Selegiline-functionalized, PEGylated poly(alkyl cyanoacrylate) nanoparticles: Investigation of interaction with amyloid-β peptide and surface reorganization

Article abstract of DOI:10.1016/j.ijpharm.2011.01.015

Alzheimer's disease (AD) is a neurodegenerative disorder for which the research of new treatments is highly challenging. Since the fibrillogenesis of amyloid-β peptide 1-42 (Aβ_1-42) peptide is considered as a major cause of neuronal degeneration, specific interest has been focused on aromatic molecules for targeting this peptide. In this paper, the synthesis of selegiline-functionalized and fluorescent poly(alkyl cyanoacrylate) nanoparticles (NPs) and their evaluation for the targeting of the Aβ_1-42 peptide are reported. The synthetic strategy relied on the design of amphiphilic copolymers by tandem Knoevenagel-Michael addition of cyanoacetate derivatives, followed by their self-assembly in aqueous solutions to give the corresponding NPs. Different cyanoacetates were used: (i) hexadecyl cyanoacetate (HDCA) to form the hydrophobic core of the NPs; (ii) rhodamine B cyanoacetate (RCA) for fluorescent purposes; (iii) methoxypoly(ethylene glycol) cyanoacetate (MePEGCA) for stealth properties and (iv) selegiline-poly(ethylene glycol) cyanoacetate (SelPEGCA) to obtain the desired functionality. Two different amphiphilic copolymers were synthesized, a selegiline-containing copolymer, P(MePEGCA-co-SelPEGCA-co-HDCA), and a rhodamine-labelled counterpart, P(MePEGCA-co-RCA-co-HDCA), further blended at variable ratios to tune the amount of selegiline moieties displayed at the surface of the NPs. Optimal formulations involving the different amphiphilic copolymers were determined by the study of the NP colloidal characteristics. Interestingly, it was shown that the zeta potential value of the selegiline-functionalized nanoparticles dramatically decreased, thus emphasizing a significant modification in the surface charge of the nanoparticles. Capillary electrophoresis has then been used to test the ability of the selegiline-functionalized NPs to interact with the Aβ_1-42 peptide. In comparison with non functionalized NPs, no increase of the interaction between these functionalized NPs and the monomeric form of the Aβ_1-42 peptide was observed, thus highlighting the lack of availability of the ligand at the surface of the nanoparticles. A mechanism explaining this result has been proposed and was mainly based on the burial of the hydrophobic selegiline ligand within the nanoparticles core.

Full text of DOI:10.1016/j.ijpharm.2011.01.015

International Journal of Pharmaceutics 416 (2011) 453–460
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Selegiline-functionalized, PEGylated poly(alkyl cyanoacrylate) nanoparticles:
Investigation of interaction with amyloid-␤ peptide and surface reorganization
Benjamin Le Droumaguet1, Hayfa Souguir1, Davide Brambilla, Romain Verpillot, Julien Nicolas∗,
Myriam Taverna, Patrick Couvreur, Karine Andrieux∗∗
Laboratoire de Physico-Chimie, Pharmacotechnie et Biopharmacie, Univ. Paris-Sud 11, UMR CNRS 8612, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément,
92296 Châtenay-Malabry, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease (AD) is a neurodegenerative disorder for which the research of new treatments
is highly challenging. Since the fibrillogenesis of amyloid-␤ peptide 1–42 (A␤1–42) peptide is con-
sidered as a major cause of neuronal degeneration, specific interest has been focused on aromatic
molecules for targeting this peptide. In this paper, the synthesis of selegiline-functionalized and fluo-
rescent poly(alkyl cyanoacrylate) nanoparticles (NPs) and their evaluation for the targeting of the A␤1–42
peptide are reported. The synthetic strategy relied on the design of amphiphilic copolymers by tandem
Knoevenagel–Michael addition of cyanoacetate derivatives, followed by their self-assembly in aqueous
solutions to give the corresponding NPs. Different cyanoacetates were used: (i) hexadecyl cyanoacetate
(HDCA) to form the hydrophobic core of the NPs; (ii) rhodamine B cyanoacetate (RCA) for fluores-
cent purposes; (iii) methoxypoly(ethylene glycol) cyanoacetate (MePEGCA) for stealth properties and
(iv) selegiline-poly(ethylene glycol) cyanoacetate (SelPEGCA) to obtain the desired functionality. Two
different amphiphilic copolymers were synthesized, a selegiline-containing copolymer, P(MePEGCA-
co-SelPEGCA-co-HDCA), and a rhodamine-labelled counterpart, P(MePEGCA-co-RCA-co-HDCA), further
blended at variable ratios to tune the amount of selegiline moieties displayed at the surface of the NPs.
