research.
We study the interaction of GB176 and GB179 with double-
hydrophilic block copolymer poly(ethylene oxide)-block-poly
(methacrylic acid), PEO–PMA, in alkaline aqueous buffers. As the
buffer, we chose a dilute solution of sodium tetraborate. Despite
the fact that it does not mimic biological conditions, we have the fol-
lowing reasons: PMA block are fully ionized and therefore soluble in
the borate buffer, and metallacarboranes do not form any insoluble
complexes with borate anions as they do with some amino-contain-
ing buffers such as Tris. Double-hydrophilic copolymers are usually
soluble in aqueous solutions [21], but the formation of spherical mi-
celle-like self-assemblies and their transformation in different mor-
phologies can be provoked by the addition of ions or other agents
[22–27]. The fluorescein moiety is not expected to interact with
PEO–PMA copolymer, but the complexation of the parent [3-cobal-
t(III) bis(1,2-dicarbollide)] cluster with PEO containing double-
hydrophilic copolymers leads to the formation of nanoparticles with
the core/shell structure, with PEO blocks very unusually located in
the core [16].
The main goal of the study was to prepare and to investigate the
nanoparticles consisting of GB176 or GB179 (inhibitors of HIV
protease) and PEO–PMA based on the rather peculiar type of interac-
tion – dihydrogen bonds [16]. Although the PMA block is not bio-
compatible, we choose the same copolymer as in our previous
study [16] in order to check whether the presence of pendant groups
of various charge attached to [3-cobalt(III) bis(1,2-dicarbollide)]
could influence the PEO/metallacarborane attraction, and to com-
pare nanoparticle morphologies of the fluorescein conjugates with
those of the parent metallacarborane. We assume that PMA blocks
can be replaced without major problems by another water-soluble
polymer, which does not interact with metallacarboranes. Along
with the change of micelle-stabilizing blocks, we can use buffers that
are closer to physiological conditions than the borate buffer. The
nanoparticle formation was monitored by static and dynamic light
scattering, SLS and DLS, and 1H NMR spectroscopy. In addition, the
time-resolved fluorescence spectroscopy was used to obtain a dee-
per insight in the studied systems. Last but not least, the morphology
of nanoparticles was studied by AFM imaging on a mica surface.
Experimental
Materials
The GB176 and GB179 preparation and characterization have
been published elsewhere [14] (structures depicted in Fig. 1). The
compounds were used in form of sodium salts.
The poly(ethylene oxide)-block-poly(methacrylic acid), PEO–
PMA, block copolymer were purchased from Polymer Source, Inc.
(Dorval, Quebec, Canada). The weight averaged relative molecular
weight of the PMA and the PEO block, provided by the manufac-
turer, are 41.0  103 and 30.7  103, respectively.
Sample preparation
The aqueous stock solutions of GB176 and GB179 were pre-
pared by mixing of 0.8 mg of the probes with 20 mL of filtered
0.05 M solution of Na2B4O7 (pH = 9.18) followed by half an hour
of sonication and stirring overnight. As estimated by UV–Vis spec-
troscopy, concentrations of clear solutions of GB176 and GB179
were 5  105 M and 1  105 M, respectively. The stock solutions
were not filtered and they were always sonicated before mixing
with other compounds.
The GB176 and GB179 solutions containing PEO–PMA in borate
buffer (pH = 9.18) were prepared as follows: (i) the GB176 and
GB179 solutions were slowly titrated by PEO–PMA solution
(c = 1 g/L) over a large span of PEO segment-to-probe ratios (0.5–
500), (ii) 2.0 mg of solid GB176 and GB179 samples were added
to 3 mL of PEO–PMA solution (c = 1 g/L) followed by stirring over-
night, and (iii) 0.5, 1.0 and 8.0 mg of solid GB176 and GB179 sam-
ples were added to 1 mL of PEO–PMA solution (c = 10 g/L) in D2O
followed by stirring overnight for 1H NMR experiments. As con-
cerns samples prepared by the titration, we could not use the titra-
tion of the copolymer solution by metallacarboranes [16], because
of significantly lower solubility of GB176 and GB179 comparing to
sodium [3-cobalt(III) bis(1,2-dicarbollide)]. We also noticed that
dialysis applied in our previous paper [16] is not a suitable method
for preparation of desired nanoparticles.
