Anticancer siRNA delivery by new anticancer molecule: A novel combination strategy for cancer cell killing

Article abstract of DOI:10.1016/j.ejmech.2012.07.035

The present report describes development of a novel, bifunctional molecule possessing both selective antiproliferative activity and siRNA transfection ability. We synthesized a series of cationic lipo-benzamides and screened for in vitro anticancer activities against a panel of cancer and non-cancer cells. The molecule with a ten carbon chain-length (C10M) significantly inhibited proliferation of cancer cells via arresting the cell cycle predominantly in the G1 phase; but did not affect non-cancerous cells. C10M effectively mediated siRNA delivery in vitro. The combined anticancer effect of the delivery of C10M together with its survivin-targeting siRNA cargo was significantly (p < 0.05) superior to that of agent alone. To our knowledge, this is the first report of a dual-purpose molecule with intrinsic anticancer activity and suitability for use in siRNA delivery.

Full text of DOI:10.1016/j.ejmech.2012.07.035

European Journal of Medicinal Chemistry 56 (2012) 400e408
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Anticancer siRNA delivery by new anticancer molecule: A novel combination
strategy for cancer cell killingq
,
,
Prathap Reddy Muktapuram a 1, Rishi Kumar Gara b 1, Komal Sharma a, Chilappa Rohit c,
Kolupula Srinivas c, Durga Prasad Mishra b, Surendar Reddy Bathula a
,
*
a Division of Pharmaceutics, CSIR-Central Drug Research Institute, Chattar Manzil Palace, 1-M.G. Marg, Lucknow 226001, India
b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India
c Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER-Hyderabad), Balanagar, Hyderabad 500037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The present report describes development of a novel, bifunctional molecule possessing both selective
antiproliferative activity and siRNA transfection ability. We synthesized a series of cationic lipo-
benzamides and screened for in vitro anticancer activities against a panel of cancer and non-cancer
cells. The molecule with a ten carbon chain-length (C10M) significantly inhibited proliferation of
cancer cells via arresting the cell cycle predominantly in the G1 phase; but did not affect non-cancerous
cells. C10M effectively mediated siRNA delivery in vitro. The combined anticancer effect of the delivery of
C10M together with its survivin-targeting siRNA cargo was significantly (p < 0.05) superior to that of
agent alone. To our knowledge, this is the first report of a dual-purpose molecule with intrinsic anti-
cancer activity and suitability for use in siRNA delivery.
Received 17 April 2012
Received in revised form
17 July 2012
Accepted 18 July 2012
Available online 27 July 2012
Keywords:
Anticancer molecule
Anticancer siRNA
Combination therapy
Survivin gene
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
to a single agent in curing cancer [11]. Developing a selective
antiproliferative agent which can deliver siRNA is challenging but
RNA interference (RNAi), an evolutionarily conserved ubiquitous
gene silencing mechanism, has potential for clinical development
of nucleic acid therapeutics [1]. Recently, several new RNAi-based
drugs have been translated into clinical testing [2]. The delivery
vector is crucial for clinical success of therapeutic RNAi [3]. To fully
exploit the therapeutic potential of RNAi in cancer therapy, various
small interfering RNA (siRNA) delivery strategies have been
developed, including stable nucleic acid-lipid particles (SNALP)
formulations that encapsulate siRNA designed to silence polo-like
kinase 1(PLK1) [4], siRNAelipoplexes made up of cationic lipid
AtuFECT01Ò and siRNA against protein kinase N3(PKN3) [5],
cyclodextrin nanoparticle carriers of siRNA for solid tumors
(CALAA-01) [6], siRNA in complex with anionic liposomes [7], self
assembled liposomeepolycationeDNA (LPD) nanoparticles [8],
cationic lipids [9], and chemically modified siRNAs [10].
has great potential toward the development of siRNA mediated
therapeutic intervention in cancer.
PEG coated polymeric nanoparticles are the most promising
nonviral vectors for systemic delivery of siRNA [12]. PEGylation not
only reduces the toxicity of cationic lipids (CL) and cationic poly-
mers such as polyethyleneimine (PEI) but also inhibits particle
aggregation [13]. Self-assembled, LPD (liposomeepolycationeDNA
complex) is a potent nanocarrier for systemic delivery of siRNA
[14]. In a recent study we have shown that lipidepolycationeDNA
(LPD) nanoparticles fabricated using an apoptosis-promoting
cationic lipid, DSGLA (N,N-distearyl-N-methyl-N-2[N0-(N2-guani-
dino-L-lysinyl)] aminoethyl ammonium chloride), significantly
inhibited tumor growth in comparison to LPD constructed using the
known cationic lipid, 1,2-di-(9Z-octadecenoyl)-3-trimethylammo-
nium-propane (DOTAP) [15]. In another study, we explored the
potential of DSAA (N,N-distearyl-N-methyl-N-2-(N0-arginyl) ami-
noethyl ammonium chloride); a cationic lipid structurally similar to
DSGLA in overcoming drug resistance in tumors [16]. The LPD
nanoparticles containing either DOTAP (DOTAP-LPD) or DSAA
(DSAA-LPD) efficiently delivered myelocytomatosis cellular onco-
gene (c-Myc) siRNA into melanoma cells. However, tumor growth
inhibition was more prominent with DSAA-LPD nanoparticles.
These siRNA delivery strategies are based on single agent ther-
apies, but combinational therapy is more effective when compared
q
CDRI Communication No-8288 2.
* Corresponding author. Tel.: þ91 522 2612411x4358; fax: þ91 522 2623405.
E-mail address: bsreddy@cdri.res.in (S.R. Bathula).