Optimal formulations involving the different amphiphilic copolymers were determined by the study of
the NP colloidal characteristics. Interestingly, it was shown that the zeta potential value of the selegiline-
functionalized nanoparticles dramatically decreased, thus emphasizing a significant modification in the
surface charge of the nanoparticles. Capillary electrophoresis has then been used to test the ability of the
selegiline-functionalized NPs to interact with the A␤1–42 peptide. In comparison with non functionalized
NPs, no increase of the interaction between these functionalized NPs and the monomeric form of the
A␤1–42 peptide was observed, thus highlighting the lack of availability of the ligand at the surface of the
nanoparticles. A mechanism explaining this result has been proposed and was mainly based on the burial
of the hydrophobic selegiline ligand within the nanoparticles core.
Received 29 October 2010
Received in revised form
28 December 2010
Accepted 5 January 2011
Available online 18 January 2011
Keywords:
Alzheimer’s disease
Selegiline
Amyloid-␤ peptide
Poly(alkyl cyanoacrylate) nanoparticles
Functionalization
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
under debate (Aliev et al., 2004; de la Torre, 2004; Korolainen
et al., 2010), the disease is physiologically characterized by two
Alzheimer’s disease (AD) is a severe neurodegenerative illness
affecting more and more aging population over the world. AD rep-
resents the most common cause of dementia and is characterized
by a progressive, but irreversible deterioration of cognitive func-
tions and a loss of memory (Querfurth and LaFerla, 2010). Although
the mechanisms leading to these dysfunctions are still unclear and
main pathological features. These hallmarks are: (i) the intracel-
lular accumulation of the hyperphosphorylated tau protein in the
neurons and (ii) the progressive production and aggregation of ␤-
amyloid peptide (A␤) (Aguzzi and O’Connor, 2010; Panza et al.,
2009), the latter being considered as the main cause of AD (Gralle
et al., 2009). Neurons produce A␤ peptides with a variable num-
ber of amino-acids and the A␤ peptide 1–42 (A␤1–42) is believed
to be the most representative and the most toxic species in AD
physiopathology due to its high tendency to spontaneously self-
aggregate (Chow et al., 2010; García-Matas et al., 2010).
∗
Corresponding author. Tel.: +33 1 46 83 58 53; fax: +33 1 46 83 59 46.
Corresponding author. Tel.: +33 1 46 83 58 12; fax: +33 1 46 83 59 46.
E-mail addresses: julien.nicolas@u-psud.fr (J. Nicolas),
∗∗
In the last decades, the pharmaceutical companies have only
attempted to combat clinical manifestations of AD. In particular,
acetylcholinesterase (AchE) inhibitors (Birks et al., 2009; Munoz-
karine.andrieux@u-psud.fr, karine.andrieux@cep.u-psud.fr (K. Andrieux).
1
These authors contributed equally to this work.
0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.01.015

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B. Le Droumaguet et al. / International Journal of Pharmaceutics 416 (2011) 453–460
Fig. 1. General approach for the synthesis of selegiline-functionalized, PEGylated poly(alkyl cyanoacrylate) nanoparticles and their possible interaction with the HiLyte
FluorTM 488 labelled amyloid-␤ 1–42 (A␤1–42) peptide.
Torrero, 2008; Sugimoto et al., 1995) and N-methyl-d-aspartate
(NMDA) receptor antagonists (Kemp and McKernan, 2002; Parsons
et al., 1999; Reisberg et al., 2003) have been widely used but
without significant success. Unfortunately, no efficient treatment
aiming at the eradication of AD has been proposed so far.
inhibitor for the treatment of AD (Sano et al., 1997; Tom,
2000; Wilcock et al., 2002). Selegiline also exhibited a cer-
tain affinity for the A␤1–42 peptide (Re et al., 2010). In this
study, we presented the synthesis of selegiline-functionalized
and fluorescent poly(alkyl cyanoacrylate) nanoparticles for A␤1–42
peptide targeting and anti-fibrillogenesis purposes. By captur-
ing monomeric peptide at the surface of these nanoparticles, we
aim to inhibit its fibrillogenesis. The synthesis strategy relied
on the dual modification of poly[methoxypoly(ethylene glycol)
cyanoacrylate-co-poly(hexadecyl cyanoacrylate)] (P(MePEGCA-co-
HDCA)) copolymers (Nicolas and Couvreur, 2009; Peracchia et al.,
1999) to introduce: (i) selegiline moieties at the extremity of
PEG chains and (ii) rhodamine B probes within the copolymer
structure. The P(MePEGCA-co-HDCA) copolymer scaffold has been
selected due to its successful ability to cross the BBB (Calvo
et al., 2001; Garcia-Garcia et al., 2005a, 2005b). Moreover, we
took advantage of a recent study that demonstrated the efficient
derivatization of such copolymers with azido-functional groups
allowing subsequent reaction with alkyne-containing ligands
through copper-catalyzed azide–alkyne cycloaddition (CuAAC)
(Nicolas et al., 2008). Functionalization was undertaken with selegi-
line by CuAAC via its native alkyne group. NPs were obtained from
the self-assembly of different ratios of selegiline-functionalized and
rhodamine B-tagged copolymers in order to tune the amount of
selegiline moieties displayed at their surface (Fig. 1). The result-
ing functionalized nanoparticles, obtained by the nanoprecipitation
technique, were characterized by dynamic light scattering (DLS)
and zeta potential (ꢀ) measurements. Finally, we used capillary
electrophoresis (CE) to monitor the interaction of the selegiline-
functionalized NPs with the A␤1–42 peptide (Brambilla et al.,
2010b).
