Monodisperse nanoparticles from self-assembling amphiphilic cyclodextrins: Modulable tools for the encapsulation and controlled release of pharmaceuticals

Article abstract of DOI:10.2174/157340612801216265

Selective chemical functionalization of cyclodextrins (CDs) is a readily amenable methodology to produce amphiphilic macromolecules endowed with modulable self-organizing capabilities. Herein, the synthesis of well-defined amphiphilic CD derivatives, with a "skirt-type" architecture, that incorporate long-chain fatty esters at the secondary hydroxyl rim and a variety of chemical functionalities (e. g. iodo, bromo, azido, cysteaminyl or isothiocyanato) at the primary hydroxyls rim is reported. Nanoprecipitation of the new CD facial amphiphiles, or binary mixtures of them, resulted in nanoparticles with average hydrodynamic diameters ranging from 100 to 240 nm that were stable in suspension for several months. The precise size, zeta potential and topology of the nanoparticles are intimately dependent on the functionalization pattern at the CD scaffold. Highly efficient molecular encapsulation capabilities of poorly bioavailable drugs such as diazepam (DZ) were demonstrated for certain derivatives, the drug release profile being dependent on the type of formulation (nanospheres or nanocapsules). The efficiency and versatility of the synthetic strategy, together with the possibility of exploiting the reactivity of the functional groups at the nanoparticle surface, offer excellent opportunities to further manipulate the carrier capabilities of this series of amphiphilic CDs from a bottom-up approach.

Full text of DOI:10.2174/157340612801216265

524
Medicinal Chemistry, 2012, 8, 524-532
Monodisperse Nanoparticles from Self-Assembling Amphiphilic Cyclo-
dextrins: Modulable Tools for the Encapsulation and Controlled Release of
Pharmaceuticals
Alejandro Mendez-Ardoy,1 Marta Gómez-García,1 Annabelle Gèze,2 Jean-Luc Putaux,3
Denis Wouessidjewe,2 Carmen Ortiz Mellet,1 Jacques Defaye,2,* José M. García Fernández,4 and
Juan M. Benito4,*
1Dpto de Química Orgánica, Facultad de Química, Universidad de Sevilla, E-41012 Sevilla, Spain
2Dépt de Pharmacochimie Moléculaire, Université de Grenoble/CNRS, UMR 5063/ICMG-FR 2607, F-38041 Grenoble,
France
3Centre de Recherches sur les Macromolécules Végétales, CNRS, LP 5301, F-38041, Grenoble, France
4Instituto de Investigaciones Químicas, CSIC – Universidad de Sevilla, E-41092 Sevilla, Spain
Abstract: Selective chemical functionalization of cyclodextrins (CDs) is a readily amenable methodology to produce am-
phiphilic macromolecules endowed with modulable self-organizing capabilities. Herein, the synthesis of well-defined am-
phiphilic CD derivatives, with a “skirt-type” architecture, that incorporate long-chain fatty esters at the secondary hy-
droxyl rim and a variety of chemical functionalities (e. g. iodo, bromo, azido, cysteaminyl or isothiocyanato) at the pri-
mary hydroxyls rim is reported. Nanoprecipitation of the new CD facial amphiphiles, or binary mixtures of them, resulted
in nanoparticles with average hydrodynamic diameters ranging from 100 to 240 nm that were stable in suspension for
several months. The precise size, zeta potential and topology of the nanoparticles are intimately dependent on the func-
tionalization pattern at the CD scaffold. Highly efficient molecular encapsulation capabilities of poorly bioavailable drugs
such as diazepam (DZ) were demonstrated for certain derivatives, the drug release profile being dependent on the type of
formulation (nanospheres or nanocapsules). The efficiency and versatility of the synthetic strategy, together with the pos-
sibility of exploiting the reactivity of the functional groups at the nanoparticle surface, offer excellent opportunities to fur-
ther manipulate the carrier capabilities of this series of amphiphilic CDs from a bottom-up approach.
Keywords: Cyclodextrins, controlled drug release, drug delivery, facial amphiphiles, nanoparticles, nanospheres, nanocapsules,
self-assembly.
1. INTRODUCTION
amphiphilic character and endows them with molecular in-
clusion capabilities, which have been extensively exploited
by the pharmaceutical industry to improve bioavailability of
poorly soluble or biodegradable drugs, to prevent undesired
effects or to enhance permeability of biological membranes
[2].
Cyclodextrins (CDs) are naturally occurring cyclic oligo-
saccharides composed by ꢀ-(1ꢀ4)-linked glucose units,
arising from enzymatic degradation of starch, that hold a
privileged position in supramolecular chemistry [1]. The
most relevant members of this family are the commercially
available hexa-, hepta- and octamers (ꢀ-, ꢁ-, and ꢂCD, re-
spectively). The three-dimensional structure resembles that
of an empty truncated cone, with the glucopyranosyl ꢀ- and
ꢁ-face pointing to the inner and outer space, respectively
(Fig. 1), and the hydroxyl groups flanking the wider (OH-2
and OH-3) and narrower rims (OH-6). As a consequence, the
hydroxylated exterior of CDs is hydrophilic, while the inte-
rior, mostly coated by methinic groups, is rather hydrophobic.
Such architecture provides CDs with a singular ‘inner-outer’
HO
O
OH
OH
OH
OH
HO
HO
n = 1, CD
n = 2, CD
n = 3, CD
O
O
O
O
HO
HO
OH
OH
OH
OH
HO
HO
HO
OH
OH
OH OH
OH
n = 2, CD
Fig. (1). Structure and schematic representation of native CDs.
