Novel self assembling nanoparticles for the oral administration of fondaparinux: Synthesis, characterization and in vivo evaluation

Article abstract of DOI:10.1016/j.jconrel.2014.07.060

Fondaparinux (Fpx) is the anticoagulant of choice in the treatment of short- and medium-term thromboembolic disease. To overcome the low oral bioavailability of Fpx, a new nanoparticulate carrier has been developed. The nanoparticles (NPs) contain squalenyl derivatives, known for their excellent oral bioavailability. They spontaneously self-assemble upon both electrostatic and hydrophobic interactions between the polyanionic Fpx and cationic squalenyl (CSq) derivatives. The preparation conditions were optimized to obtain monodisperse, stable NPs with a mean diameter in the range of 150-200 nm. The encapsulation efficiencies were around 80%. Fpx loadings reached 39 wt.%. According to structural and morphological analysis, Fpx and CSq organized in spherical multilamellar ("onion-type") nanoparticles. Furthermore, in vivo studies in rats suggested that Fpx was well absorbed from the orally administered NPs, which totally dissociated when reaching the blood stream, leading to the release of free Fpx. The Fpx:CSq NPs improved the plasmatic concentration of Fpx in a dose-dependent manner. However, the oral bioavailability of these new NPs remained low (around 0.3%) but of note, the C_max obtained after oral administration of 50 mg/kg NPs was close to the prophylactic plasma concentration needed to treat venous thromboembolism. Moreover, the oral bioavailability of Fpx could be dramatically increased up to 9% by including the nanoparticles into gastroresistant capsules. This study opens up new perspectives for the oral administration of Fpx and paves the way towards elaborating squalene-based NPs which self assemble without the need of covalently grafting the drug to Sq.

Full text of DOI:10.1016/j.jconrel.2014.07.060

Journal of Controlled Release 194 (2014) 323–331
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Novel self assembling nanoparticles for the oral administration of
fondaparinux: Synthesis, characterization and in vivo evaluation
Bettina Ralay-Ranaivo a, Didier Desmaële a, Elsa P. Bianchini b, Elise Lepeltier a, Claudie Bourgaux a,
Delphine Borgel b, Thierry Pouget c, Jean François Tranchant c, Patrick Couvreur a, Ruxandra Gref a,
⁎
a
UMR CNRS 8612, Institut Galien Paris-Sud, 5 Rue J.B. Clément, 92296 Châtenay-Malabry Cedex, France
EA 4531, Faculté de pharmacie de Châtenay-Malabry, 5 Rue J.B. Clément, 92296 Châtenay-Malabry Cedex, France
b
c
LVMH Recherche Parfums et Cosmétique, 185 Av. de Verdun, 45804 Saint Jean de Braye, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Fondaparinux (Fpx) is the anticoagulant of choice in the treatment of short- and medium-term thromboembolic
disease. To overcome the low oral bioavailability of Fpx, a new nanoparticulate carrier has been developed. The
nanoparticles (NPs) contain squalenyl derivatives, known for their excellent oral bioavailability. They spontane-
ously self-assemble upon both electrostatic and hydrophobic interactions between the polyanionic Fpx and cat-
ionic squalenyl (CSq) derivatives. The preparation conditions were optimized to obtain monodisperse, stable NPs
with a mean diameter in the range of 150–200 nm. The encapsulation efficiencies were around 80%. Fpx loadings
reached 39 wt.%. According to structural and morphological analysis, Fpx and CSq organized in spherical
multilamellar (“onion-type”) nanoparticles. Furthermore, in vivo studies in rats suggested that Fpx was well
absorbed from the orally administered NPs, which totally dissociated when reaching the blood stream, leading
to the release of free Fpx. The Fpx:CSq NPs improved the plasmatic concentration of Fpx in a dose-dependent
manner. However, the oral bioavailability of these new NPs remained low (around 0.3%) but of note, the Cmax ob-
tained after oral administration of 50 mg/kg NPs was close to the prophylactic plasma concentration needed to
treat venous thromboembolism. Moreover, the oral bioavailability of Fpx could be dramatically increased up to
9% by including the nanoparticles into gastroresistant capsules. This study opens up new perspectives for the
oral administration of Fpx and paves the way towards elaborating squalene-based NPs which self assemble with-
out the need of covalently grafting the drug to Sq.
Received 31 March 2014
Accepted 31 July 2014
Available online 13 August 2014
Keywords:
Cationic squalenyl derivative
Fondaparinux
Nanoparticle
Self-assembly
© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction
conditions in the stomach and iii) fast enzymatic degradation [4]. Due
to these drawbacks, Fpx is only administrated via subcutaneous route [5].
Since its introduction in the market in 2002, fondaparinux (Fpx, 1,
Fig. 1) became the anticoagulant of choice in the treatment of short-
and medium-term thromboembolic disease with the unfractionated hep-
arin (UFH) and low molecular weight heparin (LMWH) [1]. Fpx is a syn-
thetic analog of the antithrombin-binding pentasaccharide found in
heparin [2]. Although its molecular weight (1728 Da) is much lower
than the one of LMWH, Fpx remains a hydrophilic polyanionic macro-
molecule showing a very low bioavailability (N1%) by oral route [3].
This low oral absorption is the result of: i) poor transport through the in-
testinal epithelial barrier; ii) pronounced instability in acidic pH
It is not questionable that oral delivery is the preferred route of ad-
ministration. Few attempts have recently been made to develop emul-
sions as oral delivery forms of Fpx [6–8]. However, the preparation
methods of the Fpx-based emulsions are complicated, need heating
and up to five different excipients [6–8]. Moreover, the Fpx loadings
were low, less than 1 wt.% [6,7] or 5 wt.% [8]. In this context, the aim
of the present study was to develop an innovative oral form of Fpx,
able to associate large amounts of the drug by using a simple method
with only one excipient.
Thus, to improve the poor oral bioavailability of Fpx, we have
envisioned associating it to squalene (SQ), a natural lipid well-known
for its excellent oral absorption of more than 60% [9,10]. Moreover, SQ
derivatives have the property to self-assemble as stable nanoparticles
(NPs) in water [9,11]. It could therefore be expected that Fpx would
be protected from degradation by encapsulation into SQ-based NPs. To
form NPs, a first approach was to synthesize amphiphilic Fpx-SQ conju-
gates (data not shown). However, chemical conjugation of Fpx and SQ
Abbreviations: Fpx, fondaparinux; NPs, nanoparticles; CSq, cationic squalenyl.
