Cancer cell labeling and tracking using
fluorescent and magnetic nanodiamond
Zhi-Yi Lien
a
, Tzu-Chia Hsu
a
, Kuang-Kai Liu
b
, Wei-Siang Liao
a
, Kuo-Chu Hwang
c
, Jui-I. Chao
a
,b
,*
aInstitute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan
bDepartment of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan
cDepartment of Chemistry, National Tsing Hwa University, Hsinshu 300, Taiwan
a r t i c l e i n f o
Article history:Received 23 March 2012 Accepted 5 May 2012 Available online 5 June 2012 Keywords:
Fluorescent and magnetic nanodiamond Nanodiamond-bearing cancer cells Flow cytometer
Magnetic device Confocal microscope
a b s t r a c t
Nanodiamond, a promising carbon nanomaterial, develops for biomedical applications such as cancer cell labeling and detection. Here, we establish the nanodiamond-bearing cancer cell lines using the fluorescent and magnetic nanodiamond (FMND). Treatment with FMND particles did not significantly induce cytotoxicity and growth inhibition in HFL-1 normal lungfibroblasts and A549 lung cancer cells. Thefluorescence intensities and particle complexities were increased in a time- and concentration-dependent manner by treatment with FMND particles in lung cancer cells; however, the existence of FMND particles inside the cells did not alter cellular size distribution. The FMND-bearing lung cancer cells could be separated by thefluorescent and magnetic properties of FMNDs using the flow cytometer and magnetic device, respectively. The FMND-bearing cancer cells were identified by the existence of FMNDs usingflow cytometer and confocal microscope analysis. More importantly, the cell morphology, viability, growth ability and total protein expression profiles in the FMND-bearing cells were similar to those of the parental cells. The separated FMND-bearing cells with various generations were cryopres-ervation for further applications. After re-thawing the FMND-bearing cancer cell lines, the cells still retained the cell survival and growth ability. Additionally, a variety of human cancer types including colon (RKO), breast (MCF-7), cervical (HeLa), and bladder (BFTC905) cancer cells could be used the same strategy to prepare the FMND-bearing cancer cells. These results show that the FMND-bearing cancer cell lines, which reserve the parental cell functions, can be applied for specific cancer cell labeling and tracking.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The use of nanomaterials in biomedical applications is of great
interest since their size scale is similar to biological molecules and
structures
[1,2]
. Many nanomaterials such as quantum dots (QDs)
[2]
, gold nanobeads
[3]
, and silica nanoparticles
[4]
have been
developed for biomedical applications. The
fluorescent molecules
were covalently linked to iron oxide nanoparticles that used for the
dynamic tracking of human stem cells
[5]
. Furthermore, the human
induced pluripotent stem cells (iPS) can be generated by using
polyamidoamine dendrimer-modi
fied magnetic nanoparticle as
the transfection system for in vivo imaging and tracking
[6]
.
Besides, the silicon QDs can be used as
fluorescent probes for tumor
vasculature
targeting,
sentinel
lymph
node
mapping,
and
multicolor near infrared imaging in live mice
[4]
. In addition to
bio-labeling and -imaging, nanoparticles were studied for carrying
drugs in cancer therapies
[7
e9]
.
Nanodiamond (ND), a carbon derivative nanomaterial has
become a promising candidate in biological applications
[10
e15]
.
As substitutes for the aforesaid nanomaterials, ND is a nanomaterial
applicable in the biomedical
field, due to its excellent
biocompat-ibility as compared with other nanoscale carbon materials. It has
been corroborated that ND can be used in many cell lines without
obvious cytotoxicity such as lung
[16,17]
, neuronal
[18]
, renal
[19]
,
and cervical cells
[20,21]
. Moreover, ND did not lead to obvious
abnormality in cell division, differentiation and morphological
changes in embryonic development
[17,22]
.
ND has several advantages from its electrochemical and optical
properties. Biological molecules or therapeutic agents can be
provided, either by chemical modi
fication of NDs, or by allowing
physical attachment to NDs, which acts as a convenient binding
platform for chemical agents. The surface functionalized ND
particles have been shown to conjugated with
fluorescent
mole-cules
[18,21,23
e25]
, DNA
[26]
, siRNA
[27]
, proteins
[28
e30]
,
* Corresponding author. Department of Biological Science and Technology,
National Chiao Tung University, 75, Bo-Ai Street, Hsinchu 30068, Taiwan. Tel.:þ886
3 5712121; fax:þ886 3 5556219.
E-mail address:jichao@faculty.nctu.edu.tw(J.-I. Chao).
Contents lists available at
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0142-9612/$e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
0.5 h
A549 cells
FMND (
μ
g/ml, 24 h)
0
0.1
1
10
50
100
Cell viability (%)
0
20
40
60
80
100
DDW
PBS
0 h
2 h
4 h
12 h
24 h
PBS
DDW
A
PBS
DDW
DDW
PBS
DDW
PBS
DDW
PBS
DDW
PBS
Diameter (nm)
0 150 300 450 600 750 900 1050120013501500Number
0
20
40
60
80
100
FMND in DDW
FMND in PBS
HFL-1
FMND (
μ
g/ml, 24 h)
0
0.1
1
10
50
100
Cell viability (%)
0
20
40
60
80
100
DDW
PBS
C D
A 549 cells (n=3)
Days after FMND treatment
0
2
4
6
8
10
Cell
number
(x
10
6)
0
50
100
150
200
250
300
350
400
untreated
FMND in PBS
FMND in DDW
E
B
Fig. 1. Effect of cell viability and growth ability by treatment with FMND particles in human lung normalfibroblast and lung cancer cells. (A) Distilled deionized water (DDW) or
phosphate-buffered saline (PBS) were used as solvent for FMND particles. The stock solution of FMND particles was 2.1 mg/ml. The equal volume of 100ml FMND solution in DDW or
PBS was added in 0.5 ml eppendorf tube, and stayed for 0e24 h observation. (B) The concentration of 0.5 mg/ml FMND particles in DDW or PBS was prepared by dynamic light scattering (DLS) analysis. The green picks indicate the size distribution of FMNDs in DDW. The orange picks indicate the size distribution of FMNDs in PBS. The effects of FMNDs on
the cell viability in HFL-1 normal lungfibroblasts (C) and A549 lung carcinoma cells (D) were measured by MTT assays. The cells were treated with or without FMND (0.1e100mg/ml
for 24 h). Results were obtained from three separate experiments and the bar represents mean S.E. (E) A549 cells were plated at a density of 1 106cells/100-mm Petri dish for
24 h, and then the cells were incubated with or without 50mg/ml of FMNDs for 24 h. The cells were re-cultured in fresh medium for counting the total cell number by every 2 days
until total 10 days. Results were obtained from three separate experiments and the bar represents the mean S.E. (For interpretation of the references to color in this figure legend,
Ctrl.
