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Cite this: RSC Adv., 2017, 7, 22468
Lymphoma cell isolation using multifunctional
magnetic nanoparticles: antibody conjugation and
characterization†
Soubhagya Laxmi Sahoo,a Chi-Hsien Liu
*abcd and Wei-Chi Wude
The early detection of B-cell lymphoma and non-Hodgkin's lymphoma has a wide impact on the diagnosis and
therapy of lymphoma patients. Capturing and sorting tumour cells with magnetic nanoparticles (MNPs) has
received considerable attention in recent years. Despite these successes, the efficient isolation of circulating
tumour cells from complex biological fluids is still under development for the early diagnosis of lymphoma.
In this study, MNPs are functionalized with anti-CD20 antibodies using an avidin-biotin linkage, with the aim
of achieving specific cancer cell detection and efficient isolation. Anti-CD20 antibody-conjugated MNPs (Ab
MNPs) could specifically target CD20-expressing lymphoma cells. The capture efficiency of the Ab MNPs in
the lymphoma cell line was >95% with regard to the mixture of two cell lines, as confirmed by flow
cytometry and confocal microscopy. Transmission electron microscopy confirmed that the conjugation of
an antibody with the MNPs increased the size from 12 to 47 nm. The surface charge of the Ab MNPs was
examined by using zeta potential measurements. Furthermore, Prussian blue staining was performed to
Received 20th February 2017
Accepted 13th April 2017
confirm the interaction of Ab MNPs with the targeted lymphoma cells. Our results indicated that the
receptor recognition ability of the antibody was fully retained after conjugation with MNPs. In conclusion,
DOI: 10.1039/c7ra02084h
anti-CD20 MNPs can be used for very sensitive detection and quick isolation of CD20-positive lymphoma
rsc.li/rsc-advances
cells among mixed cells by using only a permanent magnet.
Introduction
Cancers are among the most serious diseases that can ultimately lead to death. Sensitive and rapid isolation of cancer
cells from complex bio-uids is of critical importance for cancer
research, prevention and therapy.1 Cell sorting is oen used to
enrich rare cells for further well-dened culture conditions and
to enhance the cell population. The current capture techniques
for cancer cells include ow cytometry, magnetic-based sorting
devices and microuidic chips, using for example uorescence
signals, magnetic forces and physical principles.2 Flow cytometry provides precise isolation; however, it involves a sophisticated instrument and needs expensive uorescent probes for
a
Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung
University, 259, Wen-Hwa First Road, Kwei-Shan, Tao-Yuan 333, Taiwan
b
Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic
Safety, College of Human Ecology, Chang Gung University of Science and Technology,
261, Wen-Hwa First Road, Taoyuan, Taiwan
c
Department of Chemical Engineering, Ming Chi University of Technology, 84, GungJuan Road, New Taipei City, Taiwan
d
Department of Ophthalmology, Chang Gung Memorial Hospital, 5, Fu-Hsing Street,
Taoyuan, Taiwan
e
College of Medicine, Chang Gung University, 259, Wen-Hwa First Road, Taoyuan,
Taiwan. E-mail: CHL@mail.cgu.edu.tw
† Electronic supplementary
10.1039/c7ra02084h
information
22468 | RSC Adv., 2017, 7, 22468–22478
(ESI)
available.
See
DOI:
cell labelling. A robust sorting platform is needed to isolate
tumour cells for further diagnosis and expansion. Among these
techniques, magnetic sorting utilizes targeting magnetic
nanoparticles (MNPs), which are biodegradable and have low
toxicity.3,4 The MNP-based technology has several advantages,
such as a high surface-to-volume ratio, high-binding capacity
and specic interactions between nanoparticles.5 Moreover, the
diffusion limitation within the micron-sized particles leads to
a decrease in their binding efficacy on the targeted cells in
biological uids, such as blood.6 Therefore, the nano-scaled
MNPs provide several advantages for capture applications,
including a low diffusion barrier, high surface area, stability
and specicity.
Specic targeting is a key step in realizing the full potential
of MNPs in tumour-associated diagnosis and the capture of
tumour cells. Researchers have devoted a tremendous amount
of time to develop MNPs-based models for the capture and early
detection of cancer cells that simultaneously conjugate MNPs to
active targeting moieties, such as ligands and monoclonal
antibodies. Antibodies are promising moieties for targeting
cancer cells, using high affinity and ligand-receptor specicity
for the surface antigen on the tumour.7 For example, the antiCD20 antibody (rituximab) has been applied to the treatment
of non-Hodgkin's lymphoma and inammatory diseases, such
as rheumatoid arthritis and myositis.8 The CD20 is a nonglycosylated antigen expressed on B-cell non-Hodgkin's
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lymphomas,9 and cannot be found on stem cells, pro-B cells,
normal plasma cells or other normal tissues.10 In addition, the
CD20 on the cell membrane, which is not internalized in
response to antibody binding, is a good candidate as a target for
cell isolation.
