Enhanced cytotoxicity by a benzothiazole-containing cisplatin derivative in breast cancer cells

Article abstract of DOI:10.1039/C6NJ02753A

In recent years, the concept of nanoparticles being used as an intelligent drug carrier has gained great attention. In this paper, the formulation of a liposome delivery system loaded with a novel benzothiazole-containing cisplatin derivative (CJM-Pt) was carried out. The particle size distributions were determined using dynamic light scattering, and the prepared liposomes showed a suitable size of around 98 nm. Stability studies showed that the CJM-Pt loaded liposomes were stable at 4 °C for more than four weeks. Investigation of triggered release indicated that the release performance of the prepared liposomes was controllable and the releasing effect was remarkable under low pH (42 °C). To test the suitability of the chosen formulation, CJM-Pt loaded liposomes were investigated against several tumor cell lines: MGC-803, SGC-7901, MCF-7 and MDA-MB-231. Furthermore, the cell cycle arrest was examined. The CJM-Pt loaded liposomes have the potential to be applied in drug delivery systems (DDS) for breast cancer therapy.

Full text of DOI:10.1039/C6NJ02753A

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Enhanced cytotoxicity by benzothiazole-containing cisplatin
derivative in breast cancer cells
a
a
a
a
b
c
Chaoqun You , Jia Yu , Yu Sun , Yanghui Luo , Xiangyang Zhang , Jin Zhu ,
Baiwang Sun a,*
a
School of Chemistry and Chemical Engineering, Southeast University, Nanjing
210089, PR China. Fax: +86 25 52090614, Tel: +86 25 52090614, Eꢀmail address:
chmsunbw@seu.edu.cn
b Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
c Key Laboratory of Antibody Technique of Ministry of Health, School of Pathology,
Nanjing Medical University, Nanjing 210093, PR China
Abstract
In recent years, nanoparticle as an intelligent drug carrier has caused great attention.
In this paper, formulation of liposome delivery system loaded with a novel
benzothiazoleꢀcontaining cisplatin derivative (CJMꢀPt) was carried out. The particle
size distributions were determined by dynamic light scattering and the prepared
liposomes showed a suitable size of around 98 nm. Stability study showed the CJMꢀPt
loaded liposomes were stable at 4℃ for more than four weeks. The investigation of
triggered release indicated that the release performance of prepared liposomes were
controllable and the releasing effect remarkable under low pH (< pH 6.8) and high
temperature (> 42℃). To test suitability of chosen formulation, CJMꢀPt loaded
liposomes were investigated against several tumor cell lines: MGCꢀ803, SGCꢀ7901,
MCFꢀ7 and MDAꢀMBꢀ231. Furthermore, the cell cycle arrest was examined. The
1

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CJMꢀPt loaded liposomes have the potential to be applied in the drug delivery system
(DDS) for breast cancer therapy.
KEYWORDS: cisplatin, benzothiazole, liposomes, drug delivery, synergic action, drug
release
1. Introduction
The widely used of cisplatin, carboplatin and oxaliplatin in clinical for cancer therapy
has caused great attention to search for more effective cisplatin derivatives, and as a
consequence hetaplatin and miriplatin have been successively developed and applied
in clinic 1,2. In particular, cisplatin has played a crucial role for tumor chemotherapy
among all the platinumꢀbased drugs, and has reached medicinal chemistry classic
3
around the world . However, the water insoluble and severe side effects of platinum
drugs have limited their more extensive application to a great extent. In order to
overcome these drawbacks, a series of drugꢀdesign strategies have been developed in
the past decades 4ꢀ11. Combine platinum derivatives with some other active
pharmacophore as one of the most important approaches has been put forward, and a
synergic action can be achieved when against the tumor cells 12ꢀ15
.
The synthesized molecule would break down into two original drug molecules as
18
delivery to the targeted position 16ꢀ17. Samuel G, et al
have conjugated
D(+)ꢀTryptophan to Pt(IV) for combined immunomodulation and DNA
crossꢀlinkꢀtriggered apoptosis for cancer “immuneꢀchemotherapy”. Huang Rong, et al
19
has described a novel conjugate comprising of Herceptin (an antiꢀHER2 antibody)
and platinum drug via a cathepsin B cleavable dipetide for enhancing drug
2

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accumulation and HER2ꢀpositive cancer cell specific delivery.
To overcome the water insoluble and severe side effects which limit the efficacy of
platinum drugs by countering biological compensation, synergistic combination of
platinumꢀbased derivatives with some other active pharmacophore is a promising
strategy, which allowing reduced dosage of each agent or accessing contextꢀspecific
multiple targets 20ꢀ23
.