These two authors contributed equally.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.035

P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
401
Interestingly, co-administration of Paclitaxel (PTX) and DSAA-LPD
completely inhibited the B16F10 tumor [17]. These studies
demonstrate the importance of a potentially bioactive lipid as
a delivery vector possessing intrinsic therapeutic activity.
The objective of this study was to design a bifunctional molecule
that can selectively kill cancer cells and deliver anticancer siRNA. N-
(2-Dialkylaminoalkyl)-substituted benzamides are important class
of bioactive small molecules with a wide range of biological activ-
ities such as anti-emetic (Metoclopramide) and anti-psychotic
(Tiapride); and have also been extensively investigated as diag-
nostic probes for malignant melanoma and sigma receptor target-
ing ligands [20]. Melanin-targeted, radioiodinated benzamides
such as N-(2-diethylaminoethyl)-4-iodobenzamide (BZA) and N-
The distinctive novel structural features common to the cationic
benzamide derivatives reported here include: (a) The presence of
hydrophobic groups which are directly linked to the positively
charged nitrogen atom and (b) the presence of 4-methox-
ybenzamide group. As shown in Scheme 1, anisoylchloride reacted
with mono-Boc-protected ethylenediamine to generate tert-
butyl(2-(4-methoxybenzamido)ethyl)-carbamate.
Boc
depro-
tection of the anisamide derivative using TFA/DCM followed by
alkylation with K2CO3/respective alkyl bromide offered the twin
chain tertiary amine intermediates with C2eC18 carbon atoms.
Subsequent quaternization of nitrogen with methyl iodide resulted
in the formation of target cationic methoxybenzamide amphiphiles.
All compounds were characterized using 1H NMR and mass spec-
trometry. The purity of these compounds was ascertained by TLC,
13C NMR, HRMS and differential scanning calorimetry in case of
C10M (Fig. S2). The % purity of all analogs was confirmed by HPLC
(Fig. S3) as ꢀ96%.
(2-diethylaminoethyl)-3-iodo-4-methoxybenzamide
(IMBA)
showed promise as tumor-imaging agents in pre-clinical studies for
the scintigraphic detection of melanoma [21]. Earlier we have re-
ported that the conjugation of an eight carbon twin chain cationic
lipid moiety to 17b-estradiol generated a new antiproliferative
agent that significantly inhibits the proliferation of breast cancer
cells independent of estrogen receptor expression, without harm-
ing normal cells [18]. In a separate study Pal and co-workers
developed a new class of cationic lipid-conjugated haloperidol
derivatives [19]. However, nucleic acid delivery to cytosol was not
reported for these classes of molecules.
2.2. Biological experiments
2.2.1. Cytotoxicity studies
Novel cationic benzamide derivatives with varying number
(C2eC18) of side chain carbon atoms namely C2MeC18M were
evaluated for in vitro anticancer activity using a CCK-8 assay across
We hypothesized that synthesis of cationic N-(2-
different molar concentrations (0.5,1, 2.5, 5,10, 20 mM). Briefly, cells
dialkylaminoalkyl)-substituted benzamides with
length alkyl chain (C7eC12) might yield a new anticancer mole-
cule with siRNA delivery potential. Toward this goal, we have
a medium-
(3e5 ꢁ 103 cells/well) were cultured in 96 well tissue culture plates
and treated with different concentrations of benzamide derivatives
synthesized
a
series of [2-(4-methoxy-benzoylamino)-ethyl]-
for 48 h. At the end of incubations, 10 mL of CCK-8 solution was
added to each well and incubated for 3 h. Absorbance was recorded
at 540 nm using Fluostar Omega spectrofluorimeter (BMG Labtech,
Offenburg, Germany). The growth-inhibitory effects were studied in
four human cancer cell lines, DU-145 & PC-3 (Human prostate
cancer cells), MCF-7 (breast adenocarcinoma), HepG2 (hepatocel-
lular carcinoma) and four non-cancer cell lines, WPMY-1 (prostate),
HEK-293 (Human embryonic kidney), COS-7 (African green monkey
kidney), NIH/3T3 (mouse embryo fibroblast) in order to determine
their cyto-selective nature. The results are presented in Table 1. IC50
values were based on doseeresponse curves. Compounds with
medium-length alkyl chains (C7eC12) displayed a concentration-
dependent cytotoxic profile in all four cancer cell lines. Among
the benzamides, C10M significantly inhibited the proliferation of all
four cancer cells at all concentrations (IC50 range from 3.59 to
methyl-dialkyl-ammonium iodides with varying number of side
chain carbon atoms starting from two to eighteen carbons and
evaluated as selective anticancer agents against four human cancer
cell lines and four noncancer cell lines. Further we have tested the
in vitro siRNA delivery efficacy of most active cationic lipo-
benzamide, C10M by tracking uptake of fluorescein labeled-siRNA
in DU-145 cells. Finally we have studied the combined anticancer
effect of both the antiproliferative lipo-benzamide C10M and
anticancer siRNA, using siRNA targeting to the survivin gene. Sur-
vivin is an anti-apoptotic protein that is basally-expressed in
normal tissue and overexpressed in nearly all human cancers. The
expression of survivin in tumor cell lines increases with prolifera-
tion rate and resistance to therapy. Survivin is therefore a potential
target for the development of novel anticancer therapies. It is also
known from the literature that knockdown of survivin expression
results tumor growth inhibition [22].
8.12
mM) without affecting the normal cells. The compounds having
IC50 value more than 20
mM, were considered inactive.
