Bioorganic & Medicinal Chemistry Letters 28 (2018) 1161–1165

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Nano-chemotherapy using cationic liposome that strategically targets

the cell membrane potential of pancreatic cancer cells with negative
charge
Muneaki Motomura, Hideaki Ichihara, Yoko Matsumoto ⇑
Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1, Ikeda, Nishi-ku, Kumamoto 860-0082, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Negatively charged phosphatidylserine (PS) and sialic acid-containing glycosphingolipids (GM1) were
Received 22 January 2018 observed to be over represented on the cell membranes of pancreatic cancer cells (BxPC-3) as opposed
Revised 2 March 2018 to normal pancreatic cells. Cationic liposomes (CL) were also found to selectively accumulate into the
Accepted 4 March 2018
negatively charged cell membranes of BxPC-3 cells and inhibited their growth but have no effect on
Available online 5 March 2018
the viability of normal pancreatic cells. CL induced apoptosis in BxPC-3 cells via activation of caspase-
3, -8, and -9 and mitochondrial events and inhibited tumor enlargement in xenograft mouse models of
Keywords:
pancreatic cancer.
Cationic liposomes
Pancreatic cancer
Ó 2018 Elsevier Ltd. All rights reserved.
Apoptosis
Cell membrane potential
In vivo

The early stages of pancreatic cancer are often difficult to detect
and hence most cases are detected at the advanced stages of the
disease. Surgical removal of pancreatic cancer is often carried out
at the early stages of the disease. In cases where surgery is not
possible for advanced and metastatic pancreatic cancer, radiation
therapy, chemotherapy, or both are often carried out to limit tumor
growth. FOLFIRINOX (5-fluorouracil/Folinic acid/irinotecan/
Oxaliplatin) is one of the main drugs used in the combination
chemotherapy of advanced pancreatic cancer alongside1,2 gemc-
itabine which may also be given on its own.3 However, severe side
effects may be caused by anticancer chemotherapy.4,5
Hybrid liposomes (HL) can be prepared by just the sonication of
vesicular and micellar molecules in a buffer solution.6 High inhibi-
tory effects of HL without any anti-cancer drug on the growth of
tumor cells in vitro,7,8 in vivo9,10 and for clinical application have
been reported.11 HL are fused and accumulate in membranes of
tumor cells, and then induced apoptosis via activation of caspases
and mitochondrial event.7
We have produced novel hybrid cationic liposomes (CL) com-
posed of phospholipid, cationic lipid, and PEG surfactants.12 CL
(containing cationic lipid) exerted a marked inhibitory effect on
the growth of human renal cell carcinoma and colorectal cancer
and induced both in vitro and in vivo apoptosis in the absence of
drugs.12,13
⇑ Corresponding author.
E-mail address: matumoto@life.sojo-u.ac.jp (Y. Matsumoto).
The tumor marker commonly used for pancreatic cancer is
CA19-9 which contains anionic sialic acid. However, CL therapy
which targets negatively charged cell membranes of pancreatic
cancer cells has not yet been investigated.
In this study, we investigated the inhibitory effects of CL com-
posed of 87 mol% dimyristoylphosphatidylcholine (DMPC), 8 mol
% O,O0 -ditetradecanoyl-N-(a-trimethyl-ammonioacetyl) diethano-
lamine chloride (2C14ECl), and 5 mol% polyoxyethylene(21)dodecyl
ether (C12(EO)21) on the growth of pancreatic cancer (BxPC-3) cells
as well as its ability to induce apoptosis in vitro and in vivo. We also
analyzed the difference between the membrane potential of pan-
creatic cancer cells and normal pancreatic cells.14,15 Furthermore,
the nano-chemotherapeutic effect of CL was examined in vivo
using subcutaneous mouse models of pancreatic cancer.
CL were prepared by using sonication (VS-N300; VELVO, Tokyo,
Japan) of a mixture containing DMPC (purity > 99%; NOF Co. Ltd.,
Tokyo, Japan), micellar molecules: C12(EO)21 (purity > 99%; Sogo
Pharmaceutical Co. Ltd. Tokyo, Japan) and 2C14ECl (purity > 99%;
Sogo Pharmaceutical Co. Ltd. Tokyo, Japan) in 5% glucose solution
at 45 °C with 300 W, followed by filtration with a 0.20 lm filter.
We evaluated the inhibitory effects of CL on the growth of
human pancreatic cancer (BxPC-3) cells on the basis of the WST-
1 method.16 The IC50 values of CL were determined from a plot of
concentration of CL versus cell viability recorded at each test con-
centration (Fig. 1A). The IC50 value of CL obtained for BxPC-3 cells
were lower than those of the DMPC and HL, while the IC50 values
of CL for normal pancreatic cells was over 1000 lM. IC50 values

https://doi.org/10.1016/j.bmcl.2018.03.013
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
1162 M. Motomura et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1161–1165