Benzothiazole as an active pharmacophore belong to the family of bicyclic
heterocyclic compounds having benzene nucleus fused with fiveꢀmembered ring
comprising nitrogen and sulfur atoms. Benzothiazole is an important backstop with a
wide range of interesting biological activities and therapeutic functions including
resisted some common serious diseases 24ꢀ27, remitted inflammation caused by
bacteria 28ꢀ30, anticonvulsant and analgesic 31ꢀ33, anthelmintic 34, and antitumor
activities 35. In particular, a large number of benzothiazole derivatives have shown
potent anticancer activity 36. Some of the recent literature reports are summarized in
previous literatures.
A new series of 2ꢀ(4ꢀaminophenyl)benzothiazoles has been synthesized by simple,
highꢀyielding routes through substituted in the phenyl ring has been reported 37. The
present molecule CJM126 shows potent inhibitory activity in vitro in the micromole
range against a series of human breast cancer cell lines 38, moreover, it has been even
used in clinical for tumor therapy in some case 39ꢀ40
.
Over the past few decades, there has been an increasing interest in the potential use of
nanocarriers as delivery supporter for chemotherapeutic drugs and the researches have
3

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indicated that these nanocarriers can significantly enhance the antiꢀtumor efficiency
for delivery drugs to targeted tissues 41. Recently, multifunctional delivery systems
have been developed for delivery of various guests, including liposomes 42, micelles
43ꢀ44, and inorganic nanoparticles 45.
Liposomes can release drugs in an efficacious and controllable way attributed to the
48
good biocompatibility and simple surface functionalization 46ꢀ47. Ashley, et al has
reported a nanocarrier by depositing a dioleoyl phosphatidylethanolamine (DOPE)
based liposome on mesoporous nanoparticles and observed its pHꢀdependent release
characteristics. Since both the pH value and temperature at the tumor sites are
different from those in normal tissues, designing a dualꢀstimuliꢀresponsive drug
carrier achieved the potential for clinical applications 49ꢀ58
.
In this paper, we have synthesized a novel cytotoxicity molecule by combine two drug
molecules though chemical coupling for the first time and using liposome as a carrier
for drug delivery. We look forward to the prepared drug loaded liposomes exhibit
good cytotoxicity against tumor cells owing to the antitumor property of CJM126 and
its possible synergic action with cisplatin. The prepared CJMꢀPt loaded liposomes
have the potential to be applied in the drug delivery system (DDS) for breast cancer
therapy.
2. Materials and methods
2.1 Materials. 2ꢀBenzothiazolamine was purchased from Meryer (Shanghai) Chemical
Technology Co., Ltd. Soybean phospholipids (SP), the diblock copolymer of poly
(ethylene glycol) with poly (εꢀcaprolactone)(PEG5kꢀPCL10k) were purchased from
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Polymtek Biomaterial Co., Ltd (Shenzhen, China). Cisplatin was purchased from
Shandong Boyuan Pharmaceutical Co., Ltd (Jinan, Shandong, China). FITC,
Hoechst33342 were purchased from Beyotime Biotechnology (Haimen, Jiangsu,
China). All other chemical reagents were obtained from Sinopharm Chemical Reagent
Co., Ltd.
2.2 Synthesis of benzothiazole containing cisplatin derivative (CJMꢀPt) molecules.
CJMꢀPt molecule was obtained via conjugate CJM126 to cisplatin in aqueous solution
under room temperature with three steps 59ꢀ60
.
2.3 Preparation of CJMꢀPt loaded liposomes (LipoꢀCJMꢀPt). Liposomes
encapsulating CJMꢀPt were formulated by the film hydration method as reported by
literature with slight modifications 61. Briefly, SP (45mg), PEGꢀPCL (2mg), CJMꢀPt
(15mg) was dissolved in acetonitrile (40ml), and a thin film was formed by solvent
evaporation under reduced pressure. The acetonitrile was removed completely by
placing the flask overnight in a vacuum desiccator. Then deionized water (40ml) was
added to the copolymer film and ultrasonic vibration for 5 min. Suspension was
shaken for 30 min at 30℃ to ensure well dispersed. Formulation was then centrifuged
at 3000 RCF for 15 min and filtered using a 0.22ꢁm filter (Millipore) and lyophilized.
2.4 Liposome Characterization.
Particle size analysis. Particle size distribution was estimated by dynamic light
scattering (DLS) and the sample of the stepwise extrusion through 0.22ꢁm
membranes after ultrasonic vibration for 5 min. Each sample was diluted 2ꢀfold with
ultrapure water before measurement and analyzed in triplicate at 25℃. The particle
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size distribution data were generated using the DTS nano software (version 5.2). The
liposome size polydispersity was expressed by the polydispersity index, PDI.
Zeta potential. The ζ potential was determined using a Malvern Zetasizer Nanoseries
(Malvern Instruments Zen 3600, Malvern, U.K.) and measurements were performed
at least three times after dilution in water. The ζ potential was calculated by the
electrophoretic mobility applying the HelmholtzꢀSmoluchowski equation.