10
25
50
75
100
A549 cells (n=3)
FMND (
μ
g/ml, 24 h)
Ctrl.
10
25
50
75
100
Fluorescence intensity
(fold)
0
20
40
60
80
100
*
*
*
*
*
A
C
A549 cells (n=4)
FMND (
μ
g/ml, 24 h)
Ctrl.
10
25
50
75
100
Particles complexity (fold)
0.0
0.5
1.0
1.5
2.0
*
*
*
*
*
D
Ctrl.
10
25
50
75
100
Cell counts
Fluorescence intensity
Particle complexity
Ctrl.
Particle complexity
Fluorescence intensity
10
25
50
75
100
B
E
200
160
120
80
40
0
Cell counts
200
160
120
80
40
0
0
200
400
600
800 1000
10
010
110
210
310
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
SSC-H
Fig. 2. Fluorescence intensity and particle complexity of FMND particles inside A549 lung carcinoma cells in a concentration-dependent manner. (A) The cells were incubated with
0e100mg/ml FMNDs for 24 h. At the end of treatment, the cells were trypsinized and then subjected toflow cytometer analysis. Y-axis indicates the cell counts. The fluorescence
intensity from FMND was excited with wavelength 488 nm, and the emission was collected in 515e545 nm signal range (FL1-H). (B) The fluorescence intensity was quantified from
a minimum of 10,000 cells by CellQuest software. Results were obtained from three separate experiments and the bar represents mean S.E. *p < 0.05 indicates significant
difference between untreated and FMND-treated samples. (C) The cells were incubated with 0e100mg/ml FMNDs for 24 h. Thefluorescence intensity from FMNDs was excited with
wavelength 488 nm, and the emission was collected in 515e545 nm signal range (FL1-H). SSC-H indicates the particle’s complexity. (D) Y-axis indicates the cell counts. SSC-H indicates the particle’s complexity. (E) The particle complexity of SSC-H was quantified from a minimum of 10,000 cells by CellQuest software. Results were obtained from
A549 cells
FMND (50
μ
g/ml)
Ctrl.
0.5 h
2 h
4 h
12 h
24 h
Fluorescence intensity
(f
old)
0
10
20
30
40
*
*
*
*
*
C
D
Ctrl.
0.5 h
2 h
4 h
12 h
24 h
A549 cells (n=4)
FMND (50
μ
g/ml)
Ctrl.
0.5 h
2 h
4 h
12 h
2 4 h
Particle complexity (fold)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
*
*
*
*
Ctrl.
0.5 h
2 h
4 h
12 h
24 h
A
Cell counts
Fluorescence intensity
Cell counts
Particle complexity
Ctrl.
Particle complexity
Fluorescence intensity
B
E
200
160
120
80
40
0
10
010
110
210
310
4FL1-H
SSC-H
1000
0
SSC-H
10
010
4FL1-H
0.5 h
2 h
4 h
12 h
24 h
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
SSC-H
10
010
4FL1-H
1000
0
10
010
4FL1-H
250
200
150
100
50
0
0
200
400
600
800
1000
SSC-H
Fig. 3. Fluorescence intensity and particle complexity of FMND in A549 cells via a time-dependent manner. (A) The cells were incubated with or without 50mg/ml FMNDs for
0e24 h. At the end of treatment, the cells were trypsinized and then subjected to flow cytometer analysis. Y-axis indicates the cell counts. The fluorescence intensity from FMND was excited with wavelength 488 nm and the emission was collected in 515e545 nm signal range (FL1-H). (B) The fluorescence intensity was quantified from a minimum of 10,000
cells by CellQuest software. Results were obtained from three separate experiments and the bar represents mean S.E. *p < 0.05 indicates significant difference between untreated
and treated samples. (C) The cells were incubated with or without 50mg/ml FMNDs for 0e24 h. The fluorescence intensity from FMNDs was excited with wavelength 488 nm, and
the emission was collected in 515e545 nm signal range (FL1-H). SSC-H indicates the particle’s complexity. (D) Y-axis indicates the cell counts. SSC-H indicates the particle’s complexity. (E) The particle complexity of SSC-H was quantified from a minimum of 10,000 cells by CellQuest software. Results were obtained from three separate experiments and
A
B
D
E
A549 cells (n=4)
FMND (
μ
g/ml, 24 h)
Ctrl.
10
25
50
75
100
Cell size (fold)
0.0
0.2
0.4
0.6
0.8
1.0
Ctrl.
10
25
50
75
100
Ctrl.
0.5 h
2 h
4 h
12 h
24 h
A549 cells (n=4)
FMND (50
μ
g/ml)
Ctrl.