Cancer cells have been successfully detected and isolated by
using MNPs based on antibody and antigen interactions.11,12
Cirstoiu-Hapca et al. (2007) and Xu et al. (2014) have used antiHER2 and anti-GD2 antibodies conjugated with nanoparticles to
specically isolate the tumour cells bearing the surface antigens.6,13
However, only one type of cancer cell was isolated in their targeting
studies. In contrast, Song et al. (2011) analyzed the capture efficiencies of tumour cells by using uorescent MNPs to isolate
a small number of spiked tumour cells in a large population of
normal cells.14 Magnetic particles labelled with a uorescent dye
for optical detection and conjugated with a monoclonal antibody
against the neu receptor have been demonstrated to signicantly
identify primary and metastatic breast tumours.15 The anti-EGFR
antibody-conjugated nanoparticles can be used to capture circulating tumour cells expressing EGFR and in the subsequent diagnostic analysis.16 Wu et al. have successfully developed multifunctional magnetic particles conjugated with anti-EpCAM antibody that could detect endogenous metabolites and isolate rare
tumour cell isolation.17 These antibody-conjugated nanoparticles
have been proven useful for sensitive detection and rapid isolation
of cancer cells in early diagnoses.
Our objectives are to synthesize and characterize the
antibody-conjugated MNPs (Ab MNPs) and to assay their
biocompatibility and separation efficacy. The anti-CD20 Ab
MNPs were synthesized to detect and isolate lymphoma cells
from two kinds of mixed cells via a process based on the high
affinity between antigens and antibodies. Specically, the
carboxylic group of MNPs was activated using the EDC/NHS
linker, and then avidin was conjugated onto MNPs to form
avidin MNPs. Biotin maleimide was conjugated with an antiCD20 antibody, and in the nal step, a biotinylated antibody
was added to interact with the avidin MNPs to form Ab MNPs.
The morphology and surface charge of the Ab MNPs were
examined with a transmission electron microscope and using
zeta potential measurements. The use of Ab MNPs to specically isolate the cancer cells was evaluated by ow cytometry
analysis, confocal image and Prussian blue staining.
Experimental section
Materials
All the chemical reagents were commercially purchased and used
without further purication. The materials include avidin, biotin
maleimide, ethylenediaminetetraacetic acid (EDTA), Hoechst
33342, neutral red, N-(3-dimethylaminopropyl)-ethyl carbodiimide (EDC), hydroxy succinimide (NHS), and Trypan blue solution were purchased from Sigma-Aldrich (MO, USA). Rhodamine
B was purchased from Acros Organics (NJ, USA). Potassium
thiocyanate and hydrochloric acid were purchased from J.T Baker
(PA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Haemek, Israel). Paramagnetic iron oxide
This journal is © The Royal Society of Chemistry 2017
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nanoparticles (COOH-surface modied) were purchased from
Taiwan Advance Nanotech (Taoyuan, Taiwan).
Functionalization of Ab MNPs
For the targeting approach, the carboxylic group of magnetic
nanoparticles (1 mg mL1) were activated using EDC/NHS linker
(0.64 and 1.2 mg mL1, respectively) at a constant vortex rate for 2
hours at room temperature. Next, avidin (0.2 mg mL1) was
conjugated onto magnetic nanoparticles for 1 hour at room
temperature to form avidin MNPs. The unconjugated avidin was
removed by washing three times with deionized water using
magnetic separation system (Millipore). The anti-CD20 antibodies
were puried from the spent media of BCRC 60427 hybridoma
cell line by using liquid chromatography and protein A sepharose
column (GE). Separately, the biotinylated Ab was prepared as
follows. Anti-CD20 antibody (1 mg mL1) was mixed with biotin
maleimide (0.25 mg mL1) in phosphate buffer (pH 7.2) and
reacted for 2 hours at room temperature. The detailed methods
are referred to the chemical conjugation textbook.18 In the nal
step, the biotinylated Ab was mixed with avidin MNPs to form the
Ab MNPs for 30 minutes. The Ab MNPs was washed three times to
remove unreacted biotinylated Ab and then stored at 4 C. For the
dose effect of MNP isolation, high Ab conjugated MNPs (15.86 mg
Ab per mg MNPs) and low Ab conjugated MNPs (8.07 mg Ab
per mg MNPs) were prepared by adjusting the amount of biotinylated antibody in the conjugation procedure.
Cell culture
The hybridoma cells (BCRC 60427) secreting the immunoglobulin IgG2a, which recognizes the human CD20 antigen, Chinese
hamster ovary cell line (CHO, BCRC 60185), and CD20expressing Ramos lymphoma (BCRC 60252) were obtained
from the Bioresource Collection and Research Center (BCRC,
Hsinchu, Taiwan). BCRC 60252 was maintained in RPMI-1640
medium supplemented with 10% heat inactivated fetal bovine
serum (FBS). BCRC 60427 was maintained in CD hybridoma
medium (Thermo Fisher Scientic, Sugar Land, USA). The CD20
free cell line such as CHO BCRC 60185 was maintained in Excel
medium. HaCat cells are kindly donated by Prof. Sheu, HammMing (NCKU, Taiwan) and maintained in DMEM with 10%
serum. All cells were incubated at 37 C under a humidied
atmosphere with 5% CO2. The cell morphology and growth were
monitored daily using a light microscope. Cell passage was
performed every four days to maintain an exponential growth
phase. The cell density and viability were determined using
a Beckman Coulter counter (MS3 model) and hemocytometer,
respectively, prior to all experiments.