⁎
Corresponding author. Present address: UMR CNRS 8214, Institut des Sciences
Moléculaires d'Orsay, Université Paris-Sud, 91405 Orsay Cedex, France. Tel.: +33 (0)1
69 15 82 47.
E-mail address: ruxandra.gref@u-psud.fr (R. Gref).
http://dx.doi.org/10.1016/j.jconrel.2014.07.060
0168-3659/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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B. Ralay-Ranaivo et al. / Journal of Controlled Release 194 (2014) 323–331
OSO3Na
HO
HO
O
OSO3Na
O
NH
CO2Na
NaO2C
NaO3S
OH
O
O
O
NaO3SO
O
HO
O
HO
O
NH
H
NaO3S
MeO
NHSO3Na
O
NaO3SO
OH
OSO3Na
1: Fondaparinux Sodium
NMe
3
CH SO
3
3
NMe
3
CH SO
3
3
NMe
3
Cl
3: 1,20-bis-trimethylammonium(hexanorsqualenyl)
dimethanesulfonate salt
2: Trimethyl (trisnor-squalenyl)ammonium
chloride salt
Fig. 1. Chemical structures of fondaparinux sodium (Fpx) and the cationic squalenyl derivatives salts (CSq).
was a tremendous synthetic challenge and had led to the loss of the an-
ticoagulant properties of Fpx (data not shown). Alternative strategies
based on ion-pairing to associate Fpx with SQ derivatives appeared to
be much more appealing. Therefore, two lipophilic cationic squalenyl
derivatives (CSq) (2–3, Fig. 1) were synthesized here to enable ion-
pair formation with polyanionic Fpx.
Glucose, glycerol, trehalose, sodium phosphate dibasic, sodium phos-
phate monobasic, Nile red and citrate concentrated solution were pur-
chased from Sigma-Aldrich Chemical Co. (France). Hard gelatin
capsules (size 9el) and capsule feeding needle were purchased from
Harvard Apparatus (France). Eudragit L100® was obtained as a gift
sample from IMCD (France).
Ion-pairing has previously been shown to be a potential approach
for improving the oral bioavailability of heparin [12–16]. The advan-
tage of this method is that cationic molecules such as polycationic-
lipophilic-core dendrons [13], chitosan derivatives [14,15] or
deoxycholylethylamine (DCEA) [16] efficiently interacted with heparin
without changing its chemical structure, thus avoiding the risk of reduc-
ing heparin's anticoagulant activity [16,17]. As an illustration of this strat-
egy, Lee et al. synthesized a cationic bile acid derivative to associate with
LMWH through the formation of ion pairs [16]. However, the oral bio-
availability of the complex was low (3%) with a high administered dose
of 50 mg/kg [16]. Chitosan derivatives self-assembled with LMWH in
nanocomplexes that were able to protect heparin from enzymatic degra-
dation in the gastrointestinal tract (GIT) [14,15]. Despite these interesting
results and to the best of our knowledge, no study has as yet considered
the oral administration of Fpx using the ion pairing approach.
2.2. General
IR spectra were obtained as solid or neat liquid on a Fourier Trans-
form Bruker Vector 22 spectrometer. Only significant absorptions are
listed. The 1H and 13C NMR spectra were recorded with Bruker Avance
300 (300 and 75 MHz, for 1H and 13C, respectively) or Bruker Avance
400 (400 and 100 MHz for 1H and 13C, respectively) spectrometers.
Mass spectra were recorded with a Bruker Esquire-LC instrument.
Elemental analyses were performed by the Microanalysis Service in
ICSN–CNRS, Gif-Sur-Yvette — France. Analytical thin-layer chromatog-
raphy was performed with Merck silica gel 60 F254 glass precoated
plates (0.25 mm layer) and Merck aluminum oxide 60F254 neutral
sheets. Column chromatography was performed with Merck silica gel
60 (230–400 mesh ASTM) and Fluka aluminum oxide type 507C neu-
tral. All reactions involving air- or water-sensitive compounds were
routinely conducted in oven- or flame-dried glassware under a positive
pressure of nitrogen. Except as otherwise indicated, all reactions were
carried out in distilled solvents. Triethylamine was distilled over calci-
um hydride. Chemicals obtained from commercial suppliers were used
without further purification.
We report here on the synthesis and characterization of new CSq, on
the formation and characterization of self-assemblies in aqueous media
with high Fpx loadings and on the physicochemical stability of the
resulting NPs. Finally, preliminary in vivo studies have investigated the
anticoagulant activity of this nanoparticulate system after intravenous
and oral administrations in rats.
2. Materials and methods
2.3. Synthesis and characterization of Sq+2
2.1. Drugs and chemicals
2.3.1. (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-
pentaen-1-yl methanesulfonate (5)
Fondaparinux (Fpx, Arixtra®, 10 mg/0.8 mL) was purchased from
GlaxoSmithKline (UK). Squalene (SQ) was purchased from Sigma-
Aldrich Chemical Co. (France), lithium chloride and trimethylamine hy-
drochloride from Alfa Aesar (France). Acetone, absolute ethanol, diethyl
ether, dimethylformamide and dichloromethane were obtained from
Carlo Erba (Italy). Filtered MilliQ water (Millipore®, France) was used.
To a stirred solution of trisnorsqualene alcohol 4 (582 mg,
1.5 mmol) in anhydrous CH2Cl2 (7 mL) at 0 °C, was added DMAP
(10 mg) and dropwise, triethylamine (224 mg, 2.2 mmol) followed by
methanesulfonyl chloride (205 mg, 1.8 mmol). The mixture was then
slowly raised to room temperature and stirred for 2 h. The reaction
was then quenched with brine and the mixture was extracted with

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325
CH2Cl2 (4 × 50 mL). The combined organic extracts were washed with
brine, dried over MgSO4 and concentrated in vacuo. The crude 1,1′,2-
trisnorsqualenyl methanesulfonate (5) (695 mg, 85%) was used directly
without further purification.
