Sulfonates-PMMA nanoparticles conjugates: A versatile system for multimodal application

Article abstract of DOI:10.1016/j.bmc.2012.09.023

We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates?EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs?sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems.

Full text of DOI:10.1016/j.bmc.2012.09.023

Bioorganic & Medicinal Chemistry 20 (2012) 6640–6647
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Sulfonates-PMMA nanoparticles conjugates: A versatile system
for multimodal application
Claudio Monasterolo a, Marco Ballestri a, Giovanna Sotgiu a, Andrea Guerrini a, Paolo Dambruoso a,
Katia Sparnacci d, Michele Laus d, Michelandrea De Cesare b, Assunta Pistone a, Giovanni Luca Beretta b,
Franco Zunino b, Valentina Benfenati c, Greta Varchi a,
⇑
a Istituto per la Sintesi Organica e la Fotoreattività, ‘CNR-I.S.O.F.’, Area della Ricerca di Bologna, via P. Gobetti 101, 40129 Bologna, Italy
b Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale Tumori, 20133 Milano, Italy
c Istituto per lo studio dei Materiali Nanostrutturati, ISMN Area della Ricerca di Bologna, via P. Gobetti 101, 40129 Bologna, Italy
d Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale ‘A. Avogadro’, Viale T. Michel 11, 15100 Alessandria, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2012
Revised 7 September 2012
Accepted 13 September 2012
Available online 21 September 2012
We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment,
based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged
poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of
ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 sys-
tems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated
in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides,
8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its inter-
nalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the
interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications.
In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for
negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability
of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to
the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates
is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour
or as theranostic systems.
Keywords:
PMMA
Nanoparticles
Drug delivery
Organic dyes
Theranostic systems
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
is associated with serious side effects. A number of macromolecu-
lar delivery systems are under investigation to circumvent these
Nanotechnology1 is an enabling technology that deals with
nano-meter sized objects. One of the most attractive and promis-
ing applications of nanotechnology is the use of nanomaterials in
biology. Fluorescent biological labels2 and drug and gene delivery3
are only few examples of the possible nanomaterials applications
in the field.
Improving the therapeutic index of drugs is one of the major
research areas especially for anticancer, anti-inflammatory and
anti-infective drugs. The majority of clinically used drugs are
low-molecular weight compounds (typically under 500 gmolꢀ1),
that exhibit a short half-life in the blood stream, a high overall
clearance rate and most of them show poor pharmacokinetic prop-
erties. Furthermore, they diffuse rapidly into healthy tissues and
are distributed evenly within the body. As a consequence, rela-
tively small amounts of the drug reach the target site, and therapy
limitations. Attaching drugs to polymeric systems has been proved
to considerably modify cellular uptake with respect to the free
drug. In particular this effect, which mainly consists in prolonged
drug plasma half-life and more favourable pharmacokinetic, is
due to the Enhanced Permeability and Retention (EPR) effect,
especially for non-targeted polymer carriers.4 Moreover, surface
nanoparticles charge play an important role on nanospheres’ inter-
actions with cell surfaces and it has been demonstrated that,
cationic particles generally exert stronger effect on cellular up-
take.5 On the other hand, fluorescence imaging is one of dominant
detection and sensing technologies in biomedicine. At this aim, or-
ganic fluorophores are already widely used as fluorescent markers.
However, they have some inherent drawbacks, such as poor resis-
tance to photobleaching, low quantum yields and optical proper-
ties sensitive to the surrounding environments.6 At this aim,
researchers have encapsulated organic dyes into nano-solid matri-
ces, such as silica,7 polystyrene (PS)8 and poly(methyl methacry-
late) (PMMA).9 With the protection of the nano-solid matrix,
⇑
Corresponding author. Tel.: +39 0516398283; fax: +39 0516398349.
E-mail addresses: greta.varchi@isof.cnr.it, g.varchi@isof.cnr.it (G. Varchi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.023

C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
6641
organic dyes can be protected from the surrounding environment,
2.2. Analytical measurements and methods
providing high photo-stability. Another advantage is that they
have a higher brightness due to high number of dye molecules
being loaded on one nanoparticle (NP).10 Dyes and/or drugs can
be loaded onto the nanoparticles according to different ap-
proaches, or combinations of them: (i) incorporated into the poly-
meric core, either by covalent bonds or by hydrophobic
interactions; (ii) covalently bonded on the surface or, finally, (iii)
electrostatically loaded on the external shell. Among these loading
methodologies, covalent bonding requires a higher energy to com-
plete the heterogeneous chemical reaction between the bioactive
molecules and the matrices; moreover functional groups (such as
succinimide ester or isothiocyanate or other linkers) are needed
in order to achieve covalent bond formation. In contrast, NPs elec-
trostatic post-loading needs less energy for the combination be-
tween bioactive molecules and nano-matrices, which typically
occurs, in a high reproducible manner, by simple mixing the spe-
cies and generally leads to higher loading ratio.11 Taking into ac-
count these considerations, we explored the viability of a novel
conjugated system, namely the attachment, based on ionic and
hydrophobic interactions, of different sulfonated organic salts to
positively charged biocompatible poly(methylmethacrylate)
(PMMA)-based core-shell nanoparticles (EA0) having an high den-
sity of ammonium groups on their shells. Similar nano-systems
have been previously employed for the low dose delivery of 20-
O-methyl-phosphorothioate AON, demonstrating dystrophin
expression restoration in body-wide striated muscles,12 and for
The hydrodynamic diameter of the nanospheres was deter-
mined by dynamic light scattering (DLS) at 25 °C using a Zetasizer
3000 HS system (Malvern, UK) equipped with a 10 mV He–Ne
laser. As far as the electrophoretic mobility is concerned, f-poten-
tial was measured at 25 °C by means of the same Zetasizer system.