Methods
Dynamic light scattering (DLS) and static light scattering (SLS)
The light scattering setup (ALV, Langen, Germany) consisted of a
633 nm He–Ne laser, an ALV CGS/8F goniometer, an ALV High QE
APD detector, and an ALV 5000/EPP multibit, multitau autocorrela-
tor. DLS data analysis was performed by fitting the measured nor-
malized intensity autocorrelation function g2(t) = 1 + b|g1(t)|2,
where g1(t) is the electric field correlation function, t is the lag-time
and b is a factor accounting for deviation from the ideal correlation.
An inverse Laplace transform of g1(t) with the aid of a constrained
regularization algorithm (CONTIN) provides the distribution of
relaxation times, sA(s). Effective angle- and concentration-depen-
dent hydrodynamic radii, RH(q,c), were obtained from the mean val-
ues of relaxation times, sm(q,c), of individual diffusive modes using
the Stokes–Einstein equation. To obtain true hydrodynamic radii,
the data have to be extrapolated to a zero scattering angle. The SLS
data were treated by the standard Zimm method.
Steady state and time-resolved fluorometry
Steady-state fluorescence was measured in 1 cm quartz cuvettes
with a teflon stopper using a SPEX fluorolog 3–11 fluorometer. Fluo-
rescence decays were measured by means of the time-correlated
single photon counting technique on an Edinburgh Instruments ED
 Author's personal copy
M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136
299 T fluorometer equipped with an IBH NanoLED-03 excitation
source (496 nm peak wavelength, 0.1 ns fwhm of the pulse, and
1 MHz repetition rate). The measured decays were fitted to the con-
volution of multiexponential functions with the instrument re-
sponse profile using the Marquardt–Levenberg nonlinear least
squares method. Low values of v2 and random distribution of resid-
uals were used as criteria of fit.
UV–Vis spectroscopy
UV–Vis absorption spectra were carried out with a Hewlett–
Packard 8452a diode-array spectrometer.
Atomic force microscopy (AFM)
AFM measurements were performed in the semicontact (tap-
ping) mode under ambient conditions using a scanning probe
microscope Digital Instruments NanoScope dimensions 3 equipped
with a Nanosensors silicon cantilever. The nanoparticles were
deposited from very dilute solutions (ca. 0.01 g/L) on a freshly
cleaved mica surface. The samples were left to dry in a vacuum
oven.
1
H NMR spectroscopy
1
H and 13C{1H} NMR spectra were measured on a Varian
UNITY
INOVA 400 in deuterium oxide (99.5%; Chemotrade, Leipzig,
Germany). Spectra were referenced to the solvent signal (4.80 ppm).
Results and discussion
In order to prepare polymeric micelles with fluorescein–metal-
lacarborane conjugates GB176 and GB179 within their cores, we
used an identical copolymer as in our preceding paper [16]:
poly(ethylene oxide)-block-poly(methacrylic acid), PEO–PMA, that
is molecularly soluble in borate buffer (pH = 9.18). To check if there
is any specific interaction between the fluorescent probes (GB176
and GB179) and PEO blocks, we mixed the solid probes with dilute
PEO–PMA solution (1 g/L) in borate buffer as described in the
experimental section. We immediately noticed that the solubility
of the probes (GB179 especially) increased several times when
compared with the polymer-free buffer, which indicated specific
interaction between dissolved components. DLS measurements re-
vealed the presence of nanoparticles with radius ca. 225 nm in
both samples. Nevertheless, it should be kept in mind that both
GB176 and GB179 self-assemble in water and they form aggregates
of a similar size [14]. Spectral characteristics of PEO–PMA/GB176
and PEO–PMA/GB179 mixtures are shown in Fig. 2. The absorption
and photoluminescence spectra are almost identical to those of
pure probes at alkaline conditions [14,28]. We can clearly see a
well-pronounced absorption band of fluorescein dianion (PEO–
PMA/GB176; curve 1 in Fig. 2) and a typical double-peak [14] cor-
responding to GB179 (PEO–PMA/GB179; curve 2 in Fig. 2). As will
be shown in the last paragraph, the time-resolved fluorometry data
reveal changes in fluorescein photophysics upon addition of PEO–
PMA and the increase of fluorescence intensity indicates pro-
nounced solubility of the probes in the presence of the copolymer.