30.1 (2CH2), 28.7 (CH2), 28.4 (CH2), 26.8 (CH3, HC = C(CH3)2),
23.1 (CH2), 18.7 (CH3), 17.1 (3CH3), 16.7 (CH3).
MS (ESI): m/z (%) = 428 (100).
Anal. calcd for C30H54ClN (%): C 77.62, H 11.73, N 3.02. Found: C
77.21, H 11.71, N 2.90.
1H NMR (300 MHz, CDCl3) δ: 5.14–4.96 (m,5H, CH vinyl), 4.12 (t, 2H,
J = 6.5 Hz, CH2O), 2.91 (s, 3H, CH3SO2), 2.08–1.90 (m,18H, CH2),
1.90–1.76 (m, 2H, CH2, MsOCH2CH2), 1.58 (s, 3H, HC = C(CH3)2),
1.53 (s, 3H, 15H, HC = C(CH3)CH2)
2.4. Synthesis and characterization of Sq++
13C NMR (300 MHz, CDCl3) δ: 135.1 (Cq), 134.9 (Cq), 134.8 (Cq),
132.8 (Cq), 131.2 (Cq), 125.8 (CH), 124.5 (CH), 124.4 (CH), 124.2
(2 CH), 69.7 (CH2, CH2OMs), 39.7 (2 CH2), 39.6 (CH2), 37.3 (CH3,
OSO2CH3), 35.1 (CH2), 28.2 (2 CH2), 27.2 (CH2), 26.7 (CH2), 26.6
(CH2), 26.5 (CH2), 25.6 (CH3, HC = C(CH3)2), 17.6 (CH3), 16.1
(CH3), 16.0 (3 CH3), 15.8 (CH3).
2.4.1. (4E,8E,12E,16E)-20-(methanesulfonyloxy)-4,9,13,17-tetramethylicosa-
4,8,12,16-tetraen-1-yl methanesulfonate (8)
To an ice-cooled stirred solution of 1.20-bis-trisnorsqualene alcohol
(7) (1.2 g, 3.30 mmol) and few crystals of DMAP (10 mg) in anhydrous
CH2Cl2 (20 mL) was added dropwise triethylamine (1.27 mL,
9.90 mmol) followed by methanesulfonyl chloride (564 μl, 7.92 mmol).
The reaction mixture was stirred at room temperature for 4 h and
water (40 mL) was added. The mixture was extracted with CH2Cl2
(4 × 30 mL). The combined organic extracts were washed with brine,
dried over MgSO4 and concentrated in vacuo. The residue was purified
by flash chromatography on silica gel (AcOEt/cyclohexane 1:2) to pro-
vide pure bis-mesylate 8 as colorless oil (800 mg, 67%).
2.3.2.
2,6,10,14,18-pentaene (6)
To stirred solution of trisnorsqualenyl methanesulfonate
(6E,10E,14E,18E)-22-chloro-2,6,10,15,19-pentamethyldocosa-
a
(5) (500 mg, 1.08 mmol) in anhydrous DMF (5 mL) was added LiCl
(456 mg, 10.8 mmol). The reaction mixture was heated at 80 °C for
2 h. After cooling to room temperature, the mixture was concentrated
under reduced pressure. The residue was taken in water then extracted
with diethyl ether (5 × 12 mL). The combined organic phases were
dried over MgSO4, filtered and concentrated in vacuo. The crude prod-
uct was purified by chromatography on silica gel (cyclohexane/EtOAc,
98:2 v/v) to give the 1-chlorotrisnorsqualene (6) as a colorless oil
(377 mg, 86%). IR (neat, cm−1) v = 2855–2970, 1441, 980, 832.
1H NMR (300 MHz, CDCl3) δ: 5.09–5.06 (m, 4H, CH vinyl), 4.10 (t, 4H,
J = 6.5 Hz, CH2OMs), 2.93 (s, 6H, CH3SO2), 2.05–1.89 (m, 16H, CH2),
1.73–1.82 (m, 4H, CH2CH2OMs), 1.53 (s, 12H, HC = C(CH3)CH2).
2.4.2.
Trimethyl[(4E,8E,12E,16E)-4,9,13,17-tetramethyl-20-
(trimethylazaniumyl)icosa-4,8,12,16-tetraen-1-yl]azanium
dimethanesulfonate (3)
A solution of Me3N (4.0 g, 67.7 mmol) in ethanol (9 mL) was added
to bis-trisnorsqualenylmethanesulfonate 8 (650 mg, 1.22 mmol) in a
screw cap sealed tube equipped with a stirring bar. The reaction mixture
was stirred at 100 °C for 5 days and treated as described above for com-
pound 2 to yield salt 3 (600 mg, 92%).
1H NMR (300 MHz, CDCl3) δ: 5.25–5.02 (m, 5H, CH vinyl), 3.56 (t, 2H,
J = 6.7 Hz, CH2Cl), 2.20–1.91 (m, 18H, CH2), 1.85–1.90 (m, 2H,
ClCH2CH2), 1.61 (s, 3H, HC = C(CH3)2), 1.53 (m, 15H, HC =
C(CH3)CH2).
13C NMR (300 MHz, CDCl3) δ: 135.1 (Cq), 134.9 (Cq), 134.8 (Cq),
133.0 (Cq), 131.2 (Cq), 125.6 (CH), 124.5 (CH), 124.4 (CH), 124.3
(2 CH), 44.5 (CH2, CH2Cl), 39.8 (2 CH2), 39.7 (CH2), 39.6 (CH2),
36.6 (CH2), 30.8 (CH2), 28.3 (2 CH2), 26.8 (CH2), 26.7 (CH2), 26.6
(CH2), 25.7 (CH3, HC = C(CH3)2), 17.7 (CH3), 16.0 (CH3), 15.9
(2 CH3), 15.8 (CH3).
1H NMR (300 MHz, [D4]MeOH) δ: 5.09–5.29 (m, 4H, CH vinyl), 3.29–
3.42 (m, 4H, CH2), 3.18 (s, 18H, J = 6.7 Hz, CH3N+), 2,84 (s, 6H,
CH3S), 2.01–2.33 (m, 18H, CH2), 1.82–1.89 (m, 4H, CH2), 1.41–1.76
(m, 12H, CH3).