2.2.2. Lipo-benzamide derivative with ten-carbon chain (C10M)
induces apoptosis
2. Results and discussion
We first considered induction of apoptosis as a mechanism
whereby C10M could kill cancer cells. We performed the Annexin
V/Propidium iodide (PI) binding assay to quantitatively differen-
tiate between apoptotic and necrotic cells. For this experiment,
2.1. Chemistry
The structures and general synthetic strategies adopted for
preparing cationic benzamide derivatives are outlined in Scheme 1.
cells were treated with 5 mM C10M for 24 h and harvested in PBS
Scheme 1. Outlines the synthetic strategy employed for preparing the novel cationic lipo-benzamides described in the present investigation. (a) Triethylamine, THF, rt, 5 h (b) TFA
and DCM (1:1), rt, 1.5 h (c) ReBr, ethyl acetate, K2CO3, 70 ꢂC, 12 h (d) DCM, CH3I, rt, 3 h. For C2M: R ¼ C2H5, C3M: R ¼ C3H7, C4M: R ¼ C4H9, C5M: R ¼ C5H11, C6M: R ¼ C6H13, C7M:
R ¼ C7H15, C8M: R ¼ C8H17, C9M: R ¼ C9H19, C10M: R ¼ C10H21, C11M: R ¼ C11H23, C12M: R ¼ C12H25, C13M: R ¼ C13H27, C14M: R ¼ C14H29, C15M: R ¼ C15H31, C16M: R ¼ C16H33
,
C17M: R ¼ C17H35 and C18M: R ¼ C18H37
.

402
P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
Table 1
Anticancer activity (IC50, mM) of novel lipo-benzamides.
Compound
R
Cells originated from cancer tissue
Cells originated from non-cancer tissue
(IC50 mM)
HepG2
PC-3
MCF-7
DU-145
COS-7
NIH/3T3
HEK-293
WPMY-1
C2M
C3M
C4M
C5M
C6M
C7M
C8M
C9M
C10M
C11M
C12M
C13M
C14M
C15M
C16M
C17M
C18M
C2H5
C3H7
C4H9
C5H11
C6H12
C7H15
C8H17
C9H19
>20
>20
>20
>20
>20
5.449
8.679
>20
5.309
>20
>20
13.86
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
17.63
11.91
>20
3.645
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
7.55
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
10.38
15.45
>20
3.957
7.539
11.39
6.830
10.41
>20
1.596
2.199
16.69
18.14
6.659
11.34
7.67
12.18
>20
>20
C
10H21
C11H23
C12H25
C13H27
C14H29
C15H31
C16H33
C17H35
>20
>20
>20
>20
>20
C
18H37
and analyzed using
a
flow cytometer. Dual staining clearly
did not significantly affect the phase distribution of the cell cycle in
DU-145 cells, C10M triggered a significant increase of cells in the
sub-G0 phase (Fig. 1c). In case of non-cancer cells HEK-293, (Fig. 1d)
C8M and C10M had measurable effects on the phase distribution of
the cell cycle. The above results demonstrate that the apoptosis
inducing ability of C10M in cancer cells correlates with its ability to
arrest the cell cycle in the G0/G1 phase. Further experiments are
needed and work is in progress to address the significance of the
observation that the extent of apoptosis induction and cell cycle
arrest differ in the case of DU-145 and PC-3 cells.
discriminated between early apoptotic (Annexin positive), late
apoptotic (dual positive) and necrotic (only PI positive) cells [23]. As
can be seen in Fig. 1a, exposure to C10M resulted in increased
percentage of early apoptotic cells from 3% in the untreated control
group to 23% in the treated group in DU-145 cells. In case of PC-3
cells as well, C10M increased the percentage of both early and
late apoptotic cells in comparison to the untreated control. Early
apoptotic cells rose from 1% in the untreated group to 51.48% in
C10M-treated group. Late apoptotic cells increased from 1% to
21.49% in untreated and treated groups respectively. In the non-
cancerous WPMY-1 cells, C10M treatment did not increase the
number of apoptotic cells in comparison to the untreated control
(Fig. 1a). These observations demonstrate that C10M induces
apoptosis in cancer cells but not in cells of non-cancer origin.
2.2.4. Molecular docking studies
In this study, we demonstrated that among the structurally similar
lipo-benzamides, C10M induced significant growth inhibition of all the
tested human cancer cells (Table 1). We observed induction of apoptosis
in C10M treated prostate cancer cells and G0/G1 phase arrest in C10M
treated PC-3 cells. It is well known that regulation of the cell cycle
involves series of tightly integrated events, involving cyclins and cyclin-
dependent kinases (CDKs); and inhibition of the above proteins cause
cell cycle arrest [24e26]. It is also reported that CDK2 inhibition causes
G0/G1 phase arrest [27]. We therefore attempted to dock C7M through
C12M in the active site of the human CDK2 protein structure (Pdb ID:
1CKP). Glide flexible docking simulations were performed using Glide,
version 5.5 [28]. The results are presented in Table 2. Molecular docking
results indicate that C10M has stronger binding affinity (Glide
score ꢃ1.6 kcal/mol) compared to its analogs (C7MeC9M, C11M &
C12M). These results are consistent with experimental observations of
the extent of cell cycle arrest induced by this series of compounds. The
theoretical binding mode of the proteineligand interactions in respect
of C10M is presented in Fig. 1e, which suggests hydrogen bonding
interactions with Asn 136 and Lys 291. The in silico studygives an insight
about the molecular level interactions.