The instrument calibration was checked using standard polysty-
rene latexes, supplied by Malvern Instruments with known zeta-
potential. To achieve a constant ionic background, the samples
were diluted with 1.0 mM KCl solutions to a concentration of
20 lg/ml. Each value is the average of five measurements. The
quaternary ammonium group load per gram of nanosphere was
determined by potentiometric titration of the bromide ions ob-
tained after complete ion exchange. The compound@EA0 samples
were isolated from the solution in which were suspended (both
during the synthesis, the loading and release experiments) by
means of the Eppendorf vials and a EBA 12 HETTIC centrifuge
equipped with a F-205 FALC tubes rotator. The release amount
was determined by spectrophotometric measures with a Lambda
20 Perkin Elmer spectrophotometer. Supernatants were filtered
by an Amicon Ultra 0.5 ml 100 K Millipore Filter. The positively
charged nanospheres were obtained by emulsion polymerization
of methyl methacrylate in the presence of the ionic co-monomer
2-(dimethyloctyl) ammonium ethylmethacrylate bromide as
emulsion stabilizer. A more detailed description of the NPs synthe-
sis is reported in the ESI material.
the effective delivery of
a
cisplatin derivative, namely
[PtCl3(NH3)]ꢀ, showing very promising biological activity in
C57B/6 mice bearing B16 murine melanoma.13 With the aim to ex-
tend the scope of these NPs systems for drug delivery and/or for
fluorescence imaging purpose, we report herein the electrostatic
loading of several kinds of sulfonates salts onto EA0 along with
the leakage extent of these molecules from the charged PMMA
matrices. In this context three different applications of the sulfo-
nates@EA0 systems will be described. In detail, their ability as
cytotoxic drugs and pro-drugs carriers was evaluated in vitro on
human non-small cell lung cancer carcinoma cell line NCI-H460
and in vivo against human ovarian carcinoma IGROV-1 cells. Be-
sides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt
(HPTS) was chosen for NPs loading and its internalization as bioi-
maging probe was evaluated on Hep G2 liver hepatocellular carci-
noma cells, by means of confocal microscopy. HPTS was chosen
because it is a highly water-soluble dye compound14 with low tox-
icity,15 and it is also very cheap compared with most other indica-
tors. Furthermore, this tri- or tetra-anionic dye is retained well
inside living cells at physiological pH values and it has been used
for measurement of cytoplasmic pH in many cell types,16 or acidic
organelle pH.17 The main limitation to the use of HPTS as an intra-
cellular indicator is its lack of cell permeability, and there is no
convenient pro-drug-like form to facilitate transport of this dye
into cells.18
2.3. Loading and release experiments
Loading. Briefly, sulfonates were combined with EA0 nanoparti-
cles by electrostatic interaction: a certain amount of sulfonate
derivative (typically 50–600 lg/mL in mQ H2O), was added to an
aqueous solution of positively charged EA0 (5 mg/mL) and stirred
in a Vortex apparatus for 20 s. at room temperature. Each sample
was than centrifuged (4 min, 16597 RCF) and the supernatant was
filtered with a 0.1 lm filter (1 min 10622 RCF). The filtrate was
analyzed with a Lambda 20 Perkin Elmer Spectrophotometer at
the correct wavelength, thus for each compound a calibration
curve was generated for further determination of the loading
values.
Release. Sulfonates release from EA0 was measured by spectro-
photometric analysis according to two different procedures: as a
function of the washing solution, or as a function of time by using
the same washing solution. Typically, a precise sulfonate-EA0 sam-
ple amount was treated with the washing solution, which was
added to restore the initial volume (typically 500 mL). In particular,
phosphate buffer saline (PBS) and PBS plus different amounts of
NaCl were employed as release solutions. Experiments were per-
formed either at room temperature or at 37 °C. The sample was
than re-suspended by vortexing it (20 s) and than centrifuged
(4 min, 16597RCF). The supernatant was filtered with a 0.1 lm fil-
ter (1 min 10622 RCF). The filtrate was analysed by spectrophoto-
metric measure.