(A) (C) Transmission TUNEL TO-PRO-3 Overlay

800
Control
IC50 (DMPC) [μM]

600

DMPC
400
*
200 HL

0
DMPC HL CL
CL
Normal pancreatic cells BxPC-3 cells

(B) 100
*
Apoptotic DNA rate [%]

80

60

40

20

0
Control DMPC HL CL

Fig. 1. (A) Inhibitory effects of CL on the growth of human pancreas cancer (BxPC-3) cells. The values here are represented as mean ± S.E. of three independent experiments.
Values of p < 0.05 were considered statistically significant. The p value was evaluated by Student’s t-test. *p < 0.01; versus DMPC and HL. (B) Apoptotic DNA rate for BxPC-3
cells treated with CL for 48 h. The values here are represented as mean ± S.E. of three independent experiments. Values of p < 0.05 were considered statistically significant.
The p value was evaluated by Student’s t-test. *p < 0.01; versus control, DMPC and HL. (C) Fluorescence micrographs of BxPC-3 cells using TUNEL method treated with CL for
24 h. CL:[DMPC] = 0.70 mM, [C12(EO)21] = 0.04 mM, [2C14ECl] = 0.064 mM. TUNEL (green) indicates apoptotic cells. TO-PRO-3 (red) indicates nucleus.

of CL for BxPC-3 cells was one-third or less that of normal pancre-
atic cells. DMPC, HL, and CL did not affects on the viability of nor-
mal pancreatic cells (IC50 > 1000 lM), which indicates non-toxicity
of those liposomes for normal pancreatic cells. These results indi-
cate that CL exerted a high level of selective inhibition on BxPC-3
cells without affecting the growth of normal pancreatic cells. The
induction of apoptosis in BxPC-3 cells by CL was examined using
flow cytometric analysis and the propidium iodide (PI) staining
method (Fig. 1B).17 Apoptotic DNA rates of CL for BxPC-3 cells were
higher than those of the DMPC and HL reaching a high apoptotic
rate of 90%. We observed DNA fragmentation of BxPC-3 cells
caused by CL using the TUNEL method and confocal laser micro-
scopy (Fig. 1C).18 Green color indicating apoptotic BxPC-3 cells
treated with CL was observed. No green coloring was observed
with the use of HL and DMPC. These results indicate that CL
induced apoptosis in BxPC-3 cells.
It is known that the membrane potential of cancer cells is
remarkably different from that of normal cells. We examined elec-
trostatic interactions between the cell membrane of BxPC-3 cells
and CL. We also tested for the presence of negatively charged
phosphatidylserine (PS) in the outer membrane of BxPC-3 cells
and normal pancreatic cells using the Annexin V-FITC staining
method by flow cytometry (Fig. 2A).19 Relative fluorescence
intensity of Annexin V-FITC in the cell membrane of BxPC-3 cells
was three times higher than that in normal pancreatic cells.
Subsequently, flow cytometry was used to measure the content
of gangliosides (GM1) containing sialic acid residues in the cell
membrane of BxPC-3 cells and normal pancreatic cells using the
subunit B cholera toxin (CTB) staining method (Fig. 2B).20 Relative
fluorescence intensity of GM1 for BxPC-3 cells was four times that
of normal pancreatic cells. The zeta potential of BxPC-3 cells and
normal pancreatic cells was also analyzed (Fig. 2C).21 Zeta poten-
tial in BxPC-3 cells (50 mV) was observed to be much lower than
that in normal pancreatic cells (18 mV). We also examined the
zeta potential changes of BxPC-3 cells treated with CL (Fig. 2D).21
Time-dependent increase of the zeta potential of BxPC-3 cells trea-
ted with CL from 50 mV to 20 mV was observed. These results
indicate that CL selectively targets BxPC-3 cells with negatively
charged cell membranes.
In order to understand how CL induces apoptosis in BxPC-
3 cells, we examined the fusion of CL containing NBDPC
((1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino]
dodecanoyl]-sn-glycero-3-phosphocholine (NBDPC)) as a fluores-
cence probe with the cell membrane of BxPC-3 cells using confocal
laser microscopy (Fig. 3A).22 Fusion of CL containing NBDPC with
the membrane of BxPC-3 cells was accelerated, occurring within
3 h, although less accumulation of HL was obtained. On the
contrary, there was no membrane fusion observed for BxPC-3 cells
treated with DMPC containing NBDPC. We investigated the
involvement of mitochondria in the induction of apoptosis of
BxPC-3 cells by CL (Fig. 3B).23 A decrease in mitochondrial trans-
membrane potential of BxPC-3 cells treated with CL was observed
suggesting that. CL induced apoptosis of BxPC-3 cells through the
mitochondrial pathway. We further examined the activation of
caspase-3, caspase-8, and caspase-9 to investigate the mechanism
of induction of apoptosis in BxPC-3 cells by CL using the fluores-
cence substrate assay (Fig. 3C).24 Green coloring indicating
activated caspase-8, caspase-9, and caspase-3 substrate was
observed in BxPC-3 cells treated with CL suggesting that CL
induced apoptosis of BxPC-3 cells through the activation of
 M. Motomura et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1161–1165 1163

