Efficient delivery of therapeutic small nucleic acids to prostate cancer cells using ketal nucleoside lipid nanoparticles

Article abstract of DOI:10.1016/j.jconrel.2013.09.006

A novel nucleoside lipid derived from dioleyl ketal was synthesized from uridine in three steps starting from dioleyl ketone. Electronic microscopy studies show that Ketals Nucleoside Lipids (KNL) self-assemble to form liposome-like structures in aqueous solutions. KNL is able to bind siRNA as demonstrated by electrophoresis experiment and standard ethidium bromide fluorescence displacement assay. Transfection assays of stable hepatic cell lines HupIRF, carrying a luciferase reporter gene demonstrate that KNL is able to transfect siRNA and exhibits protein knockdown more efficiently than its diester analog (DOTAU) and lipofectamine. Herein, we also report that KNLs are suitable transfecting reagents for the development of novel therapeutic approaches involving either siRNA or antisense oligonucleotide against human prostate cancer PC-3 cells resistant to chemotherapy.

Full text of DOI:10.1016/j.jconrel.2013.09.006

Efficient Delivery of Therapeutic Small Nucleic Acids to Prostate Cancer
Cells Using Ketal Nucleoside Lipid Nanoparticles
Delphine Luvino, Salim Khiati, Khalid Oumzil, Palma Rocchi, Michel
Camplo, Philippe Barth e´ l e´ my
PII:
DOI:
Reference:
S0168-3659(13)00794-3
doi: 10.1016/j.jconrel.2013.09.006
COREL 6880
To appear in:
Journal of Controlled Release
Received date:
Accepted date:
24 April 2013
5 September 2013
Please cite this article as: Delphine Luvino, Salim Khiati, Khalid Oumzil, Palma Rocchi,
Michel Camplo, Philippe Barth ´e l ´e my, Efficient Delivery of Therapeutic Small Nucleic
Acids to Prostate Cancer Cells Using Ketal Nucleoside Lipid Nanoparticles, Journal of
Controlled Release (2013), doi: 10.1016/j.jconrel.2013.09.006
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Efficient Delivery of Therapeutic Small Nucleic Acids
to Prostate Cancer Cells Using Ketal Nucleoside Lipid
Nanoparticles
Delphine Luvino,  Salim Khiati,  Khalid Oumzil, Palma Rocchi, Michel Camplo,

≠,

and Philippe Barthélémy
Univ. Bordeaux, ARNA laboratory, F-33000 Bordeaux, France
INSERM, U869, ARNA laboratory, F-33000 Bordeaux, France
≠
Keywords: siRNA, antisense, drug delivery, nucleoside lipids, prostate cancer
Abstract: A novel nucleoside lipid derived from dioleyl ketal was synthesized from uridine in three
steps starting from dioleyl ketone. Electronic microscopy studies show that Ketals Nucleoside Lipids
(KNL) self-assemble to form liposome-like structures in aqueous solutions. KNL is able to bind siRNA
as demonstrated by electrophoresis experiment and standard ethidium bromide fluorescence
displacement assay. Transfection assays of stable hepatic cell lines HupIRF, carrying a luciferase
reporter gene demonstrate that KNL is able to transfect siRNA and exhibits protein knockdown more
efficiently than its diester analogue (DOTAU) and lipofectamine. Herein, we also report that KNLs are
suitable transfecting reagents for the development of novel therapeutic approaches involving either
siRNA or antisense oligonucleotide against human prostate cancer PC-3 cells resistant to chemotherapy.

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INTRODUCTION
Among the non-viral systems developed so far, cationic lipids
remains one of the most abundant classes of transfecting reagents used nowadays in laboratories.
Recently, a rational approach to design cationic lipids was developed by Semple et al. for delivering
xiv
siRNA. In this study the transfection efficiency of a series of cationic lipids analogues to 1,2-
dilinoleyloxy-3-dimethylaminopropane (DLinDMA) was realized in vivo. The authors demonstrated
that a lipid featuring a ketal linker between the polar head and lipid chains was well tolerated in
primates; moreover, this lipid was the best performing transfecting lipid of the series investigated.
Likewise, it was recently reported that a pH-sensitive lipid featuring a ketal function facilitated the
delivery of liposomal siRNA and gene silencing activity both in vitro and in vivo.
Among cationic transfecting reagents, amphiphiles having a double functionality based on the
combination of nucleic acid and lipid characteristics have shown to be efficient transfecting reagent for
xvi
xvii,xviii
both plasmid DNA and siRNA
with poor toxicity. In parallel, our groups reported a new family
of zwitterionic ketal based nucleolipids (KNLs), which can be used to construct complementary
supramolecular systems via molecular recognition between individual nucleolipids (NLs) derived from
xix,xx
uridine and adenosine.
These studies showed that the base pair interactions between the nucleolipids
polar heads were favoured by the ketal linker,
which restricts the ribose cycle in the southern
conformation (C2’-endo). Based upon both these
results and the transfecting data obtained by Semple
et al., we hypothesized that the modification of the
chemical structure and conformations of the ribose
moiety could have an impact on the
siRNA/nucleolipid stability and siRNA delivery.
Hence, the chemical structure of the cationic
nucleolipid DOTAU (figure 1), which is a key
components in transfection formulations, was
O
O
NH
NH
MsO
Me3N
MsO
Me3N
N
O
N
O
O
O
O
O
O
O
6
O
O
7
7
6
6
7
7
6
Figure 1.
modified via the insertion of a ketal linker at the 2’ and 3’ positions.