2.2.3. Effect of lipo-benzamides on cell cycle progression
We also independently evaluated whether arrest of cell cycle
progression could be a mechanism of antiproliferative activity of the
lipo-benzamides. Cancer cells (PC-3, DU-145 & HEK-293) treated
with 5 mM each of compounds C7M, C8M, C9M, C10M, C11M and
C12M for 24 h were permeabilized, stained with PI and subjected to
flow cytometric analysis. Histograms of flow cytometric data are
shown in Fig. 1bed. The histograms were quantitatively analyzed
using a curve-fitting program to determine the percentage of cells in
each of the G0/G1, S, and G2/M phases. Consistent with the results
obtained in the cell proliferation and apoptosis induction assays,
C10M significantly caused G0/G1 phase arrest with induction of
extensive apoptosis in PC-3 cells. In Fig. 1b, cells in the G0/G1 phase
increased from 44.20% in controls to 53.83%, 62.97%, 54.06%, 69.49%,
50.20% and 50.81%, after treatment with C7M, C8M, C9M, C10M,
C11M and C12M respectively. While 5 mM of other lipo-benzamides

P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
403
Fig. 1. a). Flow cytometry was done for detection of apoptosis in DU-145, PC-3 and WPMY-1 cells after treatment with 5 mM of C10M for 24 h. Treated cells were trypsinized and
washed with PBS, and apoptosis studies were performed by annexinV-FITC kit as per manufacturer’s instructions. Stained cells were analyzed by FACScan flow cytometer (Becton
Dickinson, USA). All the experiments were performed at least three times. Values are expressed as mean ꢄ SEM of two independent experiments performed in duplicate. The
difference in the data obtained between An-control and An-treated cells is statistically significant in both DU-145 and PC-3 cells (**p < 0.001), whereas in WPMY-1 cells difference
in the data is statistically insignificant (p < 0.26). b). Cell cycle profiles of 5 mM of C7M, C8M, C9M, C10M, C11M and C12M treated PC-3 cell. For cell cycle analysis cells were fixed in
70% ethanol at ꢃ20 ꢂC for 1 h, washed twice with PBS and treated with 1 mg/mL RNase for 30 min at 37 ꢂC. Cellular DNA was stained with 50
m
g/mL PI in PBS. Stained cells were
M of
analyzed by FACScan flow cytometer (Becton Dickinson, USA). All the experiments were performed at least three times. c). Cell cycle analysis of DU-145 cells treated with 5
m
C7M, C8M, C9M, C10M, C11M and C12M compounds. Untreated cells (blank) were used as control. d). Effect of antiproliferative lipo-benzamides such as C7M, C8M, C9M, C10M,
C11M and C12M on the cell cycle of, non-cancerous, HEK-293 cell line. Cells were incubated in the presence of the lipo-benzamide compounds for 24 h. Aliquots of cells were taken
in duplicate for cell cycle analysis by flow cytometry using propidium iodide staining. The data are the average of two separate experiments using different batches of cells.
e). Docking pose of C10M molecule in the active site of CDK2 target (Pdb ID: 1CKP). C10M is depicted in ball and stick model. The key H-bond interactions between the compounds
and the residues of the binding pocket are highlighted as dotted lines.

404
P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
Fig. 1. (continued).
2.3. Transfection biology
precipitated thereafter (data not shown). The above formulation was
used in all further experiments without removing ethanol.
We tested the efficiency of siRNA delivery of the selective anti-
proliferative agent C10M in DU-145 cells by tracking uptake of fluo-
rescein labeled-siRNA (F-siRNA). We also measured the combined
anticancer efficacies of both the anticancer lipid and anti-survivin
siRNA in these cells. For that we prepared self-assembled C10M
nanostructures by two stage ethanol injection. In the first step, 2.5 mg
We measured the hydrodynamic diameters of self-assembled
C10M nanostructures alone or as siRNA/C10M complexes to
further characterize them. Strikingly, the sizes of C10M/siRNA
complexes varied within a narrow range (200e240 nm) across the
entire series of lipid/siRNA ratios and was smaller as compared to
self assembled C10M (Table S1). This decrease in size of complexes
than C10M indicates the siRNA compacting efficacy of C10M.
of C10M was dissolved in 125
ethanol solution of C10M was added rapidly to 250
solution in a 1.5 mL centrifuge tube. The aqueous phase immediately
mL of absolute ethanol and the entire
mL of 5% mannitol
2.3.1. Cellular uptake of C10M/siRNA complexes
turned milkyas a resultof self-assembly. In the next stage, 74
above milky aqueous phase was rapidly injected into 926
mannitol solution to prepare cationic vesicles of the antiproliferative
lipo-benzamide. The 1 mM formulation thus prepared was stable for
three days at ambient temperature, but the compound slowly
m
L of the
The efficiency of intracellular siRNA delivery by self-assembled
C10M was evaluated by tracking uptake of fluorescein labeled-
siRNA (F-siRNA) in DU-145 cells by flow cytometry. For this study,
mL of 5%
1
mg of F-siRNA diluted to 100
mL with Roswell Park Memorial
Institute (RPMI) culture medium was complexed with varying

P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
405
Fig. 1. (continued).
amounts of C10M cationic vesicles (1:5, 1:10, 1:20, 1:25 weight
ratio) diluted to 100 L with RPMI and incubated for 30 min. The
resultant C10M/siRNA complexes and free F-siRNA was further
of lipoplex. DU-145 cells seeded in 6 well plates were incubated
with the lipoplex for 12 h. After incubation, cells were washed
twice with PBS and analyzed by flow cytometry. Results of cellular
uptake of uncomplexed siRNA as well as C10M complexed
fluorescein-labeled siRNA in DU-145 cells are summarized in the
bar graph in Fig. 2a and the fluorescence intensity distribution
histograms are presented in Fig. S1. Free siRNA, as expected,
exhibited very poor uptake efficiency presumably due to the
repulsive interactions between negatively charged siRNA and the
negatively charged cell membrane. After complexation with C10M
higher F-siRNA uptake efficiency was observed and it was further
increased with increasing lipid concentration. It is evident from
the above results that the cationic benzamide could efficiently
deliver siRNA to the tumor cells and the delivery was concentra-
tion dependent.
m
diluted with 300 mL of RPMI containing 10% FBS to prepare 500 mL
Table 2
Molecular docking scores of molecules C7MeC12M into the active site of CDK2
target (Pdb ID: 1CKP).
