New class of squalene-based releasable nanoassemblies of paclitaxel, podophyllotoxin, camptothecin and epothilone A

Article abstract of DOI:10.1016/j.ejmech.2014.07.035

The present study reports the preparation of a novel class of squalene conjugates with paclitaxel, podophyllotoxin, camptothecin and epothilone A. The obtained compounds are characterized by a squalene tail that makes them able to self-assemble in water,

Full text of DOI:10.1016/j.ejmech.2014.07.035

European Journal of Medicinal Chemistry 85 (2014) 179e190
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New class of squalene-based releasable nanoassemblies of paclitaxel,
podophyllotoxin, camptothecin and epothilone A
Stella Borrelli a, Michael S. Christodoulou a, Ilaria Ficarra a, Alessandra Silvani a,
Graziella Cappelletti b, Daniele Cartelli b, Giovanna Damia c, Francesca Ricci c,
,
, *
Massimo Zucchetti c, Franco Dosio d *, Daniele Passarella a
a
ꢀ
Dipartimento di Chimica, Universita degli Studi di Milano, Via C. Golgi 19, 20133 Milano, Italy
b
ꢀ
Dipartimento di Bioscienze, Universita degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
c Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy
d Dipartimento di Scienza e Tecnologia del Farmaco, Via Giuria 9, 10125 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study reports the preparation of a novel class of squalene conjugates with paclitaxel,
podophyllotoxin, camptothecin and epothilone A. The obtained compounds are characterized by a
squalene tail that makes them able to self-assemble in water, and by a drug unit connected via a
disulfide-containing linker to secure the release inside the cell. All the obtained compounds were
effectively able to self-assemble and to release the parent drug in vitro. Disulfide-containing paclitaxel
esqualene derivative showed a similar biological activity when compared to the free drug. Immuno-
fluorescence assay shows that this squalene conjugate enters A549 cells and stain microtubule bundles.
The results described herein pave the way for different classes of squalene-based releasable
nanoassemblies.
Received 10 January 2014
Received in revised form
9 May 2014
Accepted 10 July 2014
Available online 27 July 2014
Keywords:
Squalene derivatives
Nanoassemblies
Paclitaxel
© 2014 Elsevier Masson SAS. All rights reserved.
Podophyllotoxin
Camptothecin
Epothilone A
1. Introduction
So, nanoparticles can be preferentially delivered to the tumor site
thanks to the enhanced permeation and retention (EPR) effect
In the treatment of cancer it is often not sufficient to find highly
active molecules. The majority of active compounds have its pri-
mary target within cells and tissues and, in order to exploit the
pharmacological activity, a minimum concentration is needed to be
reached at the site of action. Many factors may cause a decrease in
the intracellular drug concentration. Among them, we enumerate
low water solubility, low partition coefficient across cell mem-
branes and extensive exportation via efflux transporters such as
multi-drug resistance proteins and P-glycoproteins. For these rea-
sons, a selective delivery of drugs to their sites of action could result
in greater therapeutic effects and potentially minimize side-effects.
In recent years, the design, synthesis and application of nano-
technology [1] has opened up new perspectives for biological and
biomedical applications [2]. The vasculature in tumor tissues is
leaky to macromolecules, having large junctions between the cells.
[3e5], whereas the lymphatic system is usually deficient. In addi-
tion to that, nanotechnologies can improve drug properties in
several ways: by controlling release and distribution, by enhancing
drug absorption by mucosa or cells and by protecting the drug from
degradation. Therefore, they offer many possibilities of improving
the specificity of drug treatment. Despite this, there is still no
universal platform suitable for the delivery of all kinds of drugs, and
current nanotechnologies have important limitations.
A great challenge comes from the difficulty to design synthetic
materials which combine: a) low toxicity, b) lack of immunoge-
nicity and biodegradability, and c) do not accumulate in cells or
tissues [6]. The concept of lipid-drug conjugates has gained
considerable attention in recent years, and they are usually ob-
tained by a covalent coupling of the drug to biocompatible lipid
moieties. Among them, squalene, a natural precursor of many ste-
roids, showed the ability, when linked to biologically active com-
pounds, to achieve a spontaneous formation of nanoassemblies in
water. Thus, squalene, protects the drugs from environment
damaging factors and, in some cases, also improves their
* Corresponding authors.
E-mail addresses: daniele.passarella@unimi.it, Danpassarella62@gmail.com
(D. Passarella).
http://dx.doi.org/10.1016/j.ejmech.2014.07.035
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

180
S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
Scheme 1. Supposed pathway for drug release of disulfide-containing squalene conjugates.
pharmacokinetic profile by decreasing the toxicity of the associated
drugs with an increase in their therapeutic index [7]. This novel
approach progressively extended its field of application from
anticancer agents (gemcitabine, paclitaxel, cis-platin, doxorubicin)
[6e8] to antibiotics and from antiviral agents (penicillin, AZT,
acyclovir) [9,10] to nucleotides (siRNA) [11,12]. Furthermore, the
ability to spontaneously nanoassemble allowed the preparation of
nanoparticles containing both therapeutic and diagnostic agents
[13,14]. One of the key points in the realization of potent and effi-
cient squalene prodrug resides in the release of the free drug; thus,
the role of the linkage between the drug and the squalene moiety is
crucial. Recently, we have demonstrated, in paclitaxel derivatives,
the relevant influence of the linker in the cytotoxicity [6a]. Our
continuous interest in the field of the synthesis of anticancer
compounds [15e18] made us embark on the preparation of
squalene-incorporating derivatives with the use of a disulfide-
containing linker to secure the nanoassemblies and drug release.
We reported in different papers the ability of disulfide-containing
bivalent compounds to release active drugs inside tumor cells
[19] and, on the basis of the results obtained, we envisioned the
possibility to prepare a novel class of disulfide-containing squalene
conjugates (Scheme 1). The presence of the squalene tail induces
spontaneous self-assembly in water, while the disulfide bond in-
duces drug release in the intracellular environment.
We selected four knownanticancer compounds (Fig.1): paclitaxel,
podophyllotoxin, camptothecin and epothilone A. These compounds
are known to interact with tubulins (paclitaxel, podophyllotoxin and
epothilone A) and with topoisomerase I (camptothecin), but they all
suffer from many drawbacks, primarily due to the side-effects asso-
ciated with their low water solubility. The introduction of a lipophilic
tail overcomes the problem by favoring the formation of nano-
assemblies, more capable of membrane permeation.
Herein, we describe the synthesis (Scheme 2) of the designed
conjugates (Fig. 2), the characterization of the nanoassemblies,
their ability to release the drug unit, and a preliminary evaluation of
their biological activity in vitro.
described in Scheme 2. The crucial reaction is the formation of
monobromohydrine 6 that was converted into 2,3-epoxide 7 by
simple treatment with K2CO3. The oxidative cleavage of the epoxide
furnished 1,10,2-tris-norsqualene aldehyde 8 [24] and the subse-
quent NaBH4 reduction secured the alcohol 9. Condensation with
dicarboxylic acids 10aeb gave the corresponding monoesters
11aeb. These key intermediates were coupled with four different
active drugs 1e4, thus generating a small collection of squalene
conjugates. As for paclitaxel and epothilone A conjugates, the
regioselectivity of the acylation reaction was assessed on the basis
of 1H NMR spectra of the final compounds. In fact, after the for-
mation of the ester bond, the peak of the proton 20 (paclitaxel) and
7 (epothilone A) respectively is shifted to lower fields. Paclitaxel
esters derived from acylation of C20eOH have been demonstrated
to efficiently release paclitaxel [25]. The purity of the obtained
compounds was evaluated by HPLC.
2.2. Nanoassemblies: preparation and characterization
Although several articles on squalenoyl NAs are reported, a
detailed evaluation of the role of different variables involved in the
solvent-displacement precipitation are not yet totally studied. For
this reason, a detailed formulation study was carried out just
maintaining constant the kind of solvent able to dissolve up to
8 mg/mL from each compound. We considered as reference the
‘standard procedure’ applied by several authors for a variety of
compounds (2 mg/mL concentration in solvent/water 1:2). The
values of size are reported in Table 1. The parameter of size, stan-
dard error and polydispersity, monitored by DLS, allowed checking
the role of the different variables: volume of solvent, volume of
water, final concentration, ionic strength, pH and variety of salt. For
all the compounds, the role of volume of the solvent (added up to
2. Results and discussion
The squalenoylation platform discovered by Couvreur et al.
[6e8] was employed to design self-assembling nanomedicines of
both hydrophilic and hydrophobic therapeutic agents. In this work
we explore the efficacy of modifying four hydrophobic anticancer
drugs by introducing a squalene-type chain using a labile linker. A
novel class of squalene conjugates characterized by a labile disul-
fide bond has been prepared and characterized. Also, their ability to
self-immolate [23] and to release the parent drug has been verified.
2.1. Synthesis of drug-squalene conjugates
A divergent approach has been used. In particular, 1,10,2-tris-
norsqualene alcohol was obtained starting from squalene, as
Fig. 1. Paclitaxel (1), podophyllotoxin (2), camptothecin (3) and epothilone A (4), in red
the anchor points.