0.5 h
2 h
4 h
12 h
24 h
Cell size (fold)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Ctrl.
Cell size
Fluorescence intensity
10
25
50
75
100
Ctrl.
Cell size
Fluorescence intensity
0.5 h
2 h
4 h
12 h
24 h
Cell counts
Cell size distribution
Cell size distribution
Cell counts
C
F
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
1000
0
FSC-H
10
010
4FL1-H
120
100
80
40
60
20
0
0
200
400
600
800
1000
FSC-H
120
100
80
40
60
20
0
0
200
400
600
800
1000
FSC-H
10
0Fig. 4. Cell size distribution of A549 cells did not alter following exposure to FMND particles. (A) The cells were incubated with 0e100mg/ml FMND for 24 h. At the end of
lysozyme
[31]
, growth hormone
[32]
, cytochrome c
[33]
, alcohol
dehydrogenase
[34]
, antibodies
[35,36]
, anti-cancer drugs
[37
e40]
,
and dopamine derivatives
[41]
.
ND can emit bright
fluorescence, and does not bring about
photobleaching
[19,31]
. Also, the
fluorescent property can be
introduced to an ND by binding a
fluorescent molecule to the ND
surface
[18,21,23,24]
. For example, the aminated NDs, ND-NH
2,are
coupling to the reactive N-hydroxysuccinimide functionalized
tet-ramethylrhodamine (TAMRA) to form TAMRA-ND
[18,25]
.
TAMRA-ND complex is a high stable
fluorescent probe to be used as
a cellular tracking label in static images
[18]
. In addition to
fluo-rescence properties, ND particles with the magnetic property may
be developed as a contrast reagent for magnetic-resonance imaging
(MRI). It has been reported that nitrogen (
15N) and carbon (
12C) ion
implantations with implant energy of 100 keV on NDs can produce
magnetism in ND particles
[42]
. Furthermore, magnetic
nano-particles can be conjugated on the surface of ND
[21,43]
.
Although a theory postulating that endocytic ND clusters can be
segregated during cell division and remain as a single ND cluster
[17]
, there is currently no report on methods for separating
ND-labeled cells and whether such ND-labeled cell lines survive or have
the ability to be sub-cultured. In present study, we develop the
ND-bearing cancer cell lines using the
fluorescent and magnetic
nanodiamond (FMND) particles. It is successfully separated the
FMND-bearing cells by
flow cytometer or magnetic device. The
FMND-bearing cells can be characterized and detected by
flow
cytometer and confocal microscope; more importantly, these
FMND-bearing cells reserve cell viability and growth ability
without altering cellular functions by comparing with the parental
cells. The generations of FMND-bearing cancer cells can be applied
for speci
fic cancer cells on the labeling and tracking.
2. Materials and methods 2.1. Preparation of FMNDs
Thefluorescent and magnetic nanodiamond, which called FMND, was
synthe-sized as described previously[21]. Specifically, magnetic nanodiamond (MND) was
composed of pristine ND and iron nanoparticle (ferrocene) via a microwave-arcing process. The ferrocene particles and NDs formed MNDs by chemically bonding. To
introducefluorescence in MNDs, MNDs were converted into FMNDs by covalent
surface grafting with polyacrylic acids and fluorescein o-methacrylate. FMND
particles were dissolved in distilled deionized water (DDW) or phosphate-buffered saline (PBS), before the treatment of cells using FMNDs.
2.2. Dynamic light scattering
The stock solution of 2.1 mg/ml FMND particles was prepared using DDW or PBS. To examine the size distribution of FMNDs dissolved in DDW and PBS, the concentration of FMND particles in DDW or PBS (0.5 mg/ml) was prepared and analyzed by DLS (BI-200SM, Brookhaven Instruments Co., Holtsville, NY). In a particular suspension, when a beam of laser hits the particle, the particles scattered some of the laser. The measured data were subjected to the BIC dynamic light scattering software (Brookhaven Instruments Co.). The scattered light changed over time, and the average particle size was calculated by the variation of scattered light.
2.3. Cell culture
HFL-1 cells (ATCC #CCL-153) were normal lung fibroblasts derived from
a Caucasian fetus. The A549 lung epithelial cell line (ATCC #CCL-185) was derived from the lung adenocarcinoma of a 58 year old Caucasian male. RKO was colon carcinoma cell line. BFTC905 cells were derived from bladder carcinoma. MCF-7 was
a breast cancer cell line. HeLa was a cervix cancer cell line. HFL-1 and HeLa cells were maintained in DMEM medium (Invitrogen Co., Carlsbad, CA). A549, BFTC905, MCF-7 cells were maintained in RPMI-1640 medium (Invitrogen). The complete media
contained 10% fetal bovine serum (FBS), 100 unit/ml penicillin and 100mg/ml
streptomycin. These cells were incubated at 37C, and maintained in 5% CO2in
a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH). 2.4. MTT assays
The cells were plated in 96-well plates at a density 1104cells/well for 16e20 h.
The cells were treated with or without FMNDs for 24 h in complete medium. Subsequently, the medium was replaced and the cells were incubated with 0.5 mg/ ml of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO) in complete medium for 4 h. The surviving cells con-verted MTT to formazan, which generates a blue-purple color when dissolved in dimethyl sulfoxide (DMSO). The intensity of formazan was measured at 565 nm using a plate reader (VERSAmax, Molecular Dynamics Inc., CA) for enzyme-linked immunosorbent assays. Cell viability was calculated by dividing the absorbance of the cells treated with FMNDs by that of the cells not treated with FMNDs. 2.5. Cell growth assays
A549 cells were seeded at a density of 1 106cells per 100-mm Petri dish in
complete medium for 24 h. Then, the cells were treated with or without FMNDs (50mg/ml) for 24 h. Subsequently, the cells treated or untreated with FMNDs were re-cultured in fresh medium for counting the total cell number every 2 days, for a total of 10 days.