*Address correspondence to these authors at the Dépt. de Pharmacochimie
Moléculaire, Institut de Chimie Moléculaire de Grenoble (CNRS – Univ. de
Grenoble, UMR 5063, FR 2607), Bât. E André Rassat, BP 53, F-38041
Grenoble, France; Tel: +33 476635323; Fax: +33 476635313; E-mail:
jacques.defaye@ujf-grenoble.fr;
Instituto de Investigaciones Químicas, CSIC – Universidad de Sevilla, Amé-
rico Vespucio 49, Isla de la Cartuja, E-41092 Sevilla, Spain; Tel: +34
954479560; Fax: +34 954460565; E-mail: juanmab@iiq.csic.es.
A number of drug-CD complexes have been marketed
(basically small drugs and drug-like molecules) [2-7] and
applications have been extended to the agrochemical [8],
cosmetic [9] and food industry [10,11]. A main limitation for
these channels is that only the native ꢀ-, ꢁ- and ꢂCD, to-
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

Monodisperse Nanoparticles from Self-Assembling Amphiphilic
Medicinal Chemistry, 2012, Vol. 8, No. 4 525
gether with a few synthetic derivatives, are available at the
required scale to realistically consider bulk applications.
Moreover, their molecular inclusion capabilities cannot be
specifically tuned to each potential guest and, due to cavity
size restrictions, the loading capacity is limited to one guest
molecule per CD molecule in the best case. Even for suited
preliminary assessment of their self-assembling features,
their molecular encapsulation capabilities and the in vitro
release profile of loaded nanospheres and nanocapsules us-
ing the non-polar anxiolytic drug diazepam as a model.
n
n
CD:guest pairs, most of these complexes feature association
R
O
n
-1
constants (Kas) falling into the 102-104
M
range [12,13],
R
O
O
O
O
n
n
O
O
O
O
I, Br, N3
O
which might be too low to prevent complex dissociation
upon dilution.
or
O
O
R =
O
O
O
S
R
O
O
X
n
O
O
n
R
To alleviate these drawbacks, synthetic chemists have
created a number of selectively modified CD derivatives
aimed at cooperatively interact with selected guests in order
to attain improved guest complexation [14]. The traditional
strategy towards this goal involves the covalent clustering of
CD moieties into oligo- or polymeric species [3,15]. More
recently, non-covalent self-assembling strategies, which
generally involve amphiphilic CD derivatives, are taking
over [16-18]. Self-assembling features may be theoretically
tailored by modifying the amphiphilic CD structure to attain
enhanced encapsulation or scheduled dissociation and re-
lease in response to external stimuli. Such pre-organized
systems are expected to be more easily degraded in biologi-
cal media and less prone to exert toxic or immunogenic re-
sponses in vivo that covalent conjugates. Depending on their
decoration pattern, CD-based facial amphiphiles may spon-
taneously arrange into vesicles [19], nanoparticles [20], mi-
cellar systems [21] or even liquid crystals [22]. Since the
first disclosure of amphiphilic CDs by Kawabata et al. [23],
a number of promising applications for controlled drug re-
lease [17] and gene delivery [24,25], have been reported,
illustrating the potential of CD-based self-assembling sys-
tems.
O
X = NHBoc, NH2 or NCS
n = 2 or 10
O
O
O
O
O
O
O
n
O
O
n
O
R
O
O
O
O
O
O
O
O
O
R
O
O
n
n
O
R
n
n
n
Fig. (2). General structure of the facially-differentiated CDs pre-
pared in this work.
2. MATERIALS AND METHODS
2.1. Synthesis
Reagents and solvents were purchased from commercial
sources and used without further purification. Optical rota-
tions were measured at 20 ºC in 1-cm or 1-dm tubes on a
Perkin-Elmer 141 MC polarimeter. IR spectra were recorded
1
on a FTIR spectrometer. H (and 13C NMR) spectra were
recorded at 500 (125.7) and 400 (100.6) MHz. 2D COSY,
1D TOCSY, and HMQC experiments were used to assist on
NMR assignments. Thin-layer chromatography (TLC) was
carried out on aluminium sheets coated with Kieselgel 30
F245 (E. Merck), with visualization by UV light and by
charring with 10% H2SO4 in EtOH. Column chromatography
was carried out on Silica Gel 60 (E. Merck, 230-400 mesh).
Electrospray mass spectra (ESIMS) were obtained with a
Bruker Esquire6000 instrument. MALDI-TOF mass spectra
were acquired on a GSG System spectrometer operating in
the positive-ion mode with an accelerating voltage of 28
keV. Samples were dissolved in water at millimolar concen-
tration and mixed with a standard solution of 2,5-
dihydroxybenzoic acid (DHB; 10 mg mL-1 in 10% aq EtOH,
2 μL) in 1:1 v/v relative proportions; 1 μL of the mixture
was loaded onto the target plate, then allowed to air-dry at
room temperature. Elemental analyses were performed at the
Instituto de Investigaciones Químicas (Sevilla, Spain). Hep-
takis(6-deoxy-6-iodo)cyclomaltoheptaose (1), heptakis(6-
bromo-6-deoxy)cyclomaltoheptaose (2), heptakis(6-bromo-
Adjustment of the self-organizing capabilities of am-
phiphilic CDs requires a precise control of the molecular
topology, which critically depends on the availability of effi-
cient functionalization methods of the CD scaffold. The de-
velopment of strategies for the selective chemical function-
alization of CDs has allowed the elaboration of nanoarchitec-
tures engineered to perform a variety of tasks, e.g. targeting
[26], sensing [27] or catalysis [28]. Nevertheless, the most
relevant research in the field of CD-based nanoaggregates
relay on synthetic methodologies which results in heteroge-
neous products [21]. Even relatively simple reactions, such
as acylation, have been reported to lead to mixtures when
applied to CDs [29,30], which could eventually hamper re-
producibility of the aggregation and host encapsulation pro-
files from batch to batch.