S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
181
= Sq
i
Sq
HO
6
Br
ii
O
Sq
Sq
HO
Sq
iv
iii
O
H
7
8
9
O
O
X
O
Sq
Sq
X
OH
HO
X
HO
X
v
O
11a-b
O
10a-b
DRUG-OH
v
1-4
a: X = S
b: X = -CH -
2
O
DRUG
X
O
O
X
O
12-15a-b
Scheme 2. i) NBS, H2O, THF; ii) K2CO3, MeOH; iii) H5IO6, MeOH; iv) NaBH4, MeOH; v) EDC$HCl, DMAP, CH2Cl2.
1.5 mle1 ml of water) was not relevant and the values reported in
Table 1 as size and polydispersity index did not range more than
20%. Only for paclitaxel-based compounds (12a, 12b) a size increase
of 50% was observed. Increasing the final concentration to 4 mg/mL
lead to a significant decrease of size for 14a, 14b (to 103.5 nm and
110.6 nm respectively) and 13a and 13b (115.6 and 126.5 nm). No
differences in size were seen when increasing the ionic strength to
10 mM, but for most of the compounds, the increase to 100 mM
tended to cause a rapid size increase and, in some cases, a precip-
itation of the compound (12a, 12b, 13a, 13b). These data are re-
ported in Table 2 together with the data obtained regulating the pH
value and buffers. To reduce the variability, three pH values were
selected: 4.5 (for acetate and citrate buffers), 7.5 for phosphate and
10.2 for borate and carbonate buffers.
paclitaxel derivatives 12. The phosphate buffer and neutral pH
allowed obtaining NAs with size very close to that obtained in water.
At basic pH values (10.2) some compounds were unstable, for
example the epothilone derivatives 15, NAs rapidly turned to brown
color with both buffers, while carbonate buffer lead to an effective
increase of size for almost all bioconjugates (from 212 to 600 nm).
The preparation in water (phosphate buffer pH 7.2 or 10 mM NaCl),
lead to the smallest NAs which did not present signs of size increase
over time. The studied molecules do not contain any ionizable group
and, for this reason, the different behavior of some buffers citrate vs
acetate, carbonate vs borate seems directly due to the squalene-type
componentof NAs. Nosignificant differences in stability fordisulfide
vs alkyl derivatives were observed in these tests.
Taking into account the preparation in water at a standard
concentration of 2 mg/mL these aqueous NA suspensions were
found to be physically stable (by measure of particle size) for
Acidic pH (citrate buffer, Table 2) induce an increase of the NAs
(from 200 nm to 1
mm) while acetate buffer destabilized only the
O
O
O
AcO
HO
OH
X
O
Sq
O
O
N
O
X
H
H
O
O
O
O
O
Ph
NH
O
O
N
O
O
X
X
Ph
O
H
OBz
O
2'
O
OAc
O
O
O
O
X
Sq
O
O
X
O
Sq
O
14a-b
12a-b
13a-b
O
O
O
S
N
O
AcO
X
X
OH
O
O
7
Sq =
Ph
NH
O
O
O
a X = S
b X = CH2
Ph
O
H
OBz
O
OH
OAc
HO
O
O
O
O
Sq
16 (Ref. 2)
Sq
15a-b
Fig. 2. Chemical structures of the obtained conjugate compounds.

182
S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
Table 1
Physico-chemical characterization and stability of squalenoyl-drug nanoassemblies.
Compound
Sizea (nm)
PIa
Z potentiala (mV)
CACb (mg/l)
NAs stabilityc (size)
Comp. stabilityd (% unaltered )
NAs stability (GSH)e
12a
12b
13a
13b
14a
14b
15a
15b
16
103.2 0.7
107.0 0.1
184.3 2.4
179.6 1.1
195.2 6.8
154.8 7.0
161.6 6.8
124.1 2.5
173.5 3.9
0.052
0.049
0.063
0.048
0.083
0.279
0.090
0.131
0.030
ꢁ37.56 7.7
ꢁ20.78 3.5
ꢁ11.42 1.7
ꢁ28.47 2.3
ꢁ41.02 4.1
ꢁ39.14 9.2
ꢁ56.37 2.4
ꢁ25.39 4.37
ꢁ34.62 2.7
1.5
1.1
0.7
0.5
1.5
1.8
0.6
0.8
0.2
106.5 1.1
110.5 3.4
215.3 5.1
210.5 7.7
210.2 3.1
172.4 1.2
191.1 2.7
142.9 4.7
181.1 10.7
93
95
52
82
70
85
58
70
94
3
5
2
5
2
8
6
8
4
110.5 6.5
120.2 5.7
222.1 12.0
185.3 2.7
234.4 13.5
162.6 3.5
720.5 21.2
130.3 8.1
195.4 3.2
All formulations were prepared using nanoprecipitation technique at a concentration of 2 mg/mL aqueous nanodispersions.
a
Particle mean diameter, polydispersity index (P.I.) and charge, were determined using a Brookhaven Nanosizer.
b
Critical aggregation concentration (CAC) measurement by pyrene was obtained from a ratio between I373 and I384 as a function of concentration.
c
d
e
Stability was assessed by measuring the size of samples diluted with water (checked after three months at 5 ꢀC).
Stability was assessed by evaluating the ratio of HPLC area of peaks corresponding to intact compounds at T ¼ 0 and after incubation with human serum at 37 ꢀC for 48 h.
Stability was assessed by measuring the size of samples of NAs exposed to 10 mmol/L GSH for 24 h.
months when stored at 5 ꢀC (Table 1). NAs deriving from paclitaxel
were incubated at a pH 8 with a final concentration of 0.54 mM of
the reductive agent dithiotreitol (DTT). Using appropriate volume
of acetonitrile/water it was possible to avoid the NAs formation also
allowing complete solubility of DTT. The chemical stability of the
disulfide bond was determined by monitoring the peak of the
squalene derivatives. Fig. 4 demonstrates the appearance of the free
drug after treatment of 12a with DTT.
appeared smaller than NA-16 while no significant differences in
size were seen evaluating different disulfide of alkylic chain. This
unique ability of the squalene derivatives to form NAs in water was
further confirmed by the critical aggregation concentration (CAC)
measurement. It has to be noted that the observed CAC values were
higher than compound 16 [6] but still lower than those of poly-
meric micelles used for drug delivery purposes (5e10 mg/L).
Moreover, the size of the NAs of all the conjugates remained un-
changed upon dilution into water (data not shown).
Fig. 5 shows the results of derivatives with disulfide bonds. In
panel A, the size of NAs after exposure of DTT is represented (up to
78 h), while panels BeE represent the release/degradation of the
compounds (as NAs or soluble compounds) as detected by HPLC
analysis. For all the disulfide linked compounds the buffer alone did
not significantly influence the stability of disulfide bond or the ester
linkage for compounds 12b, 13b, 14b, 15b (data not shown). After
addition of DTT, a rapid break of the disulfide bond was evident
only for the compounds dissolved in organic solvent. All NAs when
treated with DTT appeared stable with very slow release of drug
during the first two days. A correlation between stability of NAs and
inner component seems to appear for podophyllotoxin derivative
13a, and in a minor percentage for epothilone 15a, where NAs
instability also produced a fast disulfide release. Further com-
pounds characterization was carried out by incubation NAs in hu-
man serum for two days at 37 ꢀC (Table 1). NAs showed good
stability but an increased release for all the disulfide linked com-
pounds appears evident. Paclitaxel derivatives were the most stable
while significant release occurred for podophyllotoxin and epo-
thilone derivatives (around 48e42% released after 48 h). Further-
more, also glutathione was used to monitor the stability of NAs.
Indeed cancer cells express high levels of detoxifying enzymes such
as glutathione-S-transferases (GSTs) and glutathione (GSH) to
protect themselves from toxic xenobiotics. In our case the NAs were
exposed to the presence of 10 mmol/L GSH (corresponding to an
2.3. Stability of squalene derivatives and NAs
The stability of NAs was initially evaluated using the same
conditions as in the preparations step, in order to compare the role
of pH and buffer type in preformed NAs. Fig. 3 represents the NAs
size after dilution in buffers and sampled after different times. For
all compounds, except those derived by paclitaxel, the citrate buffer
tended to increase the size after one day. Paclitaxel compounds
demonstrated immediate instability in carbonate buffer, with an
almost complete precipitation after 24 h. However, no instability
was observed in borate buffer, at the same pH value. NAs from
epothilone A (15a and b) showed high instability, and after 1 h, in
all conditions, a significant increase in size and a color brown
appeared. In basic conditions, NAs of compound 14a (camptothe-
cin), but not 14b, increased in size.
Contrary to the results of formulation, dilution of preformed
NAs, for all the compounds, in 100 mM NaCl did not significantly
improve their size.
In order to investigate the chemical stability of disulfide-con-
taining squalene-drug both as compound in solution and as
assembled in nanostructure, samples at a concentration of 1 mg/mL
Table 2
NAs preparation: role of pH, buffer and ionic strength.
NAs from compounds
Water/buffer
12a
12b
13a
13b
14a
14b
15a
15b
Water
100.8 1.2
462.4 71.2
107.9 2.3
391.2 31.4
185.9 8.2
428.3 29.1
275.9 9.3
201.5 14.0
202.3 9.9
242.9 18.9
676.0 44.2
180.5 9.3
629.2 81.3
287.5 7.1
193.5 8.4
211.3 15.2
315.0 19.3
720.3 33.7
195.3 7.9
376.4 9.6
224.3 11.2
198.9 9.3
160.3 9.7
250.6 11.2
339.7 9.8
160.2 5.1
375.4 12.1
275.9 21.3
201.5 13.1
202.3 11.9
242.9 20.0
461.1 15.2
165.1 4.7
197.8 9.2
201.5 5.2
235.5 15.6
207.7 15.2a
212.2 19.2a
312.9 23.5
125.3 3.5
203.2 8.6
Citrate pH 4.5
Acetate pH 4.5
Phosphate pH 7.5
Borate pH 10.2
Carbonate pH 10.2
NaCl 100 mM
>2
m
m
>2
m
m
206.0 13.7
212.9 23.9
351.1 23.2a
312.2 22.1a
502.4 41.32
162.4 10.3
181.7 14.0
723.3 34.2
125.60 9.3
161.4 11.2
635.9 52.2
>2
m
m
>2 mm
The table shows the size of NAs obtained using water or different buffers (10 mM) and at different pH values and ionic strength (due to buffer and sodium chloride) in the
solvent-displacement process (final concentration 2 mg/mL).
a
Brown color appeared.