Transmission Electrom Microscopy (TEM). Particle morphology was detected by a
JEMꢀ100CXII transmission electron microscopy (TEM).
2.5 The entrapment efficiency and drug loading capacity in LipoꢀCJMꢀPt. The
LipoꢀCJMꢀPt was prepared under specific conditions as the literature described
62
thinꢀfilm hydration method . The purified drugꢀloaded liposomes were freezeꢀdried.
The CJMꢀPt mass loaded in liposomes was calculated by a High Performance Liquid
Chromatograph at 293 nm (comparing with CJMꢀPt calibration curve) after extracting
CJMꢀPt from the dried drugꢀloaded liposomes with methanol. The entrapment
efficiency and drug loading capacity were calculated according the following
equations:
ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢈꢉꢊꢋꢌꢅ ꢂꢍ ꢎꢂꢏꢆꢐꢆꢑꢁꢐ
EE (%, CJMꢀPt) =
×100%
ꢂꢍꢂꢅꢂꢒꢎ ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢈꢉꢊꢋꢌꢅ
ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢈꢉꢊꢋꢌꢅ ꢂꢍ ꢑꢁꢅꢄꢒꢍꢆꢎ
DLC (%, CJMꢀPt) =
×100%
ꢀꢁꢂꢃꢄꢅ ꢆꢇ ꢓꢔꢕꢃ ꢎꢆꢒꢓꢁꢓ ꢎꢂꢏꢆꢐꢆꢑꢁꢐ
2.6 The stability of liposomes 63. The prepared liposomes were stored at 4℃ for up to
4 weeks. The stability of prepared liposomes was evaluated on Days 0, 7, 14, 21and
28. At each time point, the liposomal samples were centrifuged at 1200 RCF for 15
min to ensure that the leaked CJMꢀPt content was sedimented. Then 5mL aliquot was
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taken separately from each supernate, microfuged and mixed with 10mL methanol,
and then centrifuged at 1500 RCF for 15min, precipitation dissolved in methanol
10ml and the CJMꢀPt content was quantified for each time point (in triplicates) by
HPLC.
2.7 In vitro multiple triggered release of liposomes. CJMꢀPt released from
LipoꢀCJMꢀPt was performed by detecting the platinum content as released from the
liposomes 64. We tested pH (7.4, 6.8, 6.2, 5.5, 5.0) and temperature (32, 35, 37, 39, 42,
45℃) triggered release of liposomes. An amount of 2 mL of LipoꢀCJMꢀPt solution
was achieved from the flask at given condition. The solution was centrifuged at 1200
RCF for 15 min to ensure that the leaked CJMꢀPt content was sedimented. Then the
supernate was collected. After filtered using a 0.22ꢁm filter for 2 times in the dark,
the CJMꢀPt mass loaded in liposomes was calculated by a High Performance Liquid
Chromatograph at 293 nm.
To investigate the pHꢀdependent releasing efficiency, three aliquots of the CJMꢀPt
loaded nanoparticles with equal amount were immersed in 3 mL of buffer solution at
25℃ with pH = 7.4, pH = 6.8, pH = 6.2, pH = 5.7 and pH = 5.0, respectively. The
mixture was shaken at certain time intervals. The supernatant was taken out by
centrifugation to measure the absorption, and an equal volume of fresh medium was
added instead. By comparing the absorption curve of pure CJMꢀPt in different pH
solutions, the amount of released CJMꢀPt can be calculated.
To investigate the thermal stimuli release efficiency of the prepared carrier, three
aliquots of LipoꢀCJMꢀPt with equal amount were immersed in 3 mL of PBS buffer
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(pH = 7.4) at T = 25℃, T = 37℃, T = 42℃, and T = 45℃. The procedure of
absorption measurement was the same as that of a pHꢀdependent release investigation.
2.8 Cell cultures. MGCꢀ803 and SGCꢀ7901 human gastric carcinoma cells were
cultured in RPMI 1640 medium, MCFꢀ7 and MDAꢀMBꢀ231 human breast cancer
cells were cultured in Dulbecco’s Modified Eagle medium (DMEM). Both media
were supplemented with 10% heatꢀinactivated fetal bovine serum (FBS), and 1%
Antibiotic/Antimycotic solution. All cell lines were incubated at 37℃ in a humidified
incubator in a 5% carbon dioxide atmosphere.
2.9 In vitro cytotoxicity. Cytotoxicity of prepared liposomes was evaluated by MTT
assay with MGCꢀ803, SGCꢀ7901, MCFꢀ7 and MDAꢀMBꢀ231 cells. The cells were
seeded into a 96ꢀwell plate for 24 h (5×103 cells/well) and incubated at 37℃ with 5%
CO2. The supernatants were discarded, and the cells were washed twice with PBS
(pH=7.4). Free CJMꢀPt and CJMꢀPt loaded liposomes (LipoꢀCJMꢀPt) were
equivalently put into the culture medium for 24 h at a certain concentration of 3, 6, 12,
24, or 48ꢁg/mL, respectively. The complete medium without cells was put in the wells
for blank groups and the culture medium without CJMꢀPt (PBS) but contain 5%
DMSO was put in the wells for control groups. Then 20ꢁL of MTT solution (5.0
mg/mL) was added to each well, and the cells were further incubated for 4h at 37 °C.