Steady-state spectra reflect an ionization of fluorescein parts in the
borate buffer, they changed only slightly with the copolymer con-
tent and their analysis did not reveal sufficient details elucidating
the behavior at the molecular level.
The structure of nanoparticles was studied by AFM after depo-
sition on a flat mica surface. For comparison, images of mica sur-
faces treated by solutions of GB176 and GB179 in pure water are
shown in Fig. 3a and b. We can see polydisperse aggregates of
GB176 and whiskers-like structures of GB179. Pictures of PEO–
PMA/GB176 (Fig. 4a and b) and PEO–PMA/GB179 (Fig. 5a–c) nano-
particles deserve our attention. In both cases we can clearly see
131
Fig. 2. UV–Vis (curves 1 and 2) and fluorescence (curves 10 and 20 ) spectra of PEO–
PMA/GB176 (curves 1 and 10 ) and PEO–PMA/GB179 (curves 2 and 20 ) solutions in
0.05 M sodium tetraborate prepared by mixing of solid probes with PEO–PMA
solutions.
particles with the core/shell structure. We assume that GB176/
PEO and GB179/PEO complexes with beads of the solid probe can
form the core. The micelles with GB176 have rather irregular shape
and a loose structure probably because of the presence of highly
charged fluorescein part that prefers aqueous medium. One other
fact should not be neglected: the thiaamidic linker might be prot-
onized in the vicinity of superacidic boron cluster [29,30]. The
PEO–PMA/GB179 micelles appear to be more compact and spheri-
cal, and it is worth-mentioning that their morphology is similar to
the PEO–PMA micelles with the parent [3-cobalt(III) bis(1,2-dicar-
bollide)] anion [16]. This can be attributed to the presence of two
metallacarborane anions attached via diethyleneglycol spacer
and high affinity of such arrangements to encapsulate sodium cat-
ions [31]. Long whiskers-like GB179 assemblies accompany the
polymeric nanoparticles. In many cases, we detected micelles with
a core compartmentalization as shown for example in Fig. 5a–c.
Since the probes consist of fluorescein, we carried out preliminary
fluorescence lifetime imaging (FLIM) experiments in a dry state on
glass plates (not shown). Nevertheless, dimensions of the micelles
are on the edge of a detection limit of fluorescence microscopy and
obtained results are not reliable.
To confirm that the nanoparticles consist of the PEO core, we
measured 1H NMR spectra of buffered PEO–PMA solutions
(c = 10 g/L) after addition of certain amount of the solid probes
(see experimental section). We took advantage from the fact that
the NMR signal from frozen PEO segments is diminished (spectra
shown in Supporting Information; Figs. S1 and S2) [16,32]. In
Fig. 6, there is a dependence of the fraction of frozen PEO segments
calculated from the NMR spectra on the amount of GB176 (curve 1)
and GB179 (curve 2). Worth noticing are the following facts: (i)
GB179 addition results in formation of PEO–PMA/GB179 micelles
with fully frozen fluorescein moieties (no signal in the spectra)
and more than half of all PEO segments frozen at high GB179 load-
ings, (ii) PEO blocks are frozen in much lower extent (ca. 20%) in
GB176 containing particles than in micelles of GB179, (iii) curve
1 is rectangular while curve 2 has a sigmoidal shape, and (iv) in
the PEO–PMA/GB176 cores the PMA segments might be partly
immobilized and the fluorescein parts of GB176 not fully incorpo-
rated, because the PMA signal is somehow deformed and fluores-
cein signal appears in the spectrum (see Figs. S1 and S2);
therefore the fraction of frozen PEO in the PEO–PMA/GB176 mi-
celles shown in Fig. 6 could be slightly underestimated.