2. Experimental details
2.1. Materials
2.4. In vitro biological assay
The human non-small cell lung cancer carcinoma cell line NCI-
H460 (ATCC, HTB-177) was cultured in RPMI-1640 containing 10%
foetal calf serum. Cytotoxicity was assessed by growth inhibition
assay after 72 h drug exposure. Cells in the logarithmic phase of
growth were harvested and seeded in duplicates into 6-well plates.
Twenty four hours after seeding, cells were exposed to the drug for
2-(Dimethylamino)ethyl methacrylate (DMAEMA), 1-bromooc-
tane, 2,20-azobis(2-methylpropionamidine) dihydrochloride (AIBA),
methyl methacrylate (MMA, 99.0%) (distilled before use) and HTPS
were purchased from Sigma-Aldrich. Hep G2 cell line were gener-
ously provided by Istituto Scientifico Romagnolo per lo Studio e la
cura dei Tumori (IRST) of Meldola (FC, Italy).

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C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
ples have been washed for three times with PBS media before
performing imaging experiments. Optical imaging has been per-
formed with a Nikon TE 2000 inverted microscope equipped with
a 20ꢁ and 60ꢁ objective and Hamamatsu CCD camera. Fluores-
cence images were collected at 515 nm, exciting samples with
mercury lamp at 450 nm. Coverslips were then mounted by means
of a customs made chamber onto Nikon TE 2000 inverted confocal
microscope equipped with a 20ꢁ or 60ꢁ oil-objective and 400 nm
diode, 488 nm Ar+ and 543 nm He-Ne lasers as excitation sources.
Images reported are representative of 2 different experiments per-
formed in triplicate.
3. Results and discussion
3.1. Synthesis of core-shell PMMA NPs (EA0)
As reported for many emulsion polymerization systems that
include water-soluble co-monomers, the reaction starts in the
aqueous phase leading to the formation of water-soluble oligorad-
icals that are rich in the water-soluble co-monomer until they
reach the solubility limit and precipitate to form primary particles,
which are able to grow by the incorporation of monomer and co-
monomer. The water-soluble units are preferentially located at
the nanosphere surface and actively participate in latex stabiliza-
tion. Nanospheres can thus be obtained with tailored surfaces dic-
tated by the chemical structure of the co-monomer/s employed,
ultimately leading to the formation of core–shell nanospheres. In
particular, these biocompatible, surfactant-free polymeric core-
shell nanoparticles are able to bind biologically active molecules
and macromolecules on their surface.11,22 EA0 nanoparticles are
characterized by an average SEM diameter of 150 40 nm (Fig. 1)
and by a hydrodynamic diameter, evaluated by dynamic light scat-
tering (DLS), of 183 nm. The density of the cationic groups avail-
able for interaction with the organic sulfonates turned out to be
Figure 1. SEM micrograph of EA0.
72 h and counted with a Coulter counter. IC50 is defined as the
inhibitory drug concentration causing a 50% decrease of cell
growth over that of untreated control.
2.5. In vivo biological assay
Female athymic Swiss nude mice, 7–10 weeks old (Charles
River, Calco, Italy) were used. Mice were maintained in laminar
flow rooms, keeping temperature and humidity constant. Mice
had free access to food and water. Experiments were approved
by the Ethics Committee for Animal Experimentation of the Fond-
azione IRCCS Istituto Nazionale Tumori of Milan according to insti-
tutional guidelines.19 Exponentially growing human IGROV-1
ovarian carcinoma cells20 (107 per mouse) were sc injected into
the right flank of athymic nude mice. Tumor lines were achieved
by serial sc passages of fragments from growing tumors into
healthy mice as described previously.21 Tumor growth was fol-
lowed by biweekly measurements of tumor diameters with a Ver-
nier caliper. Tumor volume (TV) was calculated according to the
formula: TV (mm3) = d2 ꢁ D/2, where d and D are the shortest
and longest diameters, respectively. Drugs were delivered iv every
day for four times starting when tumors were in the range of 50 to
100 mm3. Experimental groups were eliminated when mean TV
was about 1500 mm3. The efficacy of drug treatment was assessed
as: tumor weight inhibition percentage (TWI%) in treated versus
control mice, calculated as: TWI% = 100ꢀ(mean TW treated/mean
TW control ꢁ 100). The toxicity of the drug treatment was deter-
mined as lethal toxicity. Deaths occurring in treated mice before
the death of the first control mouse were ascribed to toxic effects.
0.16 lmol of quaternary ammonium groups per nanosphere’s
gram. Finally, the analysis of f-potential (46,7 mV), which was
measured after dialysis purification, confirmed that the cationic
co-monomer employed in the polymerization reaction is cova-
lently bound to the nanoparticle surface. The difference in the
NPs radii observed with PCS and SEM techniques could be ascribed
to the different environments in which the measurements are per-
formed, for example, water and air, respectively. The Stokes diam-
eter measured with PCS corresponds to the size of the solvated
nanoparticle, which is enhanced due to the presence of the exter-
nal ammonium groups; while SEM directly measure the dry radius.