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In this contribution, we report a new non-viral transfecting system belonging to the nucleolipids
family. We demonstrate that ketal based nucleolipids, which are non-toxic cationic bioinspired
amphiphiles, provide an efficient siRNA carrier. Their ability to transfect siRNA in human
MATERIALS AND METHODS

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RESULTS AND DISCUSSIONS
Synthesis. In order to evaluate the impact of the lipid structure on the transfection properties, a novel
ketal based nucleolipid (KNL) derived from a dihexadec-9-enyl ketal was prepared from oleic acid by a
six steps synthetic pathway as shown in Scheme 1. After preparation of the dioleyl ketone 4, and its
coupling reaction with uridine, this intermediate was treated with mesyl chloride to afford the 5’-
mesylate nucleoside derivative 6. This compound was transferred to a pressure tube and treated for 2
days with trimethylamine in THF to give the final ketal derivative 7.
Scheme 1.
Physicochemical studies.
Transmission Electronic Microscopy, Dynamic Light Scatterring.
The morphology of the aggregates obtained with the new ketal nucleolipid in the absence and
presence of siRNA was first studied using TEM. First, hydration at room temperature into of the KNL at
a concentration of 0.5 mg/mL induces the spontaneous formation of spherical heterodisperse objects of
1
00 nm in size after extrusion, which are likely liposome-like structures systems (Figure 2A). Under
these conditions, at room temperature, the oleyl chains are in a “fluid” state allowing the formation of
fluid bilayers. The formation of vesicular systems is consistent with previous reports on phosphocholine
xxii
16
uridine derivatives and DOTAU .
Transmission electronic microscopy (TEM) experiments performed in the presence of siRNA,
with a mixture of KNL and siRNA (ratio = KNL/siRNA: 10) reveal the spontaneous formation of
nanoparticles (Figure 2C) with multilamellar organizations similar to lipoplexes (cationic liposome-
nucleic acid complexes) with cationic lipids (Figure 2B and 2D, incubation times of siRNA with KNL

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for 10 and 1 minutes respectively). As seen on TEM image taken at an early stage, with an incubation
time of 1 minute (Figure 2D), the formation of the KNL-siRNA lipoplexes is due to the interactions
occurring between KNL based liposomes and siRNA. The resulting self assembly involving both KNL
and siRNA molecules leads to well organized nanoparticles where the siRNA is trapped and protected
between two KNL bilayers (Figure 2G).
The Zeta potential and size distribution profile of this nucleolipid, alone and in the presence of
Figure 2.
nucleic acids (N/P ratio : KNL/siRNA= 10), were obtained via electrophoretic mobility and Dynamic
Light Scattering (DLS) measurements. The results show a decrease of the ζ-potential (Figure 2F),
accompanied by an increase of size distribution in the presence of siRNA (Figure 2E), suggesting its
complexation with KNL vesicles. The results demonstrate unambiguously that KNL formed nanometer-
scale compact spherical complexes entrapping siRNA. These complexes are ~ 100 nm in diameter

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(
Figure 2C and 2E), a size that is important for efficient endocytosis-mediated cell uptake. The size of
the complexes is known to affect delivery efficacy and small particles (up to ~ 150 nm) are typically
preferred for in vivo applications.
Fluorescence and gel migration studies.
L increases
Figure 3.
Migration in agarose gel of luciferase siRNA, 1.6 nM/well in
-
9
the presence of KNL (0.58 x 10 mole / μL). The best
complexation is observed for a N/P ratio higher than 1.8 (line
μL of KNL
Figure 4.

N/P

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Luciferase down regulation assay siRNA Transfection assays.

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Blue fluorescence image of
the nucleus
Red fluorescence image of the
labeled siRNA
Merged fluorescence image of 1
and 2
Figure 5: Study of the KNL-mediated uptake of siRNA in
PC-3 cells using red fluorescent dye siRNA.
Figure 6: Hsp27 protein levels analyzed by western blotting
3
4
days post-treatment (a) control (b) oligofectamine + OGX
27 (70 nM) (c) oligofectamine + SiRNA (40 nM) (d) KNL
+
OGX 427 N/P=3 (e) KNL + SiRNA N/P=3.

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Hsp27 protein, 27 kD
Hsp27 protein, 27 kD
siRNA 20 nM
siRNA 40 nM
KNL concentrations in nM
KNL concentrations in nM
A)
B)
Hsp27 protein, 27 kD
OGX 427 70 nM
KNL concentrations in microM (ratios with siRNA)
KNL concentrations in nM
C)
D)
Figure 7: Evaluation of protein expression demonstrates that KNL-mediated Hsp27 siRNA (A and B) or antisense (C)
delivery and gene silencing effects depend on N/P ratios. D) Cell viability was assessed using a MTT assay realized on
PC3 cells, which were incubated with KNL at different concentrations corresponding to different charge ratios.
Oligofectamine is used as positive control in these experiments.

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CONCLUSION
the ketal function inserted into the nucleolipid structure facilitates the delivery of siRNA and
gene silencing activity. Considering the high transfection efficiency observed of the KNL nanoparticles
for
, we evaluated the ability of KNL to transfect therapeutic small nucleic acids
including siRNA and antisense oligonucleotides. Importantly, in this work we demonstrate

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