The samples prepared for NMR experiments were studied also
by AFM. Typical scans of PEO–PMA/GB176 micelles and those of
PEO–PMA/GB179 are shown in Figs. 7 and 8a and b, respectively.
Figs. 7 and 8a correspond to the points at ca. 1 mg and Figs. 7
and 8b to the points at ca. 8 mg in Fig. 6. The micelles with low
 Author's personal copy

132 M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136

Fig. 3. AFM scans of (a) GB176 and (b) GB179 nanostructures deposited from dilute aqueous solutions on mica surface.

Fig. 4. AFM (a) topography and (b) phase images of PEO–PMA/GB176 nanoparticles prepared by mixing of solid GB176 with PEO–PMA solution in 0.05 M sodium tetraborate
deposited from dilute solutions on mica surface.

Fig. 5. AFM (a) topography, (b) amplitude and (c) phase images of PEO–PMA/GB179 nanoparticles prepared by mixing of solid GB179 with PEO–PMA solution in 0.05 M
sodium tetraborate deposited from dilute solutions on mica surface.

amount of GB176 are similar to those in Fig. 4a. They are loose and
asymmetrical, and we can see that mobile polymeric chains prob-
ably decorated by GB176 molecules are attached to the particles
(Fig. 7a). Further addition of GB176 causes formation of spherical
cores surrounded by diffusive polymeric coronas (Fig. 7b). Fig. 8a
demonstrates that at low GB179 concentration, the number of
the micelles is limited and it seems that PEO–PMA chains have
not been yet saturated by GB179. However, we can see fairly
monodisperse spherical particles at high GB179 loadings (Fig. 8b).
To elucidate the mechanism of micelle formation and to quan-
tify the micellar composition, we carried out a slow titration of the
probes by PEO–PMA copolymer in borate buffer (see experimental
 Author's personal copy
M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136
Fig. 6. Fractions of frozen PEO segments in PEO–PMA/GB176 (curve 1) and PEO–
PMA/GB179 (curves 2) particles calculated from 1H NMR spectra as functions of
weight amounts of GB176 or GB179 added to 1 mL of PEO–PMA solution (10 g/L) of
0.05 M sodium tetraborate in D2O.
section). We monitored the behavior by light scattering (SLS &
DLS), time-resolved fluorescence spectroscopy and AFM.
The SLS-intensity (proportional to the apparent molar mass)
and hydrodynamic radius (RH) as functions of the amount of added
copolymer are shown in Figs. 9 and 10, respectively. As already
Fig. 7. AFM topography images of PEO–PMA/GB176 nanoparticles prepared by mixing of (a) 1 mg and (b) 8 mg of solid GB176 with PEO–PMA solution (10 g/L) of 0.05 M
sodium tetraborate in D2O deposited from dilute solutions on mica surface.
Fig. 8. AFM topography images of PEO–PMA/GB179 nanoparticles prepared by mixing of (a) 1 mg and (b) 8 mg of solid GB179 with PEO–PMA solution (10 g/L) of 0.05 M
sodium tetraborate in D2O deposited from dilute solutions on mica surface.
133
mentioned, the solutions of both probes contain nanoparticles that
scatter light [14]. In this work, we did not measure (dn/dc) of
GB176 and GB179, but in our earlier study we found that the scat-
tering contrast of the parent metallacarborane is unusually high
(0.312 mL/g) and increases upon its preferential sorption within
the micellar cores of PEO–PMA (0.617 mL/g) [16]. In the titration
experiment, we measure relative intensity increase. The probe
concentration is kept constant and hence the increase in the SLS-
intensity can be therefore attributed to the massive formation of
polymeric nanoparticles. A leveling-off of the SLS-intensity in
Fig. 9 suggests that the capacity of PEO to form a complex with
metallacarborane conjugates became saturated at a certain PEO
segment-to-probe ratio. The values are ca. 20 for PEO–PMA/
GB176 micelles (curve 1) and ca. 150 for PEO–PMA/GB179 ones
(curve 2). This ratio for the parent PEO–PMA/metallacarborane mi-
celles is ca. 10 [16].
