Enhancement of hydrosolubility and in vitro antiproliferative properties of chalcones following encapsulation into β-cyclodextrin/cellulose-nanocrystal complexes

Article abstract of DOI:10.1016/j.bmcl.2019.05.056

This paper describes the preparation of two chalcone/β-cyclodextrin/cellulose-nanocrystals complexes and the study of their antiproliferative activities against two colorectal and two prostatic cancer cell lines. The aim of this work was to enhance hydros

Full text of DOI:10.1016/j.bmcl.2019.05.056

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enhancement of hydrosolubility and in vitro antiproliferative properties of
chalcones following encapsulation into β-cyclodextrin/cellulose-nanocrystal
complexes
⁎
Benjamin Riouxa, Christelle Pougeta, , Gautier M.A. Ndong-Ntoutoumeb, Robert Granetb,
Aurélie Gamonda, Aurélie Laurenta, Aline Pinona, Yves Champaviera,c, Bertrand Liagrea,
Catherine Fagnèrea, Vincent Solb
a Laboratoire PEIRENE EA 7500, Université de Limoges, Faculté de Pharmacie, 2 rue du Dr Marcland, 87025 Limoges Cedex, France
b Laboratoire PEIRENE EA 7500, Université de Limoges, Faculté des Sciences et Techniques, 123 avenue A. Thomas, 87060 Limoges Cedex, France
c BISCEm NMR platform, GEIST, Université de Limoges, 2 rue du Dr Marcland, 87025 Limoges Cedex, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
This paper describes the preparation of two chalcone/β-cyclodextrin/cellulose-nanocrystals complexes and the
study of their antiproliferative activities against two colorectal and two prostatic cancer cell lines. The aim of this
work was to enhance hydrosolubility of chalcones thanks to the hydrophilic character of cellulose nanocrystals.
These latter were linked, through ionic interactions, to a cationic derivative of β-cyclodextrins whose lipophilic
cavity allowed the encapsulation of hydrophobic chalcones: 3-hydroxy-3′,4,4′,5′-tetramethoxychalcone (1) and
3′,4,4′,5′-tetramethoxychalcone (2). First, we showed that encapsulation allowed hydrosolubilization of chal-
cones. Then, chalcone/β-cyclodextrin/cellulose-nanocrystals complexes demonstrated enhanced in vitro anti-
proliferative activities, compared to the corresponding free-chalcones.
Chalcones
β-Cyclodextrin/cellulose nanocrystals
Nanoparticles
Prostate and colorectal cancer cell lines
Hydrosolubilization of hydrophobic drugs
Cancer, which is characterized by its high incidence and mortality,
is a major health hindrance; an estimated 18.1 million new cancer cases
and 9.6 million cancer deaths occurred in 2018 worldwide.1 Despite the
availability of various chemotherapeutic protocols, associated acute
side effects of the clinically used anticancer compounds (including the
most recently introduced drugs) are still significant obstacles to effec-
tive cancer chemotherapy.2–4 Consequently, discovery and develop-
ment of new effective anticancer agents and/or therapies remain the
major goal of medicinal chemists.
and Retention (EPR) effect, and destroy them by action of the vector-
ized drug.5 In this way, Ndong- Ntoutoume et al. have demonstrated the
efficiency of curcumin I included into β-cyclodextrins/CNCs complexes,
on colorectal and prostatic cancer cells.6
CNCs are obtained after sulfuric acid-hydrolysis of cotton fibers that
results in the removal of the amorphous regions of cellulose. CNCs are
rod-like nanoparticles, 100–200 nm long, 10–20 nm wide and 5–10 nm
thick. They gather several interesting characteristics: uniform nano-rod
shape, good mechanical strength, high specific surface area, bio-
compatibility and biodegradability. Another great advantage of these
cellulose nanocrystals is the negative charges (O-sulfate ester functions
present on their surface) which enhanced their hydrophilic character
and, above all, allow the formation of ionic complexes with cationic
compounds, in our case, a cationic derivative of a β-cyclodextrin (β-
CD). β-CD are cyclic oligosaccharides made of seven D-glucopyranose
units linked by α-(1,4) glycosidic bonds. Cyclodextrins are well known
to form inclusion complexes with guest hydrophobic molecules caged
into their lipophilic inner cavity while their surface is sufficiently hy-
drophilic to endow cyclodextrins with water solubility.7
Most anti-cancer agents suffer from a lack of specificity, since they
also attack healthy cells which rapidly divide (cells from oral mucosa,
hair follicles, bone marrow, etc.), and then generate adverse side ef-
fects. Moreover, anticancer agents are also characterized by poor water
solubility and low bioavailability. To overcome these problems, many
research groups have designed nano-sized drug delivery systems.
Polymeric nanocarriers are the most widely investigated nano-
technology platform for cancer therapy, due to their easy chemical
modification. According to our projects on drugs vectorization for an-
ticancer applications, we have developed cellulose nanocrystals (CNCs)
which can specifically target tumors, through the Enhanced Permeation
Chalcones belonging to the flavonoid family, received significant
⁎ Corresponding author.
E-mail address: christelle.pouget@unilim.fr (C. Pouget).
https://doi.org/10.1016/j.bmcl.2019.05.056
Received 2 May 2019; Received in revised form 24 May 2019; Accepted 27 May 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:BenjaminRioux,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.05.056