Characterization
The size and surface property of MNPs were characterized by
transmission electron microscopy (TEM). A drop of diluted
sample was dispersed onto a 100-mesh copper grid (CF200-Cu,
Electron Microscopy Science) and the excess drop was removed
with lter paper. The sample containing copper grid was dried for
2 hours at 55 C prior to TEM analysis. The morphology of the
COOH MNPs and Ab MNPs were observed by TEM (JM-1011,
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JEOL, and Tokyo, Japan). The zeta potential was characterized
using a Zetasizer Nano ZS 90 (Malvern, Worcestershire, U.K.) at
32 C. The tested MNPs under different pH conditions were 1 mg
mL1. The different pH (pH 3, 5, 7, 9, and 11) were obtained by
adjusting the amount of HCL (0.1%) and NaOH (0.1%). Fourier
transform infrared spectroscopy (Alpha, Bruker, Germany) was
used to characterize the presence of specic chemical groups in
the modied magnetic nanoparticles. Dried samples (0.8 mg)
were mixed with KBr (IR grade, Sigma) powder (80 mg) and
compressed into a thin membrane using a desktop pellet press
(ICL, Gareld, NJ). The spectra of the samples were then processed by Bruker OPUS soware. The loading antibody on the
MNPs was determined by Nanodrop spectrophotometer (Thermo,
Wilmington, DE).
Detection and isolation of CD20 positive lymphocyte cells by
Ab MNPs
To demonstrate the abilities of Ab MNPs for detection and
isolation of cancer cell, Ramos lymphoma cells were used as
target cells. The CD20 free cells including hybridoma cells,
HaCaT and CHO cells were used as control. The CD20 positive
cells stained with Hoechst 33342 nucleic acid dye with a nal
concentration of 1 ppm and the control cells were stained with
Rhodamine B (with a nal concentration of 10 ppm) were mixed
with Ab MNPs or COOH MNPs and incubated for 20 minutes at
4 C to prevent the endocytosis. The target cells bound by their
prospective MNPs were washed three times with PBS using
a magnet and then imaged with the aid of a confocal microscope and ow cytometry. To demonstrate the Ab MNPs selectivity towards the target cells, we carried out a control
experiment with target cancer cells and non-target normal cells.
We mixed Ab MNPs with a sample containing 1 106 CD20
positive lymphoma cells and CD20 negative cells and incubated
for 20 minutes at 4 C. Aer magnetic separation, the precipitate was imaged under a confocal microscope and ow cytometry. The number of cells before and aer isolation with Ab
MNPs was imaged with the confocal microscope to know the
efficiency of the Ab MNPs to capture the target cancer cells.
Paper
free cells by stained Rhodamine B were xed at 1 106. The
mixed cells were incubated with the Ab MNPs (30 mg mL1) for
20 minutes. The isolation was performed using a magnet and
the isolated cells were analyzed by ow cytometry. The isolation efficiency of the Ab MNPs was calculated as follows.
Isolation efficiency (%) ¼ (isolated CD20 positive cells/initial
CD20 positive cells) 100
Biocompatibility studies
The CD20-expressing cells and CHO cells were employed for
cytotoxicity evaluation. Cells treated with Ab MNPs at different
concentrations were examined with Coulter counter, MTT, and
Trypan blue assay to measure cell viability. For MTT assay, cells
were seeded into 96-well plates (2 104 cells per well) with 100
mL medium and incubated overnight. Subsequently, Ab MNPs
at different concentrations (10, 30, 50 mg mL1) were added to
the well and incubated for 24 hours at 37 C. The control group
was incubated with only sterile PBS. Add 200 mL of 0.5 mg mL1
MTT reagent into each well and incubated for additional 4
hours. Aer incubation, the supernatants were removed carefully and 200 mL of DMSO was added to each well. Next, plates
were shaken on an orbital incubator for 10 minutes in order to
dissolve the formazan crystals. Finally, the absorbance of each
well was measured by a spectrophotometer. Also, coulter
counter and Trypan blue assay was performed to count the cell
number and to evaluate the toxicity effect. The cells (2 105
cells per well) were cultured in a 6 well plate. The control group
were incubated with sterile PBS, whereas different concentrations (10, 30, 50 mg mL1) of Ab MNPs were added to cells. At
each hour, 100 mL of the cell suspension was transferred to an
Eppendorf, and the cell number was evaluated by using Beckman Coulter counter (MS3 model) and femocytometer. For all
the experiments, measurements were carried out in triplicate.
The viability of the cells in the treated groups was calculated
according to the following equation.
Viability (%) ¼ (final cell population of treated group/final cell
population of control group) 100
Targeting efficiency of Ab MNPs to lymphocyte cells
Magnetic separation was performed by adding Ab MNPs to each
1 mL of sample (1 106 cells) as describe in the above procedure. The different concentration (10, 30, 50 mg mL1) of high
Ab conjugated MNPs (15.86 mg Ab per mg MNPs), and low Ab
conjugated MNPs (8.07 mg Ab per mg MNPs) were incubated
with the cell samples for 20 minutes at 4 C. Then, a magnet was
introduced to the sample tubes, and aer 3 minutes the target
cells were attached to the tube wall while the supernatant was
collected carefully using a pipet. Aer magnetic separation, the
number of isolated cells by Ab MNPs was determined with ow
cytometry to know the efficiency of the Ab MNPs to capture the
target lymphocyte cells.