S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
183
Fig. 3. Size measurement (expressed in nm) of NAs diluted in water (W), C, citrate buffer 10 mM pH 4.5; A, acetate buffer 10 mM pH 4.5; P, phosphate buffer 10 mM pH 7.5; B, borate
buffer 10 mM pH 10.2; CA, carbonate buffer 10 mM pH 10.2 after 1 (diamond), 24 (square) and 48 h (triangle) at 20 ꢀC. No correct detection was observed ** due to precipitation,
while a reduction of turbidity was represented by *.

184
S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
2.3.1. Atomic force microscopy characterization
The morphology of NAs was investigated by atomic force mi-
croscopy (AFM). A topography AFM image and the related 3D
reconstruction of compound 13a, as reference, is presented in Fig. 6.
As evident from the images, spherical soft structures with a certain
degree of aggregation appeared. It is relevant to notice that AFM
technique, even if in non-contact mode, deform the soft structure of
NAs due to the interaction between the sample and the substrate,
and the vertical force applied by the tip on the sample during the
scanning procedure. Indeed, it was observed that only using 10 mM
NaCl instead of pure water in NAs preparation, (checking the QELS
scattering intensity value of 700e800 kcps for a 0.2 mg/mL sample)
a stable signals were obtained for all the NAs.
Fig. 4. Example of HPLC analysis for compound 12a (in acetonitrile) after 1 h incu-
bation with DTT.
intracellular level) for up to 24 h. For compounds 12a, 13a, 14a
(Table 1) a progressive slight increase of size was evident while for
NA-15a the size increased dramatically and precipitation occurred.
Both DTT and GSH treatments did not significantly modify the
stability and morphology of NAs of compounds 12b, 13b, 14b, 15b.
2.4. Microtubule bundle formation (immunofluorescence)
In order to confirm the anti-microtubule effect deriving from
the use of squalene bioconjugates, by the staining of one of the
Fig. 5. Stability of disulfide linked squalene derivatives and NAs by incubation in medium plus DTT. A) size measure of the NAs obtained from compounds 12a (diamond), 13a
(square), 14a (circle), 15a (triangle). BeE) release percentage for compounds 12a, 13a, 14a, 15a respectively when maintained as native compound (diamond) in solvent þ buffer, as
NA in buffer (square), as native compound in the presence of DTT (triangle) and as NA in the presence of DTT (circle).

S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
185
Fig. 6. AFM images by non-contact mode of 13a nanoassemblies after sample deposition on mica support: topography on the top, and 3D-view.
building elements of microtubules (i.e.
investigated their structure and distribution in A549 cell line
exposed 1 h to the conjugates 12a, 13a and 15a (200 g/mL) or to
a
tubulin,
a
-TUB), we
agent did not significantly change the activity of 12b, 13b, and 14b
as it did for the corresponding NAs.
The disulfide compounds 12e15a were evaluated also against
A549 cell line. Paclitaxel derivative 12a presents the best perfor-
mance and resulted 7 times less active than paclitaxel.
m
equal concentration of parent naive drugs (1, 2 and 4). Microscopy
analyses reveal that control cells are spread with a well-organized
microtubular network, whereas treated cells are shrinking and no
obvious differences following incubation with naïve compounds
and squalene conjugates are noticeable (Fig. 7). As expected,
paclitaxel and epothilone A induce microtubule bundling; on the
other hand, podophyllotoxin completely disrupts microtubules.
Furthermore, in the presence of both naïve and squalene conjugate
compounds, there are some detaching cells, as highlighted by their
roundish morphology, due to the heavy impact of the compounds
on microtubule reorganization (Fig. 7). To verify that the observed
effects were due to intracellular action of the NA squalene conju-
gates we verified (at least for NA-12a) that NAs had retained the
ability to cross the cell membrane and probably to release the
native drug (in this case paclitaxel). Therefore, we removed all the
unassembled tubulin, leaving only the polymerized one (i.e. mi-
crotubules) and the molecules tightly bound to the microtubules.
As showed in Fig. 8, microtubule bundles (arrows) are well deco-
rated with anti-taxol antibodies in cells treated with naïve pacli-
taxel and with NA-12a as well, proving that squalene conjugate
paclitaxel enters the cells and exerts its effects through the action
on microtubule cytoskeleton.
2.5. In vitro activity
The cytotoxicity of squalene conjugates (NAs) was evaluated
using MCF7 cell line by incubation for 48 h (Table 3). In order to
check the release ability by the reducing agent the same experi-
ments were carried out after NAs treatment with DTT (10 mM).
Comparison of the doseeresponse curves obtained for each drug
and squalene conjugates NAs, revealed that the conjugation to
squalene decreased the cytotoxicity toward MCF7 cell line for each
compound (Table 3). Even if MCF7 seems very sensitive to single
drug (in nano and sub-nanomolar range), the various squalene
conjugates exhibited different IC50 values. Paclitaxel derivative 12a
appeared 100 times more toxic than the 13a, whilst the podo-
phyllotoxin is the most active free drug, indicating that there is no
direct correlation between cytotoxicity of free drug and those of
conjugates. In a scale of activity we can place 12a > 13a > 15a > 14a.
When comparing the disulfide derivatives with the alkyl chain
ones, we observed that the presence of the disulfide bond makes
the release of the toxic moiety more efficient. The alkyl chain de-
rivatives were active only at very high concentrations, ranging from
1 to 100 micromolar.
Fig. 7. Microtubule network (a-TUB staining, red) and nuclei (DAPI, blue) of A549 cells
not loaded (CONTROL) or incubated 1 h with squalene (SQUALENE) or with naïve
compounds (1, 2, and 4) or squalene bioconjugates (12a, 13a and 15a). Scale
The incubation of NAs 12a, 13a, 14a with DTT, allowed a better
release of the active molecule as detected by a tenfold increase of
activity. In particular compound 12a reached an activity similar to
that of the free paclitaxel. The treatment with the thiol reducing
bar ¼ 20
mm. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

186
S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
4. Experimental part
4.1. Chemistry. General methods
Thin layer chromatography (TLC) was performed on Merck
precoated 60F254 plates. Reactions were monitored by TLC on silica
gel, and detected by I2 and by UV light (254 nm) accordingly. Flash
chromatography was performed using silica gel (240e400 mesh,
Merck). 1H NMR and 13C NMR spectra were recorded at 400, 300
and 100, 75 MHz respectively on Bruker DRX-400 and DRX-300
spectrometers in the indicated solvents. Chemical shifts (d) for
proton and carbon resonances are quoted in parts per million
(ppm) relative to tetramethylsilane (TMS), which was used as an
internal standard. APCI mass spectra were recorded at a Thermo
Fischer instrument.
ESI mass spectra were recorded on Thermo Fischer Finnigan LCQ
Advantage Mass Spectrometer and on FT-ICR APEXII (Bruker Dal-
tonics). Quasi-elastic light scattering (QELS) and zeta potential
measurements were carried out using a 90 Plus Particle Size
Analyzer (Brookhaven Instruments, Co.) photometer. HPLC anal-
ysis: RP-18 reverse phase column (LiChrospher 100 RP 18e 5
Merck).
mm,
4.1.1. Synthesis of 2-hydroxy-3-bromosqualene (6)
To a solution of squalene (4.290 g, 10.4 mmol) in THF (30 ml)
H2O (5 ml) is added and then THF drop wise to obtain a clear so-
lution. N-bromosuccinimide (2.231 g, 12.5 mmol) is added portion
wise and the reaction mixture is stirred at room temperature for
3 h. The solvent is removed under reduced pressure, brine (100 ml)
is added and extracted with AcOEt (5 ꢂ 20 ml). The organic layers
are dried over Na2SO4 and the solvent is removed under reduced
pressure. The crude is purified by flash chromatography
(CH2Cl2:Hex 1:1) to obtain 6 as a pale-yellow oil (1.652 g, Yield:
Fig. 8. Microtubule network (a-TUB, red), microtubule-bound taxol (TAX, green) and
nuclei (DAPI, blue) of A549 cells not loaded (CONT) or incubated 1 h with naïve
paclitaxel (TAX) or NA-12a (12a). Arrows indicate microtubule bundles decorated by
anti-paclitaxel antibody. Scale bar ¼ 20
mm. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
3. Conclusions
A class of squalene-based conjugate compounds of paclitaxel,
podophyllotoxin, camptothecin and epothilone A were prepared.
The obtained compounds self-assemble and form nanoassemblies
that we characterized in this study. We demonstrated that from all
the NAs only paclitaxel derivative 12a shows moderate release
when treated with glutathione, dithiotreitol and serum. On the
contrary all the non-NA forms of compounds 12e15a present
excellent release of the corresponding drug. The in vitro activity
highlights the importance of the disulfide bond. The capacity to
permeate the cell membrane was indirectly demonstrated for
compound 12a by studying the microtubule structure and distri-
bution in A549 cell line. The results acquired from the 12e15a
derivatives can be a starting point for constructing nanoassemblies
bearing a disulfide bridge.
31%). 1H NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 5.10e5.25 (m, 5H); 4.01
(dd, J ¼ 11.3 Hz, J ¼ 1.9 Hz, 1H); 2.32e2.35 (m, 1H), 1.95e2.17 (m,
18H); 1.77e1.88 (m, 1H); 1.70 (s, 3H); 1.63 (bs, 15H); 1.37 (s, 3H);
1.36 (s, 3H). 13C NMR (CDCl3, 100 MHz):
d
(ppm) ¼ 135.1; 134.8;
132.9; 131.2; 126.0; 124.5; 124.3 (3C); 72.4; 71.0; 39.7 (3C); 38.2;
32.2; 28.3 (3C); 26.7 (2C); 25.8 (3C); 17.7; 16.0 (3C); 15.8. APCI-MS:
m/z 507.3 [M]þ.
4.1.2. Synthesis of 2,3-oxidosqualene (7)
To a solution of 6 (1.652 g, 3.2 mmol) in MeOH (60 ml) K2CO3
(0.898 g, 6.5 mmol) is added and the reaction mixture is stirred at
room temperature for 2 h, then concentrated under reduced
pressure. H2O (120 ml) is added and extracted with AcOEt
(4 ꢂ 30 ml). The organic layers are dried over Na2SO4 and the
solvent is removed under reduced pressure to obtain 7 as a yellow
oil (1.360 g, Yield: 98%) without any further purification. 1H NMR
Table 3
(CDCl3, 300 MHz):
d
(ppm) ¼ 5.06e5.17 (m, 5H); 2.69 (t, J ¼ 6.1 Hz,
Cytotoxicity of squalene conjugates and naïve compounds against MCF7 and A549.
Squalenoyl-taxol 16 has been used as reference compound. Doseeresponse curves of
12a and paclitaxel in A549 cell line.
1H); 1.95e2.19 (m, 20H); 1.68 (s, 3H); 1.60 (s, 3H); 1.58 (bs, 12H);
1.28 (s, 3H); 1.24 (s, 3H). 13C NMR (CDCl3, 100 MHz):
d
(ppm) ¼ 135.1; 134.9 (2C); 134.0; 131.2; 124.9; 124.3 (4C); 64.2;
IC50
(
m
M) MCF7
IC50
(
m
M) MCF7 (DDT)
IC50 (mM) A549
58.3; 39.7 (3C); 36.3; 28.2 (2C); 27.5; 25.7; 24.9 (2C); 18.7; 17.7; 16.0
(3C). APCI-MS: m/z 427.3 [Mþ1]þ.
12a
12b
1
0.06 0.01
1.21 0.21
0.004 0.001
9.1 0.19
0.003 0.001
1.53 0.09
0.005 0.002
8.7 0.08
0.49 0.07
e
0.082 0.0498
e
4.1.3. Synthesis of 1,10,2-tris-norsqualene aldehyde (8)
16
To a solution of H5IO6 (1.379 g, 6.0 mmol) in H2O (5 ml) a so-
lution of 7 (1.434 g, 3.4 mmol) in dioxane (12 ml) is added and the
reaction mixture is stirred at room temperature for 2 h. H2O
(150 ml) is added and extracted with AcOEt (3 ꢂ 40 ml). The organic
layers are washed with brine (100 ml) and H2O (100 ml), then dried
over Na2SO4 and concentrated under reduced pressure to obtain 8
as a pale-yellow oil (1.111 g, Yield: 86%) without any further puri-
13a
13b
2
14a
14b
3
15a
15b
4
0.4 0.11
0.06 0.005
110.8 5.6
0.0004 0.0001
0.8 0.07
20.1 0.8
0.003 0.0005
0.5 0.08
7.64 0.33
e
105.5 4.8
0.0001 0.00006
8.6 0.1
10.5 1.3
0.0005 0.0001
1.3 0.2
0.059 0.0028
>5
e
1.3 0.37
>5
e
40.2 2.8
0.0009 0.0001
45.1 1.7
0.0008 0.0002
>5
fication. 1H NMR (CDCl3, 300 MHz):
d(ppm) ¼ 9.73 (s, 1H);