B. Rioux, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Chemical structures of chalcones 1 and 2.
attention due to their wide range of anticancer activities, including
antimitotic properties, via disruption of the tubulin-microtubule
system,8–11 inhibition of NF-κB activation12 or induction of apoptosis.13
Nevertheless, in some cases, the interesting properties of some
chalcones are hindered by a poor solubility and a low bioavailability
owing to their hydrophobicity. Our encouraging results relative to the
encapsulation of curcumin into β-CD/CNCs complexes prompted us to
vectorize two chalcones (chalcones 1 and 2, Fig. 1) which, according to
literature data and our own results, already have demonstrated poten-
tial anticancer effects. These two chalcones possess a trimethoxyphenyl
ring which has been found to be of great interest for anticancer ac-
Scheme 2. a) Synthesis of CNCs; b) Synthesis of cationic β-CD.
tivity.14–16 Chalcone
1 (3-hydroxy-3′,4,4′,5′-tetramethoxychalcone)
was shown to possess a strong antiproliferative activity against K562,
A549 and MCF-7 cell lines15,17 as well as against colon (HCT-116 and
HT-29) and prostatic (PC-3 and DU145) cancer cells.18,19 Chalcone 2
(3′,4,4′,5′-tetramethoxychalcone) was found to inhibit the growth of
ovarian cancer cells20 and we also demonstrated its antiproliferative
activity against colorectal cancer cells (HT-29 and HCT-116) and pro-
static cancer cells (PC-3 and DU-145).18
In the present paper, we describe the encapsulation of chalcones 1
and 2 into β-CD/CNCs complexes and we compare the antiproliferative
properties of these complexes with those of the corresponding free
chalcones; biological tests were performed on colon (HT29 and HCT-
116) and prostate (PC3 and DU145) cancer cell lines.
The overall synthetic pathway is described in Schemes 1–3. Firstly,
chalcones 1 and 2 were prepared using the Claisen-Schmidt con-
densation (Scheme 1) from building-blocks, namely 3,4,5-trimethox-
yacetophenone and the appropriate benzaldehydes. 1H NMR spectra
showed that the (E)-stereoisomers were specifically generated since the
coupling constant between the two ethylenic protons was about
15–16 Hz. Chalcone 2 was obtained from 4-methoxybenzaldehyde in
55% yield while chalcone 1 was prepared in a good yield (69%) from 3-
hydroxy-4-methoxybenzaldehyde.
Scheme 3. a) Formation of β-CD/CNCs complexes via electrostatic coupling
between CNCs and cationic β-CD. b) Inclusion of chalcones 1 and 2 into β-CD/
CNCs complexes.
CNCs preparation was achieved by classical sulfuric acid-hydrolysis
(Scheme 2a) and characterization data match the results obtained by
6
Ndong-Ntoutoume et al. The zeta potential of CNCs, −51 mV, con-
firmed the presence of O-sulfate ester functions (Fig. S1). A transmis-
sion electronic microscopy (TEM) image of cellulose nanocrystals is
shown in Fig. 2; nanoparticles (100–200 × 10–20 nm) were observed
after uranyl acetate staining.
In parallel, cationic β-CD were obtained through reaction of glyci-
dyltrimethyl ammonium chloride (GTMAC) with β-CD in alkaline
aqueous medium (Scheme 2b) according to the Chisholm and Wenzel
procedure.21 Purification of cationic β-CD was performed by dialysis
against deionized water (MWCO: 600 Da). Chisholm and Wenzel de-
monstrated that the best inclusion capacity of cationic β-CD was
reached for a degree of substitution (DS) slightly superior to 1. In the
present study, DS is defined as the number of GTMAC units per
Fig. 2. TEM image of cellulose nanocrystals.
anhydroglucose unit of cyclodextrin. An average DS of 1.7 was calcu-
lated from NMR data as the following ratio: integral of the H1
(anomeric proton) of the glucose unit / integral of H2′ proton of the
grafted chain (Scheme 2b and Fig. S2a). The presence of quaternary
ammonium functions on cationic β-CD was revealed by FTIR spectro-
scopy, through observation of stretching vibration bands at 1478 cm−1
and 988 cm−1 (Fig. S2b).
Scheme 1. Synthesis of chalcones 1 and 2 via Claisen-Schmidt condensation.
2