3.2. Synthesis of taxol and camptothecin sulfonates
With the purpose of evaluating drugs-polymers conjugates, we
selected two conveniently tailored camptothecin (CPT) and taxol
analogues, respectively. CPT (4) is a natural product that exerts
cytotoxic activity through the inhibition of topoisomerase I leading
to cell death; unfortunately, under physiological conditions, the
2.6. HepG2 cell culturing, treatment and imaging
HepG2 cells has been cultured in T25 cell culture flasks contain-
ing Dulbecco’s Modified Eagle Media (DMEM)/HAM’s-F12 (1:1)
high in vivo reactive a-hydroxy-d-lactone pharmacophore, revers-
ibly hydrolyses to the inactive and toxic ‘ring opened’ carboxylate
form, making it inadequate for therapy.23 Paclitaxel (1) and its ana-
logs are currently recognized as the most important available
drugs for treatment of solid tumors,24 albeit these compounds
present relevant drawbacks, such as poor solubility, lack of tumor
cells selectivity25 and drug resistance expression. Following Kings-
ton’s procedure26 we first synthesized a taxol sulfonate analogue
(3, Scheme 1). Kingston and coworkers demonstrated that this
water-soluble analogue of taxol has in vitro and in vivo activity,
close to the parent compound, towards P-388 lymphocytic
supplemented with 10% v/v of Fetal Bovine Serum, 1%
1% insulin and antibiotics (penicillin/streptomycin (100 U/ml and
100 g/ml, respectively). Culture flasks were maintained in a
humidified incubator with 5% CO2 at 37 °C and cells has been split
every third day. The day before experiment cells has been re-plated
L-glutamine,
l
on 19 mm coverslips coated with poly-D-lysine, placed into 12-
multiwell plate at a concentration of 5 ꢁ 103 per sample. PMMA-
NPs, HPTS and HPTS@PMMA-NPs have been added to cell culture
media from respective sterile aqueous stock solutions at a mini-
mum dilution factor of 1:100. After 24 h of incubation, all the sam-

C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
6643
Scheme 1. Taxol and CPT sulfonate derivatives synthesis.
leukemia. CPT-sulfonate derivative 6 (Scheme 1) was straightfor-
wardly synthesized from CPT (see supplementary information for
synthesis details).
compound 3 showed a maximum loading value of 61.6 mg/mg,
while for compound the highest loading value was of
6
72.6 mg/mg, thus revealing a loading efficiency of 38% and 72%,
respectively. This parameter was determined by comparing the
highest mmol quantity of compound loaded on the nanospheres
with the effective density of cationic groups present on the
nanaoparticles (0.16 mmol/mg).
3.3. Loading and release experiments
As reported in Table 1, along with taxol and camptothecin sul-
fonate derivatives (3 and 6, respectively), we also employed some
thiophene based organic dyes (7–9), and commercially available 8-
hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS, 10)
(Table 1). Binding experiments exhibited different loading capac-
ity, mainly depending on sulfonate properties, such as medium sol-
ubility, molecular weight and steric hindrance. The release of
sulfonates from EA0 was evaluated in water, phosphate buffer
solution (PBS), which roughly mimes physiological conditions,27
and PBS containing different sodium chloride (NaCl) concentra-
tions. Release experiments provided different results depending
on the sulfonate: thiophenes based organic dyes (7–9) showed
very poor release efficiency, while HPTS was not released from
the NPs even after repeated washing, maybe due to the presence
of three sulfonate groups and/or to the system steric hindrance.
It is worth noting that very recently, Xu and coworkers reported
a similar approach for the synthesis of fluorescent dyes loaded
PMMA nanospheres by electrostatic adsorption.28 In that case fluo-
rescein sodium salt, Rhodamine 6G and Rhodamine B were em-
ployed as dyes. Compared to those examples, the use of a
reactive surfactant for EA0 synthesis, allows the formation of a
highly positively charged shell, which in turn provide a much high-
er loading capability with no release of the dye from the nano-
spheres, as also confirmed from confocal images (Fig. 4).
Moreover, compared to other micro- and nano-spheres in which
cationic surfactants are able to adsorb drugs onto particles’ sur-
face,29 our system does not suffer from inherent surface instability,
e.g. cationic surfactant desorption, thus leading to high reproduc-
ibility and lower toxic effects incidence.
The stability of 3@EA0 and 6@EA0 adducts was evaluated by
subsequent treatment with different washing solutions, such as
water, PBS and PBS containing different concentrations of NaCl.
In particular, Figure 2A shows that, starting from a sample of
3(or 6)@EA0 at a concentration of 40 lgcompound/mgparticle and per-
forming successive washes of the same samples, a gradual release
of the compound occurs. While little release is observed when
using pure water, it becomes much more pronounced when using
PBS. The chart also clearly indicates the greater tendency of the CPT
derivative to be released in phosphate buffer conditions.