Anal. calcd for C33H68N2O6S2 (%): C 60.15, H 10.41, N 4.38, O 15.02,
S 10.04. Found: C 60.06, H 10.50, N 4.38, O 15.62, S 9.03.
2.3.3.
Trimethyl[(4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-
2.5. Preparation and characterization of the NPs
4,8,12,16,20-pentaen-1-yl]azanium chloride (2)
A 50% aqueous sodium hydroxyde solution (20 mL) was added
dropwise by means of a dropping funnel to trimethylamine hydro-
chloride (3.0 g, 31.4 mmol) placed in a distilled flask. The evolved
trimethylamine gas was passed through a washed bottle containing
sodium hydroxide pellets and allowed to bubble through 4.0 g of anhy-
drous ethanol placed in another wash bottle and exactly weighed. The
obtained Me3N (2.2 g, 37.2 mmol) ethanol solution was added to
1-chlorotrisnorsqualene (6) (300 mg, 0.71 mmol) in a screw cap sealed
tube equipped with a stirring bar. The reaction mixture was stirred at
100 °C for 72 h. After cooling to room temperature, the mixture was
concentrated under reduced pressure to provide the tertiary ammoni-
um salt 2 as a pale yellow oil (294 mg, 98%). The crude product was
used without further purification.
2.5.1. Preparation of fondaparinux NPs
In order to prepare Fpx NPs, various amounts of Sq+ (0.33 to
13.43 mg) were dissolved in different volumes (0.1, 0.3, 0.5 and 1 mL)
of solvent (acetone or absolute ethanol), whereas Sq++ (0.46 to
5.55 mg) was dissolved in 0.1 mL of absolute ethanol. The Fpx:Sq+
and Fpx:Sq++ NPs were prepared by nanoprecipitation. Briefly, 0.1 to
1 mL of Sq+ or Sq++ organic solutions were added drop-wise under
stirring (500 rpm) into 1 mL of aqueous solution of Fpx (2.5 mg/mL).
NP formation occurred spontaneously. Solvent was then evaporated
using a Rotavapor® (Buchi, France). The NPs were ultracentrifuged at
230,000 ×g for 150 min at 4 °C to isolate the supernatants and deter-
mine the amount of non encapsulated Fpx.
The nanoparticle suspensions were stored at 4 °C in water until
further use.
1H NMR (300 MHz, [D4] MeOH) δ: 5.26 (t, J = 6.0 Hz, 1H, CH vinyl),
5.25–5.15 (m, 4H, CH vinyl), 3.40–3.30 (m, 2H, Me3NCH2), 3.21
(s, 9H, (CH3)3 N+), 2.20–1.80 (m, 20H, CH2), 1.69 (s, 6H, CH3), 1.62
(s, 12H, CH3).
13C NMR (300 MHz, [D4] MeOH) δ: 136.8 (2Cq), 1.36.7 (Cq), 134.7
(Cq), 132.8 (Cq), 128.1 (CH), 126.45 (CH), 126.4 (CH), 126.3
(2CH), 68.5 (CH2, CH2NMe3), 54.5 (CH3, N(CH3)3), 54.45 (CH3,
N(CH3)3), 54.4 (CH3, N(CH3)3), 41.7 (2CH2), 41.6 (CH2), 37.9 (CH2),
2.5.2. Physicochemical characterization of the NPs
2.5.2.1. Quasi-elastic light scattering (QELS). The mean size (volume
intensity) and polydispersity index of the NPs were determined at
25 °C by QELS using a nanosizer (Zetasizer Nano 6.12, Malvern Instru-
ments Ltd., UK). The measurements were performed in triplicate, after
1/10 dilution of the NPs with MilliQ® water. The zeta potential was

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2.5.4. Preparation of Fpx:Sq+ NPs-filled enteric-coated capsules
determined using a Zetasizer (Zetasizer 4, Malvern Instruments Ltd.,
UK) after dilution of the NP suspensions in an aqueous solution of KCl
(1 mM).
The optimal formulation of lyophilized Fpx:Sq+ NPs was incorporat-
ed within hard gelatin capsules size 9el (Harvard Apparatus, France),
suitable for use in rats, using a manual capsule-filling device. The NP-
filled capsules were then coated with the enteric coating polymer,
Eudragit L100® to achieve the gastroresistance characteristics. Briefly,
the capsules were immersed in a 15% (w/v) isopropanol solution of
Eudragit® L100. Trace amounts (0.2% v/v) of Nile red, a dye was incor-
porated in the coating polymer solution to visually observe the homoge-
neity of the capsule coating and further evaluate its gastroresistance.
The stability of the coatings was verified in HCl solution at pH 1.2
(SGF) for 2 h and phosphate buffer at pH 6.8 (SIF).
2.5.2.2. Cryogenic transmission electron microscopy (cryo-TEM). The mor-
phology of the optimal formulation of NPs (Fpx:Sq+ 1:6) was visualized
using cryo-TEM. 4 μL of aqueous NP suspension was placed on 200 mesh
R2/2 Quantifoil coated copper grids (Quantifoil, Germany). The excess
amount of liquid was then blotted with a Whatman no. 5 filter paper,
and the grids were rapidly plunged into a liquid ethane bath at
−180 °C using a Leica EMGP Plunge Freezer (Leica Mikrosysteme
GmbH, Austria). Afterward, the grids were constantly maintained in
liquid nitrogen and carefully transferred to a Gatan 626 DH cryoholder
(Gatan, USA). Preparations were examined at −180 °C with a cryo-
TEM Jeol 2010 F electron microscope (JEOL, USA) operating at an accel-
erating voltage of 200 kV. Images were recorded with 1–5 μm of
underfocus under low-electron-dose conditions on Digital Micrograph
2.0.
2.5.5. Stability measurements
The physicochemical stability of the NPs was monitored for their size
and polydispersity index by QELS for 5 days at 4 °C.
2.5.6. Determination of the encapsulation efficiency of Fpx into the NPs
In order to determine the amount of Fpx associated with the NPs, NP
suspensions were ultracentrifuged at 230,000 ×g for 150 min at 4 °C.