Molecule no.
Glide score (kcal/mol)
C10M
C8M
C7M
C9M
ꢃ1.6
ꢃ0.8
ꢃ0.3
1.5
C12M
1.6
C11M
2.1
Reference (Co-crystallized ligand: Purvalanol B)
ꢃ1.8

406
P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
Fig. 2. siRNA transfection and combinational cell killing affect of anticancer siRNA and bifunctional lipo-benzamide. a) Cellular uptake, as determined by flow cytometry of
Fluorescein-labeled siRNA and complexes formed with C10M compared to untreated DU-145 (control). Error bars represent the standard error of the mean. siRNA delivery efficacy
of C10M in DU-145 is statistically significant, *p < 0.05. b) Semi quantitative RT-PCR detection of endogenous survivin mRNA silencing in DU-145 cells by anticancer delivery
vehicles. C10M transfection efficiency was measured using survivin siRNA and compared with controls. Only, Survivin-siRNA and C10M used as normal controls; C10M þ control
siRNA used as negative control. c) Combined in vitro antiproliferative activity of C10M: survivin siRNA (25:1 weight ratio) complex. Cells were treated with siRNA alone, C10M
dissolved in DMSO, self assembled C10M, C10M control siRNA complex and C10M survivin siRNA complex for 12 h followed by replacement of the existing media with fresh media
and further incubation for 12 h. Antiproliferative activity was assayed by MTT reagent. The Y-axis represents % viability. Each data point is the representation of triplicate treatments.
*Represents p < 0.05.
2.3.2. Gene silencing efficiency of siRNA/C10M complex
lipoplex, 5
transferred to a 1.5 mL eppendorf tube and 95
added. To the resultant mixture, siRNA suspended in 100
RPMI was added and the whole mixture incubated for 30 min.
After incubation, 300 L of RPMI containing 10% FBS were added
to the resulting lipoplex for transfection experiments. Following
24 h of incubation with the lipoplex, cells were lysed and RNA
isolated using Trizol reagent (Invitrogen, USA) as per the manu-
facturer’s instructions. Total RNA was reverse transcribed by Super
script III, (Invitrogen, USA) cDNA synthesis kit as per the manu-
facturer’s instructions. Thereafter, RT-PCR was performed on sur-
vivin and 18 S ribosomal RNA using the PlatinumÒ PCR SuperMix
(Promega corporation, USA). The amplification conditions are
described in Supporting information. The amplified sequences
were resolved on a 1.4% agarose gel and visualized using 0.1%
m
L of, 1 mM self assembled C10M formulation was
L of RPMI was
L of
To further demonstrate the endogenous gene silencing effi-
ciency of the C10M/siRNA formulation, siRNA against survivin was
delivered to the DU-145 cells and survivin mRNA levels were
determined by semi-quantitative reverse transcription polymerase
chain reaction (RT-PCR) analysis 24 h post-transfection. Briefly,
1.5 ꢁ 105 DU-145 cells were seeded in 6 well plates, after 80%
confluence cells were treated either with 100 nM siRNA (control
m
m
m
or survivin) or 100 nM siRNA þ 2.5
mg C10M (1:25) complex for
12 h, after that siRNA and treatment containing media was care-
fully removed, fresh media was added and cells were further
incubated for 24 h. DU-145 cells were treated with 100 nM of
human survivin siRNA alone or complexed with 2.5
assembled C10M (1:25, siRNA:lipid weight ratio) to provide
a measure of target gene silencing at the mRNA level. To prepare
mg self

P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
407
ethidium bromide under UV light. The mRNA expression levels of
4.2. Statistical analysis
survivin were decreased significantly by treatment with C10M
survivin siRNA complexes, but the individual treatment of either
siRNA or cationic compound and control siRNA C10M complex did
not affect survivin levels (Fig. 2b).
All data are expressed as mean ꢄ SE. Differences between
groups were explored using analysis of variance (ANOVA). Wher-
ever the ANOVA indicated a significant difference, individual
differences were explored using t tests with a Dunnett or Turkeys
correlation multiple comparisons. Differences were considered
statistically significant for at *p < 0.05. All the statistical analysis
was performed using Graph Pad Prism version 4.00 for Windows
(Graph Pad Software, USA).
2.3.3. Combined anticancer effect of anticancer lipid and siRNA
We tested the combined effect of anticancer siRNA and lipo-
benzamide (C10M) on cancer cell killing using the MTT reduc-
tion assay. Five groups composed of 5 ꢁ 103 cells treated with
either 100 nM survivin siRNA alone, 5 mM C10M in DMSO, 5 mM
self assembled C10M, or 100 nM control or survivin siRNA
complexed with 5 M C10M complexed with 5 M C10M were
4.3. Chemical methods
m
m
incubated with DU-145 cells for 12 h. Following incubation,
siRNA and C10M containing media was carefully removed, fresh
media was added and cells were further incubated for 24 h.