In order to assess the release degree of the compound over time,
samples of 3@EA0 and 6@EA0 (40 lgcompound/mgparticle) were trea-
ted with PBS at 37 °C for 30 h. Residual loading of the compounds
has been determined by spectrophotometric analysis of aliquots of
the solutions collected at regular intervals (Fig. 2B). Time-
dependent experiments confirmed the higher tendency of the
CPT derivative to be released from the NPs with respect to the taxol
one, that is, 70% and 30% respectively after 2.5 h. NaCl addition to
the washing solution does not alter the release trend for 3@EA0,
unless it reaches a concentration of at least 2 M, when we observed
a radical release drop, maybe because an high Clꢀ concentration
prevent phosphates to replace the drug on the polymer (Fig. 2C).
Overall, experimental data showed a continuous release of the
drugs, which afterwards reaches a plateau, and a small amount
of drug seems to remain tightly associated to the nanospheres.
These analytical results prompted us to further explore the
behavior of these nano-sized systems in vitro and in vivo tumor
environment.
The opportunity to suitably modulate the bioactive molecule
release from the nanoparticle shell, represent a very interesting
characteristic in the field of theranostic nanomedicine, which
relates to the development of an integrated nano-therapeutic sys-
tem, which can diagnose, deliver targeted therapy and monitor the
response. With particular reference to taxol and CPT derivatives,
3.4. In vitro biological assay
In vitro studies were conduced on both 3@EA0 and 6@EA0 core-
shell nanoparticles conjugates to evaluate their therapeutic perfor-
mance. The human non-small cell lung cancer carcinoma cell line

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C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
Table 1
Sulfonates loading experiments on EA0 nanoparticles
Loaded compounds
Loading solutions (lg/mL)
50
100
200
300
600
9.76a
19.6
38.9
51.8
61.6
(3)
9.72
19.4
38.5
59.9
72.6
(6)
10.2
10
19.9
20
33.7
39.2
33.3
56.8
31.1
60
(7)
(8)
9.99
10
20
20
38.7
40
45.3
nab
52.2
nab
(9)
(10)
a
Amount of compound loaded onto the Nps (
Not available.
lg/mg) obtained by varying the concentration of the loading solutions.
b
NCI-H460 (ATCC, HTB-177) was used. Cytotoxicity was assessed by
growth inhibition assay after 72 h drug exposure. (Table 2) Paclit-
axel (1) and camptothecin (4) were used as references.
In comparison to the standard drugs, the two sulfonate deriva-
tives were less potent in reducing cell growth (1 vs 3, 4 vs 6). No
change in antiproliferative activity was evidenced when sulfonate
paclitaxel was loaded on nanoparticles (3 vs 3@EA0). On the con-
trary, loading of 6 on the pellets was detrimental for its potency
(6 vs 6@EA0). The very poor in vitro cytotoxicity of 6 suggests that
this derivative might act as a prodrug, thus requiring an enzyme
action to release the bioactive CPT, but more studies are needed
to establish if 6@EA0 conjugated can behave as a carrier-prodrug
system.
dose of the drug equivalent could be administrated as iv bolus
injection. With this treatment modality only 20 mg/kg (around
0.4 ml/injection) could be used, thus allowing the delivery of
15 mg/kg (Table 3). In vivo data show that the administration of
3@EA0 produced significant antitumor activity, while no effect
was found for the free taxane derivative (i.e., compound 3).
The lower efficacy of 3@EA0 in comparison to the paclitaxel
may reflect an intrinsic low efficacy of the taxane analog (3) used
for the conjugation. The in vivo lack of activity of 3 may be related
to an unfavorable pharmacokinetic behavior, which seems to be
overtaken when conjugation with EA0 nanospheres occurs.
3.6. Absorbance and fluorescence emission spectra of
HTPS@EA0
3.5. In vivo biological assay
Spectrophotometric analysis of the HPTS-doped PMMA NPs in
water solution (Fig. 3) revealed the same emission and absorption
maxima of the free fluorophore, thus indicating that the loaded dye
preserves its spectroscopic characteristics. These results, along
with cell imaging experiments (see point 3.7), suggest that the
HPTS@EA0 could be exploited as novel and effective pH responsive
nano-sensor, given that HTPS is well known for measurement of
In an attempt to explore possible therapeutic advantages of
3@EA0 adduct, we have performed comparative in vivo studies
using paclitaxel 1 as reference. The antitumor potency was evalu-
ated in nude mice bearing the human ovarian carcinoma IGROV-1
xenograft in 3 independent experiments. As a consequence of the
relatively low amount of drug linked to the nanoparticles, a limited

C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
6645
C
B
A
Figure 2. Release experiments. (A) Conditions: initial concentration [6@EA0] = [3@EA0] = 40
l
gcompound/mgparticle; washing solution: water followed by PBS; the chart reports
for 3@EA0). (B) Conditions: initial concentrations:
gcompound/mgparticle; washing solution: PBS a 37 °C. (C) Conditions: initial concentrations: [3-EA0] = 40 gcompound/mgparticle; washing solutions: PBS/
the residual amount of compound loaded on the NPs (40
[6@EA0] = [3@EA0] = 40
lg/mgparticle) after the wash (j for 6@EA0 and h
l
l
NaCl 0.5 M, 37 °C; PBS/NaCl 1 M, 37 °C; PBS/NaCl 2 M, 37 °C.