The specicity of Ab MNPs towards CD20 positive
lymphocytes was evaluated by mixing target cells with CD20
free cells in different ratios. The Hoechst 33342 stained
lymphocytes were varied from 5 104 to 1 106 and n CD20
22470 | RSC Adv., 2017, 7, 22468–22478
Localization of nanoparticles in the lymphoma cell line
Prussian blue staining and potassium thiocyanate method were
used to study iron uptake in cells. Equal volumes of 10% potassium ferrocyanide solution and 20% hydrochloric acid solution
were freshly mixed to prepare the Prussian blue solution. Ramos
lymphoma cells (1 106) were incubated with 30 mg mL1 Ab
MNPs or COOH MNPs for one hour at room temperature. Then,
washed three times with PBS and incubated 5 minutes with 150 mL
ice-cold ethanol (95%). Aer ve minutes, cells were centrifuged to
remove ethanol and washed three times with deionized water.
Then, the cells were incubated with 150 mL of Prussian blue
solution for 20 minutes in the dark. Aer washing three times in
PBS, the cells were counterstained with neutral red (1 ppm) for 2
minutes and imaged under the microscope for Prussian blue
staining.
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For potassium thiocyanate method, 1 106 cells from Ramos
lymphoma cell line, hybridoma or CHO cells were incubated with
30 mg mL1 MNPs at 37 C for 2 hours. The excess iron was
removed by washing with PBS, and the iron concentration in 1
106 cells was determined using the following thiocyanate-based
spectrophotometric assay. The cell samples were mixed with 100
mL of 12 N HCl for 4 hours at room temperature. Then, 400 mL of
5% (w/v) potassium thiocyanate was added to the solution and
incubated for 15 minutes. Samples were centrifuged at 12 000 g
for 10 min and 100 mL of the supernatant was added to a 96 well
microtiter plate. The absorbance at 480 nm was measured using
a microplate reader (Synergy HT, BioTek, Hong Kong). Two calibration curves of MNPs were prepared by using Ab MNPs and
COOH MNPs (antibody free). The detailed procedure of potassium thiocyanate method was referred to previous papers.19,20
Results and discussion
Functionalization of antibody magnetic nanoparticles (Ab
MNPs)
We rstly chose biotin maleimide to conjugate the biotins to
antibodies. The maleimide-containing biotins efficiently react to
RSC Advances
the thiol groups on the antibody and form a thio-succinimide
linkage that can maintain the antibody's targeting ability. The
disulde bonds of antibodies as conjugation sites provide
advantages over other reactive groups, such as amines and
carboxylates. Avidin is conjugated with COOH MNPs through the
EDC/NHS linker, and the biotinylated antibody is eventually
bound with COOH MNPs through a biotin–avidin non-covalent
interaction to form Ab MNPs. The strong binding affinity of
biotin towards avidin is stable during proteolysis and within
a wide range of pH and temperature levels and with a variety of
denaturing agents.14,21–23 The EDC/NHS linker directly connects
carboxylic and amino groups, for conjugating molecules with
multiple carboxylic and amino groups. The functionalization
procedure of Ab MNPs is shown in the schematic diagram (Fig. 1).
The conjugation strategy allows the functional and directional
conjugation of the Fc portion of an antibody to MNPs and is
directed outward from the surfaces of Ab MNPs, while the Fab
(capture site) is largely available for efficient targeting. Conjugating MNPs specically to the Fc portion decreases the opsonin
role of the antibody molecules that are used for targeting the cell
surface antigen.24 While conjugating the Fc-directed method, the
opsonin activity of the antibody is concealed and reduces the
Fig. 1 Schematic representation of antibody and MNPs conjugation. step 1. Avidin is conjugated with COOH MNPs in presence of EDC/NHS
linker, step 2. Biotin is conjugated with the anti-CD20 antibody to obtain biotinylated antibody, step 3. Finally, biotinylated antibody is eventually
bound with avidin modified COOH MNPs through a biotin–avidin non-covalent interaction to form antibody MNPs (Ab MNPs).
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Paper
TEM image for COOH MNPs (bare MNPs) and Ab MNPs
(conjugated with Ab). (a) COOH MNPs (b) modified Ab MNPs. The
average sizes for COOH MNPs and Ab MNPs were 12 nm and 47 nm
respectively by using image J.
Fig. 2
endocytosis of MNPs. Finally, modied MNPs with fewer cytotoxic
effects are expected. The target efficiency of the anti-CD20 antibody in relation to the CD20 antigen result in functionalized Ab
MNPs. The modied MNPs are characterized and investigated in
terms of the isolation of lymphoma cells in the following sections.