We have recently reported a reliable and diversity-
oriented approach for the synthesis of libraries of homoge-
neous polycationic amphiphilic CDs (paCDs) [31] that were
shown to self-organize in the presence of plasmid DNA
(pDNA), the mixed paCD-pDNA complexes (CDplexes)
exhibiting remarkable gene delivery capabilities [32]. The
synthetic strategy relies on the differential reactivity of pri-
mary vs secondary hydroxyls on the CD torus to homogene-
ously functionalize each rim with cationic elements and long
fatty acyl tails, respectively. Herein, we describe the imple-
mentation of this synthetic scheme to elaborate a series of
neutral facially-differentiated CD derivatives (Fig. 2) and the
2,3-di-O-hexanoyl)cyclomaltoheptaose
(5),
heptakis(6-
bromo-6-deoxy-2,3-di-O-myristoyl)cyclomaltoheptaose (6),
heptakis[6-(2-aminoethylthio)-2,3-di-O-hexanoyl]cycloma-
ltoheptaose (11), heptakis[6-(2-aminoethylthio)-2,3-di-O-
myristoyl]cyclomaltoheptaose (12), and heptakis[2,3-di-O-
hexanoyl-6-(2-isothiocyanatoethylthio)]cyclomaltoheptaose
(13) were prepared according to the reported procedures
[32,33]. Dimethylformamide, dichloromethane, tri-fluoro-
acetic acid and diazepam were named using the acronyms
DMF, DCM, TFA, and DZ, respectively.
Heptakis(6-deoxy-2,3-di-O-hexanoyl-6-iodo)cyclo-
maltoheptaose (3). To a solution of heptakis(6-deoxy-6-

526 Medicinal Chemistry, 2012, Vol. 8, No. 4
Mendez-Ardoy et al.
ether); [ꢀ]D = +115.0 (c 1.0 in DCM). IR: ꢂmax = 2117 cm-1.
1H NMR (500 MHz, CDCl3): ꢁ = 5.30 (t, 7 H, J2,3 = J3,4 = 9.2
Hz, H-3), 5.06 (d, 7 H, J1,2 = 3.8 Hz, H-1), 4.80 (dd, 7 H, H-
2), 4.00 (ddd, 7 H, J4,5 = 9.2 Hz, J5,6b = 4.9 Hz, J5,6a = 2.7 Hz,
H-5), 3.73 (t, 7 H, H-4), 3.71 (dd, 7 H, J6a,6b = 13.6 Hz, H-
6a), 3.63 (dd, 7 H, H-6b), 2.40-2.16 (m, 28 H, CH2CO), 1.59
(m, 28 H, CH2CH2CO), 1.30 (m, 56 H, CH2CH3,
CH2CH2CH3), 0.91, 0.89 (2 t, 42 H, 3JH,H = 7.3 Hz, CH3).
13C NMR (125.7 MHz, CDCl3): ꢁ = 173.2, 171.7 (CO), 96.4
(C-1), 76.7 (C-4), 70.8 (C-5), 70.1 (C-2, C-3), 51.6 (C-6),
34.0, 33.8 (CH2CO), 31.4, 31.2 (CH2CH2CH3), 24.6, 24.3
(CH2CH2CO), 22.3 (CH2CH3), 13.8 (CH3). ESIMS: m/z
2706.3 [M + Na]+. Anal. Calcd for C126H203N21O42: C 56.38,
H 7.62, N 10.96. Found: C 56.33, H 7.23, N 10.93.
iodo)cyclomaltoheptaose [33] (1, 2.0 g, 1.04 mmol) in dry
DMF (20 mL) under Ar at 0 ºC, DMAP (5.4 g, 44.0 mmol, 3
equiv) was added. Hexanoic anhydride (13.6 mL, 58.8
mmol, 4.0 equiv) was dropwise added, the reaction mixture
was stirred at room temperature for 45 min, and MeOH (25
mL) was added. After 1 h, the solution was poured into ice-
water (50 mL) and extracted with CH2Cl2 (4 x 50 mL). The
organic phase was washed with dilute H2SO4 (2 x 50 mL),
water and aqueous saturated NaHCO3 (4 x 50 mL), dried
(Na2SO4), concentrated and purified by column chromatog-
raphy (1:12 ꢀ 1:10 EtOAc-petroleum ether) to give 3.
Yield: 2.32 g (68%); Rf = 0.40 (1: 5 EtOAc-petroleum ether);
[ꢀ]D = + 63.8 (c 1.0 in CHCl3); 1H NMR (500 MHz, CDCl3):
ꢁ = 5.32 (t, 7 H, J2,3 = J3,4 = 9.5 Hz, H-3), 5.14 (d, 7 H, J1,2
=
3.5 Hz, H-1), 4.79 (dd, 7 H, H-2), 3.73 (m, 14 H, H-5, H-6a),
3.60 (dd, 7 H, J6a,6b = 14.0 Hz, J5,6b = 5.5 Hz, H-6b), 3.58 (t,
7 H, J4,5 = 9.0 Hz, H-4), 2.39-2.16 (m, 28 H, CH2CO), 1.56
(m, 28 H, CH2CH2CO), 1.29 (m, 28 H, CH33CH2), 1.23 (m,
28 H, CH3CH2CH2), 0.89, 0.87 (2 t, 42 H, JH,H = 6.7 Hz,
CH3); 13C NMR (125.7 MHz, CDCl3): ꢁ = 173.2, 171.7
(CO), 96.5 (C-1), 80.5 (C-4), 70.1 (C-2), 69.9 (C-5), 69.8 (C-
3), 34.0, 33.8 (CH2CO), 31.4, 31.3 (CH3CH2CH2), 24.4, 24.3
(CH2CH2CO), 22.4, 22.3 (CH3CH2), 13.9 (CH3), 8.6 (C-6);
MALDI-TOFMS: m/z 3300.5 [M + Na]+. Anal. Calcd for
C126H203I7O42: C 46.16, H 6.24. Found: C 45.87, H 5.95.