S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
187
5.06e5.13 (m, 5H); 2.49 (t, J ¼ 7.1 Hz, 2H); 2.30 (t, J ¼ 7.1 Hz, 2H);
1.97e2.10 (m, 16H); 1.67 (s, 3H); 1.55 (bs, 15H). 13C NMR (CDCl3,
(d, J ¼ 7.0 Hz, 2H); 2.51e2.73 (m, 6H); 2.48 (s, 3H); 2.25 (t, J ¼ 7.2 Hz,
2H); 2.25 (s, 3H); 2.15e2.22 (m, 2H); 1.87e2.13 (m, 20H); 1.68e1.79
(m, 8H); 1.63 (bs, 18H); 1.26 (s, 3H); 1.16 (bs, 3H). 13C NMR (CDCl3,
75 MHz):
d
(ppm) ¼ 202.6; 135.1; 134.9; 134.8; 132.9; 131.2; 125.4;
124.5; 124.4; 124.2 (2C); 67.1; 42.1; 39.7 (2C); 39.5; 31.8; 28.2 (2C);
100 MHz, detected signals):
d
(ppm) ¼ 204.5; 173.6; 172.6; 171.9;
26.7; 26.6; 25.7 (2C); 17.7; 16.0 (3C). APCI-MS: m/z 385.3 [Mþ1]þ.
170.5; 168.7; 167.8; 143.5; 137.7; 135.8; 135.6; 134.4; 134.3; 133.5;
132.7; 130.9; 129.8; 129.4; 129.2; 127.8; 127.2; 125.8; 125.0; 124.9;
85.1; 81.7; 79.9; 77.1; 76.3; 75.8; 74.7; 72.8; 72.5; 65.0; 59.2; 53.4;
46.3; 43.9; 40.4; 38.4; 37.8; 36.5; 36.3; 33.3; 32.7; 29.0; 27.6; 27.5;
27.4; 26.4; 24.9; 24.6; 23.4; 22.8; 21.5; 18.3; 16.7; 16.6; 15.5. HPLC
4.1.4. Synthesis of 1,10,2-tris-norsqualene alcohol (9)
To a solution of 8 (1.111 g, 2.9 mmol) in MeOH (45 ml) NaBH4
(0.055 g, 1.4 mmol) is added portion wise and the reaction mixture
is stirred at room temperature for 1 h. HCl 1 M is added to quench
the unreacted NaBH4. The solvent is the removed under reduced
pressure, H2O (150 ml) is added and extracted with AcOEt
(3 ꢂ 40 ml). The organic layers are dried over Na2SO4 and
concentrated under reduced pressure to obtain 9 as a colorless oil
(1.016 g, Yield: 91%) without any further purification. 1H NMR
(CH3CN) Rf 22 min; ESI-MS (C82H107NO17S2): m/z 1465.8 [MþNa]þ.
22
[a
]
¼ ꢁ46 (c ¼ 0.100, CHCl3).
D
4.1.6.2. 12b. 1H NMR (CD3OD-d4, 400 MHz, detected signals):
(ppm) ¼ 8.14 (d, J ¼ 8.0 Hz, 2H); 7.83 (d, J ¼ 8.0 Hz, 2H); 7.70 (t,
d
J ¼ 7.2 Hz, 1H); 7.44e7.63 (m, 10H); 7.29 (t, J ¼ 7.2 Hz, 1H); 6.48 (s,
1H); 6.09 (t, J ¼ 6.8 Hz, 1H); 5.87 (d, J ¼ 6.4 Hz, 1H); 5.66 (d,
J ¼ 7.2 Hz, 1H); 5.11e5.16 (m, 5H); 5.02 (d, J ¼ 8.0 Hz, 1H); 4.37 (dd,
J ¼ 11.2 Hz, J ¼ 6.8 Hz, 1H); 4.21 (s, 2H); 4.05 (t, J ¼ 6.8 Hz, 2H); 3.84
(d, J ¼ 7.2 Hz,1H); 2.43e2.50 (m, 5H); 2.30 (t, J ¼ 8.0 Hz, 2H); 2.19 (s,
3H); 1.99e2.15 (m, 16H); 1.95 (s, 2H); 1.72e1.88 (m, 6H); 1.68 (bs,
6H); 1.63 (bs, 15H); 1.28 (bs, 8H); 1.56 (bs, 6H). 13C NMR (CD3OD-d4,
(CDCl3, 300 MHz):
2H); 1.99e2.08 (m, 18H); 1.78e1.85 (m, 2H); 1.67 (s, 3H); 1.59 (s,
15H). 13C NMR (CDCl3, 75 MHz):
d(ppm) ¼ 5.08e5.13 (m, 5H); 3.61 (t, J ¼ 6.3 Hz,
d
(ppm) ¼ 135.1; 134.9 (2C); 134.5;
131.2; 125.8; 124.4 (2C); 124.3 (2C); 62.8; 39.7 (3C); 36.0; 30.7; 28.2
(2C); 26.6 (3C); 25.7 (2C); 17.7; 16.0 (3C). APCI-MS: m/z 387.3
[Mþ1]þ.
100 MHz, detected signals):
d
(ppm) ¼ 203.8; 174.2; 174.0; 170.2;
4.1.5. General procedure for compounds 11
169.9; 169.1; 166.3; 141.0; 137.0; 134.6; 134.4; 134.2; 133.5; 133.2;
131.5; 130.6; 130.0; 129.8; 128.7; 128.5; 128.3; 128.2; 127.2; 124.9;
124.3; 124.2; 124.1; 84.5; 80.9; 77.7; 76.1; 75.4; 74.9; 74.3; 71.5;
70.9; 63.6; 57.9; 53.9; 46.5; 43.2; 39.4; 39.3; 36.1; 35.5; 35.1; 33.8;
33.2; 28.7; 28.5; 27.8; 26.6; 26.5; 26.2; 26.1; 25.6; 24.7; 24.5; 21.9;
To a solution of 9 (0.26 mmol) in dry CH2Cl2 (2.5 ml) the
bicarboxylic acid 10 (0.52 mmol), EDC$HCl (0.31 mmol) and DMAP
(0.18 mmol) are added and the reaction mixture is stirred at room
temperature for 24 h. HCl 1 M (15 ml) is added and extracted with
CH2Cl2 (5 ꢂ 5 ml). The organic layers are dried over Na2SO4 and the
solvent is removed under reduced pressure. The crude is purified by
flash chromatography (AcOEt/Hex) to obtain 11 as a colorless oil
(Yield: 35e60%).
21.0; 19.4; 14.8; 14.6; 13.5; 9.0. HPLC (CH3CN) Rf 27 min; ESI-MS
22
(C84H111NO17): m/z 1429.9 [MþNa]þ. [
a]
¼ ꢁ24 (c ¼ 0.100, CHCl3).
D
4.1.6.3. 13a. 1H NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 6.79 (s, 1H); 6.57
(s, 1H); 6.42 (bs, 2H); 6.02 (d, J ¼ 1.0 Hz, 1H); 6.00 (d, J ¼ 1.0 Hz, 1H);