Next, the medium was removed, and then 150ꢁL of dimethyl sulfoxide (DMSO) was
put in the well to dissolve the formazan crystals formed by the living cells. The
absorption was recorded at 490 nm using a microplate reader (Thermo Scientific
Varioskan Flash, Waltham, MA, USA). The in vitro toxicity of free CJMꢀPt,
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CJMꢀPtꢀloaded and blank liposomes were expressed as cell viability, defined by the
following equation：
ꢖꢗꢋꢖꢘ
Cell viability =
×100%
ꢖꢙꢋꢖꢘ
Where A0 was the absorbance of the complete medium without cells, A1 was the
absorbance of the cells incubated with different formulations (the presence of CJMꢀPt
concentration of 3, 6, 12, 24, or 48ꢁg/mL for 24h) and A2 was the absorbance of the
control groups.
2.10 Cellular uptake study. Intracellular delivery of LipoꢀCJMꢀPt and coꢀlocalization
of CJMꢀPt and the nucleus were observed by laser confocal microscope. MGCꢀ803,
SGCꢀ7901, MCFꢀ7 and MDAꢀMBꢀ231 cells were cultured until 70~80% confluency
and then trypsinized, seeded onto confocal petri dish (BD Falcon, USA) at a density
of 1.0×105 cells/well. After the cells were incubated for 24h at 37℃, LipoꢀCJMꢀPt
with equivalent concentration of FITC at 1.0 mg/mL dissolved in 1640 or DMEM
were added and incubated for further 24h at 37℃ along with complete medium as
negative control. Hoechst 33342 in 1640 or DMEM were added to stain nuclei and
cells were incubated for 12h, respectively. All reagents were removed after incubation
and cells were washed with PBS (pH=7.4) three times. Mounting medium was added
to prevent quenched and fixed the cells. Cells were observed by laser confocal
microscope (Zeiss, Germany)
2.11 Cell apoptosis and cycle arrest.
Cell apoptosis. Annexin staining for measurement of apoptosis was performed using
the APC/7ꢀAAD Apoptosis Detection Kit II (BD Pharmingen, San Jose, USA). Briefly,
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MGCꢀ803, SGCꢀ7901, MCFꢀ7, MDAꢀMBꢀ231 cells were seeded at 1×105 cells/dish
and incubated for 24h. The cells were then treated with 50ꢁM CJMꢀPt as CJMꢀPt
loaded liposomes. Blankꢀliposomes served as blank. After 24h of treatment, cells were
washed with cold PBS and resuspended in 200ꢁL binding buffer. Cells were stained
with 10ꢁL of APC and 5ꢁL of 7ꢀAAD for 15 min in the dark before diluting with an
additional 300 ml binding buffer. Samples were analyzed by flow cytometry on a
FACS Calibur (BD Biosciences, USA).
Cell cycle arrest. 1×106 MDAꢀMBꢀ231 cells were treated with CJM126, CJMꢀPt and
cisplatin for 48h with complete medium free from drugs as negative control. Then
cells were collected and washed with PBS. After centrifugation, the cell suspensions
were fixed by addition of 70% ethanol overnight at 4℃. For staining, cells were
centrifuged, resuspended in PBS, treated with DNA staining for 30 min in the dark.
The cells were analyzed with a FACS Calibur (BD Biosciences, USA)
3. Results and discussion
3.1 Synthesis of CJMꢀPt molecules. We designed and synthesized the hybrid
compounds by combine CJM126 molecule with cisplatin via a malonyl dichloride
linker, the scheme of reaction route was show in Fig. 1. The yellowish green product
was subsequently purified from methanol, and dried in vacuo with a yield of 22%.
1HNMR displayed clearly peaks characteristic of CJM126, CJMꢀCOOH and
CJMꢀPt (Fig. S1). The data indicated that integral ratio between resonances at s
10.061 (carboxyl protons in the head of CJMꢀCOOH) and d 3.686 (methylene protons
of malonyl dichloride skeleton) was nearly to the theoretical value of 1:2(Fig. S1a, 1b),
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1
which confirming successful synthesis of CJMꢀCOOH. The HNMR data also shows
complete disappearance of signal at d 10.061 due to carboxyl protons reacted with the
equimolar barium salt, and no new set of signals come out assignable to the
formatiom of CJMꢀPt (Fig. S1c). The Mn values of CJMꢀPt could be estimated by
comparing the intensities of signals at d 10.601 and 3.965 which were attributable to
the carboxyl protons belong to the ester bonds of CJMꢀCOOH and the thiazole ring
protons next to the ester bonds of CJMꢀPt, respectively.
