Multifunctional squalene-based prodrug nanoparticles for targeted cancer therapy

Article abstract of DOI:10.1039/c3cc47427e

Fluorescent and biotinylated squalene-gemcitabine prodrug nanoparticles exhibiting high drug payloads have been prepared and successfully used to target different cancer cell lines, resulting in increased cell uptake and improved anticancer efficiency, wh

Full text of DOI:10.1039/c3cc47427e

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Multifunctional squalene-based prodrug
nanoparticles for targeted cancer therapy†
Cite this: Chem. Commun., 2014,
50, 5336
Duc Trung Bui, Julien Nicolas,* Andrei Maksimenko, Didier Desmaele and
¨
Patrick Couvreur
Received 28th September 2013,
Accepted 17th October 2013
DOI: 10.1039/c3cc47427e
www.rsc.org/chemcomm
Fluorescent and biotinylated squalene–gemcitabine prodrug nano- when the drug is hydrophilic.7 In the past few years, a novel approach
particles exhibiting high drug payloads have been prepared and suc- has emerged using squalene (Sq) – a lipidic precursor in the
cessfully used to target different cancer cell lines, resulting in increased biosynthesis of cholesterol widely distributed in nature – as a building
cell uptake and improved anticancer efficiency, which represents the block for the synthesis of drug–Sq conjugates that can self-assemble
first targeted system derived from the squalenoylation approach.
in aqueous solution to form nanoassemblies with high drug payloads
(B50 wt%).8 This approach has been applied to various drugs and
has led to promising results in vivo against several pathologies.9
However, this novel system urgently requires an efficient targeting
strategy that would enhance nanoparticle internalization by cancer
cells via a receptor-mediated mechanism, thus avoiding potential side
effects often faced when using passive drug delivery strategies.
Herein, we report an efficient and simple strategy to conceive
multifunctional Sq-based nanoparticles (i.e., therapeutic, fluorescent
and targeted) based on the co-self-assembly of the different Sq-based
functional components (Scheme 1), that is: (i) gemcitabine–squalene
(Gem–Sq, a) owing to the demonstrated activity of Gem against a
The medical application of nanotechnology, often termed nano-
medicine, has witnessed a crucial step forward with the develop-
ment of various types of drug carriers.1 The encapsulation of drugs
into colloidal nanocarriers (e.g., polymer nanoparticles, micelles,
liposomes, etc.) has, indeed, resulted in intensive research and
promising achievements in the last decade.2 However, strong
limitations still remain which may hamper their further transla-
tion to the clinic: (i) the ‘‘burst release’’, which corresponds to the
rapid release of drugs post-administration and can be harmful to
patients; (ii) the encapsulation of poorly soluble drugs, exhibiting a
high tendency to crystallization and (iii) the poor drug loadings
(generally a few percent) that require the use of a large amount of
nanocarrier materials, which can lead to prohibitive toxicity in vivo.
To resolve these issues, alternative strategies derived from the
prodrug3 concept have recently been reported and hold great hope
due to their ability to suppress the ‘‘burst release’’ and to enable easier
and more efficient incorporation of drugs into nanocarriers. For
instance, drugs can be covalently linked to amphiphilic block
copolymers (mainly on the hydrophobic block that is composed of
the core of the nanoparticle,2d at the junction of the two polymer
blocks4), or to the side chain of water-soluble polymers.5 In the latter
case, fully water-soluble conjugates or small-size aggregates are
generally formed. Additionally, it has been shown that hydrophobic
polymer chains can also be grown in a controlled fashion from drugs,
leading to either hydrophobic polymer prodrugs further stabilized by
PEG-based surfactants in the case of hydrophobic drugs,6 or amphi-
philic polymer prodrugs that can self-assemble into nanoparticles
´
´
Institut Galien Paris-Sud, Universite Paris-Sud, UMR CNRS 8612, Faculte de Pharmacie,
´
ˆ
5 rue Jean-Baptiste Clement, F-92296 Chatenay-Malabry Cedex, France.