Table 2
Antiproliferative activity of 3@EA0 and 6@EA0a
Entry
Compound
IC50 (lM)
1
2
3
4
5
6
Paclitaxel (1)
3
3@EA0
CPT (4)
6
0.046 0.012
0.076 0.011
0.080 0.062
0,0014 0.00014
0113 0.0099
0333 0142
6@EA0
a
IC50, drug concentration required for 50% reduction of cell growth as compared
with untreated controls after 72-h exposure to the drug. Means SD are reported
from at least three experiments.
Table 3
Antitumor activity of 3@EA0 on the IGROV-1 ovarian carcinoma xenografted in nude
mice
Entry
Drug
Dose (mg/kg)
Days treatment
TWIa (%)
Toxb
*
1
2
3
1
3
15
15
15
3,4,5,6
3,4,5,6
3,4,5,6
91 (13)
0
44 (13)
0/4
0/4
0/4
Figure 3. Absorption and emission spectra of HPTS@EA0 in water.
*
3@EA0
*
Fig. S3C and D) displayed mostly diffuse fluorescence, even though
granular fluorescence was also visible at higher magnification
(Fig. 4F, arrows).
P < 0.05 by Student’s t test versus control mice.
Tumor weight inhibition percentage in treated over control mice, in parenthesis
a
the day on which it was assessed.
b
Dead/treated mice.
Collectively these data demonstrated that the loading of HPTS
on PMMA-NPs enable water-soluble dye internalization in HepG2
cells and suggested that intracellular localization of the nanoparti-
cles could occur in the cytoplasm as well as in endocytic vesicles.
The main advantage of our system, compared to previously re-
ported ones,27 mainly consists in the easiness of its preparation,
e.g. by simply mixing the proper amounts of NPs and fluorophore.
More studies will be carried out to demonstrate this feature.
cytoplasmic pH in many cell types,16 or acidic organelle pH.17 The
use of this NPs system could allow to overcome the main limitation
associated with the use of free HPTS as intracellular pH indicator,
that is, the high lack of cell permeability.
3.7. HepG2 cell culturing, treatment and imaging
4. Conclusions
To verify capability of PMMA-NPs to promote cellular internal-
ization of water soluble dye such as HPTS, HepG2 cells have been
incubated with PMMA-NPs, HPTS, and HPTS@NPs for 24 h. After
washing the sample three times with PBS media, we performed
fluorescence optical microscopy and confocal microscopy. Optical
images and fluorescence images, collected at 515 nm, exciting
samples with mercury lamp at 450 nm, revealed that most of the
HepG2 cells treated with 500 ng/mg HPTS@NPs (Fig. 4A and B)
were fluorescently labelled, whereas no fluorescent signal was
emitted by cells treated with the same concentration of unloaded
HPTS (Fig. 4C and D) nor by cells treated with PMMA-NPs (See Sup-
plementary Fig. S3A and B). Confocal imaging of the same samples
indicates that HepG2 cells treated with 500 ng/mg HPTS@NPs
(Fig. 4E and F) and 200 ng/mg HPTS@NPs (See Supplementary
In summary, the available data support the interest for these
PMMA nanoparticles-sulfonates conjugated systems as a promis-
ing formulation for theranostic applications. In fact, we demon-
strated that sulfonates release can be efficiently modulated based
on molecules steric hindrance and on the number of sulfonate
groups. This behavior allows exploiting PMMA-NPs@sulfonate sys-
tems either for slow release of drugs and pro-drugs, or for efficient
cell internalization of imaging dyes or for their combined use. The
easy functionalization of bioactive molecules with sulfonates
groups by means of straightforward and high yielding synthetic
methodologies,30 paves the way to a wide range of applications
in terms of drug (pro-drugs) delivery and diagnosis tools.

6646
C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
Ph-C
Ph-C
Fluo
B
A
C
100 µm
100 µm
500 ng/ml HPTS@NP
Fluo
500 ng/ml HPTS
E
F
D
100 µm
50 µm
500 ng/ml HPTS@NP
Figure 4. (A–D) Phase Contrast (Ph-C, A,C) and relative fluorescent (fluo, B,D) micrographs collected from HepG2 cells treated with 500 ng/ml HPTS@NPs m. E) Low
(A and B) and 500 ng/ml HTPS (C and D). Scale bar is 100 single plane confocal image representative of fluorescent emission collected by HepG2 cells
magnification (20ꢁ)
l
after 24 h of incubation with 500 ng/ml HPTS@NPs. (F) higher magnification of the same sample revealed that cells exhibit mostly diffuse but also granular fluorescence
pattern (white arrows).