Heptakis(6-azido-6-deoxy-2,3-di-O-myristoyl)cyclo-
maltoheptaose (8). To a solution of either 4 or 6 (0.15
mmol) in dry DMF (20 mL), sodium azide (0.14 g, 2.1
mmol, 2 equiv) was added. The mixture was stirred at 80 ºC
for 16 h and then the solvent was removed under reduced
pressure. The resulting residue was extracted with DCM (40
mL) and washed with water (2 x 20 mL). The organic phase
was dried (Na2SO4) and concentrated and the resulting syrup
was purified by column chromatography (1:15 EtOAc-
petroleum ether). Yield: 0.55 g (81%); Rf = 0.40 (1:8 EtOAc-
1
petroleum ether); [ꢀ]D = +66.1 (c 1.0 in DCM). H NMR
Heptakis(6-deoxy-6-iodo-2,3-di-O-myristoyl)cyclo-
maltoheptaose (4). To a solution of heptakis(6-deoxy-6-
iodo)cyclomaltoheptaose [33] (1, 0.1 g, 53 μmol) in dry
DMF (5 mL) under Ar at 0 ºC, DMAP (0.27 g, 2.2 mmol, 3
equiv) was added. Myristoyl anhydride (1.29 g, 2.94 mmol,
4 equiv) was dropwise added; the reaction mixture was
stirred at room temperature for 16 h and then filtered. The
resulting solid was extensively washed with water and
MeOH. A mixture of MeOH-DCM (95:5, 50 mL) (25 mL)
was added and the solution was refluxed for 1 h, decanted
and purified by column chromatography (petroleum ether ꢀ
1:15 EtOAc-petroleum ether). Yield: 0.13 g (52%); Rf = 0.49
(1:8 EtOAc-petroleum ether); [ꢀ]D = +49.2 (c 1.0 in DCM).
1H NMR (400 MHz, CDCl3): ꢁ = 5.33 (t, 7 H, J2,3 = J3,4 = 9.0
Hz, H-3), 5.15 (d, 7 H, J1,2 = 3.0 Hz, H-1), 4.80 (dd, 7 H, H-
2), 3.74 (m, 14 H, J4,5 = J5,6a = 9.0 Hz, H-5, H-6a), 3.64 (bd,
7 H, J6a,6b = 11.8 Hz, H-6b), 3.58 (t, 7 H, H-4), 2.41-2.11 (m,
28 H, CH2CO), 1.58 (m, 28 H, CH2CH2CO), 1.27 (m, 280 H,
(500 MHz, CDCl3): ꢁ = 5.28 (t, 7 H, J2,3 = J3,4 = 9.3 Hz, H-3),
5.02 (d, 7 H, J1,2 = 3.6 Hz, H-1), 4.77 (dd, 7 H, H-2), 3.98
(m, 7 H, H-5), 3.71 (t, 7 H, J4,5 = 9.5 Hz, H-4), 3.68 (m, 7 H,
H-6a), 3.60 (dd, 7 H, J6a,6b = 13.5 Hz, J5,6b = 4.5 Hz, H-6b),
2.32, 2.21 (m, 28 H, CH2CO), 1.53 (m, 28 H, CH2CH2CO),
3
1.25 (m, 280 H, CH2), 0.86 (t, 42 H, JH,H = 7.1 Hz, CH3).
13C NMR (125.7 MHz, CDCl3): ꢁ = 173.2, 171.8 (CO), 96.4
(C-1), 76.7 (C-4), 70.8 (C-5), 70.2 (C-2), 70.1 (C-3), 51.6
(C-6), 34.1, 33.9 (CH2CO), 32.0 (CH2CH2CH3), 29.9-29.2
(CH2), 24.9, 24.8 (CH2CH2CO), 22.7 (CH2CH3), 14.0 (CH3).
MALDI-TOFMS: m/z 4523.7 [M + H]+. Anal. Calcd for
C238H427N27O42: C 67.18, H 10.11, N 6.91. Found: C 66.82,
H 9.84, N 6.66.
Heptakis[6-(2-tert-butoxycarbonylaminoethylthio)-
2,3-di-O-hexanoyl]cyclomaltoheptaose (9). To a suspen-
sion of 5 (0.29 g, 0.1 mmol) and Cs2CO3 (0.33 g, 1 mmol) in
dry DMF (3 mL), tert-butyl N-(2-mercaptoethyl)carbamate
(164 μL, 1 mmol, 1.4 equiv) was added. The suspension was
stirred under Ar atmosphere at 70 ºC for 48 h. The reaction
mixture was then cooled to rt, poured into ice-water (30 mL),
and stirred overnight. The resulting solid was filtered and
washed with water, and the residue was purified by column
chromatography (1:3 EtOAc-petroleum ether). Yield: 0.25 g
(70%). Analytical and spectroscopic data were identical to
those reported [32].