Characterization of Ab MNPs
Physical properties were measured using TEM, zeta potential,
and Prussian blue staining to conrm the quality of the Ab
MNPs. The size and morphology of the Ab MNPs were investigated by TEM (Fig. 2). The particle size was calculated with at
least 50 particles chosen at random in both the prepared
samples through an image J analysis program. From Fig. 2(a
and b), it is clear that the size of the MNPs increased from
12 nm to 47 nm aer conjugation with Ab. The properties of the
coating materials have a major effect on the diameter, and the
diameters of Ab MNPs were shown to be larger than the bare
MNPs. Further, the thickness of the coating layer around the
surface of the particle increased due to the decrease of water
molecules in the magnetic core, which proves the successful
conjugation of MNPs with Ab. The smaller size of the MNPs
might improve their ability to enter the target site and to avoid
endocytosis. Here, we have obtained Ab MNPs of about 47 nm;
in a previous study, the sizes of antibody-modied nanoparticles were 340 to 410 nm.25 The TEM images of the antibodyconjugated MNPs revealed a uniform size distribution and no
signicant change in their morphology, even aer the conjugation reaction.
Fig. 3 illustrates the zeta-potential of COOH MNPs (unconjugated Ab) and Ab MNPs (conjugated with Ab) as a function of
pH, which has been shown to conrm the presence of functional groups on the surface of MNPs. The zeta potential values
for COOH MNPs and Ab MNPs were 3 and 5 mV, respectively, at
pH 3. The zeta potential of COOH MNPs was observed at around
21 mV. However, the zeta potential of the Ab MNPs shied
from 21 mV to 10 mV at pH 11 (Fig. 3), conrming the
successful conjugation of Ab to the surface of the MNPs. The
positive zeta potential values started to decrease for both the
MNPs as the pH was raised towards 11. The negative zeta values
started to increase with the increase in pH, which indicated that
the surface charges of both conjugates had increased slightly.
The fact that the isoelectric points of COOH MNPs and Ab MNPs
22472 | RSC Adv., 2017, 7, 22468–22478
Zeta potentials of COOH MNPs (unconjugated Ab) and Ab
MNPs (conjugated with Ab) under different pH conditions. MNPs were
dispersed in water ranged from pH 3 to pH 11.
Fig. 3
were 3.6 and 4.4, respectively, was attributed to the presence of
carboxylic groups in the MNPs. The IR spectra of COOH MNPs
and Ab MNPs were presented in Fig. S1.† A prominent absorption band at 1717 cm1 in COOH group (Fig. S1A†) which was
due to C]O stretch of carboxylic group. On the other hand, in
Ab MNPs (Fig. S1B†), bands related to the presence of protein
show up, the amide I and amide II bands at 1646 and 1533
cm1, respectively. These results conrmed that the MNPs were
successfully conjugated with antibodies.
The capture and isolation of CD20-positive lymphocyte cells
by Ab MNPs
We developed a system in which the constituents are avidinlabelled MNPs, conjugated to a biotin-labelled anti-CD20 antibody (Ab MNPs) (Fig. 1). To demonstrate the specic capturing
efficiency of Ab MNPs in targeting CD20-positive lymphocyte
cells, we analyzed the MNP-targeted cells using confocal
microscopy and ow cytometry. To examine the specic or
nonspecic binding effect of MNPs, a magnetic isolation
experiment was conducted, as described in the Experimental
section. The 106 cells treated by 30 mg of MNPs per mL were
used for the cell capture assay. To test the capture efficiency, two
kinds of cells (lymphoma and hybridoma cells) were mixed and
incubated for 20 minutes with MNPs followed by isolation using
a magnet. Aer magnetic isolation, the ow cytometry analysis
indicated that COOH MNPs did not show specicity towards
any cell lines. Sequentially, an equal number of mixed cells, that
is, 48% of the hybridoma cells (CD20 negative cells) and 50% of
the lymphocyte cells (CD20-positive cells) were obtained by
COOH MNPs. In contrast, Ab MNPs separated 98% of lymphocyte cells and a negligible percentage (only 2%) of hybridoma
cells (Fig. 4b). The fact that COOH MNPs could nonspecically
capture both cell types (Fig. S3†) from mixed cells indicated that
COOH MNPs need further modication to enhance the selectivity.26 It also implies that the Ab MNPs display higher binding
sensitivity than that of bare MNPs (COOH MNPs). More
importantly, our conjugation strategy allows the quick and
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Fig. 4 Representative specific cell isolation using Ab MNPs or COOH MNPs. (a) Cell sorting by COOH MNPs and (b) cell sorting by Ab MNPs. Two
kinds of cells (BCRC 60427 and Ramos lymphoma) were incubated with (a) COOH MNPs (50 mL) or (b) Ab MNPs (50 mL). The targeted cells were
isolated with a magnet and washed three times. The results are expressed as the mean and standard deviation from two experiments, (a) 52%
2.8 lymphoma cells and 46.3% 2.3 hybridoma cells were isolated by COOH MNPs. (b) 97.5% 0.7 lymphoma cells were isolated by Ab MNPs.
efficient capture of CD20-positive cells using a low dose of Ab
MNPs. In a previous approach, more than an hour of incubation
was required for the isolation of the cells.6 The specic binding
capacity of Ab MNPs was made possible by targeting the antiCD20 Ab at the CD20 antigen on lymphocyte cells. These
results suggested that the conjugation of anti-CD20 with MNPs
can effectively enhance the specic binding of nanoparticles to
the cells.