Recently, different studies promoted the utilization of small
aromatic molecules for targeting the A␤1–42 peptide, such as cur-
cumine (Garcia-Alloza et al., 2007; Lim et al., 2001; Ono et al., 2004)
and its derivatives (Narlawar et al., 2008), Thioflavine T (Xie et al.,
2006), Congo red (Carter and Chou, 1998; Lorenzo and Yankner,
1994; Virginia, 2002) and their analogues such as Chrysamine G
(Klunk et al., 1998; Lee, 2002; Virginia, 2002) and X34 (Christopher
et al., 2001; Styren et al., 2000). These molecules have shown a cer-
tain efficiency to hinder, or even to stop, the oligomerization of the
A␤1–42 peptide and thus the production of oligomers and/or fibrils,
which are commonly considered as the toxic species for neuronal
cells. These ligands have also been extensively used as tracers of the
presence of senile plaques in the brain due to their fluorescent prop-
erties. Moreover, they can be modified with radiolabelled elements
for diagnostic purposes (Dezutter et al., 1999a, 1999b; Nordberg,
2004; Wang et al., 2002, 2004). Unfortunately, these compounds
do not overpass the blood–brain barrier (BBB). To circumvent this
crucial problem, researchers have developed three main strategies:
(i) the chemical modification of ligands to make them able to cross
the BBB, (ii) their encapsulation into nanoparticles (NPs) and (iii)
their ligation to NPs (Narlawar et al., 2008; Sun et al., 2010).
Among the pool of efficient ligands discovered so far, we
have focused our attention on selegiline, an aromatic molecule
that has been employed to slow down the progression of
the Parkinson’s disease (Tetrud and Langston, 1989), but that
has been more importantly used as monoamine oxidase-B

B. Le Droumaguet et al. / International Journal of Pharmaceutics 416 (2011) 453–460
455
2. Materials and methods
2.1. Materials
2.2.3. Capillary electrophoresis
Capillary electrophoresis (CE) was performed on a PA 800
instrument (Beckman Coulter, Roissy, France) using uncoated sil-
ica capillaries (Phymep, Paris) with an internal diameter of 50 ␮m
and 50 cm total length (40 cm effective length was employed for
the separation). All buffers were prepared with deionized water
and were filtered through a 0.22 ␮m membrane (VWR) before use.
Prior analysis, the capillaries were preconditioned by the following
rinsing sequence: 0.1 M NaOH for 5 min, 1 M NaOH for 5 min and
then deionized water for 5 min. The in-between run rinsing cycles
were carried out by pumping sequentially through the capillary:
water for 5 min, 50 mM SDS for 2 min, to inhibit the aggregation
and subsequent peptide adsorption on the capillary wall, and 0.1 M
NaOH for 5 min. The samples were introduced into the capillary
by hydrodynamic injection under 3.4 kPa. The capillary was ther-
mostated at 25 ◦C and the samples were maintained at 37 ◦C by
the storage sample module of the PA 800 apparatus. The separa-
tions were carried out at 16 kV with positive polarity at the inlet
using 80 mM phosphate buffer pH 7.4. The running electrolyte was
renewed after each run. The fluorescent peptide was detected by
a laser-induced fluorescence (LIF) detection system equipped with
3.5 mW argon-ion laser with a wavelength excitation of 488 nm,
the emission being collected through a 520 nm band-pass filter.
Peak areas were estimated using the 32 KaratTM software (Beckman
Coulter). The results are expressed as the evolution of percent-
age of monomer peak area as a function of time (Brambilla et al.,
2010b).