3.2 Preparation and characterization of liposomes. Liposomes were prepared under
sterile conditions as the literature described thinꢀfilm hydration method. The
schematic diagram of prepared LipoꢀCJMꢀPt was shown in Fig. 2. To prepare the
liposomes with a suitable size and perfect morphology, we carried out a series of
experiments with different supply proportion. Through change the supply proportion
and the reaction time of dissolutionꢀemulsification selfꢀassembled, we achieved
liposomes with relative excellent sizes suitable for in vivo transportation. The
prepared liposomes have a uniform spherical shape with a mean size of about 98nm as
shown in Fig. 3A, this result attributed to the addition of phospholipids by enhance
the stability of prepared liposomes. The TEM study indicated that the prepared
liposomes show an excellent size and a uniform spherical shape as shown in Fig. 3C.
The results also show that the average diameters of CJMꢀPt loaded liposomes were
about 98 nm and which emphasize the well dispersed state of the liposomes by
representative particle size and homogeneity morphology. The results also suggested
the surface of prepared liposomes (LipoꢀCJMꢀPt) was relatively smooth. The tested
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results of the ζ potential was suggested that the zeta potential on the surface of
LipoꢀCJMꢀPt tended to be neutral may due to not conjugated liposomes with any
ligands, such as polymer chain , micro molecule polypeptide or the antibody (Fig. 3B).
Fig. 3D shows the morphological changes of a liposomes solution of different
concentrations, liposomes solution overall brown and gray, furthermore the solution
color and the concentration were correlated positively.
The typical recipes and tested results were listed in Table S1. Table S1
demonstrated the important characteristics of the liposomes, including mean diameter,
encapsulation efficiency (EE), drug loading capacity (DLC) and polydispersity index
(PDI) value. All of the liposomes showed good homogeneity, with PDIs below 0.30
and mean particle sizes between 73.39 and 117.30 nm. From Table S1, we found that
the particle sizes of liposomes increased from 73.39 to 117.30 nm on DLS as increase
the ratio of phospholipids, the polydispersity index (PDI) of DLS was keep blow 0.3,
which meant that the liposomes show homogeneity morphology.
To investigate the entrapment efficiency and drug loading capacity in
LipoꢀCJMꢀPt, liposomes were prepared as reported previously 65ꢀ68. Then the
entrapment efficiency and drug loading capacity in LipoꢀCJMꢀPt were calculated by
detected the CJMꢀPt mass in liposomes with a High Performance Liquid
Chromatograph at 293 nm (comparing with CJMꢀPt calibration curve) after extracting
CJMꢀPt from the dried drugꢀloaded liposomes with methanol as well.
3.3 The stability of liposomes. The liposomal preparations were stored at 4℃ for up
to 4 weeks. The stability of liposomal preparations was evaluated on Days 0, 7, 14,
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and 21. At each time point, the liposome samples were centrifuged at 1200 RCF for
15 min to ensure that the leaked CJMꢀPt content was sedimented. Then 25mL
supernatant was taken out and mixed with 50mL methanol separately, centrifuged and
the CJMꢀPt content was calculated on each time point (in triplicates) by HPLC at
293nm (comparing with CJMꢀPt calibration curve), as the result show in Fig. 4.
The stability of CJMꢀPt loaded liposome formulation was monitored during
storage for four weeks at 4℃. The results demonstrated that a large portion of
liposomes retained CJMꢀPt without significant leakage and the prepared liposomes
show excellent stability as show in Fig. 4A. Generally, as for storage at 4℃，the
retention of CJMꢀPt in the liposomes was reduced to 11.29%，11.24%，11.19%，11.07%
by the 1st, 2nd, 3rd and 4ur week, respectively. At the same time the particle size and
the encapsulation efficiency showed little change, the PDI parameters remain in a
good level. This result shows that the prepared liposomal formulation can be stored
for a certain period with an excellent stability.
3.4 In vitro pH triggered release of liposomes. A CJMꢀPt loaded liposomes solution
was divided into five aliquots and the pH of each aliquot was adjusted to 5.0, 5.7, 6.2,
6.8 and 7.4. To investigate the pHꢀdependent releasing efficiency, five aliquots of
CJMꢀPt loaded liposomes with equal amount were immersed in 3mL of buffer
solution at 25℃ with pH = 7.4, pH = 6.8, pH = 6.2, pH = 5.7 and pH = 5.0,
respectively. The mixture was shaken at certain time intervals. The supernatant was
taken out to measure the CJMꢀPt content after centrifugation, and an equal volume of
fresh medium was added instead. By comparing the absorption value of the CJMꢀPt
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calibration curve, the amount of released CJMꢀPt can be calculated.