2.6. Cellularfluorescence intensity, particles complexity and size distribution by
FMNDs
A549 cells were plated at a density of 7 105cells per 60-mm Petri dish in
complete medium for 16e20 h. After treatment in medium with or without FMNDs, the cells were washed twice with PBS. The cells were trypsinized and collected by centrifugation at 1500 rpm for 5 min. Thereafter, the cell pellets were re-suspended
in PBS. To avoid cell aggregation, the cell suspension wasfiltered through a nylon
mesh membrane. Finally, the samples were analyzed byflow cytometer
(FACSCali-bur, BectoneDickinson, San Jose, CA). A minimum of 10,000 cells were analyzed. The fluorescence of FMNDs was excited at a wavelength of 488 nm, and was collected in
the green light signal range. Thefluorescence intensity, particle complexity and cell
size were quantified using a minimum of 10,000 cells by CellQuest software (BD Biosciences).
2.7. Immunofluorescence staining and confocal microscopy
The cells were cultured on cover slips, and kept in a 35-mm Petri dish for 16e20 h before treatment. After treatment with or without FMNDs, the cells were
washed with isotonic PBS (pH 7.4), and then werefixed with 4% paraformaldehyde
solution in PBS for 1 h at 37C. Thereafter, the cover slips were washed three times
with PBS and non-specific binding sites were blocked in PBS containing 10% FBS,
0.3% Triton-X-100 for 1 h. Theb-tubulin and nuclei were stained with anti-b-tubulin
Cy3 (1:100) and Hoechst 33258 (Sigma Chemical Co., St. Louis, Mo) for 30 min at
37C, respectively. Finally, the samples were examined under an OLYMPUS confocal
microscope (FV500, OLYMPUS, Japan) or Confocal Microscope System (TCS-SP5-X AOBS, Leica, Germany).
2.8. Separation of FMND-bearing cells by aflow cytometer
The protocol of separating FMND-bearing cells byflow cytometer shows in
supplementary protocol 1. Briefly, the cells were plated at a density of 2 106cells
per 100-mm Petri dish in complete medium for 24 h. After treatment with FMNDs (50mg/ml) for 24 h, the cells were washed twice with PBS. The washed cells were trypsinized and collected by centrifugation at 1500 rpm for 5 min. Thereafter, the cell pellets were re-suspended in 1e2 ml ice-cold sorting buffer, which contained
1 mMEDTA, 25 mMHEPES and 2% FBS in PBS. To avoid cell aggregation, the cell
suspension was filtered through a nylon mesh membrane. The
fluorescence-activated cell-sorting analyses were performed with a FACSCalibur with a sorter (BectoneDickinson). The FMND-bearing cells, which displayed green fluorescence
intensity inflow cytometer, were selected for separation. The separated cells were
collected in a 50 ml centrifuge tube that had been coated with 10% FBS on the wall
was collected in 515e545 nm signal range (FL1-H). FSC-H indicates the cell size distribution. (B) Y-axis indicates the cell counts. FSC-H indicates the cell size distribution. (C) The cell size distribution of FSC-H was quantified from a minimum of 10,000 cells by CellQuest software. Results were obtained from three separate experiments and the bar represents
mean S.E. (D) The cells were incubated with or without 50mg/ml FMND for 0e24 h. The fluorescence intensity from FMNDs was excited with wavelength 488 nm and the
emission was collected in 515e545 nm signal range (FL1-H). FSC-H indicates the cell size distribution. (E) Y-axis indicates the cell counts. FSC-H indicates the cell size distribution. (F) The cell size distribution of FSC-H was quantified from a minimum of 10,000 cells by CellQuest software. Results were obtained from three separate experiments and the bar
and contained 15e20 ml of complete medium inside. After separation, the cell suspensions were centrifuged at 1000 rpm for 10 min. Then, the cell pellets were
re-suspended in complete medium. The cells were incubated at 37C, in 5% CO2in
a humidified incubator, or added 10% DMSO for storage in liquid nitrogen. 2.9. Separation of FMND-bearing cells by a magnetic device
The protocol of separating FMND-bearing cells by magnetic device shows in
supplementary protocol 2. Briefly, the cells were plated at a density of 2 106
cells
per 100-mm Petri dish in complete medium for 24 h. After treatment with 50mg/ml
FMNDs for 24 h, the cells were washed twice with PBS. The washed cells were trypsinized, and collected by centrifugation at 1500 rpm for 5 min. The cell pellets were re-suspended in 1 ml PBS and transferred to 1.5 ml eppendorf tubes. The eppendorf tubes were placed onto a magnetic rack (Magna GrIP Rack, Millipore, Bedford, MA) for at least 3 min, until the cell pellets were absorbed on the tube walls. Then, the suspensions were removed, and the cell pellets were dissolved in complete medium. The cell suspensions with the eppendorf tubes were repeatedly placed onto the magnetic rack 5 times. Finally, the FMND-bearing cells were
incu-bated at 37C, and in 5% CO2in a humidified incubator, or added 10% DMSO for
storage in liquid nitrogen. 2.10. SDS-PAGE analysis
To compare the total protein expression profiles between the parental and FMND-bearing cells, the cells were subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The separated FMND-bearing cells were lysed in the ice-cold cell extraction buffer (pH 7.6), which
con-tained 0.5 mMDTT, 0.2 mMEDTA, 20 mMHEPES, 2.5 mMMgCl2, 75 mMNaCl, 0.1 mM
Na3VO4, 50 mMNaF, 0.1% Triton-X-100. The protease inhibitors included 1mg/ml
aprotinin, 0.5mg/ml leupeptin and 100mg/ml 4-(2-aminoethyl)
benzenesulfonyl-fluoride were added to the cell suspension. The cell extracts were gently rotated at
4C for 30 min. After centrifugation, the pellets were discarded, and the supernatant
protein concentrations were determined by a BCA protein assay kit (Pierce,
Rock-ford, IL). Equal amounts of proteins (40mg) were subjected to electrophoresis by 12%
SDS-PAGE. After electrophoresis, the gel was stained with a coomassie blue buffer (0.1% coomassie blue, 10% acetic acid and 45% methanol) for 1 h.