(A) (B) 30

100 * 25 *
PS (Fluorescent Intensity)[-]

GM1 (Fluorescent intensity [-]

80
20

60 15

40 10

20 5

0 0
Normal pancreatic cells BxPC-3 cells Normal pancreatic cells BxPC-3 cells

(C)
Normal pancreatic cells BxPC-3 cells (D) 0
0 CL 1h CL 3h
0

Zeta potenal (mV)

Zeta potential (mV)

-20
-20
*
-40
*
-40

-60
-60 *
Fig. 2. (A) Fluorescent intensity of PS for normal pancreatic and BxPC-3 cells. The values here are represented as mean ± S.E. of three independent experiments. Values of p <
0.05 were considered statistically significant. The p value was evaluated by Student’s t-test. *p < 0.01; versus normal pancreatic cells. (B) Fluorescent intensity of GM1 for
normal pancreatic and BxPC-3 cells. The values here are represented as mean ± S.E. of three independent experiments. Values of p < 0.05 were considered statistically
significant. The p value was evaluated by Student’s t-test. *p < 0.01; versus normal pancreatic cells. (C) Zeta potential of normal pancreatic and BxPC-3 cells. The values here
are represented as mean ± S.E. of three independent experiments. Values of p < 0.05 were considered statistically significant. The p value was evaluated by Student’s t-test.
*
p < 0.05; versus normal pancreatic cells. (D) Zeta potential change of BxPC-3 cells after the treatment with CL for 1 and 3 h. The values here are represented as mean ± S.E. of
three independent experiments. Values of p < 0.05 were considered statistically significant. The p value was evaluated by Student’s t-test. *p < 0.01; versus non-treated BxPC-cells.

(A) 1h 2h 3h
(B)
: Control
DMPC : CL
Count

HL

DiOC6(3) ( ψ )
CL (C)
Control DMPC HL CL

Caspase-3

Caspase-8

Caspase-9

Fig. 3. (A) Fluorescence micrographs of BxPC-3 cells after treatment with CL including NBDPC. CL:[DMPC] = 0.70 mM, [C12(EO)21] = 0.04 mM, [2C14ECl] = 0.064 mM. (B)
Mitochondrial transmembrane potential (Dwm) disruption of mitochondria in BxPC-3 cells treated with CL for 48 h. (C) Activation of caspase-3, -8, and -9 in BxPC-3 cells after
the treatment with CL for 48 h. CL:[DMPC] = 0.70 mM, [C12(EO)21] = 0.04 mM, [2C14ECl] = 0.064 mM.
1164 M. Motomura et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1161–1165