E-mail: julien.nicolas@u-psud.fr; Fax: +33 1 46 83 59 46; Tel: +33 1 46 83 55 11
† Electronic supplementary information (ESI) available: Experimental details, biological
activity and endocytosis in the presence of inhibitors. See DOI: 10.1039/c3cc47427e
Scheme 1 Structure of gemcitabine–squalene (Gem–Sq, a), rhodamine–
squalene (Rho–Sq, b) and biotin–squalene (Biotin–Sq, c), and their co-self-
assembly to prepare multifunctional nanoparticles for cancer cell targeting.
5336 | Chem. Commun., 2014, 50, 5336--5338
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Fig. 1 Evolution of the average diameter, the particle size distribution and
the zeta (z) potential with time (a), and cryogenic transmission electron
microscopy images (b) of Gem–Sq/Biotin–Sq/Rho–Sq (86 : 9 : 5 wt%)
multifunctional nanoparticles N1*.
wide range of solid tumors;10 (ii) rhodamine–squalene (Rho–Sq, b)
due to advantageous properties of Rho (e.g., high water solubility,
good photostability, etc.) and the retained fluorescence emission at a
broad range of pH of its tertiary amide derivative;11 and (iii) biotin–
squalene (Biotin–Sq, c) in order to selectively target cancer cells via
biotin receptors overexpressed at the surface of many cancer cells.12
Rho–Sq was simply achieved in 90% yield by direct acylation of the
piperazine-functionalized rhodamine B11 with the chloroformate mixed
anhydride of trisnor-squalenic acid.† On the other hand, Biotin–Sq was
Fig. 2 Kinetics of cell capture of non-functionalized (Gem–Sq/Rho–Sq)
N1 and biotin-functionalized (Gem–Sq/Biotin–Sq/Rho–Sq) N1* nano-
particles in MCF7 (a), M109 (b) and HeLa (c) cells. Fluorescence of cells
after incubation of MCF7, M109 and HeLa cells with nanoparticles N1 and
N1* for 4 h at 4 1C or 37 1C (d).
The use of specific endocytosis inhibitors (chlorpromazine,
obtained from conjugation of biotin to trisnorsqualenol through a short filipin III and amiloride, respectively, associated with clathrin-
hydrophilic triethylene glycol linker (to promote surface ligand display mediated endocytosis, caveolae-mediated endocytosis and macro-
from the resulting nanoparticles) via Mitsunobu reaction.†
pinocytosis)14 suggested that the biotinylated nanoparticles were
Multifunctional nanoparticles N1* were prepared by co-self- internalized by endocytosis; clathrin and caveolae-mediated endo-
assembly of Gem–Sq, Biotin–Sq and Rho–Sq (86 : 9 : 5 wt%) in cytotic pathways being both involved (Fig. S2).† Conversely, non-
aqueous solution via the nanoprecipitation technique. The average functionalized nanoparticles were not affected by endocytosis
diameter, Dz, was 149 Æ 3 nm with a narrow particle size distribu- blockers, in agreement with previous literature.15
tion (PSD) of B0.15, and a zeta (z) potential value of À25 Æ 3 mV.
Importantly, in order to demonstrate the integrity of the nano-
Their colloidal stability was assessed over a period of at least 7 days, particles during in vitro experiments (i.e., the absence of colloidal
during which little variations in their colloidal characteristics were disassembly that would split up Rho–Sq, Gem–Sq and Biotin–Sq), a
noticed (Fig. 1a). Cryogenic-transmission electron microscopy similar experiment was performed with the double fluorescence
(Cryo-TEM) showed spherical morphologies and average diameters labelling by using Rho–Sq and Chol–BODIPY (Table S1).† Incubation
in good agreement with DLS data (Fig. 1b). Interestingly, a thorough of HeLa cells with the resulting dual fluorescent nanoparticles; either
inspection of Cryo-TEM images showed internal organization of the targeted N2* (Gem–Sq/Biotin–Sq/Rho–Sq/Chol–BODIPY) or N2 (Gem–
nanoparticles, similarly to what has been previously observed with Sq/Rho–Sq/Chol–BODIPY), was monitored by flow cytometry through
Gem–Sq nanoassemblies8b (see the inset in Fig. 1b).
both fluorescence channels associated with rhodamine B and BODIPY.