2.11. Statistic analysis
Data was analyzed using Student’s t-test, and a p value < 0.05 was considered as statistically significant in the experiments.
3. Results
3.1. Size distribution of FMNDs in DDW and PBS
The stock solution of 2.1 mg/ml FMND particles was prepared
using DDW or PBS in tubes, and observed by immobilized for
A
Phase
contrast
FMND
fluorescence
Parental cells
FMND-bearing cells
B
Particle complexity
SSC-H
Fluorescence intensity
FL1-H
FL1-H
untreated
FMND-treated
1000
800
600
200
400
0
10
010
110
210
310
4SSC-H
1000
800
600
200
400
0
10
010
110
210
310
4Fig. 5. Separation of the FMND-bearing cells byflow cytometer with a sorter. (A) A549 cells were plated at a density of 2 106cells per 100-mm Petri dish for 24 h. Then the cells
were incubated with 50mg/ml of FMND for 24 h. After FMNDs treatment, the cells were collected in sorting buffer, and the FMND-bearing cells were separated byflow cytometer.
The R1 gate indicates the region of control (greenfluorescence-negative) cells. The R2 gate indicates the region of FMND-bearing (fluorescence-activated) cells. (B) After separation
byflow cytometer, the cells were immediately observed under a living cell imaging system with a fluorescent and phase contrast microscope (40 magnification). The fluorescence
0
e24 h.
Fig. 1
A shows that FMNDs in DDW formed precipitates in
the bottom of tubes after 2 h observation. FMNDs in PBS did not
signi
ficantly cause aggregates after 24 h observation (
Fig. 1
A). To
measure the size distribution of FMNDs, 0.5 mg/ml of FMND
solution prepared in DDW or PBS was analyzed by DLS. The
first
peak of FMNDs in PBS was from 95.22 to 116.34 nm, and the second
peak was from 272.58 to 333.04 nm (
Fig. 1
B, orange lines). The
average size of FMNDs in PBS was 131.7 nm. The
first peak of
FMNDs in DDW was from 137.24 to 176.71 nm, and the second peak
was from 805.15 to 1127.86 nm (
Fig. 1
B, green lines). The average
size of FMNDs in DDW was 277.7 nm.
3.2. No signi
ficant cytotoxicity and cell growth inhibition by FMNDs
in HFL-1 normal
fibroblasts and A549 lung cancer cells
To examine cytotoxicity following treatment with FMMDs in
human lung cells, HFL-1 normal
fibroblasts and A549 human lung
carcinoma cells were used. The cells were treated with FMNDs, and
analyzed by MTT assays. As shown in
Fig. 1
C, HFL-1 normal
fibro-blasts treated with FMNDs (0.1
e100
m
g/ml, 24 h) did not signi
fi-cantly reduce cell viability. Similarly, FMND particles did not induce
cytotoxicity in A549 cells (
Fig. 1
D). The cell growth ability was
analyzed after treatment with or without 50
m
g/ml FMND particles
for 24 h in A549 cells, and then further cultured for another 10 days.
The total cell number was counted every 2 days.
Fig. 1
E shows that
FMND particles did not alter the cell growth ability in A549 cells.
Using DDW or PBS as an FMND solvent had no signi
ficantly altered
the cell viability and cell growth ability (
Fig. 1
C
eE). To prepare the
FMND-bearing cancer cells, PBS was used as a solvent for FMND
particles in this study.
3.3. Fluorescence intensity, particle complexity and size distribution
of A549 lung cancer cells after treatment with FMNDs
The
fluorescence intensity of FMNDs in A549 cells was
examined by
flow cytometer. Treatment with FMNDs (10e100
m
g/
Parental cells
FMND-bearing cells
A
B C
Parental cells
FMND-bearing cells
181
122
80
51
40
26
Protein marker
(kDa)
Parental cells
FMND-bearing cells
Cell counts
Fluorescence intensity
200
160
120
80
40
0
FL1-H
10
010
110
210
310
4Fig. 6. Comparison of cell viability and total protein expression profiles between the parental and FMND-bearing cancer cells. (A) A549 cells were plated at a density of 2 106cells
per 100-mm Petri dish for 24 h. Then the cells were incubated with 50mg/ml of FMND for 24 h. At the end of treatment, the FMND-bearing cells were separated byflow cytometer.