The supernatant was withdrawn and the concentration of Fpx was
determined (see following paragraph).
2.5.2.3. Electron microscopy after freeze-fracture (FFEM). The Fpx:Sq+ NPs
(ratio 1:6), previously incubated with glycerol (30% v/v) used as a cryo-
protectant, were visualized by TEM after freeze-fracture. Briefly, a drop
of NP sample was placed on a Cooper support and immediately frozen in
liquid propane cooled with liquid hydrogen, and the NPs kept in liquid
nitrogen. Before TEM observations, NPs were fractured and the cut
surface was sprayed with platinum (4 nm) and carbon (30 nm).
The encapsulation efficiency (EE) is defined as:
Q
Fpxsediment
EE ¼
ꢀ 100
Q
Fpxtotal
where QFpx
is the percentage of the drug which was associated
sediment
with the NPs and QFpx is the total amount of Fpx in the colloidal
suspension.
2.5.2.4. Small-angle X-ray scattering (SAXS). The structure of the Fpx:Sq+
NPs (ratio 1:6) was further investigated by SAXS. Suspensions of nano-
particles (2.5 mg/mL) were loaded into quartz capillaries (diameter
1.5 mm, Glas Müller, Berlin, Germany). The top of the capillaries was
sealed with a drop of paraffin to prevent water evaporation.
X-ray scattering experiments were performed on the Austrian syn-
chrotron beamline at ELETTRA and on the SWING beamline at SOLEIL.
The scattered intensity was reported as a function of the scattering vec-
tor q = 4π sinθ/λ, where 2θ was the scattering angle and λ the wave-
length of the incident beam. For both instruments the calibration of
the q range was carried out with silver behenate.
total
Fpx was measured using the Azure A colorimetric method [3,10].
This method is based on the measurement of the metachromatic activ-
ity of Fpx with the dye, Azure A. Azure dye tightly binds to Fpx, thereby
shifting color from blue to violet. Practically, Azure A was dissolved in
water (0.04 mg/mL) and 100 μL of this solution was added to 40 μL of
the supernatants recovered after NP ultracentrifugation into 96-well
plate. The optical density of the solution was measured with a micro-
plate spectra PR 2100 reader at 490 nm and the extinction was related
to the Fpx concentration using a calibration curve.
The Austrian SAXS beamline was operated at 8 keV. SAXS patterns
were recorded using a position sensitive linear gas detector, argon-
ethane filled, with sample-detector distance of 1 m. Exposure times
were typically 300 s. On SWING beamline operated at 11 keV, the data
were collected by a two-dimensional CCD detector. Intensity values
were normalized to account for beam intensity, acquisition time and
sample transmission. Each powder-like diffraction pattern, displaying
a series of concentric rings, was then integrated circularly to yield the
intensity as a function of q.
2.5.7. Elemental analysis
Elemental analyses were performed by the Microanalysis Service of
ICSN–CNRS, Gif-Sur-Yvette — France.
Composition of the Fpx:Sq+ NPs (%): C 64.65, H 9.72, N 2.90, S 4.91
which correspond to [C31H43N3Na2O49S8]·[C30H54N]8·NaCl, featuring
1 Fpx for 8 Sq+ (theoretical values: C 64.68; H 9.51; N 3.06; S 5.10).
2.6. In vivo studies
2.5.3. Preparation and characterization of freeze-dried Fpx:Sq+
nanoparticles
The animal experiments were carried out according to the principles
of laboratory animal care and European legislation (recommendation
2007/526/EC). The protocol ethics were institutionally approved. Male
Sprague-Dawley rats (250–300 g) were purchased from Harlan Labora-
tories and used for the in vivo studies.
The following formulations were administrated orally to the rats
(n = 4) with the help of feeding needles (Harvard Apparatus, France):
NPs at 25 mg/kg (eq. Fpx) and 50 mg/kg (eq. Fpx); Fpx aqueous solu-
tion at 50 mg/kg and isotonic sodium chloride solution were used as
controls. The volume of the administrated NPs was 1 mL per 100 g of an-
imal body weight. Additionally, a saline solution of Fpx and Fpx:Sq+
NPs, both at 200 μg/kg (eq. Fpx) were injected intravenously to the
rats (n = 3) through the tail vein.
The freeze-dried NPs were prepared using trehalose as cryoprotec-
tant. Briefly, trehalose at a concentration of 20 wt.% was added to an
aqueous solution of Fpx (5 mg/mL). At this aqueous solution, Sq+
(8 mg) dissolved in 0.1 mL acetone was added drop-wise under stirring
(500 rpm). NP formation immediately occurred. Acetone was then
evaporated using a Rotavapor®. 1 mL of NP suspension was then incor-
porated in glass vials with flat bottom and frozen at −8 °C for 15 h. Vials
containing the frozen NP dispersions were lyophilized using an Alpha
1-2 LO plus freeze-drier to obtain the freeze-dried Fpx:Sq+ NPs in pow-
der form. The mean particle size and the PDI of the freeze-dried NPs
after rehydration were measured by QELS using a nanosizer (Zetasizer
Nano 6.12, Malvern Instruments Ltd., UK) as previously indicated. The
morphology of the freeze-dried NPs was investigated by cryo-TEM as
previously described.
Blood samples (450 μL) were taken from the jugular veins and
mixed directly in the syringe with 50 μL 3.8% (m/v) aqueous sodium
citrate solution to prevent coagulation. Plasma was collected after

B. Ralay-Ranaivo et al. / Journal of Controlled Release 194 (2014) 323–331
327
chloride salt (Sq+) 2 in 98% yield. In a similar manner, the known
1,20-hexanorsqualene diol
7 available from SQ via 2,3,22,23-
dioxidosqualene [19] was converted to Sq++ 3, via the symmetrical
bismesylate 8 upon treatment with trimethylamine in 92% yield as de-
scribed above.