3
CH2), 0.87 (t, 42 H, JH,H = 7.0 Hz, CH3). 13C NMR (100.6
MHz, CDCl3): ꢁ = 173.3, 171.7 (CO), 96.4 (C-1), 80.4 (C-4),
70.2 (C-2), 69.9 (C-5), 69.7 (C-3), 34.1, 33.9 (CH2CO), 32.0
(CH2CH2CH3), 29.9-29.2 (CH2), 24.9, 24.8 (CH2CH2CO),
22.7 (CH2CH3), 14.1 (CH3), 8.6 (C-6). MALDITOF-MS: m/z
4842.6 [M + H]+. Anal. Calcd for C238H427I7O42: C 58.95, H
8.88. Found: C 58.76, H 8.57.
Heptakis(6-azido-6-deoxy-2,3-di-O-hexanoyl)cyclo-
maltoheptaose (7). To a solution of either 3 or 5 (0.2 mmol)
in dry DMF (20 mL), sodium azide (0.18 g, 2.8 mmol, 2
equiv) was added. The mixture was stirred at 80 ºC for 16 h
and then the solvent was removed under reduced pressure.
The resulting residue was extracted with DCM (40 mL) and
washed with water (2 x 20 mL). The organic phase was dried
(Na2SO4) and concentrated and the resulting syrup was puri-
fied by column chromatography (1:9 EtOAc-petroleum
ether). Yield: 0.46 g (85%); Rf = 0.42 (1:9 EtOAc-petroleum
Heptakis[6-(2-tert-butoxycarbonylaminoethylthio)-2,3
-di-O-myristoyl]cyclomaltoheptaose (10). To a suspension
of 6 (0.45 g, 0.1 mmol) and Cs2CO3 (0.33 g, 1 mmol) in dry
DMF (3 mL), tert-butyl N-(2-mercaptoethyl) carbamate (165
μL, 1 mmol, 1.4 equiv) was added. The suspension was
stirred under Ar atmosphere at 70 ºC for 48 h. The reaction
mixture was then cooled to rt, poured into ice-water (30 mL),
and stirred overnight. The resulting solid was filtered and
washed with water, and the residue was purified by column
chromatography (1:2 EtOAc-petroleum ether). Yield: 0.37 g

Monodisperse Nanoparticles from Self-Assembling Amphiphilic
Medicinal Chemistry, 2012, Vol. 8, No. 4 527
(72%). Analytical and spectroscopic data were identical to
those reported [32].
duced pressure at 35 ºC. The nanoparticle suspensions were
prepared in triplicate and were stored in closed vials at +6
°C.
Heptakis[6-(2-isothiocyanatoethylthio)-2,3-di-O-
myristoyl]cyclomaltoheptaose (14). To a solution of hep-
taamine 12 [32] (102 mg, 21.2 μmol) in a mixture of DCM
(1.6 mL) and water (1.6 mL), CaCO3 (59 mg, 0.59 mmol, 4
equiv) and CSCl2 (23 μL, 0.30 mmol, 2 equiv) were added.
The reaction mixture was vigorously stirred for 16 h and
then concentrated. The residue was dissolved in DCM (15
mL) and washed with water (6 mL). The organic phase was
decanted, dried (Na2SO4) and concentrated. The residue was
subjected to column chromatography (1:6 EtOAc-petroleum
ether). Yield: 35 mg (35%); Rf = 0.38 (1:4 EtOAc-petroleum
2.2.3. Preparation of Homogeneous DZ-Loaded Nano-
spheres and Nanocapsules
The nanoparticle suspensions were prepared following
the above described procedures respectively, from acetone
solutions containing both, the corresponding ꢂCD derivative
and DZ (0.5 mg·mL-1 for nanospheres and 1 mg·mL-1 for
nanocapsules). To formulate DZ loaded-nanospheres, poly-
sorbate 80 (Montanox® 80) was added to the aqueous phase
at a concentration of 1 mg·mL-1 before nanoprecipitation.
The nanoparticle suspensions were prepared in triplicate and
were stored in closed vials at rt. The recovery of DZ in the
colloidal suspensions was assayed spectrophotometrically at
285 nm and 366 nm following solubilization of a known
volume of the suspension in MeOH/H2SO4 (5 g·L-1).
1
ether); [ꢀ]D = +72.3 (c 0.6 in CHCl3). H NMR (500 MHz,
CDCl3): ꢁ = 5.27 (t, 7 H, J2,3 = J3,4 = 9.4 Hz, H-3), 5.05 (d, 7
H, J1,2 = 3.4 Hz, H-1), 4.77 (dd, 7 H, H-2), 4.14 (dbd, 7 H,
J5,6b = 2.5 Hz, H-5), 3.82 (t, 7 H, J4,5 = 8.7 Hz, H-4), 3.78 (t,
3
14 H, JH,H = 6.5 Hz, CH2Ncyst), 3.17 (bd, 7 H, J6a,6b = 13.1
Hz, H-6a), 3.07 (dd, 7 H, J5,6b = 2.5 Hz, H-6b), 3.00, 2.92
2.2.4. Size and Zeta Potential Measurements
2
3
(2 dt, 14 H, JH,H = 13.0 Hz, JH,H = 6.0 Hz, CH2Scyst), 2.38-
2.17 (m, 28 H, CH2CO), 1.54 (m, 28 H, CH2CH2CO), 1.27
A Zetasizer 3000 instrument (10mWHeNelaser at 632.8
nm) with a K7132 correlator was used at the fixed angle of
90º and at 25 ± 0.1 ºC for size analysis and zeta potential (ꢃ)
determination (Malvern Instruments, Orsay, France). The
average hydrodynamic diameter Dh (Z average mean) and
polydispersity index (PI) were calculated in intensity using
the Cumulant analysis mode. Dh values were derived from
the measured translational diffusion coefficient of the parti-
cles moving under Brownian motion. The values of merit for
size analysis varied from 60 to 70%. ꢃ potential values were
evaluated by laser Doppler anemometry. In both determina-
tions, the colloidal suspensions were analyzed following
appropriate dilution with NaCl (0.01% w/w).