Azidopoly(ethylene
glycol)
cyanoacetate
glycol)
(N3PEGCA,
cyanoacetate
Mn = 2 kDa),
methoxypoly(ethylene
(MePEGCA, Mn = 2 kDa) and hexadecyl cyanoacetate (HDCA)
were obtained from already published synthetic procedures
(Nicolas et al., 2008). Poly[hexadecyl cyanoacrylate-co-rhodamine
B
cyanoacrylate-co-methoxypoly(ethylene glycol) cyanoacry-
late] (P(HDCA-co-RCA-co-MePEGCA), C1) copolymer containing
5% rhodamine cyanoacetate (with respect to HDCA monomer)
and poly[hexadecyl cyanoacrylate-co-azidopoly(ethylene gly-
col) cyanoacrylate] P(HDCA-co-N3PEGCA) copolymers were
obtained by tandem Knoevenagel–Michael addition of cyanoac-
etate (Brambilla et al., 2010a; Nicolas et al., 2008). NaH2PO4
(>99%) was purchased from Merck
& Co (Fontenay-sous-
Bois, France). Na2HPO4 (>98%) was obtained from Prolabo
(Strasbourg, France). Pluronic F-68 (99%), sodium carbon-
ate (Na2CO3, BioUltra, minimum 99.5%) and sodium dodecyl
sulfate (SDS, 99%), R-(−)-Deprenyl hydrochloride (selegiline,
≥98%), N-dicyclohexylcarbodiimide (DCC, Fluka, ≥99% GC), 4-
dimethylaminopyridine (DMAP, 99%), formaldehyde solution
(37 wt.% in water, contains 10–15% methanol as stabilizer), pyrro-
lidine (99% GC, T), copper sulfate pentahydrate (CuSO4·5H2O,
≥98%), (+)-sodium l-ascorbate (crystalline, 98%), ethylene-
diaminetetraacetic acid tetrasodium salt hydrate (EDTA, practical
grade ∼95%) were purchased from Sigma–Aldrich (St. Quentin
Fallavier, France). Sodium hydroxide (NaOH) solution (1 M) was
obtained from VWR (Fontenay-sous-Bois, France). All solvents
were purchased at the highest grade from Carlo Erba (Val de
Reuil, France). Deuterated chloroform (CDCl3, ≥99.8 atom% D) and
dimethyl sulfoxide (DMSO-d6, ≥99.8 atom% D) were obtained from
Sigma–Aldrich and used as received.
2.3. Synthesis of P(MePEGCA-co-SelPEGCA-co-HDCA) copolymers
2.3.1. Neutral form of selegiline
Selegiline·HCl (100 mg, 0.45 mmol) was dispersed in a 1 M
Na2CO3 solution (5 mL, 5 mmol). The solution was vigorously
stirred for 1 h and transferred into a separation funnel. Neutralized
selegiline was extracted with diethylether (3× 15 mL). The joined
organic fractions were dried over MgSO4, filtered off and the solvent
was removed under reduced pressure to give the desired product
as a colourless oil in a quantitative yield. 1H NMR (d6-DMSO, 298 K)
ı (ppm): 0.87 (d, 3H, J = 6.6 Hz), 2.27 (s, 3H), 2.90 (td, 2H, J = 4.1 and
12.6 Hz), 3.10 (t, 1H, J = 2.5 Hz), 3.35 (t, 2H, J = 2.45 Hz), 7.13–7.20
(m, 3H), 7.22–7.30 (m, 2H).
Lyophilized HiLyte FluorTM 488 labelled amyloid-␤ 1–42
(A␤1–42) peptide was provided by ANASPEC (Le Perray en Yvelines,
France). Spectra/Por dialysis bags (2 kDa molecular weight cut off)
were purchased from Spectrum Laboratories Inc. and used after
washing in deionized water for 1 h.
2.2. Analytical techniques
2.2.1. 1H NMR spectroscopy
All 1H NMR spectra were performed in deuterated solvents
(CDCl3 or d6-DMSO) at ambient temperature on a Bruker Avance
300 MHz spectrometer.
2.3.2. Selegiline-poly(ethylene glycol) (SelPEG)
Selegiline-poly(ethylene glycol) was obtained by click chem-
istry (Kolb et al., 2001). In
azidopoly(ethylene glycol) (200 mg, 0.1 mmol) and neutralized
selegiline (37.5 mg, 0.2 mmol) were dissolved in 2 mL of
a 10 mL round bottom flask,
a
1:1 tBuOH/deionized water mixture. The solution was bub-
bled for 10 min with nitrogen at room temperature. Then,
CuSO4·5H2O (2.5 mg, 0.01 mmol) and (+)-sodium l-ascorbate
(4.0 mg, 0.02 mmol) were dissolved in 1 mL of deionized water and
the resulting solution was immediately added to the reaction mix-
ture, followed by nitrogen bubbling for another 20 min and finally
stirred overnight at room temperature under nitrogen atmosphere.
tBuOH was then removed under reduced pressure and the aqueous
solution was diluted with 1 mL of a 1 M EDTA aqueous solution and
extensively dialyzed against deionized water using a 2 kDa dialy-
sis bags. After dialysis, water was removed under reduced pressure
and the product, dissolved in DCM, was dried over MgSO4. After
removal of the solvent, the pure product was obtained as a slightly
brown powder with 75% yield. 1H NMR (298 K, CDCl3) ı (ppm): 1.13
(d, 3H, J = 5.5 Hz), 2.55 (br s, 3H), 3.35–3.94 (m, 188H), 4.20 (s, 2H),
4.57 (t, 2H, J = 5.1 Hz), 7.19–7.35 (m, 5H), 8.06 (s, 1H).