5.92 (dd, J ¼ 9.0 Hz, J ¼ 4.0 Hz, 1H); 5.10e5.17 (m, 5H); 4.63 (d,
J ¼ 4.2 Hz, 1H); 4.37e4.42 (m, 1H); 4.19e4.25 (m, 1H); 4.07 (t,
J ¼ 6.7 Hz, 2H); 3.83 (s, 3H); 3.79 (s, 6); 2.83e2.98 (m, 2H);
2.73e2.80 (m, 4H); 2.55e2.64 (m, 2H); 2.46 (t, J ¼ 6.8 Hz, 2H);
1.95e2.28 (m, 20H); 1.74e1.80 (m, 4H); 1.70 (s, 3H); 1.65 (s, 15H).
4.1.5.1. 11a. 1H NMR (CDCl3, 300 MHz):
d
(ppm) ¼ 5.06e5.12 (m,
5H); 4.03 (t, J ¼ 6.0 Hz, 2H); 2.68e2.74 (m, 4H); 2.40e2.51 (m, 4H);
1.99e2.07 (m, 22H); 1.71e1.79 (m, 2H); 1.68 (s, 3H); 1.59 (s, 15H).
13C NMR (CDCl3, 75 MHz):
d
(ppm) ¼ 178.0; 173.1; 135.1; 134.9;
133.6; 125.1; 124.3 (4C); 64.3; 39.7 (3C); 37.8; 37.6; 35.8; 32.6; 32.1;
28.2 (3C); 26.8; 26.7 (3C); 25.7; 24.3 (2C); 23.9; 17.7; 16.0 (3C); 15.9.
ESI-MS: m/z 606.2 [Mꢁ1]þ.
13C NMR (CDCl3, 100 MHz):
108.3 (2C); 107.00; 101.6; 73.8; 71.3; 64.3; 60.7; 56.2; 45.6; 43.8;
39.7 (2C); 38.7; 37.8; 37.7; 35.8; 33.1; 32.6 (2C); 28.3; 26.7; 26.0;
d
(ppm) ¼ 125.2; 124.3 (2C); 109.8;
4.1.5.2. 11b. 1H NMR (CDCl3, 300 MHz):
d
(ppm) ¼ 5.06e5.13 (m,
25.2; 24.5 (2C); 24.3; 24.1 (2C); 16.0 (5C). HPLC (CH3CN) Rf 21 min;
22
5H); 4.02 (t, J ¼ 9.0 Hz, 2H); 2.25e2.36 (m, 4H); 1.97e2.08 (m, 20H);
ESI-MS (C57H78O11S2): m/z 1025.6 [MþNa]þ. [
a
]
¼ ꢁ51 (c ¼ 0.900,
D
1.59e1.84 (m, 22H); 1.30 (bs, 8H). 13C NMR (CDCl3, 75 MHz):
CHCl3).
d
(ppm) ¼ 178.9; 173.9; 135.1; 134.9; 1337.7; 125.1; 124.3 (4C); 64.0;
39.7 (3C); 35.8; 34.3; 33.8; 29.7; 29.0 (4C); 28.2 (3C); 26.9; 26.7
(2C); 25.6; 24.9 (2C); 24.6; 17.7; 16.0 (4C). ESI-MS: m/z 507.3
[Mꢁ1]þ.
4.1.6.4. 13b. 1H NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 6.77 (s, 1H); 6.57
(s, 1H); 6.42 (s, 2H); 6.01 (d, J ¼ 6.5 Hz, 2H); 5.91 (d, J ¼ 9.0 Hz, 1H);
5.11e5.21 (m, 5H); 4.63 (d, J ¼ 4.1 Hz, 1H); 4.39 (t, J ¼ 7.8 Hz, 1H);
4.23 (t, J ¼ 7.8 Hz, 1H); 4.06 (t, J ¼ 6.7 Hz, 2H); 3.84 (s, 3H); 3.77 (s,
6H); 2.96 (dd, J ¼ 9.0 Hz, J ¼ 4.5 Hz, 1H); 2.79e2.90 (m, 1H);
2.30e2.47 (m, 16H); 1.52e1.78 (m, 24H); 1.35 (bs, 8H). 13C NMR
4.1.6. General procedure for compounds 12e15
To a solution of 11 (0.04 mmol), in dry CH2Cl2 (1 ml) EDC$HCl
(0.06 mmol) and DMAP (0.03 mmol) are added. The drug
(0.04 mmol) is added and the reaction mixture is stirred at room
temperature for 3e96 h. HCl 1 M (15 ml) is added and extracted
with CH2Cl2 (5 ꢂ 5 ml). The organic layers are dried over Na2SO4
and the solvent is removed under reduced pressure. The crude is
purified by flash chromatography (AcOEt/Hex) to obtain 12e15 as a
solid (Yield: 15e70%).
(CDCl3, 100 MHz, detected signals):
d
(ppm) ¼ 152.7; 135.0; 134.9;
132.4; 131.8; 128.5; 125.1; 124.4 (2C); 124.3 (2C); 109.8; 108.2 (2C);
107.0; 101.6 (2C); 73.4 (2C); 71.4; 64.0; 60.8; 56.2 (2C); 45.6; 43.8;
39.8 (3C); 38.8; 35.8; 34.4 (2C); 29.7; 29.1 (4C); 28.3 (3C); 27.0; 26.7
(2C); 25.0 (3C). HPLC (CH3CN) Rf 23 min. ESI-MS (C59H82O11): m/z
990.6 [MþNa]þ. [
a
]
¼ ꢁ53 (c ¼ 0.100, CHCl3).
22
D
4.1.6.5. 14a. 1H NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 8.42 (s, 1H); 8.25
4.1.6.1. 12a. 1H NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 8.17 (d, J ¼ 7.1 Hz,
(d, J ¼ 8.0 Hz, 1H); 7.96 (d, J ¼ 8.0 Hz, 1H); 7.86 (t, J ¼ 8.0 Hz, 1H);
7.70 (t, J ¼ 8.0 Hz, 1H); 7.24 (s, 1H); 5.70 (d, J ¼ 17.2 Hz, 1H); 5.42 (d,
J ¼ 17.2 Hz, 1H); 5.31 (bs, 2H); 5.10e5.16 (m, 5H); 4.04 (t, J ¼ 6.8 Hz,
2H); 2.61e2.76 (m, 6H); 2.40 (t, J ¼ 7.3 Hz, 2H); 2.28e2.33 (m, 1H);
2.15e2.20 (m, 1H); 1.98e2.09 (m, 22H); 1.62e1.76 (m, 20H); 1.00 (t,
2H); 7.77 (d, J ¼ 7.1 Hz, 2H); 7.63 (t, J ¼ 7.1 Hz, 1H); 7.51e7.56 (m,
3H); 7.35e7.47 (m,10H); 6.93 (d, J ¼ 9.3 Hz,1H); 6.32 (s,1H); 6.28 (t,
J ¼ 9.0 Hz,1H); 6.00 (dd, J ¼ 9.2 Hz, J ¼ 3.1 Hz,1H); 5.71 (d, J ¼ 7.1 Hz,
1H); 5.53 (d, J ¼ 3.1 Hz, 1H); 5.32 (s, 2H); 5.10e5.16 (m, 5H); 5.00 (d,
J ¼ 7.6 Hz, 1H); 4.50 (dd, J ¼ 10.9 Hz, J ¼ 6.6 Hz, 1H); 4.34 (d,
J ¼ 8.4 Hz, 1H); 4.23 (d, J ¼ 8.4 Hz, 1H); 4.05 (t, J ¼ 6.7 Hz, 2H); 3.84
J ¼ 7.5 Hz, 3H). 13C NMR (CDCl3, 100 MHz):
(ppm) ¼ 173.6; 172.6;
d
168.2; 158.0; 153.0; 149.6; 147.0; 146.6; 135.8; 135.7; 134.3; 131.9;