Ref 18
OH
The 1H NMR spectra of Sq+ and Sq++ showed single peaks at
3.21 ppm and 3.18 ppm respectively, attributed to the trimethylamino
group and additionally for Sq++ the mesylate peak was observed at
2.84 ppm. The presence of these signals confirmed the successful syn-
thesis of the two CSq derivatives. These two salts were fully character-
ized by 1H, 13C NMR, mass spectrometry and their purity was assessed
by elemental analysis including dosage of sulfur and oxygen for salt 3.
4
Squalene
Ref 16
a,b,c
OH
3.2. Optimization of the formulation of Fpx: Sq+ and Fpx: Sq++ NPs
OH
7
d,e
NPs based on the formation of an ion-pair complex between CSq
and polyanionic Fpx were prepared by nanoprecipitation (Fig. 3).
Nanoprecipitation is the most common technique used to prepare
X
X
5: X = OMs
6: X = Cl
-
2: X= Cl , Me N-
3
X
8: X = OMs
3: X= CH SO , Me N-
-
3
3
3
Fig. 2. Synthesis of the CSq. Reagents and conditions: a) CH3SO2Cl, Et3N, CH2Cl2, 0 °C, 2 h,
85%; b)LiCl, DMF, 80 °C, 2 h, 86%; c) Me3N, EtOH, squalenyl, 100 °C, 72 h, 98%; d) CH3SO2Cl,
Et3N, CH2Cl2, 0 °C, 2 h, 67%; e) Me3N, EtOH, 100 °C, 120 h, 92%.
centrifugation at 2300 ×g for 15 min and stored at −20 °C until analy-
sis. The anti-factor Xa activity in plasma was determined using the chro-
mogenic substrate assay (STA Rotachrom® Heparin 8, Diagnostica
Stago, France). This assay is based on the FXa binding with the Fpx-
antithrombin complex. The FXa activity was measured using a specific
chromogenic substrate in the kit STA Rotachrom® Heparin
8
(Diagnostica Stago, France). In brief, 10 μL of plasma from treated or
control rat was introduced in each well of a 96-well plate, before adding
95 μL of substrate (830 μM). After incubation at room temperature for
3 min, the reaction was initiated by the addition of 75 μL FXa (to a
final concentration of 4 nM in a total volume of 180 μL) and was moni-
tored continuously at 405 nm using a microplate spectra PR2100 reader
for 20 min. The Fpx plasma concentration in the sample was directly
deduced from a calibration curve. Statistical tests were performed
using Student test.
In order to prevent the NP degradation in gastric medium, freeze-
dried Fpx:Sq+ NPs were filled into Eudragit L100® coated capsules.
The capsules were administrated orally in fed male Sprague-Dawley
rats as described above.
3. Results and discussion
3.1. Synthesis and characterization of CSq derivatives
Trimethyl (squalenyl) ammonium chloride salt (Sq+, 2) and 1,20-
bis-trimethylammonium (hexanorsqualenyl) dimethanesulfonate salt
(Sq++, 3) were synthesized from SQ according to Fig. 2. Briefly, to
obtain Sq+ 2, the trisnorsqualene alcohol 4 was prepared by sodium
borohydride reduction of the corresponding aldehyde obtained by
periodic acid cleavage of 2,3-epoxysqualene according to van Tamelen
[18]. Alcohol 4 was activated as methanesulfonate by reaction with
methanesulfonyl chloride in the presence of triethylamine. The latter
was then converted into 1-chlorotrisnorsqualene 6 by nucleophilic
substitution with lithium chloride in dimethylformamide. Finally,
heating of chloride 6 with a large excess of trimethylamine at 100 °C
in ethanol in a sealed tube gave the desired trimethylammonium
Fig. 3. Schematic representation of the formation of Fpx-Sq+ NPs by nanoprecipitation.
The organic phase containing SqC is added dropwise into the aqueous phase containing
Fpx, leading to the instantaneous formation of nanoparticles. Solvent is removed using a
rotary evaporator, forming stable suspensions of nanoparticles.

328
B. Ralay-Ranaivo et al. / Journal of Controlled Release 194 (2014) 323–331
100
Table 1
Physicochemical characterization of the NPs of Fpx:Sq+ and Fpx:Sq++ with different mo-
90
80
70
60
50
40
30
20
10
0
lar ratios (mean values
(d), zeta potential (z) and polydispersity index (PDI).
standard deviation, n = 6): measurement of mean diameter
Type of NPs Molar ratio Charge ratio z [mV]
d [nm]
PDI
Fpx:Sq+
1:0.5
1:1
1:2
1:3
1:4
1:5
1:6
1:10
1:20
1:0.5
1:1
10:0.5
10:1
10:2
10:3
10:4
10:5
10:6
10:10
10:20
10:1
10:2
10:4
10:6
10:8
10:12
−54
−61
−61
−67
−58
−60
−60
−1
−0.5
−52
−60
−60
−30
−10
−5
5
5
1
4
4
1
3
118
129
139
145
146
143
149
2
1
1
3
5
3
1
1
5
3
2
12
8
0.12
0.15
0.14
0.14
0.15
0.12
0.13
0.11
0.18
0.14
0.17
0.11
0.28
0.36
0.01
0.01
0.03
0.01
0.02
0.02
0.01
0.01
0.06
0.02
0.04
0.03
0.02
0.03
0.10
12 147
184
10 133
5
1:0.5
1:1
1:2
1:3
1:4
1:5
1:6
1:10
Fpx:Sq++
Molar ratio Fpx: Sq+
5
2
2
5
191
382
519
725
1:2
1:3
1:4
1:6
Fig. 4. Encapsulation efficiency (EE) of Fpx within the Fpx:Sq+ NPs. The studies were
carried out at a Fpx concentration of 2.5 mg/mL. Data represent mean values standard
deviation (n = 6).
87
10 772
151 0.92
NPs. It combines the advantages of a one-step preparation, a facile scale-
up procedure and the use of less toxic solvents as compared to other
fabrication methods [20].
into account that Fpx has ten negative charges, at molar ratios Fpx:
Sq+ of 1:10 and 1:20 (corresponding to charge ratios of 1:1 and 1:2,
respectively), the number of Sq+ per Fpx chain was likely sufficient to
neutralize the Fpx charges, leading to a neutral global charge of the
resulting Fpx:Sq+ NPs. As a consequence, at these values, the stability
of the system was poor (see for further details Supporting information,
§ 2). (See Table 2.)