3
(m, 280 H, CH2), 0.86 (t, 42 H, JH,H = 7.0 Hz, CH3). 13C
NMR (125.7 MHz, CDCl3): ꢁ = 173.4, 171.8 (CO), 132.3
(NCS), 96.7 (C-1), 78.5 (C-4), 71.6 (C-5), 70.3 (C-2), 70.2
(C-3), 45.6 (CH2Ncyst), 34.1 (C-6, CH2CO, CH2Scyst), 33.8
(CH2CO), 32.0 (CH2CH2CH3), 30.0-29.3 (CH2), 24.9, 24.8
(CH2CH2CO), 22.7 (CH2CH3), 14.1 (CH3). MALDITOF-
MS: m/z 4779.7 [M
+
Na]+. Anal. Calcd for
C259H455N7O42S14: C 64.97, H 9.58, N 2.05. Found: C 64.87,
H 9.53, N 1.96.
2.2. Nanoparticle Formation and Characterization
2.2.1. Preparation of Homogeneous and Mixed Unloaded
Nanospheres
2.2.5. Cryo-Transmission Electron Microscopy (Cryo-
TEM).
The nanoparticle suspensions were prepared in triplicate
using the nanoprecipitation technique [34,35] taking advan-
tage of the spontaneous self-assembling capabilities of am-
phiphilic ꢂCD derivatives when dispersed into an aqueous
phase. Briefly, the corresponding amphiphilic ꢂCD was dis-
solved in anhydrous acetone to a final concentration of 1
mg·mL-1 and this solution was incorporated into an equal
volume of distilled water with magnetic stirring (500 rpm) at
rt. Nanoparticles were formed spontaneously and the organic
solvent was removed under reduced pressure at 35 ºC. The
aqueous suspension was filtered through 0.8 μm membrane
(Millex AA, Millipore, France), characterized, and stored in
closed vials.
Thin vitrified films of CD nanoparticle suspensions were
prepared as previously described [36]. Droplets of 0.1%
(w/v) suspensions were deposited on ‘lacey’ carbon films.
After blotting the liquid in excess with filter paper, the grid
was quench-frozen into liquefied ethane. The specimens
were then mounted in a Gatan 626 cryo-holder, transferred
into a Philips CM200 ‘Cryo’ microscope and observed at
low temperature (-180 ºC), under reduced illumination, at an
accelerating voltage of 80 kV. Images were recorded on Ko-
dak SO163 films.
2.2.6. In Vitro Release Kinetics
In vitro release kinetics studies were performed in tripli-
cate, by dialysis (cutoff of 8kDa, Spectra/Por® Dialysis,
Fisher Bioblock, France), in phosphate buffer saline medium
(0.1M, pH 7.4, 37°C) using a paddle apparatus (Sotax AT6,
Sotax, Switzerland). The rotating paddle was set at 50 rpm.
All the drug release experiments were performed to ensure
that the maximum concentration corresponding to 100% DZ
release did not exceed 25% of the saturating DZ concentra-
tion in PBS 0.1M at 37 °C (experimentally determined at
0.045 g/L). Drug quantification over time in the release me-
dium was assayed spectrophotometrically at 280 nm in PBS
0.1M.
2.2.2. Preparation of Homogeneous Unloaded Nanocap-
sules
The corresponding amphiphilic ꢂCD was dissolved in
anhydrous acetone (2 mg·mL-1) containing a small amount
(100ꢀL) of capric/caprylic triglycerides (Miglyol® 812), a
non-ionic hydrophobic surfactant (Montane® 80, 2 mg·mL-1)
and an aqueous phase with a non-ionic hydrophilic surfactant
(Montanox® 80, 2 mg·mL-1). This solution was incorporated
into distilled water (twice the volume of acetone) under
magnetic stirring (500 rpm) at rt. Nanoparticles were formed
spontaneously and organic solvent was removed under re-

528 Medicinal Chemistry, 2012, Vol. 8, No. 4
Mendez-Ardoy et al.
Br
O
3. RESULTS AND DISCUSSION
3.1. Synthesis
NaN3, DMF
HS
NHBoc
80 ˚C
O
O
O
O
Cs2CO3
DMF, 70 ˚C
While functionalization at the primary hydroxyl positions
of CDs is relatively straightforward taking advantage of the
differential reactivity of primary vs. secondary hydroxyls,
particularly by using the per-C6-halogenation methodology
reported by Gadelle and Defaye [33], subsequent acylation
of the secondary hydroxyls with fatty acid chains is nontriv-
ial [37]. We recently found that the use of the acyl anhy-
dride/4-(N,N-dimethylamino)pyridine (DMAP) system effi-
ciently furnished homogeneous per-(O2,O3)-esters. Tet-
radecahexanoates 3 and 5 were prepared accordingly in
multigram scale from per-C6-iodo (1) and per-C6-bromo (2)
ꢀCD, respectively, in 68-72% yield after column chromatog-
raphy purification (Scheme 1). The corresponding tetrade-
camyristoylates 4 (52%) and 6 (67%) were synthesized
analogously, the slightly lower yields obtained being attrib-
uted to difficulties in the separation of the pure compounds
from the excess of acylating reagent rather than to lower
reactivity. The homogeneity of compounds 3-6 was demon-
strated by NMR, ESI-MS and combustion analysis (see ex-
perimental section).