After separation, the cells were re-cultured in fresh medium for 24 h. Representative phase contrast photomicrographs (40 magnification) show that cell morphology and viability in the parental cells and FMND-bearing cells. (B) Total protein expression profiles were compared between the parental and FMND-bearing cells. At the end of incubation, the total protein extracts were prepared for SDS-PAGE analysis. The left lane indicates the loading marker of proteins. (C) After separation, the FMND-bearing cells were re-cultured in fresh
1st
2nd
3rd
4th
Generations
A
Parental
cells
1st FMND-bearing cells
2nd FM
3rd FMND-bearing cells
ND-bearing cell
s
4th FMND-bearing cells
181
122
13
19
80
51
40
26
Protein marker
(kDa)
Parental cells
2nd FMND-bearing cells
3rd FMND-bearing cells
4th FMND-bearing cells
1st FMND-bearing cells
C
FMND-bearing cells
Generations
1st
2nd
3rd
4th
Separating ratio (%)
0
20
40
60
80
100
D
Cell counts
Fluorescence intensity
FL1-H
Parental cells
FMND-bearing
cells
B
phase contrast
FMND
merge
200
160
120
80
40
0
10
010
110
210
310
4Fig. 7. Comparison of cell morphology, viability, total protein expression profile, and fluorescence intensity in the parental and FMND-bearing cells with various generations. (A)
A549 cells were plated at a density of 2 106cells per 100-mm Petri dish for 24 h. Then the cells were incubated with 50mg/ml of FMNDs for 24 h. At the end of treatment, the 1st
generation of FMND-bearing cells was separated by magnetic device. After separation, the FMND-bearing cells were re-cultured in fresh medium for 24 h. Thereafter, the cells were subjected to magnetic device for separation of the 2nd generation of FMND-bearing cells. The same protocol was repeated to separate the 3rd and 4th generations of FMND-bearing
ml for 24 h) in A549 cells increased the
fluorescence intensities of
the cells (
Fig. 2
A). The quanti
fied data showed that the
fluores-cence intensities of FMNDs inside A549 cells were in a
concen-tration-dependent manner (
Fig. 2
B). The intracellular particle
complexities of FMNDs in A549 cells were examined by SSC-H
(lateral light scatter) using
flow cytometer analysis. Treatment
with FMNDs (10
e100
m
g/ml for 24 h) increased the intracellular
particle complexities of A549 cells (
Fig. 2
C and D). The quanti
fied
data showed that the intracellular particle complexities of FMNDs
in A549 cells were in a concentration-dependent manner
(
Fig. 2
E). Besides, treatment with FMNDs (50
m
g/ml) for a
time-course (0.5
e24 h) in A549 cells increased both the fluorescence
intensities (
Fig. 3
A and C) and the intracellular particle
complexities (
Fig. 3
C and D). The quanti
fied data showed that
cells. The cell morphology, viability, and growth ability of FMND-bearing cells with different generations were observed under a phase contrast photomicroscope (40 magnifi-cation). (B) The total protein extracts from the parental cells and FMND-bearing cells were prepared for SDS-PAGE analysis. The left lane indicated the loading marker of proteins. (C)
The different generations of FMND-bearing cells were separated by magnetic device. The greenfluorescence of FMNDs in cells was detected by a living cell imaging system or flow
cytometer. (D) A549 cells were plated at a density of 2 106cells per 100-mm Petri dish for 24 h. Then the cells were incubated with 50mg/ml of FMNDs for 24 h. Different
generations of FMND-bearing cells were separated by magnetic device. The separating ratio of FMND-bearing cells is calculated by separated cell number from magnetic device dividing to total counted cell number before separation.
nuclei
β
- tubulin
FMND
merge
phase
contrast
10
μ
m
FMND-bearing cells
(separated by flow cytometer)
Parental cells
FMND-bearing cells
(separated by magnetic device)
B
40
μ
m
A
10
μ
m
10μm
nuclei
β
- tubulin
FMND
merge
separated by
flow cytometer
separated by
magnetic device
C
Fig. 8. Comparison of cell morphology and viability between the parental cells and re-thawed FMND-bearing cells. (A) A549 cells were plated at a density of 2 106cells per
100-mm Petri dish for 24 h. Then the cells were incubated with 50mg/ml of FMNDs for 24 h. At the end of treatment, the FMND-bearing cells were separated by magnetic device. After
separation, the FMND-bearing cells were re-cultured on cover slips with fresh medium for 24 h. At the end of incubation, the cells werefixed and stained with the Cy3-labeled
anti-b-tubulin and Hoechst 33258. The greenfluorescence from the FMNDs was excited with 488 nm, and the emission collected in the range of 510e530 nm. Theb-tubulin protein
displayed redfluorescence. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. (B) The FMND-bearing A549 cell lines separated by flow cytometer or
magnetic device were stored in liquid nitrogen. The cryopreservation of FMND-bearing cells was re-thawed in complete medium. The cell morphology, viability and growth ability were observed under a phase contrast microscope (40 magnification). The round-up cells (the arrows) indicate that the cells are undergoing cell division. (C) The FMND-bearing
fluorescence intensities (
Fig. 3
B) and particle complexities
(
Fig. 3
E) were signi
ficantly increased following FMNDs in A549
cells via a time-dependent manner. Nonetheless, treatment with
FMNDs (10
e100
m
g/ml for 24 h) in A549 cells did not alter the cell
size distribution by FSC-H (forward light scatter) analysis using
flow cytometer (
Fig. 4
A and B). Consistently, FMNDs (50
m
g/ml)
for a time-course (0.5
e24 h) did not alter the cell size distribution
of A549 cells (
Fig. 4
D and E). The quanti
fied data showed that the
cell size distribution of A549 cells was not signi
ficantly altered
after treatment with FMNDs (
Fig. 4
C and F, p
> 0.05).
3.4. Identi
fication of the FMND-bearing cells separated by flow
cytometer
Separation of the FMND-bearing cells by
flow cytometer was
described in supporting information (please see the supplementary
protocol 1). To separate the FMND-bearing cells, A549 cells were
treated with or without FMNDs (50
m
g/ml for 24 h). The R1 gate
(green
fluorescence-negative) indicates the region of parental cells
(
Fig. 5
A). The R2 gate (green
fluorescence-active) indicates the
region of the FMND-bearing cells that displayed the green
fluo-rescence intensities (
Fig. 5
A). The FMND-bearing cells in the R2
region were collected by the
flow cytometer with a sorter. After
separation, the FMND-bearing cells were immediately examined
under a living cell imaging system with a
fluorescent and phase
contrast microscope. Comparison with the parental cells shows
that the separated FMND-bearing cells exhibited signi
ficant
fluo-rescence intensity under a
fluorescent microscope (
Fig. 5
B, right
lower picture). Moreover, the FMND-bearing cells were re-cultured
for a further 24 h. The cell morphology and viability were observed
under a phase contrast microscope. The viability and growth ability
of FMND-bearing cells were similar to the parental cells by
observing con
fluent cells in culture dishes (
Fig. 6
A). Subsequently,
the total protein expression pro
files of the FMND-bearing cells and
parental cells were examined by SDS-PAGE analysis. The protein
expression patterns on the SDS-PAGE were not signi
ficantly altered
between the parental cells and FMND-bearing cells (
Fig. 6
B).