caspase-3, -8, and -9. These results indicate that CL is capable of
fusing with the cell membrane of BxPC-3 cells inducing apoptosis
via activation of mitochondrial events and caspase-3, -8, and -9.
After the subcutaneous inoculation of BxPC-3 cells was com-
pleted, we examined the therapeutic effects of subcutaneous
administration of CL for tumor growth in xenograft mouse models
of pancreatic cancer. During the study, the animals were handled
according to the guidelines for animal experimentation under Japa-
nese law. This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory
Animals issued by the Sojo University. The protocol was approved
by the Committee on the Ethics of Animal Experiments at Sojo
University (Fig. 4A).25,26 Marked inhibition of tumor enlargement
(20% vs. control, p < 0.05) was obtained in model mice treated with
CL, in contrast with 90% (vs. control) obtained in mouse models of
pancreatic cancer treated with HL (Fig. 4A). Reduction in tumor
growth was observed by analyzing the autopsy photographs of
tumor in mouse models of pancreatic cancer treated with CL. Using
the TUNEL assay, we investigated the induction of apoptosis by CL
using sections of subcutaneous tumor in mouse models of pancre-
atic cancer treated with CL (Fig. 4C).27 Apoptotic cells (brown
color) were detected among the tumor cells of the tissue section
obtained from model mice treated with CL. In contrast, no apop-
totic cells were observed in group of control, DMPC, and HL. These
results indicate that CL are remarkably effective for inhibiting the
growth of BxPC-3 cells and inducing apoptosis in mouse models
of pancreatic cancer. The inhibitory effects of DMPC and HL on
the growth of BxPC-3 cells on the basis of IC50 (Fig. 1A) and
propidium iodide staining (Fig. 1B) were observed, although less
effective than CL. However, CL with positive charge selectively
fused into BxPC-3 cells with negative charge as compared with
DMPC and HL (Fig. 3A), and then immediately induced apoptosis
(Fig. 1C) via mitochondrial event (Fig. 2B) and activation of
caspases (Fig. 2C). Remarkably high therapeutic effects of CL for
subcutaneous mouse models of pancreatic cancer along with
apoptosis compared with DMPC and HL were obtained (Fig. 4).
No toxicity of CL was observed in normal mice in vivo nor were
there any side-effects.28 These results suggest that CL have phar-
macological selectivity for BxPC-3 cells.
In summary, this is the first study to report that CL are able to
inhibit the growth of BxPC-3 cells through the induction of apopto-
sis in vitro and in vivo. We also observed that negatively charged
phosphatidylserine and sialic acid-containing glycosphingolipids
were over represented on the cell membranes of pancreatic cancer
cells (BxPC-3) but not on normal pancreatic cells. Further observa-
tions showed that CL selectively fused with the negatively charged
cell membranes of BxPC-3 cells inhibiting the growth of BxPC-3
cells and inducing apoptosis, but had no effect on the viability of
normal pancreatic cells. CL induced apoptosis for BxPC-3 cells via
the activation of caspase-3, -8, and -9, and mitochondrial events.

(A) Control
DMPC
HL
400 CL
Tumor volume(mm3)

200
*

0
0 7 14 21 28
Time(day)
(B)
Control DMPC HL CL

(C)