Moreover, the great versatility of the nanoparticles synthesis, al-
lows to easily obtain nano-matrix with different degree of external
also supported by Italian MIUR project FIRB-RBPR05JH2
(ITALNANONET).
charges and with sizes ranging from 20 nm to 1 lm. Furthermore, a
fine tuning of the polymerization parameters, such as the nature of
the monomer, of the co-monomer and their relative amounts, al-
lows the further external shell functionalization, which could be
extremely useful for the covalent attachment of target moieties,
for example, peptides, antibodies, etc. Overall, in vivo biological
data strongly support the potential value of these core-shell nano-
particle as delivery system for negatively charged drugs or biolog-
ically active molecules. The administration of the nanoparticles by
slow infusion is expected to improve the efficacy. Relevant to this
point is the good tolerability of this novel drug formulation. These
results confirm the potential of the PMMA core-shell nanoparticles
as drug carriers, previously reported by Osella and coworkers,11 by
extending the scope of these systems to a more wide range of mol-
ecules. Additionally, we have demonstrated the ability of these
PMMA core-shell nanoparticles to act as efficient carriers of fluoro-
phores. In particular, the electrostatic irreversible attachment of
HPTS on the outer NPs shell, allows the efficient internalization
of the dye, thus introducing new chances for cellular uptake of neg-
atively charged fluorophores and their possible use as nanosensors.
In principle, thanks to the high PMMA NPs external charge density,
sequential post-loading of different sulfonates is achievable, thus
allowing the preparation of nanocarriers either with bi-modal drug
delivery behaviour, e.g., loading two different drugs with diverse
action modes, or as theranostic systems, for example, for diagnosis,
drug-delivery and response monitoring. Investigation of these pos-
sibilities is actively underway in our laboratories.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.023.
References and notes
1. Feynman, R. Science 1991, 254, 1300.
2. (a) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016; (b) Wang, S.; Mamedova,
N.; Kotov, A.; Chen, W.; Studer, J. Nano Lett. 2002, 2, 817.
3. Panatarotto, D.; Prtidos, C. D.; Hoebeke, J.; Brown, F.; Kramer, E.; Briand, J. P.;
Muller, S.; Prato, M.; Bianco, A. Chem. Biol. 2003, 10, 961.
4. (a) Matsumura, Y.; Maeda, H. Cancer Res. 1986, 46, 6387; (b) Iyer, A.; Khaled, G.;
Fang, J.; Maeda, H. Drug Discovery Today 2006, 11, 812.
5. (a) Nel, A. E.; Mädler, L.; Velegol, D.; Xia, T. E.; Hoek, M.; Somasundaran, P.;
Klaessig, F.; Castranove, V.; Thompson, M. Nat. Mater. 2009, 8, 543; (b) Fleck, C.
C.; Netz, R. R. Europhys. Lett. 2004, 67, 314.
6. (a) Zhou, X.; Zhou, J. Anal. Chem. 2004, 76, 5302; (b) Ma, D. L.; Kell, A. J.; Tan, S.;
Jakubek, Z. J.; Simard, B. J. Phys. Chem. C 2009, 113, 15974.
7. (a) Andrew, A. B.; Jelena, V.; Hooisweng, O.; Erik, H.; Oula, P. M.; Martin, B.;
Steven, M. L.; Ulrich, W.; Michelle, B. Nano Lett. 2009, 9, 442; (b) Liang, S.; John,
C. L.; Xu, S. P.; Chen, J.; Jin, Y. H.; Yuan, Q.; Tan, W. H.; Zhao, J. X. J. In Silica-Based
Nanoparticles: Design and Properties Advanced Fluorescence Reporters in
Chemistry and Biology II; Demchenko, A., Ed.; Springer: Berlin, 2010; p 229.
8. Wittmershaus, B. P.; Skibicki, J. J.; McLafferty, J. B.; Zhang, Y. Z.; Swan, S. J.
Fluoresc. 2001, 11, 119.
9. (a) Melanie, B.; Muthupandian, A.; Franz, G. J. Am. Chem. Soc. 2003, 125, 525; (b)
Bosma, G.; Pathmamanoharan, C.; Hoog, E. H. A. D.; Kegel, W. K.; Blaaderen, A.
V.; Lekkerkerker, H. N. W. J. Colloid Interface Sci. 2002, 245, 292.
10. (a) Yao, G.; Wang, L.; Wu, Y. R.; Smith, J.; Xu, J. S.; Zhao, W. J.; Lee, E. J.; Tan, W.