Besides, the green
fluorescence intensities in the FMND-bearing
cells could be distinguished by
flow cytometer after the cells
were cultured for a further 24 h (
Fig. 6
C, red peak in web version).
3.5. Identi
fication of the FMND-bearing cells separated by magnetic
device
Separation of the FMND-bearing cells by magnetic device was
described in supporting information (please see the supplementary
protocol 2). To collect the FMND-bearing cells, A549 cells were
treated with FMNDs (50
m
g/ml for 24 h), and separated by magnetic
device. The cell morphology, viability and growth ability of the
FMND-bearing cells with different generations separated by
magnetic device were similar to the parental cells (
Fig. 7
A). In
addition, the protein expression patterns of FMND-bearing cells on
the SDS-PAGE were not signi
ficantly altered (
Fig. 7
B). Moreover, the
FMND-bearing cells still carried the
fluorescence intensities of
FMNDs, and that these
fluorescence intensities could be detected
by
flow cytometer (
Fig. 7
C, lower picture) or confocal microscope
(
Fig. 7
C, upper picture).
The separating ratio of the generations of FMND-bearing cells
was calculated by dividing the separated FMND-bearing cell
number by total counted cell number before separation. The
separating ratio of the
first generation had an average of 75.89%,
and the separating ratio in the fourth generation had an average of
w60% (
Fig. 7
D).
3.6. Cryopreservation and characterization of the generations of
FMND-bearing cells separated by
flow cytometer or magnetic device
The
first generation of the FMND-bearing cells separated by flow
cytometer or magnetic device was cryopreservation in liquid
nitrogen. After re-thawing the FMND-bearing cells, the cells were
observed by laser scanning confocal microscope. The nuclei were
stained with Hoechst 33258 that presented with blue color. The red
fluorescence (Cy3) exhibited by
b
-tubulin that presented the cell
morphology (cytoskeleton) of A549 cells. The FMND particles
exhibited green
fluorescence in A549 cells at wavelength 488 nm,
and the emission collected in the range of 510
e530 nm (
Fig. 8
A).
The images of phase contrast showed the location of FMNDs
retained in A549 cells (
Fig. 8
A, dark black spots). The cell
morphology and viability of the FMND-bearing cells were still
similar to those of the parental cells (
Fig. 8
B). The arrows in
Fig. 8
B
indicate round-up cells undergoing cell division. Moreover, the
FMND
’s particles retained inside of the FMND-bearing cells after
the cells were cultured for a further 7 days (
Fig. 8
C).
3.7. Separation and identi
fication of various FMND-bearing cancer
cell types
A variety of cancer cell types have been examined for illustrating
universal of the FMND-bearing cells. Human cancer cell lines
including human colon (RKO), bladder (BFTC905), breast (MCF-7),
and cervix (HeLa) were incubated with FMND particles (50
m
g/ml
for 24 h), and then the FMND-bearing cells were separated by
magnetic device. The
fluorescence intensities of FMNDs in various
cancer cell types were examined by
flow cytometer. All cell lines
were increased the
fluorescence intensities following treatment
with FMNDs (
Fig. 9
A). The quanti
fied data showed that the
fluo-rescence intensities of FMNDs were signi
ficantly increased in all
cell lines (
Fig. 9
A). The FMND-bearing cancer cell lines were
con
firmed by confocal microscope by which retained the FMND
particles that exhibited green
fluorescence (
Fig. 9
B).
4. Discussion
NDs have been developed in biological applications in recent
years. In this study, we demonstrate the FMND-bearing cancer cell
lines, which can be separated by
flow cytometer and magnetic
device. The FMND-bearing cells can be characterized and detected
by
flow cytometer and confocal microscopy. More importantly, the
FMND-bearing cells reserved cellular functions, including cell
morphology, cell viability and growth ability by comparison with
the parental cells. In addition, a variety of human cancer types
including lung (A549), colon (RKO), breast (MCF-7), cervical
(HeLa), and bladder (BFTC905) cancer cells can be used the
strategy to prepare the FMbearing cancer cell lines. These
ND-bearing cell lines can be cryopreservation for further biomedical
applications such as cellular labeling and tracking in cancer or
stem cells.
NDs can be taken into A549 lung cancer cells by endocytosis
[17]
. The endocytic ND
’s clusters in cells were separated by cell
division;
finally, the cell retained a single ND’s cluster
[17]
.