4.3.1. Synthesis of the cationic lipo-benzamide derivatives
Scheme
1 outlines the synthetic strategy employed for
preparing the cationic lipo-benzamide derivatives described in the
present study. Detailed experimental procedure for the synthesis of
C10M is delineated below.
Cultures were treated with 10 mL of MTT for a final 3 h, formazan
solubilized with DMSO-methanol 1:1 and absorbance read at
540 nm. The data indicate that survivin siRNA delivered by C10M
had significantly higher antiproliferative activity than C10M
(p < 0.05) and control siRNA had no effect on cell survival
(Fig. 2c). Further, free survivin siRNA did not induce any cyto-
toxicity due to the lack of spontaneous cellular entry. Interest-
ingly, self assembled C10M had higher antiproliferative activity
than its DMSO solution.
4.3.1.1. Synthesis of tert-butyl 2-(4-methoxybenzamido)ethyl-
carbamate (1). To a solution of tert-butyl 2-aminoethylcarbamate
(2.01 g, 12.56 mmol) in a mixture of triethylamine (3.49 mL,
25.1 mmol) and THF (20 mL), anisoylchloride (1.93 mL,
14.25 mmol) was added drop wise and reaction mixture was
stirred at room temperature for 3 h. After completion of the
reaction whole reaction mixture was diluted with 50 mL of ethyl
acetate, washed with water (1
ꢁ
50 mL), brine solution
3. Conclusions
(1 ꢁ 50 mL) and finally dried with anhydrous sodium sulfate.
Column chromatographic purification (using 60e120 mesh silica
gel and 4% methanol in chloroform as eluting solvent mixture) of
the residue afforded 1 (Scheme 1) (1.7 g, 46% yield, Rf: 0.8 in 10%
methanol in chloroform).
The present report describes the discovery of a novel bifunc-
tional, cationic lipo-benzamide (C10M) which induces apoptosis
selectively in prostate cancer cells similar to targeted anticancer
agents and also self assembles and delivers siRNA. The compound is
easy to synthesize and both the antiproliferative activity and siRNA
delivery capability are highly efficient and consistent. To verify the
potential of C10M as a combinational antitumor agent in vivo, we
are currently pursuing the targeted delivery of anticancer siRNA
and paclitaxel in mouse models of breast and prostate cancer. Our
recent research effort is also focused toward the development of
bifunctional cationic molecule (serving as both, a formulation
component as well as a therapeutic agent) containing multifunc-
tional nanoparticles to effectively treat cancer.
1H NMR (200 MHz, CDCl3)
d
¼ 1.39 [m, 9H{NHCOOC(CH3)3}],
3.08e3.2 {m, 2H(NH2CH2CH2)}, 3.3e3.4 {m, 2H(NH2CH2CH2)}, 3.85
{s, 3H(OCH3)}, 6.9e7.13 {m, 2H(m-Ph)}, 7.74e7.8{m, 2H(o-Ph)}, 8.32
{s, 1H(CH2CH2NH)}.
FABMS: m/z: 295 (M þ H)þ.
4.3.1.2. Synthesis of N-(2-didecylamino-ethyl)-4-methoxy-benza-
mide (2). Product obtained in the above step was dissolved in (1,
1.7 g, 1.78 mmol) a 1:1 mixture of trifluoroacetic acid and
dichloromethane. The reaction mixture was stirred at room
temperature for 90 min. TFA was removed under reduced pressure
by co-evaporating with toluene. Column chromatographic purifi-
cation (using 60e120 mesh silica gel and 8% methanol in chloro-
form as eluting solvent mixture) of the dried residue afforded
primary amine derivative (0.67 g, 66% yield, Rf: 0.4 in 10% methanol
in chloroform).
To, ethyl acetate (10 mL) solution of above free amine derivative
(0.67 g, 3.45 mmol) K2CO3 (2.34 g, 13.8 mmol) and 1-decyl bromide
(1.82 mL, 8.8 mmol) were added. The whole mixture was refluxed
at 70 ꢂC for 12 h. After completion of the reaction, the reaction
mixture further diluted with 20 mL of ethyl acetate and washed
with water (2 ꢁ 20 mL). The organic layer dried with anhydrous
Na2SO4 and concentrated in vacuum. Column chromatographic
purification (using 60e120 mesh silica gel and 35% ethyl acetate in
hexane as eluting solvent) of the residue afforded alkylated ben-
zamides 2 (Scheme 1) (0.73 g, 45% yield, Rf: 0.4 in 10% methanol in
chloroform).
4. Experimental protocols
4.1. Biological methods
The size of the self assembled C10M was obtained from Malvern
Instruments Zetasizer nano-ZS. The human cancer cell lines e.g.
prostate cancer cells (PC-3 and DU-145), MCF-7, HepG2 and normal
non-transformed cell types WPMY-1, NIH/3T3, COS-7 used in the
present study were obtained from the ATCC (American Type Culture
Collection, USA). Cell viability measurement kit (CCK-8), was
purchased from the Dojinando Laboratories (Kumamoto, Japan). The
sense 50-fluorescein-modified siRNA duplex was obtained from
Dharmacon. Sequence of 50-fluorescein siRNA: Sense: 50-CUU ACG
CUG AGU ACU UCG AdTdT-30, Antisense: 50-UCG AAG UAC UCA GCG
UAA GdTdT-30. Control siRNAs, and survivin siRNA were procured
from SigmaeAldrich. Sequence of siRNA against human survivin:
Sense: 50-GGA CCA CCG CAU CUC UAC AdTdT-30, Antisense: 50-UGU
AGA GAU GCG GUG GUC CdTdT-30. Sequence of scrambled siRNA:
Sense strand: 50-ACGCGUAACGCGGGAAUUUdTdT30 Antisense
strand: 50-AAAUUCCCGCGUUACGCGUdTdT-30. All detailed proce-
dures of biology experiments were included in Supporting
information.
