drugs, cis-diamminedichloroplatinum (II) (cisplatin).13 Qi et al.14
demonstrated that tumor hypoxia promotes oral squamous cell
carcinoma resistance to the treatment of cisplatin. Currently,
clinical combination studies were developed using platinum
drugs with resistance modulators and new molecularly targeteddrugs. Our previous study developed in vitro delivery andin vivo transfection systems for bleomycin and the cdtB gene
into oral squamous cell carcinoma using sonoporation.15 In
addition, we also have developed anti-epidermal growth factor
receptor antibody-conjugate microbubbles and evaluated theircapacity to enhance anti-cancer drug cytotoxicity in vitro andin vivo.16
In this study, we evaluated the anticancer effect of thenovel platinum nanocomposite beads (PtNCP beads) developed by New Material Development Center in Nippon Steel &Sumikin Chemical Co., Ltd. in vivo and in vitro. It was demonstrated that PtNCP beads dramatically showed cytotoxicity
in HSC-3-M3 cells, and tumor growth inhibition in HSC-3-M3
xenografts, with extending histopathological necrosis lesion.
MATERIALS AND METHODS

Cell lines and culture conditions
HSC-3-M3, human oral squamous carcinoma cell lines weremaintained in Eagle’s Minimum Essential Medium (EMEM)(Wako, Osaka, Japan) supplemented with 10% fetal bovine
serum (FBS) (CORNING, NY, USA), penicillin G potassium(100 U/mL, Nacalai Tesque, Kyoto, Japan) and streptomycin
sulfate (100 mg) (Nacalai Tesque) at 37 C under 5% CO2
conditions.
Reagents
The latex beads (P2VPs) and PtNCP beads were provided by
New Material Development Center in Nippon Steel & Sumikin Chemical Co., Ltd (Chiba, Japan). The PtNCP beads nuclei
are composed of P2VPs and the surroundings are covered
with platinum. For preparation of P2VPs decorated withplatinum, the aqueous dispersion of P2VPs, 3.0 wt % 1.5 g,
was mixed with H2PtCl6 aqueous solution, 40 mM, 5.0 mL atroom temperature for 3 h. After removing the free metal
ions from these mixtures, these mixtures were redisposed indeionized water. Dimethylamine borane (DMAB) aqueous
solution (13 mM, 14 mL) was mixed with the aqueous
dispersion of precursor particles (0.073 wt %, 48.2 g). And
then, PtNCP beads were dialysis using deionized water.17
The size distribution of PtNCP beads did not change for8 months. PtNCP beads have long term dispersion stability.
In this study, P2VPs and the PtNCP beads were used at
5 mg/mL and at 10 mg/mL in vivo experiments (the dose of
PtNCP beads and P2VPs was determined according to thereport by Matsumura et al.17 and in vitro experiments
Figure 3).
Viability and cytotoxicity tests
Cell counting kit-8 (CCK-8) (Dojindo Laboratories Co., Kumamoto, Japan) was conducted to evaluate the effect of the
PtNCP beads on HSC-3-M3 cell viability. HSC-3-M3 cells werecollected by trypsinization, washed twice in phosphatebuffered saline (PBS: pH 7.2) and resuspended at 2.0 × 104

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TANAKA ET AL.