The results showed the liposomes express sensitively pH trigged release as
demonstrated in Fig. 4B. The release process of the LipoꢀCJMꢀPt was monitored at
certain time intervals within 24h at 25℃. As show in Fig. 4B, the amounts of released
CJMꢀPt increased along with reduced the pH value. Specifically, about 41.27% of
CJMꢀPt was released at pH = 7.4 when about 83.00% of CJMꢀPt was released at pH =
6.8 in 24h. Furthermore, the released amount of CJMꢀPt significantly increased to
about 97.65% at pH = 5.0, which more than 2ꢀfold as compared with it at pH = 7.4.
This is because lipid bilayer of the phospholipids added little PEGꢀPCL structure
exhibits a compact arrange state at some degree under neutral pH conditions, which
transforms to a hydrophilic chain structure while reduced the pH value. Such a change
of morphological structure on the phospholipid bilayer impacts the barrier property of
liposomes. The release efficiency in 24h was 41.27%, 83.00%, 88.50%, 94.72% and
97.65% corresponding to pH 7.4, pH 6.8, pH 6.2, pH 5.5 and pH 5.0, respectively.
Thus, the different pH value demonstrated obviously effect on the releasing of
LipoꢀCJMꢀPt. As a consequent, the prepared CJMꢀPt loaded liposomes exhibited a
quite remarkable pHꢀcontrollable property with the PEGꢀPCL mediated lipid bilayer.
This is of great valuable in vivo applications of pHꢀresponsive nanocarriers because
of the low pH environment of cancer cells than that of the normal ones.
3.5 In vitro temperature triggered release of liposomes. The investigated of
temperature triggered release of liposomes was the same procedure as that of the pH
triggered. Investigated with different temperature degree by 25℃, 30℃, 37℃, 42℃,
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45℃of buffer solution at pH = 7.4.
As the pHꢀcontrollable release performance was achieved previously, the
thermoꢀcontrollable release characteristic was studied. As shown in Fig. 4C, the phase
transition temperature (Tm) of the prepared liposome is around 42℃ at pH 7.4.
Therefore, the release curves at four different temperatures (25℃, 37℃ (T < Tm), 42℃
(T = Tm), and 45℃ (T > Tm)) were obtained with certain pH value at 7.4 in our study.
The achieved results are show in Fig. 4C. The releasing efficiency of CJMꢀPt at 42℃
(Tm) reached 85.90% after 24 h, which is remarkable higher than those at the lower
temperatures (57.52% at 25℃ and 81.28% at 37℃). Moreover the releasing
efficiency of CJMꢀPt at 45℃ reached 92.59% after the same time. The results
consistent with the property of the liposomes as described by the previously literatures.
The possible explanation may as follows. Briefly, as the temperature is below Tm, the
contained CJMꢀPt was leaked from the gap of the composite bilayer. However, when
the temperature reaches the Tm value of the composite bilayer, the PEGꢀPCL
mediated phospholipids structure could be depolymerized and fall to pieces under
high temperature. When the temperature increased to be higher than Tm, the
hydrophobic structure of liposomes become more depolymerized and even to the
surface, which accelerated the release of CJMꢀPt from the liposomes core. The
achieved results indicated that the prepared liposomes show a thermoꢀresponsive
releasing ability.
3.6 In vitro cytotoxicity. A remarkable attention to nanomaterials for bioꢀapplication is
their toxicity, therefore, the cytotoxicity of CJM126 and CJMꢀPt toward tumor cells
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were investigated with the standard MTT assays, and then the IC50 (ꢁg/mL) for each
cell culture were calculated as show in Table S2. Each sample was diluted 2ꢀfold with
ultrapure water before measurement and analyzed in triplicate at 25℃.
The IC50 (ꢁg/mL) for MGCꢀ803, SGCꢀ7901, MCFꢀ7 and MDAꢀMBꢀ231 cell
lines were investigated and the results were shown in Table S2. Among these cell lines,
CJM126 was more toxicity to MDAꢀMBꢀ231 particularly. The IC50 of CJMꢀPt was
38.36% less than cisplatin while the IC50 of CJM126 was nearly double of cisplatin,
which indicated that CJM126 and cisplatin enhance synergistically in cytotoxicity
against MDAꢀMBꢀ231 through the combination.
Furthermore, to investigate whether the cancer cells can be efficiently inhibited
by LipoꢀCJMꢀPt, we incubated the CJMꢀPt loaded liposomes with MGCꢀ803,
SGCꢀ7901, MCFꢀ7, MDAꢀMBꢀ231 cells for 24h at 37℃, and then the cytotoxicity of
LipoꢀCJMꢀPt towards tumor cells was investigated with the standard MTT assays (Fig.