O
7
5, n = 2
6, n = 10
NHBoc
n
n
N3
S
O
O
O
O
O
O
O
O
O
O
O
O
7
7
n
n
7, n = 2
8, n = 10
9, n = 2
10, n = 10
n
n
NCS
NH2
TFA-CH2Cl2
rt, quant
S
S
Cl2CS, CaCO3
H2O-CH2Cl2, rt
O
O
O
O
O
O
O
O
O
O
O
O
7
7
n
n
n
n
13, n = 2
14, n = 10
11, n = 2
12, n = 10
OH
I2, TPP
DMF, 80 ˚C
NBS, TPP
DMF, 80 ˚C
O
HO
Scheme 2. Synthesis of face-differentiated CDs 7-14.
HO
O
7
Further on, derivatives 9 and 10 were transformed into
the hepta-isothiocyanates 13 and 14 following the previously
described tandem acid-catalysed carbamate cleav-
ageꢀthiophosgene-mediated isothiocyanation protocol [32].
The functional groups at the primary face of the CD plat-
form, i.e. azide and isothiocyanate, were purposely selected
to be compatible with “click” type conjugation by either the
copper(I)-catalyzed azide-alkyne cycloaddition [38] or the
thiourea-forming reaction [39], opening the possibility for
surface modification of nanoparticles following self-
assembly.
I
ꢀCD
Br
O
O
HO
HO
HO
O
HO
O
7
7
1
2
acyl anhydride
DMAP, DMF
acyl anhydride
DMAP, DMF
Br
O
I
O
O
O
O
O
O
O
O
O
O
3.2. Self-Assembling Capabilities
O
7
7
The ability of the new hexanoylated and myristoylated
CDs to self-assemble into submicronic particles in aqueous
media was subsequently evaluated in the conditions of nano-
precipitation [34,35]. Dispersion of acetone solutions of the
neutral CDs into bulk water instantaneously formed nano-
spheres in all cases. Dynamic light scattering (DLS) moni-
toring of nanoparticle size revealed unimodal distributions,
with little differences in the average hydrodynamic diameter
between hexanoylated and myristoylated compounds. The
particle size was more sensitive to the nature of the func-
tional group at the primary rim of the CD (Table 1). Thus,
halogenated derivatives yielded larger nanospheres, while
the less bulky azide and isothiocyanate substituents gave rise
to the smallest aggregates. The ꢁ potentials, slightly negative
for all formulations, were also affected by the nature of the
functional groups at the primary rim, but to a lesser extent.
3, n = 2
4, n = 10
5, n = 2
6, n = 10
n
n
n
n
Scheme 1. Synthesis of face-differentiated CDs 3-6.
Compounds 3-6 were thus obtained in only two steps
from commercial ꢀCD. They are themselves interesting fa-
cially-differentiated CDs with self-organizing capabilities
(see hereinafter). Most interestingly, they are also excellent
precursors for further chemical elaboration since the halogen
groups can be displaced by a variety of nucleophiles. For
instance, the reaction of the per-C6-bromo ꢀCD derivatives 5
and 6 with sodium azide furnished tetradecahexanoate 7 [38]
and tetradecamyristoylate 8 [38], respectively, with excellent
yields (85 and 81%, respectively) (Scheme 2). Cesium car-
bonate-promoted nucleophilic displacement of bromine in 5
and 6 by N-tert-butoxycarbonyl (Boc)-cysteamine afforded 9
[32] and 10 [32], respectively, in 70 and 72% yields. Analo-
gous results were obtained using the corresponding iodides 3
and 4 as starting materials instead.
The low polydispersity index (PI) measured by DLS was
further confirmed by transmission electron microscopy. Fig.
(3) shows the cryo-TEM micrograph for the highly homoge-
neous and spherical nanoparticles obtained from compound

Monodisperse Nanoparticles from Self-Assembling Amphiphilic
Medicinal Chemistry, 2012, Vol. 8, No. 4 529
Table 1. Hydrodynamic Size, Polydispersity and ꢀ Potential of Homogeneous Blank Nanospheres
ꢀ Potential ± SD
Compound
Acyl Chain
Functional Group
Z Average Mean ± SD (nm)
PI
(mV)
3
5
Hex
Hex
Hex
Hex
Hex
Myr
Myr
Myr
Myr
Myr
I
240 ± 54
238 ± 29
181 ± 5
0.08
0.02
0.08
0.04
0.04
0.04
0.13
0.06
0.03
0.04
-36 ± 4
-36 ± 4
-34 ± 2
-25 ± 5
-43 ± 8
-39 ± 8
-54 ± 2
-33 ± 2
-24 ± 4
-42 ± 2
Br
7
N3
S(CH2)2NHBoc
S(CH2)2NCS
I
9
197 ± 4
13
4
123 ± 2
217 ± 17
244 ± 13
170 ± 10
211 ± 10
111 ± 2
6
Br
8
N3
10
14
S(CH2)2NHBoc
S(CH2)2NCS
When co-formulated with pharmaceutically-approved
synthetic triglycerides, these face-differentiated CDs were
able to form nanocapsules. In general, nanocapsules were
larger in size but also very homogeneous, which might be
interesting in order to increase their loading capacity. For
instance, dispersion of a solution containing a mixture of CD
derivative 7, Montane® 80 and Miglyol® 812 into water
containing a non-ionic surfactant (Montanox® 80) furnished
stable nanocapsules with an average hydrodynamic diameter
of 240 ± 10 nm (PI, 0.05). Remarkably, formulations of ei-
ther nanospheres or nanocapsules were found highly stable
in water. No significant changes in the measured properties
were observed after 8-month storage at 6 ºC (data not
shown).
Nanoprecipitation of equimolar mixtures of halogenated
CDs 3 and 5 furnished nanospheres with similar size and ꢀ
potential than those obtained from 3 and 5 independently
(Table 2, line 1). This is very sound since the self-
aggregation behaviours of 3 and 5 were virtually identical
(Table 1). A similar situation was observed after co-
formulation of 3 and 5 with derivative 7 (Table 2, lines 2 and
3), thereby providing a way to access multifunctional
Fig. (3). Cryo-TEM micrograph of nanospheres prepared from 3.