N-(2-Didecylamino-ethyl)-4-methoxy-benzamide: Yield: 45%.
1H NMR N-(2-didecylamino-ethyl)-4-methoxy-benzamide
(300 MHz, CDCl3)
1.14e1.37 [m, 28H {NCH2CH2(CH2)7CH3}]; 1.63e1.74 [m, 4H,
{NCH2CH2(CH2)7CH3}]; 2.44 [t, 4H, 7.19 Hz, {NCH2CH2
(CH2)7CH3}]; 2.62 {t, 2H, J ¼ 5.46 Hz, (NCH2CH2)}; 3.46 {q, 2H,
d
¼ 0.87 [t, 6H, J ¼ 6.62 Hz, {N(CH2)9CH3}];
J
¼

408
P.R. Muktapuram et al. / European Journal of Medicinal Chemistry 56 (2012) 400e408
J ¼ 5.52 Hz, (NCH2CH2)}; 3.84 {s, 3H, (OCH3)}; 6.89e6.91 {m, 3H,
(m-Ph & NH)}; 7.74 {d, 2H, J ¼ 8.85 Hz, (o-Ph)}.
ESIMS: m/z: 475 (M þ H)þ.
References
[1] (a) K.A. Whitehead, R. Langer, D.G. Anderson, Nat. Rev. Drug Discov. 8 (2009)
129e138;
(b) K. Gao, L. Huang, Mol. Pharm. 6 (2009) 651.
4.3.1.3. Synthesis of didecyl-[2-(4-methoxy-benzoylamino)-ethyl]-
methyl-ammonium iodide (C10M). To the tertiary benzamide ob-
tained from above step (2, 0.73 g,1.54 mmol) in DCM (5 mL), methyl
iodide (15e20 equivalents) was added. The reaction mixture was
stirred at room temperature for 3 h. Solvent was removed under
[2] J.C. Burnett, J.J. Rossi, K. Tiemann, Biotechnol. J. 6 (2011) 1130e1146.
[3] S.C. Semple, A. Akinc, J. Chen, A.P. Sandhu, B.L. Mui, C.K. Cho, D.W. Sah,
D. Stebbing, E.J. Crosley, E. Yaworski, I.M. Hafez, J.R. Dorkin, J. Qin, K. Lam,
K.G. Rajeev, K.F. Wong, L.B. Jeffs, L. Nechev, M.L. Eisenhardt, M. Jayaraman,
M. Kazem, M.A. Maier, M. Srinivasulu, M.J. Weinstein, Q. Chen, R. Alvarez,
S.A. Barros, S. De, S.K. Klimuk, T. Borland, V. Kosovrasti, W.L. Cantley, Y.K. Tam,
M. Manoharan, M.A. Ciufolini, M.A. Tracy, A. de Fougerolles, I. MacLachlan,
P.R. Cullis, T.D. Madden, M.J. Hope, Nat. Biotechnol. 28 (2010) 172e176.
[4] B.L. Davidson, P.B. McCray Jr., Nat. Rev. Genet. 12 (2011) 329e340.
[5] D. Strumberg, B. Schultheis, U. Traugott, C. Vank, A. Santel, O. Keil, K. Giese,
J. Kaufmann, J. Drevs, Int. J. Clin. Pharmacol. Ther. 50 (2012) 76e78.
[6] M.E. Davis, J.E. Zuckerman, C.H. Choi, D. Seligson, A. Tolcher, C.A. Alabi, Y. Yen,
J.D. Heidel, A. Ribas, Nature 464 (2010) 1067e1070.
[7] M. Scherr, K. Battmer, T. Winkler, O. Heidenreich, A. Ganser, M. Eder, Blood
101 (2003) 1566e1569.
[8] S.D. Li, L. Huang, Ann. N. Y. Acad. Sci. 1082 (2006) 1e8.
[9] R. Srinivas, S. Samanta, A. Chaudhuri, Chem. Soc. Rev. 38 (2009)
3326e3338.
[10] (a) M. Nothisen, M. Kotera, E. Voirin, J.S. Remy, J.P. Behr, J. Am. Chem. Soc. 131
(2009) 17730e17731;
(b) O. Nakagawa, X. Ming, L. Huang, R.L. Juliano, J. Am. Chem. Soc. 132 (2010)
8848e8849.
[11] D. Lane, Nat. Biotechnol. 24 (2006) 163e164.
vacuum.
Column
chromatographic
purification
(using
60e120 mesh silica gel and 30% acetone in hexane as eluting
solvent) of the residue afforded quaternary benzamide C10M
(Scheme 1 representative structure indicated as CM) as a solid
materials (0.628 g, 83.5% Yield, Rf: 0.71 in 60% acetone in hexane).
1H NMR (300 MHz, CDCl3)
d
¼ 0.87 [t, 6H, {N(CH2)9CH3}];
1.13e1.37 [m, 28H, {NCH2CH2(CH2)7CH3}]; 1.63e1.72 [m, 4H,
{NCH2CH2(CH2)7CH3}]; 3.28 {s, 3H, (N þ CH3)}; 3.43e3.48 [m, 4H,
{NCH2CH2(CH2)7CH3}]; 3.81e3.84 {m, 5H, (OCH3 & NCH2CH2)};
3.99 {d, 2H, J ¼ 4.32 Hz, (NCH2CH2)}; 6.92 {d, 2H, J ¼ 8.75 Hz, (m-
Ph)}; 8.07 {d, 2H, J ¼ 8.84 Hz, (o-Ph)}; 8.53 {t, 1H, J ¼ 5.4 Hz,
(NHCH2CH2)}.