188
S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
131.4; 130.3; 129.2; 128.9; 128.7; 125.8; 125.0 (4C); 120.9; 96.6;
67.8; 64.9; 50.6; 40.4 (2C); 38.4; 37.9; 36.5; 33.3; 32.8; 32.5; 28.9
(2C); 27.6; 27.5 (2C); 26.4; 24.9; 24.6; 18.37; 16.7 (3C); 16.6; 8.3.
carbonate) with a 500 mL of solvent, aqueous solution 1 ml at 2 mg/
mL. The stability of NAs was evaluated by analyzing the mean
particle size using a nanosizer, after incubation at 20 ꢀC for different
times (zero, 24 h, 48 h) at a concentration of 0.2 mg/mL in water
and 10 mM buffers: acetate pH 4.5, citrate pH 4.5, phosphate pH 7.2,
borate pH 10.2, carbonate pH 10.2 .
HPLC (CH3CN) Rf 27 min. ESI-MS (C55H72N2O7S2): m/z 960.3
20
[MþNa]þ. [
a
]
¼ ꢁ36 (c ¼ 0.145, CHCl3).
D
4.1.6.6. 14b. 1H NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 8.42 (s, 1H); 8.24
(d, J ¼ 8.0 Hz, 1H); 7.97 (d, J ¼ 8.0 Hz, 1H); 7.86 (t, J ¼ 8.0 Hz, 1H);
7.69 (t, J ¼ 8.0 Hz, 1H); 7.24 (s, 1H); 5.70 (d, 16.0 Hz, 1H); 5.42 (d,
J ¼ 16.0 Hz, 1H); 5.31 (bs, 2H); 5.12e5.18 (m, 5H); 4.04(t, J ¼ 7.5 Hz,
2H); 2.48e2.59 (m, 2H); 2.27e2.36 (m, 1H); 2.21e2.25 (m, 2H);
2.14e2.20 (m, 1H); 1.95e2.14 (m, 20H); 1.59e1.77 (m, 22H); 1.28
(bs, 8H); 1.00 (t, J ¼ 8.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz, detected
4.1.8. Size and zeta potential measurements
The average diameter and polydispersity index of the nano-
assemblies was determined using quasi-elastic light scattering
(QELS) at a fixed angle of 90 ꢀC and at a temperature of 25 ꢀC. The
NAs dispersions were diluted 1:20 with MilliQ water before anal-
ysis. Each value reported is the average of five measurements. To
determine the electrophoretic mobility, the NAs samples were
diluted with 0.1 mM KCl and placed in the electrophoretic cell,
where an electric field of 15.24 V/cm was established. Each sample
was analyzed in triplicate. The zeta potential values were calculated
using the Smolochowski equation.
signals):
d
(ppm) ¼ 167.5; 146.0; 131.2; 130.6; 129.6; 128.5; 128.2;
128.0; 125.0; 124.4; 124.3; 120.4; 96.0; 67.1; 64.0; 49.9; 39.7; 35.8;
34.3; 33.8; 31.9; 29.7; 29.1; 29.0; 28.3; 26.9; 26.7; 25.7; 24.9; 24.6;
16.0; 7.6. HPLC (CH3CN) Rf 28 min. ESI-MS (C57H76N2O7): m/z 924.2
20
[MþNa]þ. [
a
]
¼ ꢁ21 (c ¼ 0.175, CHCl3).
D
4.1.6.7. 15a. 1H NMR (CDCl3, 400 MHz):
d(ppm) ¼ 7.05 (s, 1H); 6.73
4.1.9. Atomic force microscopy characterization
(s, 1H); 5.71 (dd, J ¼ 11.4 Hz, J ¼ 2.4 Hz, 1H); 5.55 (d, J ¼ 7.9 Hz, 1H);
5.46 (d, J ¼ 6.2 Hz, 1H); 5.37 (d, J ¼ 7.1 Hz, 1H); 5.10e5.17 (m, 5H);
4.15e4.21 (m, 1H); 4.07 (t, J ¼ 6.7 Hz, 2H); 3.28e3.44 (m, 1H);
3.00e3.11 (m, 1H); 2.89e2.97 (m, 1H); 2.65e2.82 (m, 8H);
2.38e2.64 (m, 6H); 1.98e2.18 (m, 20H); 1.80e1.95 (m, 2H);
1.71e1.78 (m, 4H); 1.70 (s, 3H); 1.49e1.68 (m, 21H); 1.41 (s, 3H);
0.90e1.33 (m, 9H). 13C NMR (CDCl3, 100 MHz, detected signals):
AFM observation was done using a Park XE 100 atomic force
microscope (Park Instruments, Sunnyvale, CA, USA). AFM images
were obtained by measurement of the interaction forces between
the tip (Mikromash NSC15) and the sample surface. The experi-
ments were conducted at room temperature (20 ꢀC) and at atmo-
spheric pressure (760 mmHg) operating in non-contact mode, in
which the space between the tip and the sample is between 10 and
100 Å and the total force is very low. The resonant frequencies of
this cantilever were found to be about 250 kHz. Immediately before
the analysis, the samples were diluted in water (1:10 v/v) to obtain
d(ppm) ¼ 216.4; 172.9; 172.5; 170.6; 135.1; 135.0; 133.6; 125.2;
124.4; 75.8; 71.2; 64.3; 57.6; 57.3; 54.9; 54.4; 43.3; 42.5; 39.7; 38.5;
37.9; 37.6; 36.1; 35.8; 34.6; 32.7; 30.9; 29.7; 28.3; 26.9; 26.7; 25.7;
24.3; 23.9; 16.9; 16.0; 15.5. HPLC (CH3CN) Rf 16 min. APCI-MS
a less sticky fluid to analyze. Droplets of constant volume (20 ml) of
22
(C61H95NO9S3): m/z 1082.6 [M]þ. [
a
]
¼ ꢁ7 (c ¼ 0.100, CHCl3).
NAs were deposited onto a small mica disk with a diameter of 1 cm.
After 2 min, excess water was removed using a paper filter.
In order to obtain stable and reproducible signals the NAs were
prepared using 10 mM solution of NaCl, checking the QELS scat-
tering intensity values to 700e900 kcps for 0.2 mg/mL NAs
preparation.
D
4.1.6.8. 15b. 1H NMR (CDCl3, 400 MHz, detected signals):
d
(ppm) ¼ 7.05 (s, 1H); 6.75 (s, 1H); 5.72 (dd, J ¼ 11.4 Hz, J ¼ 2.5 Hz,
1H); 5.56 (dd, J ¼ 9.9 Hz, J ¼ 2.2 Hz, 1H); 5.44 (m, 1H); 5.36 (d,
J ¼ 7.4 Hz, 1H); 5.10e5.14 (m, 5H); 4.20 (m, 1H); 4.05 (t, J ¼ 6.7 Hz,
2H); 3.34e3.40 (m,1H); 3.05e3.09 (m,1H); 2.90e2.94 (m,1H); 2.82
(m, 2H); 2.50e2.72 (m, 5H); 2.28e2.38 (m, 6H); 2.97e3.18 (m,
20H); 1.55e1.80 (m, 20H); 0.98e1.47 (m, 26H). 13C NMR (CDCl3,
4.1.10. Stability assessment of drug-squalene conjugates (Cs) and
nanoassemblies (NAs) in the presence of dithiothreitol (DTT),
glutathione (GSH) and serum
The stability of NAs was evaluated by analyzing the mean par-
ticle size, after incubation at 20 ꢀC for different times (1 h, 24 h,
48 h) at a concentration of 0.2 mg/mL in water and in 10 mM
buffers: acetate pH 4.5, citrate pH 4.5, phosphate pH 7.2, borate pH
10.2, carbonate pH 10.2.
100 MHz, detected signals):
135.0; 133.7; 125.1; 124.4; 75.8; 70.8; 64.0; 57.7; 54.9; 43.6; 39.8;
38.8; 37.5; 36.1; 35.8; 34.4; 31.6; 29.7; 29.1; 28.3; 26.9; 26.7; 25.7;
d
(ppm) ¼ 173.9; 173.4; 170.6; 135.1;
25.1; 25.0; 24.0; 22.7; 17.5; 16.1; 15.9; 15.6. HPLC (CH3CN) Rf 17 min.
20
ESI-MS (C63H99NO9S): m/z 1069.4 [MþNa]þ. [
a]
¼ ꢁ20 (c ¼ 0.080,
D
CHCl3).
Dithiothreitol (DTT): To define the stability of disulphide bonds
inside compounds and NAs the method described by Satyam was
essentially used, with slight modifications [20]. Each drug conju-
gates was dissolved in acetonitrile or ethanol/acetonitrile mixture
4.1.7. Preparation of nanoassemblies (NAs)
In a standard procedure the nanoassemblies (NAs) were pre-
pared by first dissolving the drug-squalene conjugates (Cs) in a
volume of appropriate solvent (ethanol, acetone or THF). The so-
lution was drop wise added under stirring (500 rpm) into a volume
of MilliQ grade water. Finally, the organic solvent was completely
evaporated using a Rotavapor® at 30 ꢀC under vacuum. The aqueous
suspension of squalenoyl-drug nanoassemblies were stored at 5 ꢀC
in the dark. To evaluate the optimal conditions to prepare stable
nanoassemblies the influence of different variables to the process
to 1 mg/mL solution (0.5 ml) and were added 10
ml of borate buffer
0.2 M pH 9, 50 l water and 20 l of DTT (dissolved in acetonitrile)
m
m
solution 16 mM. The vials were capped and kept at RT under ni-
trogen with occasional shaking by hand. For comparison the same
solutions without DTT were prepared. Samples (20 ml) were spiked
and analyzed by HPLC, as described above, at various time points.
For evaluation of disulfide release data was considered the disap-
pearance of the major peak related to the pure compound. The
same proportions of buffer and DTT (dissolved in water) were used
to analyze the stability of NAs. Samples kept at different times were
were considered: A) volume of solvent (500, 750
organic solvent) and a water volume of 1 ml and final concentration
of 2 mg/mL; B) volume of water (500, 750 L, 1, 1.5 and 2 ml) and a
solvent volume of 500 L, final concentration of 2 mg/mL; C) final
mL, 1 and 1.5 ml
m
m
diluted with acetonitrile (50 ml) just before HPLC injection. Serum:
drug-squalene conjugate concentration (0.5, 2, 4 mg/mL) in a final
volume of 1 ml; D) the ionic strength of water solution (10, 100 mM
NaCl) in a volume of 1 ml and at 2 mg/mL; E) the role of pH values
(4.5, 7.2, 10.2) and buffer salts (acetate, citrate, phosphate, borate,
The stability of the conjugates was also analyzed in the presence of
serum. Incubation in human serum was performed at 37 ꢀC with a
final concentration of NAs of 0.33 mg/mL. Aliquots (60e80
ml) were
removed after 48 h of incubation. After addition of 0.8 ml of