The ability of Ab MNPs to recognize the target cells was
evaluated in the following experiments. The Ab MNPs were
incubated with mixed cells (Fig. 4b), as mentioned in the
Experimental section. For the specic isolation, the hybridoma
cells (BCRC 60427) and HaCaT cells (skin cells) were used as the
negative control and lymphocyte cells expressing CD20 were the
target cells. Because the control cells had been stained with
Rhodamine B and the target cancer cells with Hoechst dyes,
they could be visualized by their different uorescent qualities
when excited under various levels of excitation. If blue uorescence from the target cell nucleus and red uorescence on the
surface of the cells appeared, we could conclude that the
respective cells were detected by the Ab MNPs. The cell mixtures
were incubated with Ab MNPs for 20 minutes and isolated by
a magnet. Confocal microscopic images of the precipitates were
then taken. Fig. S3† shows the experimental results in which the
Ab MNPs isolated only CD20-positive lymphoma cells stained
with blue uorescence (from Hoechst 33342), while CD20-free
cells stained with red uorescence (Rhodamine B) were found
in the supernatant (as presented in Fig. S4 in the ESI† section).
Ab MNPs binding to CD20-negative cells (such as BCRC 60427
cells or HaCaT cells) was negligible, indicating the ability to
recognize CD20 antigens with high specicity. The binding of
Ab MNPs to CD20-positive cells was conrmed in the confocal
images (Fig. S3†). For the recognition and isolation of markers
on the cell surface, MNPs are advantageous because the high
volume ratio offers a greater surface area for attaching specic
ligands and for capturing markers. Most importantly, the size of
the MNPs is smaller by orders of magnitude (approximately)
than that of a cell, which allows multiple MNPs to be attached to
a cell to facilitate magnetic isolation. Hence, the advantages of
using the surface-modied MNPs with a target ligand for targeting and isolating cells are that they require only a short
incubation time (20 minutes) and a simple washing process
with a PBS buffer. The above experiments suggested that the
mechanism of interaction between CD20-expressing cells and
Ab MNPs is cell surface clustering.27 The high target specicity
makes these MNPs ideal as isolation tools for recognizing and
sorting cells.
Aer showing the expected binding, we tested the feasibility
of Ab MNPs to isolate target cells from cell mixtures by rst
preparing mixed samples of CD20-positive cells (lymphoma)
and CD20 negative cells, such as hybridoma cells (BCRC 60427),
CHO (Chinese hamster ovary cells) and HaCaT cells. The
Flow cytometric analysis for cell separation efficacy using Ab MNPs in different cell mixtures. (a) Lymphoma and hybridoma, (b) lymphoma
and CHO cells and (c) lymphoma and HaCaT. 30 mg mL1 Ab MNPs were added to each cell samples and incubated for 20 minutes at 4 C. The
results are obtained as the mean and standard deviation of two determinations, 96.5% 0.7 (a) 94.5% 0.7 (b) and 94.8 2.6 (c).
Fig. 5
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sample was prepared by mixing approximately 1 106 target
cells and 1 106 of other kinds of cells, such as hybridoma,
CHO or HaCaT cells, and then incubated with 30 mg mL1 of Ab
MNPs. The lymphoma cells were stained with Hoechst and
BCRC 60427, and CHO or HaCaT cells were stained with
Rhodamine B to distinguish the target and non-target cells.
Aer incubation and magnetic separation, the magnetically
isolated cells were analyzed by ow cytometry. When lymphoma
cells mixed with CD20-negative hybridoma cells, the isolated
cells contained 96% of CD20-positive cells, as shown in Fig. 5.
Similarly, when mixed with either CHO or HaCaT cells, 95% of
CD20 positive cells could be isolated by using the Ab MNPs. We
also examined the cells that remained in the supernatant, and
most of these cells were CD20 negative, as shown in Fig. S4.†
Dose effect of Ab MNPs on the isolation of target lymphoma
cells
To evaluate the effects of different concentrations, different
doses of antibody were conjugated with MNPs to isolate CD20positive cells and quantied by ow cytometry. A comparison of
the high Ab-loaded MNPs (15.86 mg Ab per mg MNPs) and the
low Ab-loaded MNPs (8.07 mg Ab per mg MNPs) was evaluated
with mixed cells having both CD20-positive cells and CD20negative cells. Different amounts (10, 30, and 50 mg mL1) of
both high and low Ab-loaded MNPs were incubated with
lymphoma (CD20 positive) and CD20-negative cells for 20
Paper
minutes and isolated by 3 minutes of magnetic separation. The
CD20-positive cell binding increased with increasing concentrations of Ab MNPs. Both the high and low Ab-loaded MNPs
showed good separation ability (Fig. 6a–f). The high Ab-loaded
MNPs (10 mg mL1) could efficiently isolate 95% of the CD20positive cells from CD20-negative hybridoma cells. Furthermore, 30 mg and 50 mg mL1 of Ab MNPs separated 96% and
98% of CD20-positive cells, respectively. In contrast, the low Abloaded MNPs showed that 10 mg mL1 of MNPs separated 88%
of CD20-positive cells (Fig. 6d). The cell-capture rate of high
concentrations of Ab MNPs had higher separation efficacy even
at a low concentration (10 mg mL1) of Ab MNP treatment of
cells. Whereas, with a low concentration of Ab MNPs, at 10 mg,
30 mg and 50 mg mL1, separated 88%, 90% and 95% of CD20positive cells, respectively, and resulted in a few more CD20
negative cells compared with the high Ab MNPs (Fig. 6a–f). This
proves the successful Ab conjugation on MNPs since lower
concentrations of Ab MNPs can specically bind to CD20positive cells. The isolation of the CD20 cells demonstrated
some differences in binding, further indicating the Ab effect on
MNPs. However, in the studied concentration range, Ab MNPs
with high Ab conjugation exhibited more specic isolation of
lymphocyte cells than those with low Ab modication on MNPs.