3. The diameter of nanoparticles observed by TEM (ca. 150
nm for 3) is significantly smaller than that measured by
DLS, the particle size discrepancy probably arising from the
different operational principles, as observed previously [36].
Table 2. Hydrodynamic Size, Polydispersity and ꢀ Potential of Mixed Blank Nanospheres
Compounds (ratio)
Functional Group
Z Average Mean ± SD (nm)
PI
ꢀ Potential ± SD (mV)
3 + 5 (1:1)
3 + 7 (1:1)
5 + 7 (1:1)
3 + 11 (4:1)
5 + 11 (4:1)
7 + 11 (4:1)
I + Br
I + N3
225 ± 31
204a
0.04
0.02
0.02
0.04
0.16
0.02
0.15
-40 ± 10
n. d.
Br + N3
200a
n. d.
+
I + S(CH2)2NH3
98 ± 14
122 ± 14
105 ± 2
117 ± 39
+30 ± 9
+35 ± 6
+35 ± 5
+39 ± 6
+
Br + S(CH2)2NH3
+
N3 + S(CH2)2NH3
+
5 + 11 (7:3)
Br + S(CH2)2NH3
n. d. Not determined, a Results from one batch

530 Medicinal Chemistry, 2012, Vol. 8, No. 4
Mendez-Ardoy et al.
tained from CD 7 when co-formulated with 11, illustrating
the potential to modulate nanoparticle properties using rela-
tively simple tools.
15
10
5
3.3. Drug Encapsulation and Release
To assess the potential of these CD derivatives as encap-
sulation and release systems, the anxiolytic drug diazepam
(DZ) was chosen as a model guest. DZ is a benzodiazepine
used for the treatment of nervous disorders and muscle
spasms. The low solubility of diazepam in water is a limita-
tion for certain administration routes (e.g. parenteral). To
overcome this hurdle, a number of carrier systems have been
developed [40-42], and this makes DZ an appropriate com-
parative model.
0
10
100
Size (nm)
1000
15
10
5
DZ-charged nanospheres and nanocapsules were pre-
pared from hepta-azide 7 (7:DZ NS and 7:DZ NC, respec-
tively) as above described using a mixture of both compo-
nents. The average particle size and ꢀ potential did not differ
significantly from those measured for unloaded NS and NC.
The DZ recovery in the aqueous suspension was determined
spectrophotometrically and found to be 75% and 93% for NS
and NC, respectively. The high solubility of DZ in Miglyol®
812 used in NC formulation probably explains the higher
efficiency observed for the latter system and is consistent
with the lipophilicity of the drug [43]. These results parallel
those reported for other types of amphiphilic CD [44] and
are remarkable considering that there is no need of perform-
ing the CD:drug complex [45].
0
-150
-50
50
150
 (mV)
Fig. (4). Particle size (above) and ꢀ potential (below) distribution
for 7 (solid line) and mixed 7:11 (4:1 ratio, scattered line) nano-
spheres obtained by DLS.
nanoparticles exposing different functional groups at the
surface.
In contrast with the behaviour of neutral CD derivatives,
the cationic compounds 11 and 12 were unable to form de-
tectable nanoparticles (DLS) using the nanoprecipitation
method. Apparently, disordered aggregation takes place pro-
ducing a fast precipitation in spite of their expected higher
solubility. The scenario is completely different when cationic
CDs such as compound 11 are co-formulated with the neu-
tral amphiphilic CDs 3, 5 or 7. Nanospheres turned much
smaller (ca. 120 nm) even using small amounts of 11 (Table
2, entries 4 to 6) and the ꢀ potential switched to positive.
Increasing the cationic CD ratio resulted in slightly higher
compaction, furnishing particles with smaller size and higher
ꢀ potential (Table 2, entry 7). Fig. (4) exemplifies the dra-
matic switch in size and ꢀ potential for nanoparticles ob-
Concerning the in vitro drug release from DZ-loaded
nanoparticles, it was shown that DZ recovery gradually in-
creased upon stirring according to a single-mode release
(hyperbolic) profile in both NS and NC. However, the re-
lease kinetics were rather contrasting: while DZ is com-
pletely released from NS in approximately 6 h, in NC two
days were required for complete DZ recovery (Fig. 5). The
rapid DZ release phase (during the first 4 hours) observed in
the case of nanospheres strongly suggests that the drug is
partly adsorbed at the surface of particles, this drug localiza-
tion being responsible for this pronounced burst effect. The
subsequent slower release phase (from time 4h) may be at-
tributed to a fraction of DZ more deeply anchored within the
100
80
60
40
20
0
0
10
20
30
time (h)
40
50
Fig. (5). In vitro release profiles of DZ from nanospheres (NS, ꢀ) and nanocapsules (NC, ꢀ) formulated with CD 7 in PBS (pH 7.4) at 37 ºC
under sustained stirring (50 rpm).

Monodisperse Nanoparticles from Self-Assembling Amphiphilic
Medicinal Chemistry, 2012, Vol. 8, No. 4 531
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4. CONCLUSIONS
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ACKNOWLEDGEMENTS
This work was supported by the Spanish Ministerio de
Innovación
y
Ciencia (MICINN, contract numbers
SAF2010-15670, and CTQ2010-15848, the Junta de An-
dalucía. A.M.-A. is grateful to the MICINN (FPU program)
for a pre-doctoral fellowship. The authors are grateful for the
technical assistance of C. Brunet-Manquat.
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Received: March 31, 2011
Revised: August 01, 2011
Accepted: August 01, 2011