2.2.2. Mean average diameter and zeta potential measurements
Nanoparticles average diameter (Dz) was measured by dynamic
light scattering (DLS) with a Nano ZS from Malvern (173◦ scat-
tering angle) at a temperature of 25 ◦C. Colloidal stability was
evaluated as a function of time in different buffers (cell culture
medium, phosphate buffer saline) at 25 ◦C. The zeta potential (ꢀ)
was calculated with the same apparatus from the electrophoretic
mobility (u) using the Smoluchowsky relationship ꢀ = ꢁu/εf(Äa),
where it is assumed that Äa ꢀ 1, where ꢁ is the solution viscos-
ity, ε is the dielectric constant of the medium, and Ä and a are the
Debye–Hückel parameter and the particle radius, respectively, f(Äa)
is the Henry’s function and its value is 1.5 (Smoluchowski approxi-
mation) when the electrophoretic determinations of zeta potential
are made in aqueous media and moderate electrolyte concentra-
tion. Zeta-potential measurements of nanoparticle dilute aqueous
solutions were carried out at 25 ◦C in 10−3 M KCl.

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B. Le Droumaguet et al. / International Journal of Pharmaceutics 416 (2011) 453–460
nanoparticles, aliquots of HiLyte FluorTM 488 labelled A␤1–42 pep-
tide stock solutions were diluted in 20 mM phosphate buffer
(NaH2PO4) pH 7.4 containing 20 ␮M of P(MePEGCA-co-RCA-
2.3.3. Synthesis of selegiline-poly(ethylene glycol) cyanoacetate
(SelPEGCA)
In a 10 mL round bottom flask containing the selegiline-
poly(ethylene glycol) (150 mg, 68 ␮mol) and cyanoacetic acid
(11.5 mg, 136 ␮mol), were added 2 mL of DCM and 1 mL of ethylac-
etate. The resulting solution was cooled down with ice and placed
under nitrogen atmosphere. DCC (21 mg, 102 ␮mol) and a cat-
alytic amount of DMAP were dissolved in 1 mL of DCM and added
dropwise. The reaction mixture was stirred at room temperature
overnight under nitrogen atmosphere. After filtration to discard
the insoluble dicyclohexylurea (DCU), the solvents were removed
under reduced pressure and the resulting residue was dissolved in
a minimum of DCM and precipitated in cold diethyl ether to give
the pure product as a slightly brown powder with 86% yield. 1H
NMR (298 K, CDCl3) ı (ppm): 1.15 (d, 3H, J = 5.5 Hz), 2.52 (br s, 3H),
3.52 (s, 2H), 3.35–3.94 (m, 188H), 4.19 (s, 2H), 4.35 (t, 2H, J = 4.7 Hz),
4.59 (t, 2H, J = 5.1 Hz), 7.19–7.35 (m, 5H), 8.06 (s, 1H).
co-HDCA) nanoparticles N3 or N8 to obtain
a final peptide
concentration of 5 ␮M. The samples were then incubated at 37 ◦C
and analyzed by CE approximately every 2 h using LIF detection
(Brambilla et al., 2010b).
3. Results and discussion
As previously mentioned, the “amyloid hypothesis” (i.e. the
hypothesis that the amyloid fibrils formed by oligomerization of
A␤ peptides and especially of the A␤1–42 peptide are the major
causes of the neuronal degeneration), is so far considered by
researchers as the most probable cause of AD. Consequently,
more and more research groups have focused their efforts on the
inhibition of this fibrillogenesis with suitable compounds/ligands.
Among these ligands, selegiline has been chosen due to its termi-
nal alkyne functionality that allowed for further covalent linkage
on azide-containing copolymers/nanoparticles via CuAAC reaction.
The synthesis of selegiline-functionalized poly(alkyl cyanoacry-
late) nanoparticles was therefore undertaken.