5). The liposomes were diluted with 1640 or Dulbecco's Modified Eagle's medium
(DMEM) and incubated with MGCꢀ803, SGCꢀ7901, MCFꢀ7 and MDAꢀMBꢀ231 cells
in 5% CO2 at 37℃ for 24h. According to all the Blankꢀliposomes, the cytotoxicity
slightly increases with increasing liposomes concentration. As for human gastric
cancer cells, no significant cytotoxicity was observed in cells when the concentration
of Blankꢀliposomes was less than 48ꢁg/mL, and the cell viability remained above 80%
except 70.82% of MGCꢀ803 at 48ꢁg/mL. The results indicate that the prepared
liposomes have low cytotoxicity for gastric cancer cells under the current drug
concentration.
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Next, we further investigated the cytotoxicity of LipoꢀCJMꢀPt against human
breast cancer cells with the MCFꢀ7 and MDAꢀMBꢀ231cells. The cytotoxicity of free
cisplatin, Blankꢀliposomes and LipoꢀCJMꢀPt was investigated in MCFꢀ7 and
MDAꢀMBꢀ231 cell lines, and the anticancer effect was studied by MTT assay as well.
CJMꢀPt shows concentration of positive correlations on the cell viability of both
MCFꢀ7 and MDAꢀMBꢀ231 cells lines after dose for 24h as show in Figs. 5C and 5D.
However, the liposomes shell structure reduced the cytotoxicity of CJMꢀPt
against MGCꢀ803 cells and maintained the cell viability to over 45% even increasing
the equivalent concentration of LipoꢀCJMꢀPt (Fig. 5A). Furthermore, the inhibitory
effect on the cell viability of SGCꢀ7901 cells was timeꢀdependent. There was no
remarkable viability change in SGCꢀ7901 cells when treated with Blankꢀliposomes
(Fig. 5B). Although the cytotoxicity was diminished using CJMꢀPt loaded liposomes
against SGCꢀ7901 cells, LipoꢀCJMꢀPt showed doseꢀdependent inhibitory effect and
inhibited the proliferation of MCFꢀ7 cells to less than 57% after treatment for 24h in
48mg/mL of equivalent concentration of LipoꢀCJMꢀPt (Fig. 5C). The inhibitory effect
on the proliferation of MDAꢀMBꢀ231 maintained at 74% for 24h (Fig. 5D). The
results indicated that when LipoꢀCJMꢀPt came into contact with the MDAꢀMBꢀ231,
more quantity were absorbed by cells and demonstrated higher cell inhibition.
However the inhibitory effect of LipoꢀCJMꢀPt was obviously cannot be comparable to
free CJMꢀPt in 24h.
3.7 In vitro cellular uptake assay. In this study, the cellular uptake efficiency of
LipoꢀCJMꢀPt was evaluated by confocal laser scanning microscopy (CLSM). As show
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in Fig. 6, only a few of LipoꢀCJMꢀPt (green fluorescence signed) was focused on the
nucleus in MGCꢀ803 cells and SGCꢀ7901 cells after incubated for 12h, which was
consistent with the cytotoxicity results. The remarkable strong green fluorescence in
the nucleus of MCFꢀ7 cells with LipoꢀCJMꢀPt treated (compared to the human gastric
cancer cells) indicated the intracellular uptake was enhanced by LipoꢀCJMꢀPt. In
particular, LipoꢀCJMꢀPt showed the highest intracellular uptake in MDAꢀMBꢀ231
cells (with strongest green fluorescence intensity), which was almost cover the whole
field of view. However, the fluorescence intensity indicated no remarkable difference
between MCFꢀ7 and MDAꢀMBꢀ231 for human breast cancer cells. These results
indicated that human breast cancer cells effectively internalized the CJMꢀPt loaded
liposomes by the cell endocytosis.
Based on the previous cellular uptake experiments, the possible mechanism of
cellular uptake enhancement for the CJMꢀPt loaded liposomes was as follows: Due to
the good biocompatibility of composite material liposomes, it can release drugs in an
efficient way. The excellent biocompatibility of the CJMꢀPt loaded liposomes was
delivery to the tumor cells; the drug in liposomes rapidly released when subjected to
low pH or hyperpyrexia and then spread into the tumor along the drug concentration
gradient.
3.8 Cell apoptosis and cycle arrest. To further evaluate the induced apoptosis effect of
the LipoꢀCJMꢀPt on tumor cells, the annexin staining for measurement of apoptosis
was performed with the APC/7ꢀAAD Apoptosis Detection Kit, the prepared cells were
sorted by flow cytometry. To compare the apoptosis rate between human gastric
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cancer cells and breast cancer cells, MGCꢀ803, SGCꢀ7901, MCFꢀ7 and
MDAꢀMBꢀ231 cells were cultivated. After the cell cultures were treated with
LipoꢀCJMꢀPt for 24 h, the apoptotic cells were detected by flow cytometry as shown
in Fig. 7, Blankꢀliposomes did not induce obvious apoptosis for all and only induced
less than 10% apoptosis especially in MCFꢀ7 cells the data only 6.0%. Meanwhile, the
blank liposomes treatment to MGCꢀ803 induced alone only around 10.6%.