Quantum measurement and orientation tracking of
fluorescent
NDs were used by the controlled single spin probes for
nano-magnetometry inside living cells
[44]
. We found that treatment
with FMNDs increased the
fluorescence intensity and particle
complexity of cancer cells in a concentration- and time-dependent
manner but did not alter the cellular size distribution. Several
studies show that NDs do not induce cytotoxicity in a variety of cell
types
[16
e21]
. It has been found that NDs did not cause signi
ficant
HeLa cells (n=3)
Fluore
sce
nce
intensity
(fold)
0
1
2
3
4
5
6
**
MCF-7 cells (n=3)
control FMND
control FMND
Fluore
sce
nce
intensity
(fold)
0
5
10
15
20
25
30
**
B
nuclei
FMND
β-tubulin
merge
BFTC905
MCF-7
RKO
HeLa
FMND-bearing
cell lines
5
μ
m
5
μ
m
5
μ
m
5
μ
m
BFTC905 cells (n=5)
control FMND
0
2
4
6
8
10
12
14
**
RKO cells (n=3)
control FMND
Fluore
sce
nce
intensity
(fold)
0
1
2
3
4
5
6
7
**
Fluore
sce
nce
intensity
(fold)
A
RKO cells
BFTC905 cells
Control
FMND-bearing
Control
FMND-bearing
200
160
120
40
80
0
Counts
10
010
110
210
310
4FL1-H
200
160
40
80
0
Counts
10
010
110
210
310
4FL1-H
120
MCF-7 cells
HeLa cells
Control
FMND-bearing
Control
FMND-bearing
200
160
40
80
0
Counts
10
010
110
210
310
4FL1-H
120
200
160
40
80
0
Counts
10
010
110
210
310
4FL1-H
120
Fig. 9. Separation and identification of various FMND-bearing cancer cell types. (A) A variety of cancer cell lines including human colon (RKO), bladder (BFTC905), breast (MCF-7)
and cervix (HeLa) cancer cells were plated at density of 2 106cells per 100-mm Petri dish for 24 h. Then the cells were treated with or without 50mg/ml FMNDs for 24 h. At the
end of treatment, the cells were separated by magnetic device. Thefluorescent intensity of FMNDs in the cells was detected by flow cytometer. The fluorescence intensity from
FMNDs was excited with wavelength 488 nm, and emission was collected in 515e545 nm signal range. The fluorescence intensity was quantified from a minimum of 10,000 cells by
CellQuest software. Result were obtained from 3 to 5 separate experiment and bar represent mean S.E. *p < 0.05 indicates significant different between control and FMND-treated
samples. (B) The FMND-bearing cells were subjected to nuclear and microtubule staining, and observed by laser scanning confocal microscope. The microtubule was stained with
anti-b-tubulin Cy3 that presented with red color. The nuclei were stained with Hoechst 33258 that presented with blue color. The greenfluorescence from FMND’s particles was
excited with wavelength 488 nm, and the emission was collected in the range of 510e530 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
differentiation
[17]
and morphogenesis during embryogenesis
[22]
.
FMNDs did not induce cytotoxicity and growth alteration in HFL-1
normal lung
fibroblast and A549 lung cancer cells. These findings
provided that FMNDs were carried inside of cells without inducing
cellular damage. Accordingly, FMND is a biocompatible
nano-material for establishing these ND-bearing cell lines.
FMNDs were with an aqueous solubility by stock solution at
2.1 mg/ml
[21]
. We found that PBS was more suitable solvent for
FMNDs in the present study. The average size of FMNDs in PBS was
131.7 nm. We suggest that the functional groups on the surface of
FMND particles may provide more solubility for FMNDs in the PBS
buffer system. According to solubility, PBS was used as a solvent for
FMND particles for preparing the FMND-bearing cells. Moreover,
using DDW or PBS as an FMND solvent did not alter the cell viability
and growth ability in human cells. The results indicate that
different size of FMNDs does not induce cytotoxicity in human cells.
FMNDs contained both the advantages of
fluorescence and
magnetic properties for cancer cell labeling and detection. The
fluorescence intensity of FMNDs provides for the separation of the
ND-bearing cells by
flow cytometer with cell-sorting function. The
FMND-bearing cells were distinguished by
fluorescence intensity of
FMND particles using
flow cytometer. In addition, the
FMND-bearing cells can be separated by magnetic device. It has been
used
flow cytometer to get the fluorophore-conjugated iron oxide
nanoparticle (Feridex)-labeled human hematopoietic stem cells
[5]
.
After separation by
flow cytometer or magnetic device, these
FMND-bearing cells displayed cell survival and growth ability.
Moreover, the morphology and total protein expression pro
files of
the FMND-bearing cells were similar to those of the parental cells.
The labeled or separated cells have cell morphology, viability
and growth ability similar to those of the parent cells after
sub-culturing or cryopreservation. The separating ratio of the
genera-tions of FMND-bearing cells was calculated by dividing the
sepa-rated FMND-bearing cell number by total counted cell number
before separation. We found that the separating ratio of the
generations of FMND-bearing cells around 60
e75%. It is possible
that partial FMND-bearing cells may be lost during separation
procedure. However, over 60% separation ratio of the fourth
generation of the FMND-bearing cells can provide enough cell
number for identi
fication and application.
The characterization of FMND-bearing cells separated by
flow
cytometer and magnetic device was summarized in
Table 1
. These
FMND-bearing cell lines can be cryopreservation and stored in
liquid nitrogen for further biological applications such as speci
fic
cancer cell labeling and tracking. The FMND-bearing cancer cells
may provide for cancer tracking by optical imaging or MRI
detec-tion in animals. Moreover, the FMND-bearing cancer cell lines can
be used for anti-cancer drugs screening or other biomedical
applications.
5. Conclusions
We have established the FMND-bearing cancer cell lines
sepa-rated by
flow cytometer and magnetic device without inducing
cellular damages. The FMND-bearing cancer cell lines reserve the
cellular functions similar to those of the parental cells that can
provide for speci
fic cancer cell labeling and tracking.
Acknowledgments
This work was supported by grants from the National Science
Council (NSC 96-2311-B-320-006-MY3 and NSC
99-2311-B-009-003-MY3) and the National Chiao-Tung University (100W976) in
Taiwan. The authors also thank the core facility of Multiphoton and
Confocal Microscope System in National Chiao University, Hsinchu,
Taiwan.
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at
doi:10.1016/j.biomaterials.2012.
05.009
.
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