B. Rioux, et al.
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Table 1
Table 2
Size (DLS), polydispersity index (PDI) and zeta potential (ζ) characteristics of
CNCs intermediates and final β-CD/CNCs/drug complexes in deionized water.
Loading ratios of chalcones into β-CD/CNCs complexes.
Complexes
Loading ratio (w/w)
Chalcone-loaded β-CD/CNCs (mol·L−1
)
CNCs Type
DLS (nm)
PDI
ζ-potential (mV)
β-CD/CNCs/1
β-CD/CNCs/2
13.5%
23.3%
7.84.10-4
1.41.10-3
CNCs
141.5
198.1
206.8
205.8
0.2
2.1
1.6
0.9
0.239
0.122
0.151
0.141
0.015
0.018
0.013
0.021
−51.0
−41.0
−29.2
−30.1
0.7
1.1
1.8
1.5
β-CD/CNCs
β-CD/CNCs/1
β-CD/CNCs/2
Then, cationic β-CD were brought into contact with cellulose na-
nocrystals in aqueous medium (Scheme 3a), under ultrasonic stirring. β-
CD/CNCs complexes, resulting from an electrostatic coupling, were
centrifuged at 13,000 rpm for 10 min and thus separated from free ca-
tionic β-CD. FTIR (KBr disc) spectra of β-CD/CNCs and CNCs differ by
the presence of a new signal at 1478 cm−1 corresponding to the –N+
-
(CH3)3 group of cationic β-CD (Fig. S3). A significant increase of zeta
potential was also observed due to the positive charge of cationic β-CD:
−41 mV for β-CD/CNCs vs −51 mV for CNCs (Table 1).
These β-CD/CNCs complexes, obtained as an opalescent aqueous
suspension, were very stable, due to the repulsion between the nano-
crystal particles which bear the negatively charged O-sulfate ester
functions (Schemes 2 and 3). Therefore, they were ready for the in-
clusion of non-polar drugs, i.e. chalcones 1–2, into the β-CD hydro-
phobic cavity. Acetone solutions of chalcones were mixed with β-CD/
CNCs suspensions (Scheme 3b), and β-CD/CNCs/chalcone complexes
were purified by centrifugation. These complexes were obviously much
more soluble in water than free chalcones as shown in Fig. 3. Finally,
the loading of β-CD/CNCs with chalcones 1 and 2 increased, at the
same time, the size of the complexes and ζ-potential (Table 1).22
Loading ratios of drug into β-CD/CNCs were evaluated by
UV–Visible titration, for each one of the two chalcones (Table 2): a
small volume of β-CD/CNCs/chalcone was taken and the drug was
extracted by addition of chloroform. Then, concentration of the chal-
cone in the organic phase was determined by a UV–Visible titration.
Cell viability of cancer cell lines was evaluated in presence of this
nanoformulation. We first checked that β-CD/CNCs nanocarriers alone
only slightly reduced cell proliferation (Fig. 4). Then, cell lines were
incubated 48 h in presence of chalcones, either complexed or free, in
the 0.05–50 µM concentration range. IC50 values are presented in
Table 3. We evaluated the cytotoxic properties of the complexed chal-
cones and compared the results with those obtained for equivalent
concentrations of free chalcones to show the interest of the en-
capsulation (Fig. 5).
Fig. 4. Effect of β-CD/CNCs complexes on cancer cells proliferation.
Table 3
*
IC50 values (µM) from MTT assay for β-CD/CNCs/chalcones complexes and
free chalcones 1 and 2.
Drugs
HT-29
HCT-116
ND**
PC-3
DU-145
β-CD/CNCs
ND**
13.98
27.8
4.5
ND**
0.77
0.10
6.3
ND**
0.72
β-CD/CNCs/1
0.74
1.7
0.5
0.6
0.83
0.8
0.01
0.05
0.01
0.02
1.2
0.4
1.7
1
0.1
1.0
6.3
β-CD/CNCs/2
2
8.9
1.5
7.9
7.4
10.7
0.6
7.8
0.5
13.8
* IC50 values are the average of three determinations at least.
** IC50 undetermined, cell viability is superior to 84% at 50 µM.
(0.72
0.02 μM vs 6.3