2.3.4. Synthesis of selegiline-functionalized copolymer
The procedure for the synthesis of 10% selegiline-functionalized
copolymer (P(MePEGCA-co-SelPEGCA-co-HDCA)) is as follows. In
a 10 mL round bottom flask were introduced HDCA (120 mg,
0.38 mmol), MePEGCA (180 mg, 0.864 mmol) and SelPEGCA (22 mg,
0.096 mmol) in 2 mL of DCM and 1 mL of EtOH. To this solution were
consecutively added dropwise formalin (0.2 mL, 4.43 mmol) and
pyrrolidine (10 ␮L, 0.122 mmol). The reaction was stirred overnight
under nitrogen atmosphere. The solvents were removed under
reduced pressure and the residue was redissolved in DCM. The
organic phase was washed with 3 portions of deionized water,
once with 1 M HCl solution, once with brine and finally dried over
MgSO4. The solvent was removed under reduced pressure to afford
the resulting copolymer as a slightly brown sticky solid (92% yield).
1H NMR (298 K, CDCl3, 300 MHz) ı (ppm): 0.81 (t, 12H, J = 6.6 Hz),
0.96–1.41 (m, 104H), 1.66 (m, 8H), 2.25–2.77 (br m, 8H), 3.30 (s,
2.7H), 3.33–3.84 (m, 188H), 4.19 (br s, 10H), 4.59 (t, 0.2H, J = 5.2 Hz),
7.11–7.29 (m, 0.5H), 8.04 (s, 0.1H).
3.1. Synthesis and characterization of selegiline-functionalized
copolymers
Prior functionalization of PEGCA with selegiline, its hydrochlo-
ric salt was turned into its neutral form using Na2CO3 in order to
avoid any alteration of the copper catalyst involved in the CuAAC.
Indeed, some preliminary studies (data not shown) yielded no cou-
pling when selegiline, hydrochloric salt was used under standard
click conditions. Neutral selegiline was obtained quantitatively as
confirmed by 1H NMR spectroscopy that displayed all protons
accounting for the molecule (see Section 2).
We took advantage of a recent study which demonstrated
the covalent linkage of model ligands, either at the surface of
azido-functionalized nanoparticles or directly to the correspond-
ing copolymer (Nicolas et al., 2008). However, in the present study,
a slightly different approach was used whereby a functionalized
poly(ethylene glycol) cyanoacetate was first prepared prior syn-
thesis of the corresponding copolymer. Selegiline-poly(ethylene
glycol) cyanoacetate (SelPEGCA) was copolymerized with HDCA
and MePEGCA in the presence of ethanol, formalin and pyrrolidine
to give the selegiline-functionalized P(MePEGCA-co-SelPEGCA-co-
HDCA) copolymer. It was characterized by 1H NMR spectroscopy
which confirmed the presence of selegiline moieties within the
copolymer structure, via its aromatic protons (see Section 2).
2.4. Nanoparticle preparation
A
typical procedure for the preparation of selegiline-
functionalized nanoparticles (Table 1, N6) is as follows. 5 mg
of 10% selegiline-functionalized P(MePEGCA-co-SelPEGCA-co-
HDCA) copolymer and 5 mg of P(MePEGCA-co-RCA-co-HDCA)
copolymer were dissolved in 2 mL of acetone. This solution was
added dropwise to an aqueous solution of 0.5% (w/v) of Pluronic
F-68 (4 mL) under vigorous stirring. Nanoparticle suspension
formed immediately. Acetone was then removed under reduced
pressure and the nanoparticles were purified by ultracentrifuga-
tion (150,000 × g, 1 h, 4 ◦C, Beckman Coulter, Inc.). The supernatant
was discarded and the pellet was resuspended in the appropriate
volume of deionized water to yield a 2.5 mg mL−1 nanoparticle
suspension. The colloidal characteristics of the nanoparticles were
then analyzed by DLS and zeta potential measurement.
3.2. Formation and characterization of selegiline-functionalized
nanoparticles
The ability of the selegiline-functionalized poly(alkyl
cyanoacrylate) copolymers to form nanoparticles by self-assembly
was assessed and the suspensions of nanoparticles were then
characterized by DLS and zeta potential measurements as stability
studies.
P(MePEGCA-co-SelPEGCA-co-HDCA) nanoparticles N1 and N2
containing respectively 10 or 50% SelPEGCA (with respect to the
overall PEGCA content of the copolymer) were prepared by the
nanoprecipitation technique. Their characterization revealed that
nanoparticles N2 obtained by including 50% SelPEGCA exhib-
ited a poor colloidal stability. Indeed, several minutes after their
formation, the average diameter dramatically increased from
approximately 150 to 200 nm, together with an increase of the
particle size distribution (PSD) from less than 0.2 to 0.4. The
2.5. Peptide sample preparation and storage
Lyophilized HiLyte FluorTM 488 labelled A␤1–42 peptide was
dissolved in 0.16% (w/v) of ammonium hydroxide aqueous solu-
tion to reach a concentration of 0.5 mg mL−1. The peptide solution
was then divided into aliquots, individually stored at −20 ◦C freshly
thawed prior analysis.