cells/ml in a 96-well plate. These cells were cultured at 37 C
under 5% CO2 for 24 h. Subsequently, the supernatant was
aspirated, changed to a new medium with PtNCP beads(0, 6.25, 12.5, 25, 50, and 100 μg/mL), and cultured for 48 hunder the same conditions. After that, CCK-8 assay were
performed according to the manufacturer’s recommended
protocol and absorbance was measured at 450 nm. Next, cell
cytotoxicity was tested by cytotoxicity lactate dehydrogenase(LDH) assay kit (Dojindo laboratories Co.). Culture and the
PtNCP beads pretreatment in the LDH assay were performed
under the same conditions as in the CCK-8 assay. The assay
was performed with the recovered supernatant according
to the manufacture’s protocol and the absorbance wasmeasured at 490 nm.
Scanning electron microscope analysis
For preparing scanning electron microscope (SEM) specimens,
HSC-3-M3 cells were seeded at 3 × 105 cells with 5% FBS (+)
EMEM into 35 mm dish (Asahi Glass Co., Tokyo, Japan) containing φ 14 mm plastic sheet (Wako). Thereafter, the PtNCP beads(4.0 × 103 beads/cell) was suspended and cultured at 37 C
under 5% CO2 for 48 h. After completion of the culture, thesupernatant was aspirated, washed twice with PBS and preﬁxed
with 2% glutaraldehyde ﬁxed solution for 1 h. Washed twicewith 0.1 M sucrose/0.1 M phosphate buffer and postﬁxed with
1% osmium. Dehydration with 20–100% ethanol and substitution with t-butanol were carried out. After lyophilization, platinum deposition was carried out to prepare a sample of SEM.
SEM observation was carried out with S-3300 N (HITACHI,Tokyo, Japan) operated with an acceleration voltage of 10 kV.
Animals and tumor xenograft model
A male of 6-week-old KSN/slc mice (body weight: 20–25 g)
were obtained from SLC (Shizuoka, Japan). All mice were fed
with standard feed and water, and individually reared in plastic cages in a monitored environment (temperature 22  1 C,
humidity 50  5%, 12 h light/12 h dark cycle). HSC-3-M3
cells (5.0 × 106 cells/200 μL in serum-free EMEM) were
injected into the back of mice. No signiﬁcant weight loss wasobserved in all mice. This animal experiment was approvedby the Kyushu Dental University Experimental Animal Careand Use Committee (Permit number: 17-018).
Measurement of tumor volume
In order to comprehend the inﬂuence on the tumor by administration of the PtNCP beads, beads corresponding to the experimental group were administered to the tumor, and then thevolume was measured to evaluate. Tumor xenograft model mice
were randomly divided into three groups, the untreated control
group (n = 6), the P2VPs-treated group (5 mg/mL) (n = 6) and
the PtNCP beads-treated group (10 mg/mL) (n = 6). Each bead
was administered at 50 μL/day/1 tumor on day 3, 4, and 5. Thelong and short diameters of a tumor and the body weight were
measured once every 2 or 3 days, and the tumor volume wascalculated by the following formula: volume (mm3) = a (mm) ×b2 (mm2)/2, where a and b are long and short diameters,
respectively measured with a digital caliper.15,18

ANTICANCER EFFECT OF PLATINUM NANOCOMPOSITE BEADSORIGINAL RESEARCH REPORTHistological staining
After the observation period has ended, mice were sacriﬁced
by cervical dislocation, the tumor was resected. The resected
tumor was ﬁxed in 10% formalin for 48 h, and then embedded in parafﬁn. The parafﬁn blocks were sliced in the vertical direction to the epithelium (4 μm thick sections), they
were stained with hematoxylin and eosin for histopathological analysis.
Statistical analysis
All data were expressed by mean value and  standard deviations (SDs) using Excel and analyzed by one-way ANOVA
test by Tukey method.
RESULTS