1.2 μM). Concerning HCT-116 cells, IC50
values for the complex and the free chalcone are identical (0.83 μM).
IC50 against PC-3 cells only is not improved, but nevertheless remains
less than 1 μM (0.77
0.05 μM vs 0.10
0.01 μM). Therefore, en-
capsulation of chalcone 1 into β-CD/CNCs proves to be a relevant
choice since it makes it possible to significantly improve its anti-
proliferative potential on HT-29 and DU-145 cells, while preserving the
antiproliferative activity on HCT-116 and PC-3 cells.
Samples of β-CD/CNCs/chalcone complexes were studied in water
while free chalcone solutions had to be made in DMSO. This confirmed
hydrosolubilization of chalcones through encapsulation into β-CD/
CNCs complexes.
Concerning the β-CD/CNCs/2 complex, the IC50 values are slightly
lower than those of chalcone 2 against the four cell lines studied. As for
chalcone 1, we can say that encapsulation of this chalcone by the β-CD/
CNCs nanoformulation is a relevant strategy to improve the anti-
proliferative effect.
The β-CD/CNCs/1 complex presents the lowest IC50 in this series.
When tested against HT-29 cells, the IC50 value is almost divided by 2
In conclusion, we described the preparation of β-cyclodextrin/cel-
lulose nanocrystals complexes, stabilized by ionic interaction and able
to encapsulate hydrophobic chalcones which were selected for their
antiproliferative potency. The first aim of the chalcone hydro-
solubilization was achieved through these β-CD/CNCs/chalcone com-
plexes, which allowed biological evaluation in aqueous medium. Then,
β-CD/CNCs were found to be suitable for efficient drug loading and
release, as described in our previous studies.5,6 Loading ratio was
around 20% for the two complexes. Then, β-CD/CNCs/chalcone com-
plexes demonstrated in vitro antiproliferative effect against colorectal
and prostatic cancer cell lines. As β-CD/CNCs alone were shown to have
no effect on cell proliferation, the antiproliferative activity was due to
the release of drugs from the encapsulated form. Considering chalcones
1 and 2, the use of nanocarriers enhanced the antiproliferative effect
observed with corresponding free chalcones. As demonstrated by
Ndong-Ntoutoume et al. encapsulation may increase internalization of
(13.98
0.74 μM for β-CD/CNCs/1 vs 27.8
1.7 μM for 1). Against
DU-145 cells, the IC50 is approximately 9-fold lower than for 1
Fig. 3. Chalcone 1 on the left and β-CD/CNCs/chalcone 1 complex on the right,
both in aqueous medium.
3

B. Rioux, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
hydrophobic chalcones into cancer cells.6 In vivo studies have to be
performed to evidence the role of these nanocarriers in the targeting of
tumor cells via the Enhanced Permeation and Retention (EPR) effect.
The use of these complexes (β-CD/CNCs/chalcone) may therefore be a
relevant strategy in cancer therapies.
Acknowledgments
The authors thank the ‘Conseil Régional du Limousin’ for financial
support, Dr. Michel Guilloton for help in manuscript editing and Dr.
Cyril Colas, from the “Fédération de Recherche” ICOA/CBM (FR2708)
platform, for the HRMS analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.05.056.
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Fig. 5. In vitro antiproliferative effect of free chalcones and β-CD/CNCs/chal-
cones complexes against HT-29 (a), HCT-116 (b), PC-3 (c) and DU-145 (d)
cancer cell lines after 48 h treatment.
4