The synthesized Sq+ and Sq++ cationic derivatives were compara-
tively used for the formation of Fpx-loaded NPs. It was observed that
the two SqC alone were not able to self-assemble as NPs in water, but
formed large aggregates (when used alone in the nanoprecipitation
technique). However, when used in combination with Fpx, the sponta-
neous formation of NPs in water was observed (Fig. 3), likely due to
electrostatic CSq/Fpx and hydrophobic CSq–CSq interactions.
Particle size is an important parameter determining the in vivo fate
of the NPs after oral administration and as a rule thumb, sizes smaller
than 500 nm are considered to facilitate the interactions with the epi-
thelia [15,21]. In this respect, the NP preparation was optimized to ob-
tain stable monodisperse colloidal suspensions together with high
drug loadings. For this purpose, different parameters were tested:
i) the nature of the organic solvent (acetone or ethanol), ii) the volume
ratio of the two phases (organic/aqueous phases) and iii) the concentra-
tion of CSq.
In contrast to Sq+, Sq++ failed to produce monodisperse NPs except
at a ratio below 1:2. Indeed, aggregates or NPs with large sizes up to
772 nm were obtained in other cases (Table 1). Moreover, the NPs
prepared with Sq++ were less stable than the Sq+ ones over time and
precipitated within two days at 4 °C (see Supporting information, § 2).
3.3. Determination of the amount of Fpx associated with the NPs
3.3.1. Extraction studies
In a first approach, to determine the amount of incorporated Fpx,
Fpx:Sq+ NPs were submitted to destructuration studies in the presence
surfactants such as sodium cholate, a bile salt (see Supporting informa-
tion, § 2). Whatever the experimental conditions, the nanoparticles
could not be destructured, but only broken into smaller ones (10 to
50 nm). Therefore, the amount of Fpx inside the particles was deter-
mined by assessing the amount of non-encapsulated Fpx in the super-
natants after NP centrifugation (for more details see the Materials and
methods section).
The first parameter needed to be identified was a solvent of CSq, also
miscible with water. Since Sq++ was insoluble in acetone, NPs could be
obtained only by using ethanol. In the case of Sq+, the best solvent to
obtain small (around 200 nm) and monodisperse NPs was acetone.
The influence of the volume ratio acetone/water was further investi-
gated for optimization of Fpx:Sq+ NPs (see Supporting information,
§ 1). The lowest volume ratio organic to aqueous phase of 1:10 was
found to be the best to obtain monodisperse NPs (polydispersity of
3.3.2. Encapsulation efficiency of the Fpx:Sq+ NPs
0.07
0.01) with smaller mean diameter (145
6 nm). Thus, the
The Azure A assay was an effective method to determine the amount
of non-associated Fpx in the supernatants after NP ultracentrifugation.
volume ratio of 1:10 was chosen for further evaluation of the influence
of the amount of CSq (Sq+ and Sq++) on the formation of the NPs.
Table 1 reports the physicochemical characteristics for the two types
of NPs at various Fpx–CSq ratios. In the case of NPs prepared using
Sq+, whatever the experimental conditions, nanoprecipitation was suc-
cessful, leading to the formation of NPs with mean diameter lower than
200 nm and polydispersity index lower than 0.2. Zeta potential values
were approximately −60 5 mV. Above the threshold of molar ratio
Fpx:Sq+ of 1:6, the zeta potential reached values close to zero. Taking
100
90
80
70
60
50
40
30
20
10
0
Table 2
Physicochemical characterization of the Fpx:Sq+ NPs at a molar ratio of 1:3 as a function of
the volume ratios (acetone/water) (mean values
ment of mean diameter (d) and polydispersity index (PDI).
standard deviation, n = 6): measure-
1:0,5
1:1
1:2
1:3
Volume ratio (acetone/water)
d [nm]
PDI
Molar ratio Fpx: Sq++
0.1
0.3
0.5
1
145
153
170
372
3
9
23
32
0.14
0.09
0.08
0.20
0.01
0.02
0.01
0.05
Fig. 5. Encapsulation efficiency (EE) (%) of Fpx within the Fpx:Sq++ NPs with different ra-
tios. Fpx concentration in the NP suspension was 2.5 mg/mL. Data represent mean
values standard deviation (n = 6).

B. Ralay-Ranaivo et al. / Journal of Controlled Release 194 (2014) 323–331
329
Fig. 4 presents the encapsulation efficiencies (EE) obtained using this
assay. The best EE were obtained with molar ratios (Fpx to Sq+) of 1:6
and 1:10, showing that 85% and 87%, respectively, of Fpx was successful-
ly associated with Sq+ under the form of NPs. Calculated corresponding
loadings reached 38.5% at the ratio 1:6, which is the optimized ratio in
this study. Indeed, although the NPs with the 1:10 ratio encapsulated
large Fpx amounts too, they were unstable as already discussed before
(see also Supporting Information, § 2).
To confirm these results, a direct method (elemental analysis) was
developed to determine the amount of Fpx associated with the NPs in
the pellets obtained after ultracentrifugation. Elemental analysis
enabled determining four elements (carbon, hydrogen, nitrogen and
sulfur) which were assayed in the pellet and in the supernatant. Carbon,
hydrogen and nitrogen are common to both Sq+ and Fpx, while only
sulfur is present in Fpx. In the elemental analysis of the optimized NPs
(ratio 1:6), all the four elements were present, which clearly confirmed
the presence of Fpx. Calculations have shown that 80% of Fpx was asso-
ciated with the NPs, whereas 20% remained in the supernatants. This is
in agreement with the dosage of Fpx in the supernatant using the Azure
A assay (ie. 85%, Fig. 4). From these data, it could be concluded that in
the NPs, one Fpx molecule was associated with 8 Sq+ molecules, where-
as in the supernatants, one Fpx molecule was associated with one Sq+
(see the Material and methodssection, § 2.5.5). Hence, the existence of
strong interactions between Fpx and Sq+ could be confirmed, as these
two components were always associated, even in the supernatants.
NPs could be released quickly in the blood, resulting in an anticoagulant
activity similar to free Fpx. These data prove that Fpx could be effective-
ly dissociated from the Sq+ moieties in the blood stream.