S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
189
acetonitrile, the aliquots were centrifuged and the solution was
analyzed. All the tests were repeated thrice. The chemical stability
of the squalenoyl conjugates was determined by HPLC analysis.
DABCO (SigmaeAldrich) and examined with a Zeiss Axiovert 200
microscope equipped with a 63ꢂ Neofluor lens (Zeiss, Oberkochen,
Germany).
Samples in a range of 5e15
a LiChrospher 100 RP 18e (4.6 ꢂ 250 mm, 5
m
g of the compounds were injected into
m) equipped with a
Furthermore, to ascertain that NA-12a is able to permeate cell
membrane and bind microtubules, we removed unassembled
tubulin, using previously described procedures [21] with minor
modifications. With more detail, to avoid the administration of
further paclitaxel, we used a microtubule-optimized PEM buffer
(85 mM PIPES, pH 6.94, 10 mM EGTA, 1 mM MgCl2, 2 M glycerol,
m
C18 guard column. The column was eluted with acetonitrile/water
(40:60 and, after 5 min, linear gradient to 100% acetonitrile, 35 min)
at flow rate 1 ml/min, and the column effluent was detected at
227 nm for paclitaxel and camptothecin derivatives and 217 nm for
podophyllotoxin and epothilone A derivatives. The assay was linear
1 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 1
mM
over the tested concentration range (1e50
mg as paclitaxel, podo-
pepstatin, 2 g/mL aprotinin). After fixation and permeabilization,
m
phyllotoxin, camptothecin and epothilone A). Eluted peaks were
also characterized using a Waters HPLC-MS Micromass ZQ with ESI
detector, eluting the column with methanol and 0.05% trifluoro-
acetic acid in water, with the same gradient as described above.
Glutathione (GSH): (stability of compounds) a solution of GSH
(0.0058 mmol) in H2O (0.300 ml) was added to a solution of the
squalene conjugate (0.0058 mmol) in MeOH (3 ml) and the reaction
mixture was stirred at room temperature for 24 h. A sample of the
reaction mixture was submitted to an ESI-MS spectrometry analysis
and in each case the peaks due to the free drugs were
detected (stability of compounds as NAs). Incubation at 1 mg/mL
with reduced glutathione (10 mM final concentration) at 37 ꢀC
sampling the size of NAs after different times (0, 1, 6, 24, 48, 78 h).
All the tests were repeated thrice.
cells were double immunostained using a polyclonal anti-taxol
antibody (abcam, Cambridge, UK) 1:1000 in PBS for 1 h at 37 ꢀC
and then a monoclonal anti-a-tubulin antibody. Afterward we used
a mix of secondary antibodies, goat anti-mouse Alexa Fluor568 and
donkey anti-rabbit Alexa Fluor488 (Life Technologies Italia, Monza,
Italy). All other procedures were made as described above.
4.2.2. Cytotoxicity assay
The cytotoxicity assay of drug-squalene conjugates was carried
out on MCF7 human breast cancer cell line and A549 human lung
cancer. MCF7: Cell culture medium was used as dilution medium to
make relevant dilutions of the nanoassemblies for this assay.
Cytotoxicity was evaluated by incubating the cells with different
concentrations of nanoassemblies for 48 h at 37 ꢀC/5% CO2. To
evaluate the activity of NA after reduction of disulfides bridges,
dithiotrithreitol was added to reach a 10 mM concentration and
sample (at a 1 mM concentration) was incubated for 1 h at 37 ꢀC.
For comparison purposes, paclitaxel, podophyllotoxin, camptothe-
cin and epothilone A were used as a control. In brief, MCF7 cells
(25,000/well) were cultured in RPMI 1640 supplemented with 10%
4.1.11. Self-aggregation behavior of nanoassemblies
The critical aggregation concentration test (CAC) was performed
by steady state fluorescence spectra as previously described [6].
Briefly: samples of the NAs in water, with concentrations ranging
from 0.1 mg/L to 0.5 g/L, were incubated with a constant pyrene
concentration of 6 ꢂ 10e7ꢁM for 12 h at room temperature under
stirring. Fluorescence emission spectra were recorded from 360 to
550 nm, using 343 nm excitation wavelength. In the emission
spectra of unstacked pyrene, the intensity ratio of the first band
(I373) to the third band (I384) was analyzed as a function of NAs
concentration. A CAC value was determined from the intersection
of the tangent to the curve at the inflection with the horizontal
tangent through the points at low concentration.
fetal calf serum, 50 U/mL penicillin, 50
mg/mL streptomycin and
2 mM -glutamine. The cytotoxicity of drugs and drug-squalene
L
NAs was determined using sulforhodamine B (SRB) assay essen-
tially as described by Voigt [22]. MCF-7 cells were seeded at a
density of 25 ꢂ 103 cells/well in a 96 well plate and incubated for
24 h. Then various concentrations of drug-squalene NAs were
added to the medium. After 48 h, the cell viability was determined
by the SRB assay. Briefly, the medium was removed, and then cells
were fixed with trichloroacetic acid, washed and stained with SRB
4.2. Biological assays
(100
200
m
m
l/well SRB 0.057% w/v in acetic acid 1% v/v). After washings
l/well di Tris base 10 mM pH 10.5 were added and, after 300
4.2.1. Cell cultures and fluorescence microscopy
the absorbance was measured at 492 nm using a 96 well plate
reader (Titertek Multiskan PLUS MKII). The survival percentages
were calculated using the following formula: Survival
% ¼ (A492 nm for the treated cells/A492 nm for the control
cells) ꢂ 100%. The experiment was repeated thrice in triplicate for
each concentration of the tested compounds. Finally, doseeeffect
curves were made and IC 50 values were calculated. A549: The
cytotoxicity of 12e15a and 1e4 was evaluated also on A549 cells.
Briefly, 25 ꢂ 102 cells/well in a 96 well plate and incubated for 24 h.
Cells were then treated for 96 h with different concentrations of
nanoassemblies. Cytotoxic activity was evaluated by MTS and the
IC50 calculated by interpolating the doseeresponse curve.
Human lung carcinoma cell line A549 (CCL-185; American Type
Culture Collection, Rockville, MD, USA) was grown in minimal
essential medium with Earle's (E-MEM), supplemented with 10%
fetal bovine serum (Hyclone Europe, Oud-Beijerland, Holland),
2 mM L-glutamine, 100 U/mL penicillin, and non-essential amino
acids. Cells were maintained at 37 ꢀC in a humidified atmosphere at
5% CO2. To test the ability of squalene bioconjugated to affect
microtubule cytoskeleton, A549 cells (50,000 cell/ml) were seeded
on glass coverslips and grown in control medium. After 24 h, cells
were incubated for 1 h with 200
mg/mL of the squalene bio-
conjugated, or with equal concentration of the parent compounds
(paclitaxel, epothilone A, podophyllotoxin). Cells were fixed and
permeabilized for 6 min with methanol at ꢁ20 ꢀC, washed with
PBS, and blocked in PBS þ 5% bovine serum albumin (BSA) for
15 min at room temperature. To localize tubulin, the cells were
Acknowledgments
This research has been developed under the umbrella of CM1106
COST Action “Chemical Approaches for Targeting Drug Resistance in
Cancer Stem Cells”. The authors express their gratitude to Ettore
Bernardi (Dep. Physic, Univ. degli Studi di Torino) for AFM experi-
ments. A. Silvani thanks PRIN 2009 “Natural products and bio-
inspired molecules interfering with biological targets involved in
control of tumor growth”. The authors express their gratitude to Dr.
incubated with monoclonal anti-a-tubulin antibody (clone B-
5e1e2, SigmaeAldrich, St. Louis, MO) 1:500 in PBS for 1 h at 37 ꢀC.
We used goat anti-mouse Alexa Fluor568 (Molecular Probes,
Eugene, OR) 1:1000 in PBS þ 1% BSA for 45 min at 37 ꢀC as sec-
ondary antibodies. Nuclei staining was performed by incubation
with DAPI (0.25
mg/mL in PBS) for 15 min at room temperature. The
ꢀ
coverslips were mounted in Mowiol (Calbiochem, San Diego, CA)-
Anna Daghetti (Dipartimento di Chimica, Universita degli Studi di