2.6. Nanoparticle interaction with monomeric Aˇ1–42
To study the interaction between the monomeric form of
the fluorescent A␤1–42 peptide and the selegiline-functionalized

B. Le Droumaguet et al. / International Journal of Pharmaceutics 416 (2011) 453–460
457
Table 1
Compositions and initial colloidal properties of selegiline-functionalized poly(alkyl cyanoacrylate) nanoparticles.
Expt.
P(MePEGCA-
co-SelPEGCA-
co-HDCA)
P(MePEGCA-
co-RCA-co-
HDCA)
copolymer C2
(wt.%)
SelPEGCAb
(mol.%)
Average
particle
Particle size
Zeta potentiald
(ꢀ) (mV)
distributionc,d
diameterd (Dz)
(nm)
copolymer C1a
(wt.%)
1 (N8)
2 (N7)
3 (N6)
4 (N5)
5 (N4)
6 (N3)
90
70
50
10
5
10
30
50
90
95
98
9
7
5
1
0.5
0.2
93
92
96
104
110
113
0.16
0.13
0.16
0.18
0.14
0.18
9
7.9
−2.4
−12.9
−11.1
−14.9
2
a
Copolymer containing 10% SelPEGCA with respect to PEGCA overall content.
With respect to PEGCA overall content.
Given by the DLS apparatus.
b
c
d
Values measured immediately after purification of the nanoparticles.
nanoparticles N1 synthesized with only 10% SelPEGCA displayed
a mean average diameter of around 100 nm (PSD ∼ 0.2). But, within
2 days, their size also increased significantly together with an
increase of their particle size distribution above 0.3. Even after
some efforts to change the nanoprecipitation parameters (such
as the organic phase/aqueous phase ratio or the percentage of
Pluronic F-68 surfactant added to the aqueous solution), the
poor P(MePEGCA-co-SelPEGCA-co-HDCA) nanoparticle stability
remained unchanged. Therefore, a co-nanoprecipitation approach
with P(MePEGCA-co-SelPEGCA-co-HDCA) (C1) and P(MePEGCA-
co-RCA-co-HDCA) (C2) copolymers was investigated. Indeed,
P(MePEGCA-co-RCA-co-HDCA) copolymer C2 exhibited a great col-
loidal stability over time and a rather low zeta potential value
(around −40.6 0.3 mV) (Brambilla et al., 2010a). Variable blends
of these two copolymers C1 and C2 were tested (see Table 1).
These new formulations of selegiline-functionalized nanopar-
ticles N3 to N8, incorporating from 0.2 to 9% selegiline PEGCA,
respectively, were characterized by DLS and zeta potential
measurement (Table 1). These nanoparticles displayed average
diameters in the 90–120 nm range with narrow particle size dis-
tribution (PSD < 0.2), together with zeta potential values varying
from 10 to −15 mV. It was observed that the more selegiline-
functionalized copolymer incorporated in the formulation, the
higher the zeta potential. This demonstrated the presence of the
selegiline ligands at the surface of the nanoparticles, rendering their
surface more positively charged than the non-functionalized ones.
The NPs were then subjected to DLS measurements over a period
of 8 days in deionized water at 25 ◦C (Fig. 2). It was shown that
the mean average diameter remained rather stable (the variation
observed for N6 at day 7 should not be considered relevant).
However, as shown in Fig. 3, the zeta potential values
phosphate buffer allowed further studies of their interactions with
the A␤1–42 peptide by CE.
3.3. Interaction of the nanoparticles with the Aˇ1–42 peptide
CE is an analytical technique that has been recently used for
the screening and identification of efficient ligands towards their
inhibition properties against A␤1–42 peptide aggregation (Colombo
et al., 2009; Kato et al., 2007; Sabella et al., 2004). Thereby,
we recently developed an innovative protocol based on capil-
lary electrophoresis coupled to laser-induced fluorescence (CE-LIF)
detection to monitor the interactions of P(MePEGCA-co-RCA-co-
HDCA) nanoparticles with the A␤1–42 peptide (Brambilla et al.,
2010b). Interestingly, it was demonstrated that the P(MePEGCA-co-
of nanoparticles N3–N8 dramatically decreased within
1 or
2 days, whereas it remained rather constant for rhodamine
B-labelled P(MePEGCA-co-RCA-co-HDCA) NPs as previously pub-
lished (Brambilla et al., 2010a). Indeed, an average drop of ∼15 mV
was observed, indicating a change in the physico-chemical prop-
erties at the surface of the nanoparticles. Following this marked
decrease, the zeta potential remained stable, close to values
usually observed for P(MePEGCA-co-RCA-co-HDCA) nanoparticles
(Brambilla et al., 2010a).