Fig. 7B shows the plasma concentration–time profile of free
Fpx (saline solution) as compared to encapsulated Fpx after oral admin-
istration. As expected, Fpx in saline solution was not absorbed. The
concentration-profiles were similar to those of the control group. By
contrast, administration of nanoparticulate suspensions at two distinct
concentrations resulted in an increase in plasma concentration of Fpx
(Fig. 7B). The NPs containing Fpx doses of 25 mg/kg and 50 mg/kg
showed Cmax of about 0.11 mg/L and 0.22 mg/L, respectively with a
time to maximum plasma concentration (Tmax) of around 60 min in
both cases. These results revealed a dose-dependent increase in Cmax
whereas the time to reach maximal concentration (Tmax) was dose-
independent. Of note, the Cmax obtained after oral administration of
50 mg/kg Fpx:Sq+ was close to the prophylactic plasma concentration
needed in venous thromboembolism [3].
It was concluded that the newly developed nanoparticulate system
could improve the oral delivery of Fpx as compared to the free Fpx.
However, the absolute bioavailability still remained low (around 0.3%)
probably due to the instability of Fpx:Sq+ at the acidic pH of the stom-
ach. To avoid this, the nanoparticles were entrapped into Eudragit
L100®-coated capsules, resistant to the pH in the stomach. For this,
nanoparticles have been lyophilized in the presence of trehalose, recov-
ering the same features as before (“onion-type” structures, mean diam-
eter and zeta potential preserved, as assessed by cryo-TEM and
3.3.3. Encapsulation efficiency of Fpx:Sq++ NPs
For all the experimental conditions studied using the second CSq,
Sq++, less than 60% of Fpx was effectively incorporated into the NPs
(Fig. 5) and the resulting NPs were less stable especially at ratios N1:3.
In conclusion, among the two CSq synthesized here, Sq+ was the most
adapted to encapsulate efficiently Fpx (EE over 80%).
3.4. Supramolecular organization of the Fpx:Sq+ NPs
The morphology of the optimized Fpx:Sq+ NPs (molar ratio Fpx to
Sq+ of 1:6) was investigated by cryo-TEM (Fig. 6a). The Fpx:Sq+ NPs
had a regular spherical shape with an inner “onion-type” structure. Syn-
chrotron small-angle X-ray scattering (SAXS) was used in conjunction
to cryo-TEM to provide a better insight into the supramolecular organi-
zation of the NPs. A (repeat order) correlation peak at q = 0.13 Å−1 and
a very weak second order at about q = 0.26 Å−1 were observed on the
scattering curve of the NPs (Fig. 6c). Supposing a lamellar organization,
a thickness of about 48 Å of the lamella could be estimated from the
peak at 0.13 Å−1. This pattern is consistent with the interlamellar dis-
tance measured on the cryo-TEM images (45–50 Å, Fig. 6a). Taking
into account these considerations, the hypothesized supramolecular
organization of the Fpx:Sq+ NPs is represented in Fig. 6b. Likely, the
“onion”-like structure consists of hydrated layers of Fpx interacting
with layers of Sq+. Presumably, both Fpx-Sq+ electrostatic interactions
and Sq+-Sq+ hydrophobic interactions play a role for maintaining the
cohesion of these assemblies. However, further studies would be need-
ed to fully unravel the supramolecular organization of these NPs.
3.5. In vivo anticoagulant activity
The anticoagulant activity of the optimized Fpx:Sq+ aqueous
nanoparticulate suspensions was evaluated in vivo after oral and intra-
venous (IV) administration in male Sprague-Dawley rats. Blood samples
were periodically taken and the plasma concentration of active Fpx was
estimated using an anti-FXa assay.
Fig. 7A shows the plasma concentration–time profile of free Fpx
(saline solution) as compared to encapsulated Fpx after IV administra-
tion. Both profiles are very similar, with maximum plasma concentra-
tion (Cmax) of 0.16 mg/L and 0.12 mg/L for free Fpx and encapsulated
Fpx, respectively. These results showed that the Fpx associated to the
Fig. 6. Supramolecular organization of Fpx:Sq+ NPs at a ratio 1:6: A) cryo-TEM image;
B) electron microscopy after freeze-fracture; C) SAXS pattern and D) hypothetical organi-
zation of Fpx (dark blue) and Sq+ (pink) in aqueous phase showing ion pair formation
(Fpx:Sq+) and stabilization by interaction between lipophilic squalenyl derivatives. Bars
represent 100 nm. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

330
B. Ralay-Ranaivo et al. / Journal of Controlled Release 194 (2014) 323–331
(p b 0.05) were found between fasted and fed rats. Food increases
around 48% the maximal plasma concentration of Fpx.
With further studies, this nanoparticulate system holds promises for
the oral delivery of Fpx.
4. Conclusion
This study proposes a new approach to the oral administration of the
anticoagulant Fpx using self-assembled NPs obtained by electrostatic in-
teractions between cationic squalenyl derivatives and the polyanionic
pentasaccharide. Optimized nanoformulations were monodisperse,
stable over storage and possessed a mean diameter in the range of
150–200 nm suitable for the oral route. A multilamellar supramolecular
organization has been shown by cryo-TEM and SAXS.
The absolute bioavailability of Fondaparinux could be increased
up to 9% and the described nanoparticles allowed improved pharma-
cokinetics in a dose-dependent profile and therapeutic range. This
approach opens up challenging perspectives for the oral delivery of
Fpx in the future and paves the way towards elaborating Sq-based
NPs which self assemble without the need of covalently grafting
the drug to Sq.
Acknowledgments
The authors would like to thank G. Pehaud-Arnaudet (Institut
Pasteur) for the cryo-TEM experiments and V. Domergue-Dupont
(Faculty of Pharmacy, Châtenay Malabry) for her technical help with
the animal experiments (University of Paris-Sud, Faculty of Pharmacy).
The research leading to these results has received funding from the
French Ministry of National Education, Research and Technology
(grant to B.R.R.) and from the European Research Council under the
European Community's Seventh Framework Programme FP7/2007-
2013 (grant agreement no. 249835).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2014.07.060.
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