Three cancer cell lines; human breast adenocarcinoma cells Whereas Rho–Sq/Chol–BODIPY dyes alone (N3) resulted in poor cell
(MCF7), murine lung cancer cells (M109) and human cervix capture, nanoparticles N2* and N2 led to similar cell internalization
carcinoma cells (HeLa), which overexpress biotin receptors,12,13 patterns two-by-two, whatever the fluorescence channel (i.e., by follow-
were chosen to evaluate the tumor targeting ability of the multi- ing each dye individually) (Fig. S1).† Moreover, the improved inter-
functional nanoparticles N1* and compared to non-biotinylated nalization, at 37 1C, of biotin-functionalized nanoparticles N2*
Gem–Sq/Rho–Sq nanoparticles N1 (Dz = 120 nm, PSD = 0.19). After compared to non-targeted counterparts N2 was confirmed for both
incubation at different time intervals, the cells were collected for channels. At 4 1C, very low cell internalization was again noted.
analysis of rhodamine B fluorescence by flow cytometry. The results
Additionally, confocal microscopy investigation showed that
showed a higher cell fluorescence intensity of all three cell lines multifunctional nanoparticles N2* are localized intracellularly as
when biotin-decorated nanoparticles N1* were employed, as endocellular and perinuclear fluorescent spots, suggesting an endo-
opposed to the treatment with N1 (Fig. 2a–c). This demonstrated lysosomal distribution (Fig. 3). Noteworthy is the fact that the nearly
the surface availability of biotin and the effectiveness of the perfect co-localization of the two fluorochromes, as attested by the
targeting. When incubation was achieved for 4 h at 4 1C, the cell overlay of the red and green fluorescence channels, suggested that
fluorescence intensity of all the three cell lines dramatically the nanoparticles are likely to be still intact after 24 h of incubation.
decreased down to very low values with both types of nanoparticles
In order to assess the therapeutic effect of the targeted Sq-based
(Fig. 2d), suggesting internalization rather governed by endocytosis. nanoparticles N1*, they were then tested for their in vitro anticancer
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on the concomitant self-assembly of the different Sq-based building
blocks to furnish stable multifunctional nanoparticles. They demon-
strated improved internalization in different cancer cell lines as well
as greater anticancer activity than non-functionalized Gem–Sq nano-
particles. This approach could be easily applied to other anticancer
drugs (e.g., nucleoside analogues, antifolic acid compounds, etc.),
fluorescent dyes or biologically active ligands (e.g., folic acid, anisa-
mide, small peptidic sequences, etc.). Therefore, it paves the way to
the design of various multifunctional Sq-based nanoparticles simply
by changing the nature of the functional moiety linked to the Sq.
The authors thank V. Nicolas (Plateforme Imagerie Cellulaire,
IFR 141) for help with the CLSM. The research leading to these
results received funding from the European Research Council
under the European Community’s Seventh Framework Programme
FP7/2007-2013 (Grant Agreement No. 249835). The PhD training
program in France of the University of Science and Technology of
Hanoi is acknowledged for the financial support provided to D.T.B.
The authors also thank the CNRS for financial support.
Fig. 3 Confocal microscopy images [red (Rho, a) and green (BODIPY, b)
fluorescence images] and merge of red and green fluorescence images with
the Nomarski image (c) after a 24 h incubation of HeLa cells with dual
fluorescent Gem–Sq/Biotin–Sq/Rho–Sq/Chol–BODIPY N2* nanoparticles.
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5338 | Chem. Commun., 2014, 50, 5336--5338
This journal is ©The Royal Society of Chemistry 2014