The stability of these NPs was also assessed in phosphate buffer
saline (pH 7.4) used in CE analysis and in cell culture medium.
As observed in Fig. 4, the average diameter of the nanoparticles
remained rather stable up to one week with a particle size distri-
bution below 0.3 in both buffers. The decrease in zeta potential was
observed in both media, still confirming the surface charge evolu-
tion of the nanoparticles with time (data not shown). However, this
good stability of the average diameter for nanoparticles N3–N8 in
Fig. 2. Evolution with time of the average diameter and of the particle size distribu-
tion (PSD) in deionized water at 25 ◦C of the selegiline-functionalized nanoparticles
as a function of the selegiline content within the copolymer mixture: (᭹) N3 (0.2%);
(O) N4 (0.5%); ( ) N5 (1%); (+) N6 (5%); ( ) N7 (7%); (×) N8 (9%).

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B. Le Droumaguet et al. / International Journal of Pharmaceutics 416 (2011) 453–460
Fig. 5. Comparative evolution of area under curve of HiLyte FluorTM 488 labelled
A␤1–42 peptide monomeric peak alone (blank, H), and in presence of non-
functionalized ( ) or selegiline-functionalized nanoparticles N3 (᭹) and N8 ( ).
Fig. 3. Evolution with time of the zeta-potential values in deionized water at 25 ◦C of
selegiline-functionalized nanoparticles as a function of the selegiline content within
the copolymer mixture: (᭹) N3 (0.2%); (O) N4 (0.5%); ( ) N5 (1%); (+) N6 (5%); ( )
N7 (7%); (×) N8 (9%).
(from 100 to 20% after 6 h) whereas this value remained unchanged
in the absence of any polymeric nanocarrier (control experiment,
AUC ∼ 100% after 14 h) as observed in Fig. 5. These data combined
with other results have been related to the adsorption of the pep-
tide at the surface of P(MePEGCA-co-RCA-co-HDCA) nanoparticles
(Brambilla et al., 2010b). Regarding A␤ peptide affinity, the roles of
PEG and rhodamine are still unclear but are under investigation.
However, under identical experimental conditions, no impor-
tant decrease of the A␤1–42 peptide monomer peak was observed
in the case of selegiline-functionalized nanoparticles (N3 and
N8) when compared to their non-functionalized counterparts
(Fig. 5). The effect was even lower, which confirmed an impor-
tant evolution of the positioning of the ligand at the surface of
RCA-co-HDCA) nanoparticles were able to bind the A␤1–42 peptide
under its so-called “monomeric” form. We claimed that this pro-
tocol could be relevant to screen NPs for A␤1–42 targeting and to
discover suitable candidates.
Herein, we investigated the interaction of such a functionaliza-
tion of a similar polymeric scaffold with selegiline, with the A␤1–42
peptide using CE coupled to LIF detection. Samples of fluorescently
tagged A␤1–42 peptide incubated with nanoparticles N3 and N8
were analyzed every 2 h using a LIF detection system that allowed
monitoring the relative concentration of the peptide. P(MePEGCA-
co-RCA-co-HDCA) nanoparticles dramatically decrease the area
under the curve (AUC) of the A␤1–42 peptide monomeric peak
Fig. 4. Evolution of the average diameter and of the particle size distribution of selegiline-functionalized nanoparticles N3 (᭹, 0.2% SelPEGCA) and N8 (×, 9% SelPEGCA) at
25 ◦C in (A) cell culture medium and in (B) phosphate buffer saline pH 7.4 as a function of time.

B. Le Droumaguet et al. / International Journal of Pharmaceutics 416 (2011) 453–460
459
leading to their complete inaccessibility towards A␤1–42. More
importantly, this study points out the importance of the hydropho-
bicity/hydrophilicity of the selected ligand displayed at the surface
of polymeric NPs.
Acknowledgments
The research leading to these results has received funding
from the European Community’s Seventh Framework Programme
(FP7/2007-2013) under agreement no. 212043. The CNRS and the
French Ministry of Research are also warmly acknowledged for
financial support.
Fig. 6. Schematic representation describing the rearrangement of the seligiline lig-
ands at the surface of the seligiline-functionalized nanoparticles N3–N8.
the P(MePEGCA-co-SelPEGCA-co-HDCA) nanoparticles. This ineffi-
ciency of selegiline-functionalized nanoparticles towards binding
to the A␤1–42 peptide clearly demonstrated that the ligand was
not well exposed and probably inaccessible for efficient interac-
tion with A␤1–42 peptide which thus confirmed the hypothesis
postulated earlier in this study.
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