Fig. 4. (A) Tumor volume changes in xenograft mouse models of pancreas cancer treated with CL after the inoculation of BxPC-3 cells (n = 6). Values of p < 0.05 were
considered statistically significant. The p value was evaluated by Student’s t-test. *p < 0.05; versus control. (B) Photographs of tumor in xenograft mouse models of pancreatic
cancer treated with CL. Scale bar: 1 cm. (C) Induction of apoptosis in the tissue section of tumor in xenograft mouse models of pancreas cancer after the treatment with CL.
Red arrows and lines indicate apoptotic cells. Scale bar: 0.1 mm.
 M. Motomura et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 1161–1165
CL also inhibited tumor enlargement of xenograft mouse models of
pancreatic cancer by inducing apoptosis. The results of this study
could be potentially advantageous for future clinical applications
of CL therapy in patients with pancreatic cancer.
Acknowledgment
We thank Kosuke Kuriyama for technical assistance. This work was
supported in part by a Grant-in Aid for Science Research from
the Ministry of Education, Science, and Culture of Japan (No.
17K01383, 17K05944).
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15. Rat pancreatic normal cells were obtained from normal healthy rat according to
previous method14 and cultured using DMEM medium supplemented with 20%
of fetal bovine serum, 1% sodium pyruvate (100 mM), 1% non-essential amino
acids (10 mM) and 1% penicillin/streptomycin solution (Invitrogen, Carlsbad,
CA, USA).
16. Cells were maintained at 37 °C at 5% CO2 atmosphere. The 50% inhibitory
concentration (IC50) on the growth of tumor cells was determined on the basis
of WST-1 [2-methoxy-4-nitrophenyl-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-
2H-tetrazolium, monosodium salt] assay (Cell Counting Kit-1, Dojindo
Laboratories, Kumamoto, Japan). Cells treated with DMPC, HL or CL were
incubated in medium adding WST-1 solution. The absorbance at a wavelength
of 450 nm was measured by spectrophotometer. The inhibitory effects of CL on
the growth of tumor cells were evaluated by Amean/Acontrol, where Amean and
Acontrol denote the absorbance of water-soluble formazan, in the presence and
absence of CL, respectively.
17. BXPC-3 cells treated with CL were centrifuged and suspended in PBS ()
containing propidium iodide (PI, Molecular Probes, Eugene, OR, USA), 1 mg/ml
RNase and 0.1% Triton X-100. The DNA contents and percentage of cells in each
phase of cell cycle were analyzed by a flow cytometer.
1165
18. Detection of apoptotic cells was performed by the TUNEL method using an In
Situ Cell Death Detection Kit (Roche Diagnostics K.K.). BXPC-3 cells treated with
CL processed for TUNEL assay according to the manufacturer’s instructions. The
nuclei of BXPC-3 cells were also stained with a fluorescent dye TO-PRO-3
(Molecular Probes, Inc., OR, USA). The stained cells were observed using a
confocal laser microscopy.
19. The amount of PS in the plasma membranes of BXPC-3 cells was analyzed using
Annexin-V binding assay. BXPC-3 cells were processed for an Annexin-V-FLUOS
Staining Kit (Roche Diagnostics, Basel, Switzerland) according to the
manufacturer’s instruction. The stained cells were analyzed using a flow
cytometer.
20. The amount of GM1 in the plasma membranes of tumor cells was observed
using a confocal laser microscope with a fluorescent reagent cholera toxin
subunit B (CTB) conjugates Alexa Fluor 647 (Invitrogen, Carlsbad, CA, USA).
BXPC-3 cells incubated in the culture media containing CTB, and observed
using a confocal laser microscope.
21. Zeta potential of cells (BXPC-3 and pancreatic normal cells) was measured
using a zeta potential spectrophotometer. All measurements were made at pH
7 to simulate mascara application. The measurements were repeated three
times.
22. The fusion and accumulation of CL including a fluorescence probe (1-palmitoyl-
2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-glycero-3-
phosphocholine (NBDPC; Avanti Polar Lipids, Alabama, U.S.A.) into the
membrane of BXPC-3 cells was performed using confocal laser microscope.
The cells were treated with CL including NBDPC for 3 h and were observed
using confocal laser microscope.
23. Mitochondrial membrane potential of BXPC-3 cells was determined by
measuring the retention of the fluorescent dye 3,30 -dihexy1oxacarbocyanine
[DiOC6(3)] (Molecular Probes, Oregon, USA) in the cellular mitochondria. The
quantification of the cells retaining DiOC6(3) in the mitochondria was
performed with a flow cytometer.
24. The caspase activity was measured as the protease activity of caspase using the
cell-permeable substrate of PhiPhiLux G1D2 (caspase-3), CaspaLux 8-L1D2
(caspase-8) and CaspaLux 9-M1D2 (caspase-9) (OncoImmunin, Inc.,
Gaithersburg, MD, USA) using a confocal laser microscope according to the
manufacturer’s instructions. BXPC-3 cells were treated with CL. The stained-
cells were observed using confocal laser microscope with excitation 488 nm of
15 mW Ar laser (detection at 505–555 nm).
25. Balb/c Rag-2/Jak3 double-deficient (Balb/c-R/J) mice were provided by Dr. Seiji
Okada (Center for AIDS Research, Kumamoto University, Japan).27 BxPC-3
cells (5.0  106 cells) suspended into matrigel (BD Co., NJ, USA) were
subcutaneously inoculated to dorsal of mice. Number of mice was six in each
group. The tumor volume reached 100 mm3 at day 7 after the inoculation of
BxPC3 cells, and then CL were subcutaneously administered once each day for
14 days from day 7. The tumor volume was measured using Vernier caliper and
calculated using the equation of V = 0.5  a2  b, where a and b denote the
smallest and longest superficial diameter, respectively.
26. Ono A, Hattori S, Kariya R, et al. J Biomed Biotechnol. 2011;2011:539748.
27. Detection of apoptotic cells was performed on the basis of the TUNEL method
using apoptag peroxidase in situ apoptosis detection kit (S7100, Merck
Millipore, Darmstadt, Germany) according to the manufacturer’s directions.
After the experimental period, tumor was removed from anaesthetized mice
after the treatment with CL. Paraffin-embedded sections were made, and the
detection of apoptosis of a solid tumor was performed on the basis of TUNEL
assay according to the conventional method. The tumor sections were stained
with 3,30 -DAB chromogen and observed by an optical microscope.
28. Ichihara H, Motomura M, Matsumoto YJ. Carcinog Mutagen. 2016;7:1000267.