Antitumor activity of PtNCP beads in subcutaneous
HSC-3-M3 xenografts models
In initial experiments, we examined the effect of PtNCP beads ontumor growth in KSN/slc nude mice model, in vivo. Equal numbers of HSC-3-M3 cells (5.0 × 106 cells) were injected in threegroups of mice. One group received the cells alone, and otherwith 0.75 μg P2VPs or 1.5 μg PtNCP beads. Figure 1 shows
tumor images (A) and tumor growth curves (B) in the untreatedgroup, the P2VPs group and the PtNCP beads group. HSC-3-M3
cells were injected into back of KSN/slc nude mice. After 2 daysof inoculation, established subcutaneous tumor was visible (volume, 23–26 mm3). And then, mice were treated with P2VPs or
PtNCP beads for three times once every 1 day. At 14 days sacriﬁce, the mean tumor volumes in HSC-3-M3 cells xenografts were
91.38  19.71 mm3 (long diameter: 7.25  1.13 mm, short
diameter: 5.02  0.79 mm) in PtNCP beads treatment group,217.89  18.84 mm3 (long diameter: 8.27  0.37 mm, short
diameter: 7.25  0.37 mm) in P2VPs treatment group, and206.18  37.89 mm3 (long diameter: 9.64  2.26 mm, short
diameter 6.61  0.61 mm) in untreated group, respectively.
Tumor growth and tumor volume in PtNCP beads-treated groupwas signiﬁcantly different in P2VPs-treated group (*p < 0.05).
The PtNCP beads inhibition rate on tumor volume was 44.32%
compared to untreated group and 41.94% compared to P2VPs
treated group. Tumors in each group were analyzed histologically by hematoxylin and eosin staining. Necrotic areas were
often observed in the PtNCP beads-treated group (Fig. 2(G), (H),and (I)), compared to the untreated group (Fig. 2(A), (B), and(C)) and the P2VPs-treated group (Fig. 2(D), (E), and (F)). Whenthe necrosis area was magniﬁed and observed (Fig. 2(I), ×400),
transplanted tumor cells existed in the surroundings, and someof them had pyknosis and disappeared cell nuclei, and they had
necrosis. Mouse neutrophil inﬁltration was also observed
around the necrotic cells (Fig. 2(I), ×400).
In vitro cell viability of HSC-3-M3 cells after PtNCP
beads treatment
We examined the effects of PtNCP beads treatment on cell viability in HSC-3-M3 cells. The 50 and 100 μg/mL PtNCP beads
caused a decrease in cell viability compared to untreated
HSC-3-M3 cells at 48 h (*p < 0.005)(Fig. 3(A)). P2VPs treatment had no effect on cell viability (Fig. 3(B)). LDH assay
revealed that cytotoxicity in HSC-3-M3 cells remarkably

increased at concentrations above 50 μg/mL the PtNCP beads
compared to untreated HSC-3-M3 cells (*p < 0.005 and **p <0.001). In addition, we conﬁrmed an increase of the extracellular LDH level in HSC-3-M3 cells depending on the concentration of the PtNCP beads (Fig. 3(C)).
Cell damage of HSC-3-M3 cells induced by PtNCP beads
treatment
We examined cell shape and membrane surface morphology
in PtNCP beads-treated HSC-3-M3 cells utilizing SEM. The
membrane surface of HSC-3-M3 cells exhibited extensions of
the plasma membrane in untreated HSC-3-M3 cell (Fig. 4(A)). As shown in Figure 4(B), the PtNCP beads aggregatedaround cell membrane. The cytoskeleton became unclear
and the cell size expanded in PtNCP beads-treated HSC-3-M3
cells. The cell membrane was destroyed in which the PtNCP
beads are aggregated, with numerous small and variable
sized globular structures or blebs appearing at the cellsurface.
DISCUSSION