The LipoꢀCJMꢀPt mediated apoptosis, which was remarkable higher than treatment
with blank liposomes alone. All the induced apoptosis occurred at the early apoptosis,
related to the inductive effect on the apoptosis of cancer cells was timeꢀdependent.
The apoptosis rate of MDAꢀMBꢀ231 reached 60.2% as higher than MCFꢀ7 (47.8%)
and MGCꢀ803 (46.6%). In particular, this obtained results demonstrated that the
LipoꢀCJMꢀPt show the minimal impact to SGCꢀ7901 with only 40.8% degree. These
results suggest that treatment with CJMꢀPt loaded liposomes can overcome resistance
of breast cancer cells. However, the LipoꢀCJMꢀPt treatment induced more than
1.2ꢀfold increase in apoptosis for breast cancer cells when compared with
LipoꢀCJMꢀPt treatment of gastric cancer cells on the whole. This suggested that the
prepared liposomes more suitable for resistance of human breast cancer cells.
To further investigate the cell cycle arrest induced by CJM126 and CJMꢀPt, the
cell cycle distribution of MDAꢀMBꢀ231 cells treated with CJM126 and CJMꢀPt were
detected by flow cytometry under the PI staining with cisplatin for comparison. As
shown in Fig. 8, cisplatin strongly interfere with the cell cycle in S phase. The cell
percentage at S phase obviously increases from 19.01% to 23.10%, with a decrease of
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cell percentage at G0/G1 phase from 75.83% to 74.81% which highly consistent with
the general consensus that cisplatin acts as a DNA binding agent to prevent DNA
replication 69. CJMꢀPt also induces the cell cycle remarkably as compared with
control group. In particularly, the cell percentage at G0/G1 phase decreased and those
at G2/M phase decrease from 5.16% to 1.83% as well. As for CJM126, the cell
percentage at G0/G1 phase decrease, while those at G2/M phase increase from 5.16%
to 6.63%. It indicates that CJM126 remarkable induces cell cycle arrest mainly at
G2/M phase. Based on the previous cell cycle experiments, the proposed mechanism
of effect on cell cycle for the CJMꢀPt molecule was as follows: As the CJMꢀPt
released from LipoꢀCJMꢀPt in the cancer cells, the CJMꢀPt molecule break down into
CJM126 and cisplatin with the cleavage of chemical bonds. So it suggested that
CJMꢀPt acted as a derivative from cisplatin may have changed its DNA binding mode
and result in a different cell cycle arrest from cisplatin. It also indicated that it
achieved the synergistic effect from the CJMꢀPt molecule.
4. Conclusions
A synergic actional cisplatin derivative via conjugated CJM126 with cisplatin was
successfully synthesized and delivery by liposomes for cancer treatment in this study.
The prepared drug loaded liposomes show suitable size around 98nm and a spherical
morphology. The entrapment efficiency and drug loading capacity in prepared
liposomes reached 75% and 31%, respectively. And the present liposomes also
demonstrated an excellent stability by retained cisplatin derivative without significant
leakage for four weeks.
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The investigated of pH and temperature triggered release of liposomes indicated
that the release of prepared liposomes was controllable and the releasing effect
remarkable under low pH (< pH 6.8) and high temperature (> 42℃). The cisplatin
derivative molecule demonstrated good cytotoxicity as compared with cisplatin,
especially the IC50 (ꢁg/mL) less than 17.47 ( the IC50 of cisplatin) for MDAꢀMBꢀ231.
As compared with human gastric cancer cells, the prepared drug loaded liposomes
showed more cytotoxicity to human breast cancer cells. The cellular uptake in vitro
result showed that the prepared nanocarriers were internalized by tumor cells
obviously owing to the good biocompatibility of liposomes. The cisplatin derivative
molecule induced more than 1.2ꢀfold increase in apoptosis for breast cancer cells
when compared with the treatment of gastric cancer cells on the whole. This suggests
that the prepared cisplatin derivative loaded liposomes could achieve better
chemotherapy efficiency on human breast cancer cells. The cell cycle study indicates
that CJM126 obviously induces cell cycle arrest mainly at G2/M phase. To sum up,
the current studies indicate that the cisplatin derivative loaded liposomes have the
potential to be applied in the drug delivery system (DDS) for human breast cancer
therapy.
Acknowledgments
We are highly grateful to the financial support from the National Natural Science
Foundation of China (Nos. 21628101 and 21371031), the International S&T
Cooperation Program of China (No. 2015DFG42240) and the Priority Academic
Program Development （PAPD）of Jiangsu Higher Education Institutions.
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Supporting Information
Synthesis, characterization, IC50 values and relevant testing curve.
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Fig. 1. Overview of synthetic for the synthesis of CJM-Pt.
181x145mm (600 x 600 DPI)