H. Anal. Bioanal. Chem. 2006, 385, 518; (b) Rampazzo, E.; Bonacchi, S.; Montalti,
M.; Prodi, L.; Zaccheroni, N. J. Am. Chem. Soc. 2007, 129, 14251.
11. Wang, S.; Fan, W.; Kim, G.; Jin Hah, H.; Koo Lee, Y.-E.; Kopelman, R.; Ethirajan,
M.; Gupta, A.; Goswami, L. N.; Pera, P.; Morgan, J.; Pandey, R. K. Lasers Surg.
Med. 2011, 43, 686.
12. Rimessi, P.; Sabatelli, P.; Fabris, M.; Braghetta, P.; Bassi, E.; Spitali, P.; Vattemi, G.;
Tomelleri, G.; Mari, L.; Perrone, D.; Medici, A.; Neri, M.; Bovolenta, M.; Martoni,
E.; Maraldi, N. M.; Gualandi, F.; Merlini, L.; Ballestri, M.; Tondelli, L.; Sparnacci,
K.; Bonaldo, P.; Caputo, A.; Laus, M.; Ferlini, A. Mol. Ther. 2009, 17, 820.
Acknowledgments
Mediteknology S.r.l, Area Ricerca CNR, Via Gobetti 101, I-40129
Bologna, Italy for kindly providing compounds 6–8. This work was

C. Monasterolo et al. / Bioorg. Med. Chem. 20 (2012) 6640–6647
6647
13. Monti, E.; Gariboldi, M. B.; Ravizza, R.; Molteni, R.; Sparnacci, K.; Laus, M.;
Gabano, E.; Ravera, M.; Osella, D. Inorg. Chim. Acta 2009, 362, 4099.
14. Zhang, Z.; Seitz, W. R. Anal. Chim. Acta 1984, 160, 47.
15. Wolfbeis, O. S.; Fuerlinger, E.; Kroneis, H.; Marsoner, H.; Fresenius’, Z. Anal.
Chem. 1983, 314, 119.
16. Giuliano, K. A.; Gillies, R. J. Anal. Biochem. 1987, 167, 362.
17. Overly, C. C.; Lee, K. D.; Berthiaume, E.; Hollenbeck, P. J. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 3156.
23. Wall, M. E. In Biochemie und Physiologie der Alkaloide; Mothes, K., Schreiber, K.,
Eds.; Akademie Verlag: Berlin, 1969; p 77.
24. (a) Nicolau, K. C.; Dai, W. M.; Guy, R. K. Angew. Chem., Int. Ed. Engl. 1994, 33, 15;
(b) Menzin, A. W.; King, S. A.; Aikins, J. K.; Mikuta, J. J.; Rubin, S. C. Cancer
Gynecol. Oncol. 1994, 54, 103; (c) Rowinski, E. K. Annu. Rev. Med. 1997, 48, 353.
25. Chari, R. V. J. Adv. Drug Delivery Rev. 1998, 31, 89.
26. Zhao, Z.; Kingston, D. G. I.; Crosswell, A. R. J. Nat. Prod. 1991, 54, 1607.
27. Matthews, G. G. Cellular Physiology of Nerve and Muscle, IV ed.; Blackwell
Publishing Malden: MA, 2003. pp. 5–6.
18. Han, J.; Burgess, K. Chem. Rev. 2010, 110, 2709.
19. Workman, P.; Balmain, A.; Hickman, J. A.; McNally, N. J.; Mitchison, N. A.;
Pierrepoint, C. G.; Raymond, R.; Rowlatt, C.; Stephens, T. C.; Wallace, J. Br. J.
Cancer 1988, 58, 109.
20. Soranzo, C.; Pratesi, G.; Zumino, F. Anticancer Drugs 1990, 1, 23.
21. Polizzi, D.; Pratesi, G.; Tortoreto, M.; Supino, R.; Riva, A.; Bombardelli, E.;
Zumino, F. Cancer Res. 1999, 59, 1036.
28. Wang, X.; Xu, S.; Liang, C.; Li, H.; Sun, F.; Xu, W. Nanotechnology 2011, 22,
275608.
29. (a) O’Hagan, D.; Singh, M.; Ugozzoli, M.; Wild, C.; Barnett, S.; Chen, M.;
Schaefer, M.; Doe, B.; Otten, G. R.; Ulmer, J. B. J. Virol. 2001, 75, 9037; (b) Oster,
C. G.; Kim, N.; Grode, L.; Barbu-Tudoran, L.; Schaper, A. K.; Kaufmann, S. H. E.;
Kissel, T. J. Control Release 2005, 104, 359.
22. Laus, M.; Sparnacci, K.; Lelli, M.; Vannini, R.; Tondelli, L. J. Polym. Sci., Part A:
Polym. Chem. 2000, 38, 1110.
30. Matthews, G. G. Cellular Physiology of Nerve and Muscle, fourth ed.; Blackwell
Publishing Malden: MA, 2003. pp. 5–6.