Metal nanoparticles, such as gold, silver and platinum nanoparticles, are being used in a wide array of applications thatreach far beyond therapeutics. Gold nanoparticles enhanced
radiosensitization in mouse tumor model. Glucose and PEGcoated gold nanoparticles were shown to effectively enhance
uptake and radiotherapy in breast adenocarcinoma cells,in vitro and in vivo.19 Silver nanoparticles have been widelyused as the therapeutic agents in anti-inﬂammatory or
wound healing.20 Platinum nanoparticles have been shown
to possess the capacity to enter human colon carcinoma
cells,21 and compromised DNA integrity and decrease cellular glutathione,12 leading to the hypothesis that platinum
ions from nanoparticles could be the cause. The platinumbased compounds cisplatin and carboplatin belong to thegroup of most frequently used anti-cancer drugs in clinicalpractice. However, their use is limited by severe organ toxicity. In this study, we used platinum nanoparticles-coated
P2VPs, PtNCP beads, and investigated the effect of PtNCP
beads on anticancer activity against oral squamous cell carcinoma cells.
As shown to Figures 1 and 2, we performed in vivo
experiments using subcutaneously xenografting mice withhuman squamous cell carcinoma cells, HSC-3-M3 cells.
PtNCP beads treatment against mouse xenograft decreased
the tumor volume compared to P2VPs treatment with histological necrotic lesion. Previous study reported that platinum
nanoparticles (500–2000 mg/kg) have suppressive effect ontumor growth,22,23 similar with our results. PtNCP beads
were suggested that Pt nanoparticles are formed not only on
the surface of P2VP particles but also inside P2VP cores.17
Further experiments will be needed to investigate that the
inhibitory effect of PtNCP beads on tumor growth by the surface and inside platinum nanoparticles.
Based on in vivo results, we performed in vitro experiments
using HSC-3-M3 cells to investigate the tumor growth inhibitory
effect of PtNCP beads. There is some evidence that nanoparticle

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FIGURE 1. Effect of PtNCP beads on tumor growth in HSC-3-M3 cell xenografts. HSC-3-M3 cells were injected into back of KSN/slc nude mice. After
2 days of inoculation, mice were treated with 5 mg/mL P2VPs or 10 mg/mL PtNCP beads for three times once every 1 day. All mice were divided
into three experimental groups: control (n = 6), P2VPs beads (n = 6), and PtNCP beads (n = 6). (A) Images of xenograft at the back of mice on threeexperiment groups. Bar =10 mm. (B) Growth inhibition of HSC-3-M3 cells in KSN/slc nude mice by PtNCP beads treatment. Arrows indicate theinjection days (Day 3, 4, and 5) of P2VPs or PtNCP beads. Closed square: P2VPs treatment. Closed diamond: non treatment. Closed triangle: PtNCP
beads treatment. Data were expressed as mean  SDs of three independent experiments made in three replicates. Data were analyzed by one-way
ANOVA test followed by Tukey’s test. *p < 0.05.

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TANAKA ET AL.

ANTICANCER EFFECT OF PLATINUM NANOCOMPOSITE BEADSORIGINAL RESEARCH REPORTFIGURE 2. Histological analysis of xenograft tumors. Hematoxylin & Eosin staining on each group; untreated group (A), P2VPs treated group(B) and PtNCP beads treated group (C) (Magniﬁcation beginning at the top, ×12.5, ×40, and ×400). Black squares indicate enlarged areas. Arrows
point to necrotic carcinoma cells. Arrowhead points to normal carcinoma cells.

pass through cell membranes and interact with cellular structures, and then have a direct impact on cell viability.24,25 Bendale et al.26 also have reported the cytotoxicity of platinum
nanoparticles in ovarian, lung and pancreatic cancer cell lines.
In contrast, no signiﬁcant cytotoxic effect was observed in thenormal human peripheral blood mononucleocyte.27 In agreement with our in vivo results, the results of WST-8 assay andLDH assay revealed the suppression of cell proliferation and theextracellular leakage of intracellular LDH in PtNCP beadstreated HSC-3-M3 cells (Fig. 3). LDH is a soluble cytoplasmic
enzyme that is present in cells and is released into extracellularspace when the cell plasma membrane is damaged.26 Necrotic
cell death is evaluated by determining damage of the plasma
membrane. In contrast to apoptosis, necrosis was once considered as a passive and accidental form of cell death. However,recent studies have clearly established that necrosis can be a
programmed event, called necroptosis.28 The SEM image(Fig. 4) in HSC-3-M3 cells suggested the possibility of cell membrane destruction by PtNCP beads. In addition, PtNCP beads
treatment induced cytotoxicity with extracellular LDH releasein HSC-3-M3 cells, in vitro. These results suggested that cell
membrane destruction by PtNCP beads treatment may strongly
be supported the induction of necroptosis.
Since platinum nanoparticles have been reported toinduce apoptosis signaling,28,29 we also investigated the relationship between inhibitory effect on cell proliferation andcell death induction in PtNCP beads-treated HSC-3-M3 cells.
Although a necrotic rather than an apoptotic mechanism ofkilling is suggested, the detail by which PtNCP beads had
cytotoxicity to oral squamous cell carcinoma cells is still not
fully understood. To better understand the cell death mechanism, we conﬁrmed apoptotic mechanism, such as caspase-3,−8, −9, and p53 in PtNCP beads-treated HSC-3-M3 cells.
However, there was no marked difference in apoptotic