The DLS results are presented in Fig. 10. In case of the GB176
micelles, we noticed that the dimension of polymeric particles re-
mains almost constant after PEO–PMA incorporation within the
experimental error (curve 1) and their size is close to that of
GB176 aggregates in polymer-free solutions. It suggests that poly-
meric chains penetrate into metallacarborane pre-aggregates. At
the PEO-to-probe ratio ca. 20, the distribution of relaxation times
becomes bimodal and a new fast mode appears, which corresponds
to the free PEO–PMA chains. It indicates that the GB176 aggregates
 Author's personal copy
134 M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136
Fig. 9. Dependence of relative light scattering intensity (extrapolated to zero
scattering angle) of GB176 (curve 1) and GB179 (curve 2) on the PEO segment-to-
probe molecule ratio after addition of PEO–PMA solution in 0.05 M sodium
tetraborate buffer.
Fig. 10. Dependence of hydrodynamic radius (RH) of GB176 system (curves 1 and
10 ) and GB179 system (curve 2) on the PEO segment-to-probe molecule ratio after
addition of PEO–PMA solution in 0.05 M sodium tetraborate buffer.
become saturated by the copolymer (curve 10 ). As already shown,
the GB179 containing micelles are more rigid and regular. The for-
mation of micelles requires much larger amount of copolymer (the
ratio value ca. 150, see curve 2) than in case of GB176 and the
shape of curve 2 is therefore sigmoidal. This is due to presence of
two metallacarborane anions in GB179 and its bulkiness. The
Fig. 11. AFM topography images of (a) PEO–PMA/GB176 nanoparticles and (b) PEO–PMA/GB179 nanoparticles prepared by slow titration of probes solutions with PEO–PMA
solution in 0.05 M sodium tetraborate buffer water deposited from dilute solutions on mica surface; both systems are saturated by PEO–PMA: the PEO segment-to-probe
molecule ratio is (a) 200 and (b) 500.
micelles are therefore larger than the probe pre-aggregates. This
behavior, which is consistent with NMR and AFM data (see Figs.
6 and 8a and b), reminds the critical micellar concentration at cer-
tain copolymer concentration. We did not detect any traces of free
copolymer chains, because the contribution of small unimers is
hidden in the intensive scattering caused by large PEO–PMA/
GB179 micelles with a much higher contrast.
Morphology of the micelles formed by consecutive PEO–PMA
addition can differ when compared to that created by mixing of
PEO–PMA with solid probes. AFM scans of GB176 and GB179 sam-
ples prepared by the titration are shown in Fig. 11a and b,
respectively. Both samples imaged by AFM are fully saturated by
PEO–PMA (see plateau in Fig. 9). From Fig. 11a, it is evident that
individual polymeric chains destabilized by attaching of GB176
join together step-by-step resulting in non-spherical grained parti-
cles. On the contrary, GB179 micelles (Fig. 11b) are compact and
spherical, as already observed. However, a slow titration leads to
increase of nanoparticle polydispersity [16].
Careful inspection of DLS data reveals that the distribution of
correlation times of the polymeric nanoparticles is monomodal
but fairly broad with typical polydispersities around 1.5. The nano-
particles polydispersity is clearly demonstrated even by AFM scans
(see corresponding figures). Strikingly, the size distributions of the
micelles and the corresponding probe pre-aggregates are almost
identical. It supports our assumption that the shape, dimension
and number of the micelles are predetermined by the aggregation
of pure metallacarborane conjugates. This could be one of the pos-
sibilities to control the micelle bulkiness. Furthermore, for tuning
the metallacarborane content within the micelles, we can take
advantage from the stimuli-responsibility of PEO/metallacarbora-
ne composite to changes of salt concentration [16]. Also changes
in pH could influence the association and micellization behavior
of the probes.