A similar result was obtained when CD20-positive cells were
mixed with CD20-negative CHO cells, as indicated in Fig. S7.† In
a previous report, it was suggested that an optimal
Fig. 6 Representative dose-dependent capture efficacy by two kinds of Ab MNPs. CD20 positive cells (BCRC 60252) and CD20 negative cells
(BCRC 60427) were isolated by using high Ab conjugated or low Ab conjugated MNPs at different concentrations. Panel (a–c): high Ab
conjugated MNPs (15.86 mg Ab per mg MNPs). Panel (d–f): low Ab conjugated MNPs (8.07 mg Ab per mg MNPs) at 10, 30, 50 mg mL1 MNPs. The
flow cytometry data are reported as the mean and standard deviations (n ¼ 2). The separation efficacy was 94.2 1.3% (a) 96 0.92% (b) or 98
0.68% (c) for high Ab conjugated MNPs. The separation efficacy was 86.5 2.1% (d) 94.47 5.5% (e) or 93.6 1.0% (f) for low Ab conjugated
MNPs.
22474 | RSC Adv., 2017, 7, 22468–22478
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(i.e. only 5 104 target lymphocyte cells mixed with 106 nontarget cells), only 31% of target cells were isolated.
Biocompatibility studies
Fig. 7 The isolation efficacy of targeted lymphoma cells from a higher
population of hybridoma (CD20 negative) cells by using Ab MNPs. A
small number of spiked lymphoma cells ranged from 5 104 to 1
106 were mixed with hybridoma cells fixed at 1 106 cells. Each bar
represents the standard deviation (n ¼ 2).
concentration of Ab would be necessary to achieve efficient
cellular targeting of MNPs.25 The above studies showed that
both Ab concentrations (high and low) showed specicity
towards the target cells.
Generally, in the early stage of cancer, very few cancer cells
are present within a large number of normal cells in the blood.
To determine the specicity of Ab MNPs towards less CD20positive lymphocyte cells, the target cells were mixed with
non-target cells in different ratios (Fig. 7); stained (Hoechst
33342) target cells ratio were varied (5 104–1 106), and
stained (Rhodamine B) non-target cells ratio were xed (1
106). The mixed cells were incubated with xed concentrations
(30 mg mL1) of Ab MNPs for 20 minutes and then isolated using
a magnet and analyzed by ow cytometry. The results showed
that even if we used low concentrations of target cells, the Ab
MNPs could recognize the specic target cells against the large
population of normal cells. When the target cells were very rare
Factors such as surface conjugation, charges and sizes of MNPS
are key aspects in understanding the biocompatibility of the
functionalized MNPs. A high concentration of MNPs has been
reported to be toxic to in vitro cells due to the generation of
reactive oxygen species.28 On the contrary, trivial effects on the
viability of various cell lines have been reported for these MNPs
by other authors.29,30 Herein, we evaluated the biocompatibility
of modied Ab MNPs using several complementary approaches,
such as Trypan blue, cell counting and MTT assay for CD20positive lymphocyte cells. The results of Trypan blue assay
and cell counting (Fig. 8a and b) conrmed the biocompatibility
of Ab MNPs. The cell viability was above 90% aer 8 hours of
incubation. Trypan blue test has been proposed as the standard
method to validate cell viability aer MNP incubation. The
results of cell counting showed that cell density remained the
same aer 8 hours of incubation with Ab MNPs. The MTT assay
(Fig. 8) showed that cell activity was not signicantly affected by
the presence of Ab MNPs at 24 hours of treatment (>80%
viability in relation to the control sample), even at the highest
concentration (50 mg mL1). Moreover, Ab MNPs toxicity effect
was also evaluated with normal cell (CHO cells) and presented
in Fig. S8.† Higher viability has been observed for the two cell
types when compared to the control conditions. MTT assay
shows that Ab MNPs do not exert acute adverse effect on CHO
cells and lymphoma cells even at high dosage, suggesting that it
can be applicable in vivo applications. The MTT assay is based
on the reduction of soluble yellow tetrazolium into insoluble
purple formazan crystals by mitochondrial succinate dehydrogenase of the viable cell. Thus, the rate of formazan crystal
formation is directly proportional to the total number of viable
cells, which is measured in terms of absorbance.31 A modied
MTT assay has been adopted here to avoid the inuence of
MNPs on the colorimetric assay.32 Fig. 8c, shows that the
viability of cells exhibited a slightly decreasing trend from 1
Fig. 8 Biocompatibility of Ab MNPs in lymphoma cell line evaluated by hemocytometer (a) cell counting (b) and MTT assay (c) with different Ab
MNPs concentrations ranging from 10–50 mg mL1 for 8 hours. In the MTT assay, cells with Ab MNPs or COOH MNPs were incubated for 24
hours. The coefficient of variation (SD/mean) is around 4%.