190
S. Borrelli et al. / European Journal of Medicinal Chemistry 85 (2014) 179e190
(c) D. Passarella, D. Comi, G. Cappelletti, D. Cartelli, J. Gertsch, A.R. Quesada,
J. Borlak, K.-H. Altmann, Synthesis and biological evaluation of epothilone A
dimeric compounds, Bioorg. Med. Chem. 17 (2009) 7435e7440;
(d) D. Passarella, A. Giardini, B. Peretto, G. Fontana, A. Sacchetti, A. Silvani,
C. Ronchi, G. Cappelletti, D. Cartelli, J. Borlak, B. Danieli, Inhibitors of tubulin
polymerization: synthesis and biological evaluation of hybrids of vindoline,
anhydrovinblastine and vinorelbine with thiocolchicine, podophyllotoxin and
baccatin III, Bioorg. Med. Chem. 16 (2008) 6269e6285.
Milano) for her meticulous efforts in developing useful analytical
methods for MS analysis and Ms. Ioana Stupariu for the revision of
the manuscript.
References
[1] A.H. Faraji, P. Wipf, Nanoparticles in cellular drug delivery, Bioorg. Med. Chem.
17 (2009) 2950e2962.
[2] C. Liang, Y. Yang, Y. Ling, Y. Huang, T. Li, X. Li, Improved therapeutic effect of
folate-decorated PLGA-PEG nanoparticles for endometrial carcinoma, Bioorg.
Med. Chem. 19 (2011) 4057e4066.
[3] M. Talekar, J. Kendall, W. Denny, S. Garg, Targeting of nanoparticles in cancer:
drug delivery and diagnostic, Anti-Cancer Drugs 22 (2011) 949e962.
[4] H. Hillaireau, P. Couvreur, Nanocarriers' entry into the cell: relevance to drug
delivery, Cell. Mol. Life Sci. 66 (2009) 2873e2896.
[5] U. Schroeder, P. Sommerfeld, S. Ulrich, B.A. Sabel, Nanoparticle technology for
delivery of drugs across the bloodebrain barrier, J. Pharm. Sci. 87 (1998)
1305e1307.
[16] Synthesis of topoisomerase inhibitors:
(a) M.S. Christodoulou, F. Zunino, V. Zuco, S. Borrelli, D. Comi, G. Fontana,
M. Martinelli, J.B. Lorens, L. Evensen, M. Sironi, S. Pieraccini, L. Dalla Via,
O.M. Gia, D. Passarella, Camptothecin-7-yl-methanthiole: semisynthesis and
biological evaluation, ChemMedChem 7 (2012) 2134e2143.
[17] Synthesis of tyrosine kinases inhibitors:
(a) C. Peruzzotti, S. Borrelli, M. Ventura, R. Pantano, G. Fumagalli,
M.S. Christodoulou, D. Monticelli, M. Luzzani, A.L. Fallacara, C. Tintori, M. Botta,
D. Passarella, Probing the binding site of Abl tyrosine kinase using in situ click
chemistry, ACS Med. Chem. Lett. 4 (2013) 274;
(b) F. Colombo, C. Tintori, A. Furlan, S. Borrelli, M.S. Christodoulou, R. Dono,
F. Maina, M. Botta, M. Amat, J. Bosch, D. Passarella, ‘Click’ synthesis of a triazole-
based inhibitor of Met functions in cancer cells, Bioorg. Med. Chem. Lett. 22
(2012) 4693;
(c) A. Furlan, F. Colombo, A. Kover, N. Issaly, C. Tintori, L. Angeli, V. Leroux,
S. Letard, M. Amat, Y. Asses, B. Maigret, P. Dubreuil, M. Botta, R. Dono, J. Bosch,
O. Piccolo, D. Passarella, F. Maina, Identification of new aminoacid amides
containing the imidazo[2,1-b]benzothiazol-2-ylphenyl moiety as inhibitors of
tumorigenesis by oncogenic Met signalling, Eur. J. Med. Chem. 47 (2012) 239;
(d) F. Arioli, S. Borrelli, F. Colombo, F. Falchi, I. Filippi, E. Crespan, A. Naldini,
G. Scalia, A. Silvani, G. Maga, F. Carraro, M. Botta, D. Passarella, N-[2-methyl-5-
(triazol-1-yl)phenyl]pyrimidin-2-amine as a Scaffold for the synthesis of in-
hibitors of Bcr-Abl, ChemMedChem 6 (2011) 2009.
[6] a) F. Dosio, L.H. Reddy, A. Ferrero, B. Stella, L. Cattel, P. Couvreur, Novel
nanoassemblies composed of squalenoyl-paclitaxel derivatives: synthesis,
characterization and biological evaluation, Bioconjug. Chem. 21 (2010)
1349e1361;
b) J. Caron, A. Maksimenko, S. Wack, E. Lepeltier, C. Bourgaux, E. Morvan,
€
K. Leblanc, P. Couvreur, D. Desmaele, Improving the antitumor activity of
squalenoyl-paclitaxel conjugate nanoassemblies by manipulating the linker
between paclitaxel and squalene, Adv. Healthc. Mater. 2 (2013) 172e185.
€
[7] D. Desmaele, R. Gref, P. Couvreur, Squalenoylation: a generic platform for
nanoparticular drug delivery, J. Control. Release 161 (2012) 609e618.
[8] a) A. Maksimenko, J. Mougin, S. Mura, E. Sliwinski, E. Lepeltier, C. Bourgaux,
S. Lepetre, F. Zouhiri, D. Desmaele, P. Couvreur, Polyisoprenoyl gemcitabine
conjugates self assemble as nanoparticles, useful for cancer therapy, Cancer
Lett. 334 (2013) 346e353;
[18] Synthesis of miscellaneous inhibitors:
(a) M.S. Christodoulou, N. Fokialakis, D. Passarella, A.N. García-Argaez, O.M. Gia,
ꢁ
I. Pongratz, L. Lisa Dalla Via, S.A. Haroutounian, Synthesis and biological eval-
uation of novel tamoxifen analogues, Bioorg. Med. Chem. 21 (2013)
4120e4131;
(b) M.S. Christodoulou, F. Colombo, D. Passarella, G. Ieronimo, V. Zuco, M. De
Cesare, F. Zunino, Synthesis and biological evaluation of imidazolo[2,1-b]
benzothiazole derivatives, as potential p53 inhibitors, Bioorg. Med. Chem. 19
(2011) 1649.
€
b) P. Couvreur, B. Stella, L.H. Reddy, H. Hillaireau, C. Dubernet, D. Desmaele,
^
S. Lepetre-Mouelhi, F. Rocco, N. Dereuddre-Bosquet, P. Clayette, V. Rosilio,
V. Marsaud, J.M. Renoir, L. Cattel, Squalenoyl nanomedicines as potential
therapeutics, Nano Lett. 6 (2006) 2544e2548;
c) L.H. Reddy, H. Khoury, A. Paci, A. Deroussent, H. Ferreira, C. Dubernet,
ꢀ
^
€
X. Decleves, M. Besnard, H. Chacun, S. Lepetre-Mouelhi, D. Desmaele,
B. Rousseau, C. Laugier, J.C. Cintrat, G. Vassal, P. Couvreur, Squalenoylation
favorably modified the in vivo pharmakokinetics and biodistribution of
gemcitabine in mice, Drug Metab. Dispos. 36 (2008) 1570e1577.
[19] Synthesis of disulfide containing compounds:
(a) G. Cappelletti, D. Cartelli, B. Peretto, M. Ventura, M. Riccioli, F. Colombo,
J.S. Snaith, S. Borrelli, D. Passarella, Tubulin-guided dynamic combinatorial li-
brary of thiocolchicine-podophyllotoxin conjugates, Tetrahedron 67 (2011)
7354e7357;
ꢁ
[9] N. Semiramoth, C. Di Meo, F. Zouhiri, F. Saïd-Hassane, S. Valetti, R. Gorges,
€
V. Nicolas, J.H. Poupaert, S. Chollet-Martin, D. Desmaele, R. Gref, P. Couvreur,
Self-assembled squalenoylated penicillin bioconjugates: an original approach
for the treatment of intracellular infections, ACS Nano 6 (2012) 3820e3831.
[10] M.G. Sarpietro, D. Micieli, F. Rocco, M. Ceruti, F. Castelli, Conjugation of
squalene to acyclovir improves the affinity for biomembrane models, Int. J.
Pharm. 382 (2009) 73e79.
(b) D. Passarella, B. Peretto, R. Blasco
y Yepes, G. Cappelletti, D. Cartelli,
C. Ronchi, J.S. Snaith, G. Fontana, B. Danieli, J. Borlak, Synthesis and biological
evaluation of novel thiocolchicine-podophyllotoxin conjugates, Eur. J. Med.
Chem. 45 (2010) 219e226;
(c) D. Passarella, D. Comi, A. Vanossi, G. Paganini, F. Colombo, L. Ferrante,
V. Zuco, B. Danieli, F. Zunino, Histone deacetylase and microtubules as targets
for the synthesis of releasable conjugate compounds, Bioorg. Med. Chem. Lett.
19 (2009) 6358;
(d) E. Riva, D. Comi, S. Borrelli, F. Colombo, B. Danieli, J. Borlak, L. Evensen,
J.B. Lorens, G. Fontana, O.M. Gia, L. Dalla Via, D. Passarella, Synthesis and bio-
logical evaluation of new camptothecin derivatives obtained by modification of
position 20, Bioorg. Med. Chem. 18 (2010) 8660e8668.
[11] V. Allain, C. Bourgaux, P. Couvreur, Self-assembled nucleolipids: from supra-
molecular structure to soft nucleic acid and drug delivery devices, Nucleic
Acids Res. 40 (2012) 1891e1903.
€
[12] M. Raouane, D. Desmaele, M. Gilbert-Sirieix, C. Gueutin, F. Zouhiri,
C. Bourgaux, E. Lepeltier, R. Gref, R. Ben Salah, G. Clayman, L. Massaad-mas-
sade, P. Couvreur, Synthesis, characterization, and in vivo delivery of siRNA-
squalene nanoparticles targeting fusion oncogene in papillary thyroid carci-
noma, J. Med. Chem. 54 (2011) 4067e4076.
[20] A. Satyam, Design and synthesis of releasable folate-drug conjugates using a
novel heterobifunctional disulfide-containing linker, Bioorg. Med. Chem. Lett.
18 (2008) 3196e3209.
€
[13] J.L. Arias, L.H. Reddy, M. Othman, B. Gillet, D. Desmaele, F. Zouhiri, F. Dosio,
R. Gref, P. Couvreur, Squalene based nanocomposites: a new platform for the
design of multifunctional pharmaceutical theragnostics, ACS Nano 5 (2011)
1513e1521.
[21] G. Cappelletti, G. Tedeschi, M.G. Maggioni, A. Negri, S. Nonnis, R. Maci, The
nitration of
t protein in neurone-like PC12 cells, FEBBS 562 (2004) 35e39.
[14] F. Dosio, B. Stella, A. Ferrero, C. Garino, D. Zonari, S. Arpicco, L. Cattel,
S. Giordano, R. Gobetto, Ruthenium polypyridyl squalene derivative: a novel
self-assembling lipophilic probe for cellular imaging, Int. J. Pharm. 440 (2013)
221e228.
[22] W. Voigt, Sulforhodamine B assay and chemosensitivity, Methods Mol. Med.
110 (2005) 39e48.
[23] C.A. Blencowe, A.T. Russel, F. Greco, W. Hayes, D.W. Thornthwaite, Self-
immolative linkers in polymeric delivery systems, Polym. Chem. 2 (2011)
773e790.
[24] M. Ceruti, G. Balliano, F. Viola, L. Cattel, N. Gerst, F. Schuber, Synthesis and
biological activity of azasqualenes, bis-azasqualenes and derivatives, Eur. J.
Med. Chem. 22 (1987) 199e208.
[25] Z. Zhao, D.G.I. Kingston, A.R. Crosswell, Modified taxols, 6. Preparation of
water-soluble prodrugs of taxol, J. Nat. Prod. 54 (1991) 1607e1611.
[15] Synthesis of tubulin binders:
(a) F. Calogero, S. Borrelli, G. Speciale, M.S. Christodoulou, D. Cartelli, D. Ballinari,
F. Sola, C. Albanese, A. Ciavolella, D. Passarella, G. Cappelletti, S. Pieraccini,
M. Sironi, 9-Fluorenone-2-carboxylic acid as scaffold for new tubulin inter-
acting compounds, ChemPlusChem 78 (2013) 663e669;
(b) E. Riva, M. Mattarella, S. Borrelli, M.S. Christodoulou, D. Cartelli, M. Main,
S. Faulkner, D. Sykes, G. Cappelletti, J.S. Snaith, D. Passarella, Reparation of
fluorescent tubulin binders, ChemPlusChem 78 (2013) 222;