It is worth-mentioning that mean fluorescence lifetimes only
slightly changed with gradual saturation of the probes by PEO–
PMA copolymer (dependence not shown). As we recently pub-
lished [14], the fluorescence emission kinetics of GB176 and
GB179 is double exponential and complexation of the probes for
example with cyclodextrins manifests in changes of fraction inten-
sities of the individual lifetime components. Here, we observed
small but sudden decrease of mean lifetimes (difference ca.
0.1 ns) as compared to polymer-free solution where smean is
around 2.5 ns. The drop in smean occurred at ca. 5 and 30 PEO seg-
ments per probe molecule for GB176 and GB179, respectively.
Hence the fluorescence response occurred much earlier than
 Author's personal copy
M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136
Scheme 1. Proposed structure of (a) PEO–PMA/GB176 micelles and (b) PEO–PMA/GB179 micelles in alkaline buffer.
changes in the nanoparticle morphology. These fluorescence data,
changes in NMR spectra and shapes of DLS distributions support
our assumption on the continuous penetration of PEO–PMA chains
into the metallacarborane pre-aggregates.
Conclusions
We prepared stable micelle-like nanoparticles of block
copolymer PEO–PMA with two types of sparingly soluble fluores-
cein–metallacarborane conjugates, GB176 and GB179 (see Fig. 1),
which exhibit properties of HIV protease inhibitors [20]. The self-
assembly is a result of an attractive interaction between metalla-
carborane molecules and PEO blocks. We believe, that polymeric
nanoparticles with embedded metallacarborane conjugates of
proper shape, size, biological activity, biocompatibility and stim-
uli-responsibility could be potentially exploited as suitable drug
delivery carriers [33–35]. From the above-presented results of LS,
fluorescence, NMR and AFM experiments, we propose the follow-
ing scenario of the micelle formation, even though the procedure
of sample preparation can partly influence the nanoparticle
morphology:
(I) PEO–PMA/GB176 micelles (Scheme 1a): In an alkaline buf-
fer, the fluorescein part of GB176 molecule is fully ionized.
The metallacarborane part (one per GB176 molecule)
attached via thiaamidic bond induces a formation of large
pre-aggregates due to its amphiphilicity. After the addition
of a double-hydrophilic PEO–PMA copolymer, the PEO
blocks interact with the probe molecules. The copolymer
chains penetrate by the PEO ends into the GB176 pre-aggre-
gates. The polymeric associates form gradually as the PEO–
PMA unimers anchor in pre-aggregates. The nanoparticles
are quite loose due to the presence of highly hydrophilic
fluorescein moieties. The PEO blocks are only partly immobi-
lized. Irregular shape of the micelles causes a hindrance in
PMA segments motion and the micelles probably have not
distinct core/shell structure. The nanoparticles contain one
GB176 molecule per roughly 20 PEO segments.
(II) PEO–PMA/GB179 micelles (Scheme 1b): Due to the chemical
structure (two metallacarborane moieties per GB179 mole-
cule attached to fluorescein via diethyleneglycol linkers)
the fluorescein part cannot lose protons even in alkaline buf-
fers. The probe is sparingly soluble and large pre-aggregates
can be detected in aqueous solutions. After addition of
135
double-hydrophilic PEO–PMA copolymer, the PEO blocks
interact with the probe molecules. The copolymer chains
penetrate by the PEO ends into the GB179 pre-aggregates,
but the nanoparticles have to reorganize due to the bulki-
ness of GB179 at certain copolymer concentration reminding
the critical micellar concentration. The nanoparticles are
spherical and compact with well-pronounced core/shell
architecture. The PEO blocks are considerably immobilized
within the micellar core, which may contain the GB179 nan-
obeads. The fraction of frozen PEO (ca. 50%) is however smal-
ler than in the case of micelles containing the parent cobalt
bis(dicarbollide) (75%) [16]. This is due to the lower weight
fraction, higher charge and bulkiness of GB179 within the
micelles. The nanoparticles contain one GB179 molecule
per roughly 150 PEO segments.