13C NMR (300 MHz, CDCl3)
d
¼ 167.7, 162.6, 129.6, 124.8, 113.7,
[12] L. Huang, Y. Liu, Annu. Rev. Biomed. Eng. 13 (2011) 507e530.
62.8, 60.5, 55.3, 49.4, 34, 31.8, 29.3, 29.2, 29.1, 26.2, 22.6, 22.5, 14.
ꢀ
[13] (a) G. Wang, C. Kucharski, X. Lin, H. Uludag, J. Drug Target. 18 (2010) 611;
ESIMS: m/z: 489 (M)þ.
(b) M. Ogris, S. Brunner, S. Schüller, R. Kircheis, E. Wagner, Gene Ther. 6 (1999)
595e605.
[14] J. Gao, W. Liu, Y. Xia, W. Li, J. Sun, H. Chen, B. Li, D. Zhang, W. Qian, Y. Meng,
L. Deng, H. Wang, J. Chen, Y. Guo, Biomaterials 32 (2011) 3459e3470.
[15] Y. Chen, J. Sen, S.R. Bathula, Q. Yang, R. Fittipaldi, L. Huang, Mol. Pharm. 6
(2009) 696e705.
[16] Y. Chen, S.R. Bathula, J. Li, L. Huang, J. Biol. Chem. 285 (2010) 22639e22650.
[17] Y. Chen, S.R. Bathula, Q. Yang, L. Huang, J. Invest. Dermatol. 130 (2010)
2790e2798.
[18] S. Sinha, S. Roy, S.R. Bathula, K. Pal, G. Sudhakar, S. Iyer, S. Dutta, E. Wang,
P.K. Vohra, K.R. Roy, P. Reddanna, D. Mukhopadhyay, R. Banerjee, Mol. Cancer
Res. 9 (2011) 364e374.
IR (KBr) values: 3282.3, 2925.4, 2855.2, 1652.3, 1535.7, 1254.3,
1038.2, 844.2, 769.1, 603 cmꢃ1
.
HRMS
(ESI)
calculated
for
C31H57N2O2:
489.7899
found ¼ 489.4486.
M.P: 120e122 ꢂC.
Other alkyl chain derivatives are prepared following the same
procedure and the details of the 1H NMR chemical shifts and
molecular ion peaks were included in Supporting information.
[19] K. Pal, S. Pore, S. Sinha, R. Janardhanan, D. Mukhopadhyay, R. Banerjee, J. Med.
Chem. 54 (2011) 2378e2390.
Acknowledgments
[20] R. Banerjee, P. Tyagi, S. Li, L. Huang, Int. J. Cancer 112 (2004) 693e700.
[21] M.F. Moreau, J. Michelot, J. Papon, M. Bayle, P. Labarre, J.C. Madelmont,
D. Parry, J.Y. Boire, N. Moins, H. Seguin, Nucl. Med. Biol. 22 (1995) 731e741.
[22] (a) G.Y. Miao, Q.M. Lu, X.L. Zhang, World J. Gastroenterol. 13 (2007)
1170e1174;
Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. This is CDRIeCSIR communication and Prathap
Reddy and R.K.G. thanks Council of Scientific Industrial Research
(CSIR), Government of India for a Ph.D. fellowship. Komal Sharma
thanks Department of Science and Technology (DST) for the
INSPIRE fellowship. Durga Prasad Mishra acknowledges financial
assistance from Ministry of Health, Government of India in form of
a grant ‘GAP-0001’. Surendar Reddy Bathula acknowledges finan-
cial assistance from CSIR, in a form of EMPOWER project. Amit
Misra’s help in manuscript preparation is acknowledged.
(b) X. Liu, R. Gao, Y. Dong, L. Gao, Y. Zhao, L. Zhao, X. Zhao, H. Zhang, BMC
Cancer 10 (2010) 1e13;
(c) H. Caldas, L.E. Honsey, R.A. Altura, Mol. Cancer 4 (2005) 1e9;
(d) D.C. Altieri, Oncogene 22 (2003) 8581e8589;
(e) D.C. Altieri, Cancer Lett., in press.
[23] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, J. Immunol.
Methods 184 (1995) 39e51.
[24] M.F. Tsou, H.F. Lu, S.C. Chen, L.T. Wu, Y.S. Chen, H.M. Kuo, S.S. Lin, J.G. Chung,
Anticancer Res. 26 (2006) 1965e1972.
[25] J.K. Buolamwini, Curr. Pharm. Des. 6 (2000) 379e392.
[26] J. Sridhar, N. Akula, N. Pattabiraman, AAPS J. 8 (2006) E204eE221.
[27] (a) T. Hashimoto, Z. He, W.Y. Ma, P.C. Schmid, A.M. Bode, C.S. Yang, Z. Dong,
Cancer Res. 1 (2004) 3344e3349;
Appendix A. Supporting information
(b) W. Qi, D. Qiao, J.D. Martinez, Radiat. Res. 157 (2002) 166e174;
(c) M.H. Lee, H.Y. Yang, Cancer Metastasis Rev. 22 (2003) 435e449.
[28] Glide, Version 5.5, Schrödinger, Inc., New York, NY, 2009.
Supporting information related to this article can be found
online at http://dx.doi.org/10.1016/j.ejmech.2012.07.035.