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RSC Advances
Paper
hour to 24 hours of co-culture for both bare and Ab MNPs in
a similar manner to that of the control sample (without MNPs),
and the viability remained more than 80%. All the Trypan blue
dye, MTT assay, and cell counting results conrmed the
compatibility of the Ab MNPs developed in this study. Cell
viability could be maintained within the 24 h incubation using
50 mg mL1 of MNPs.
Localization of nanoparticles in the lymphoma cell line
The Prussian blue staining test was employed to detect the
localization of the nanoparticles with antibody-conjugated and
unconjugated MNPs on CD20-positive lymphocyte cells (Fig. S9†).
This method detects iron within the treated cells through the
reduction of ferric ions into the ferrous state, which is shown as
a blue colour.33 The cells treated with COOH MNPs (antibody free)
showed occasional blue spots outside of the cells (Fig. S9a†),
whereas the cells treated with antibody-conjugated nanoparticles
showed clear blue shells around the cells, conrming the presence of Ab MNPs around the cell membranes (Fig. S9b†). There is
abundant literature on MNPs conjugation efficiency; however, the
results are difficult to compare due to the different experimental
protocols, such as size, incubation time, surface coating, longer
incubation time and higher concentration. Moreover, longer
incubation time and higher concentrations of MNPs enable high
interactions with the cell surface and increase the detection efficiency.34 High concentrations of MNPs for an extended incubation
may reduce viability and enhance cell aggregation.35 Therefore,
the required quantity of MNPs for cellular uptake must be
balanced with their biocompatibility for efficient diagnosis or
treatment.29 To evaluate the selectivity of Ab MNPs, the iron
concentration was also obtained by the potassium thiocyanate
method (Fig. 9). In brief, the iron content of different cells (1
106) was measured aer 2 hours of incubation with 30 mg of Ab
MNPs or COOH MNPs at 37 C. The results showed that a small
difference in the amount of accumulated iron in the two cell lines
used as controls could be due to either different MNP sizes or the
type of cell line. Our potassium thiocyanate data indicated that Ab
MNPs bound effectively at the level of 16.8 mg per 106 cells on the
cell surface of lymphocytes. In contrast, half the quantity of Ab
MNPs was deposited on CD20-free hybridoma and CHO cells at
the level of 7.9 and 8.8 mg per 106 cells, respectively. Nonspecic
iron binding (13–20 mg per 106 cells) was observed in the three cell
lines when isolation was performed by using COOH MNPs. This
result conrmed the nonspecic binding of COOH MNPs to cells.
Recently, the location of functionalized MNPs in MCF-7 cells aer
24 h incubation has been observed by optical microscopy and
TEM. Most of the MNPs were distributed in the periplasmic area.30
In addition, their results for Prussian blue staining and ferrozinebased assay also indicated that MNPs (21 mg per 106 cells) can be
internalized effectively (21 mg per 106 cells) by MCF-7 cells. In
contrast, the major location of our Ab MNPs or COOH MNPs was
on the surface of cellular membranes in the lymphoma cells, as
indicated in Fig. S9b.† It is important to point out the dose of our
MNPs was 13-fold lower than in Calero's paper, which might have
resulted in the difference.30
22476 | RSC Adv., 2017, 7, 22468–22478
Quantification of intracellular iron content. Thiocyanate-based
iron uptake among the cell lines. The three cell lines were incubated
with 30 mg mL1 of Ab MNPs or COOH MNPs at 37 C for 2 hours. The
excess iron was washed with PBS, and the iron concentration in 1
106 cells from each cell line was determined using a thiocyanatebased spectrophotometric assay. The error bars in the figure represent
the mean and standard deviation (SD) of four repeats.
Fig. 9
Conclusion
In this study, we demonstrated a simple and robust conjugation
method by using antibodies and MNPs. The specicity for targeting the thiols of the Fc fragment in an antibody can be
improved by using maleimide conjugation. The synthetic platform described herein was efficient. It has the potential for
further optimization for antibody conjugation on different
nanoparticles. In addition, the lymphocytes cells were
successfully detected and isolated from mixed samples containing other cells and target lymphocytes cells. The Ab MNPs
fully retained their antibody binding capacity and could detect
and isolate the CD20-positive cells from the mixed cells. The
capture efficiency of Ab MNPs for lymphoma cells was above
95% under our experimental conditions. The capture efficiency
was conrmed by complementary techniques, such as ow
cytometry and confocal microscopy. Based on this simple
antibody conjugation on MNPs, this platform enables robust
cell sorting by using only a permanent magnet. The present
approach demonstrated a facile, time saving and economical
synthesis of Ab MNPs that can be used as an efficient capture
platform for tumour cells.
Conflict of interest
The authors would like to declare that no potential conicts of
interest exist.
Acknowledgements
We express gratitude to Ministry of Science and Technology
(MOST 105-2221-E-182-070), Chang Gung University (BMRP
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758) and Chang Gung Memorial Hospital (CMRPD 1E0352) for
funding and supporting this research.
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