Acknowledgments
Authors would like to acknowledge the financial support of the
Grant Agency of the Academy of Sciences of the Czech Republic
(IAAX00320901). The authors were supported by long term Re-
search Plans of the Ministry of Education of the Czech Republic
MSM 0021620857, AV0Z40320502 and partly by Research Center
LC 523. MU and KP acknowledge the financial support by the Marie
Curie Research and Training Network (Grant No. 505 027, POLY-
AMPHI). Dr. Milena Spirkova is gratefully acknowledged for fruitful
discussions on (not only) AFM technique in MBS, Prague, Czech
Republic. The authors thank Dr. Jiri Zednik for assistance with
NMR experiments, Prof. Jan Konvalinka for HIV protease inhibition
data and Dr. Jana Humpolickova for fluorescence lifetime imaging
(FLIM) and fluorescence correlation spectroscopy (FCS) data that
have not been included to the manuscript.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jcis.2010.04.037.
References
[1] Z.J. Lesnikowski, Collect. Czech. Chem. Commun. 72 (2007) 1646–1658.
[2] I.B. Sivaev, V.V. Bregadze, Eur. J. Inorg. Chem. 2009 (2009) 1433–1450.
 Author's personal copy
136 M. Uchman et al. / Journal of Colloid and Interface Science 348 (2010) 129–136
[3] P. Cigler, M. Kozisek, P. Rezacova, J. Brynda, Z. Otwinowski, J. Pokorna, J. Plesek,
B. Gruner, L. Doleckova-Maresova, M. Masa, J. Sedlacek, J. Bodem, H.G.
Krausslich, V. Kral, J. Konvalinka, Proc. Natl. Acad. Sci. U.S.A. 102 (2005)
15394–15399.
[4] M. Kozisek, P. Cigler, M. Lepsik, J. Fanfrlik, P. Rezacova, J. Brynda, J. Pokorna, J.
Plesek, B. Gruner, K. Grantz Saskova, J. Vaclavikova, V. Kral, J. Konvalinka, J.
Med. Chem. 51 (2008) 4839–4843.
[5] P. Rezacova, J. Pokorna, J. Brynda, M. Kozisek, P. Cigler, M. Lepsik, J. Fanfrlik, J.
Rezac, K. Grantz Saskova, I. Sieglova, J. Plesek, V. Sicha, B. Gruner, H.
Oberwinkler, J. Sedlacek, H.G. Krausslich, P. Hobza, V. Kral, J. Konvalinka, J.
Med. Chem. 52 (2009) 7132–7141.
[6] E. Hao, M. Sibrian-Vazquez, W. Serem, J.C. Garno, F.R. Fronczek, M.G.H. Vicente,
Chem. Eur. J. 13 (2007) 9035–9042.
[7] J.G. Planas, F. Teixidor, C. Vinas, M.E. Light, M.B. Hursthouse, Chem. Eur. J. 13
(2007) 2493–2502.
[8] J.G. Planas, C. Vinas, F. Teixidor, A. Comas-Vives, G. Ujaque, A. Lledos, M.E.
Light, M.B. Hursthouse, J. Am. Chem. Soc. 127 (2005) 15976–15982.
[9] R. Khattar, C.B. Knobler, M.F. Hawthorne, Inorg. Chem. 29 (1990) 2191–2192.
[10] G. Chevrot, R. Schurhammer, G. Wipff, J. Phys. Chem. B 110 (2006) 9488–9498.
[11] P. Matejicek, P. Cigler, K. Prochazka, V. Kral, Langmuir 22 (2006) 575–581.
[12] P. Kubat, K. Lang, P. Cigler, M. Kozisek, P. Matejicek, P. Janda, Z. Zelinger, K.
Prochazka, V. Kral, J. Phys. Chem. B 111 (2007) 4539–4546.