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FIGURE 3. Effect of PtNCP beads on cell viability and cytotoxicity of HSC-3-M3 cells. HSC-3-M3 cells were treated with P2VPs and PtNCP beads for
48 h. (A, B) Cell viability of HSC-3-M3 cells with PtNCP beads treatment (A) (n = 3, *p < 0.005 vs. untreated group) and P2VPs treatment (B) was
measured using CCK-8 assay. (C, D) Cell cytotoxicity of HSC-3-M3 cells with PtNCP beads treatment (C) (n = 3,*p < 0.005, and **p < 0.001
vs. untreated group) and P2VPs treatment (D) treatment was measured using LDH assay. Data were expressed as mean  SDs of three independent
experiments made in three replicates. Data were analyzed by one-way ANOVA test followed by Tukey’s test.

protein expression level under PtNCP beads treatment (datanot shown). Considering expansion of cell morphology andleakage of LDH in SEM, other cell death will be suggested,such as necroptosis. In the future, it is necessary to further
investigation of signal induction for several cell death types
in PtNCP beads treatment.

In conclusion, PtNCP beads exhibited cytotoxic activity
against oral squamous cell carcinoma cells with necrotic cell
death. The antitumor efﬁcacy of PtNCP beads for treating oral
squamous cell carcinoma in xenograft mouse lends support
for its use in various traditional systems of medicine. PtNCP
beads can also be expected to be used as a drug transport

FIGURE 4. SEM images in HSC-3-M3 cells (magniﬁcation × 2000). (A) Untreated HSC-M3 cells, (B) PtNCP beads-treated HSC-3-M3 cells. Bar = 20
μm. Arrows points to a PtNCP bead and an arrowhead points to the destruction of cell membrane.

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TANAKA ET AL.

ANTICANCER EFFECT OF PLATINUM NANOCOMPOSITE BEADSORIGINAL RESEARCH REPORT

carrier and as an application for improving therapeutic effects.
In addition, we have developed the intracellular drug delivery
method using ultrasound and cancer speciﬁc antibodiesconjugated microbubbles.15,16 Further studies are required
to attach PtNCP speciﬁcally to cancer cells, developing cancer
speciﬁc antibodies-modiﬁed PtNCP beads. The strong membranolitic effect of PtNCP beads should make it difﬁcultfor the cancer cell to develop resistance to widespread used
platinum drugs. These therapeutic combinations should be
explored additionally for therapeutic use.
ACKNOWLEDGMENTS

We would like to acknowledge Dr. Matsuo Kou and
Dr. Toyono Takashi for technical advice during the experiments. We also thank to the researchers of Nippon Steel &Sumikin Chemical Co., Ltd. for providing P2VPs and PtNCP
beads. This study was supported by a Grant-in-Aid for
Scientiﬁc Research (15 K11083 and 16 K11698), from the
Ministry of Education, Culture and Science of Japan.
CONFLICT OF INTEREST

The authors declared no potential conﬂicts of interest withrespect to research, authorship, and/or publication of thisarticle.
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