[13] P. Matejicek, P. Cigler, A.B. Olejniczak, A. Andrysiak, B. Wojtczak, K. Prochazka,
Z.J. Lesnikowski, Langmuir 24 (2008) 2625–2630.
[14] M. Uchman, P. Jurkiewicz, P. Cigler, B. Gruner, M. Hof, K. Prochazka, P.
Matejicek, Langmuir (2010), doi:10.1021/la904047k.
[15] J. Rak, M. Tkadlecovka, P. Cigler, V. Kral, Chem. Listy 102 (2008) 209–212.
[16] P. Matejicek, J. Zednik, K. Uselova, J. Plestil, J. Fanfrlik, A. Nykanen, J.
Ruokolainen, P. Hobza, K. Prochazka, Macromolecules 42 (2009) 4829–4837.
[17] Y. Bae, W.-D. Jang, N. Nishiyama, S. Fukushima, K. Kataoka, Mol. BioSyst. 1
(2005) 242–250.
[18] W.J. King, J.S. Mohammed, W.L. Murphy, Soft Matter 5 (2009) 2399–2406.
[19] A. Lavasanifar, J. Samuel, G.S. Kwon, Adv. Drug Deliv. Rev. 54 (2002) 169–190.
[20] Private communication of Prof. Jan Konvalinka, Institute of Organic Chemistry
and Biochemistry, ASCR, Prague, April 19th 2009: the half maximal inhibitory
concentration (IC50) in vitro of GB176 and GB179 4.0 lM and 100 nM,
respectively.
[21] H. Colfen, Macromol. Rapid Commun. 22 (2001) 219–252.
[22] L.M. Bronstein, S.N. Sidorov, A.Y. Gourkova, P.M. Valetsky, J. Hartmann, M.
Breulman, H. Colfen, M. Antonietti, Inorg. Chim. Acta 280 (1998) 348–354.
[23] M. Sedlak, M. Antonietti, H. Colfen, Makromol. Chem. Phys. 199 (1998) 247–
254.
[24] L.M. Bronstein, S.N. Sidorov, A.Y. Gourkova, P.M. Valetsky, J. Hartmann, M.
Breulman, H. Colfen, M. Antonietti, Langmuir 15 (1999) 6256–6262.
[25] S. Forster, N. Hermsdorf, W. Leube, H. Schnablegger, M. Regenbrecht, S. Akari,
P. Lindner, C. Böttcher, J. Phys. Chem. B 103 (1999) 6657–6668.
[26] T.K. Bronich, P.A. Keifer, L.S. Shlyakhtenko, A.V. Kabanov, J. Am. Chem. Soc. 127
(2005) 8236–8237.
[27] K.D. Gatsouli, S. Pispas, E.I. Kamitsos, J. Phys. Chem. C 111 (2007) 15201–
15209.
[28] R. Sjoback, J. Nygren, M. Kubista, Spectrochim. Acta A 51 (1995) L7–L21.
[29] C.A. Reed, Acc. Chem. Res. 31 (1998) 133–139.
[30] M. Juhasz, S. Hoffmann, E. Stoyanov, K.C. Kim, C.A. Reed, Angew. Chem. Int. Ed.
43 (2004) 5352–5355.
[31] J. Plesek, B. Gruner, S. Hermanek, J. Baca, V. Marecek, J. Janchenova, A. Lhotsky,
K. Holub, P. Selucky, J. Rais, I. Cisarova, J. Caslavsky, Polyhedron 21 (2002) 975–
986.
[32] S. Kawaguchi, M.A. Winnik, K. Ito, Macromolecules 29 (1996) 4465–4472.
[33] A. Harada, K. Kataoka, Prog. Polym. Sci. 31 (2006) 949–982.
[34] J. Rodriguez-Hernandez, F. Checot, Y. Gnanou, S. Lecommandoux, Prog. Polym.
Sci. 30 (2005) 691–724.
[35] E.S. Gil, S.A. Hudson, Prog. Polym. Sci. 29 (2004) 1173–1222.