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Open Access Article. Published on 24 April 2017. Downloaded on 18/11/2017 00:29:16.
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RSC Advances
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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 oen used to
enrich rare cells for further well-dened culture conditions and
to enhance the cell population. The current capture techniques
for cancer cells include ow cytometry, magnetic-based sorting
devices and microuidic 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 specic 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 specicity.
Specic 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 specicity
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 inammatory 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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Paper
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
specically 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 signicantly
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. Specically, 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 specically 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 purication. 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
RSC Advances
nanoparticles (COOH-surface modied) were purchased from
Taiwan Advance Nanotech (Taoyuan, Taiwan).
Functionalization of Ab MNPs
For the targeting approach, the carboxylic group of magnetic
nanoparticles (1 mg mL 1) were activated using EDC/NHS linker
(0.64 and 1.2 mg mL 1, respectively) at a constant vortex rate for 2
hours at room temperature. Next, avidin (0.2 mg mL 1) 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 puried 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 mL 1) was mixed with biotin
maleimide (0.25 mg mL 1) 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 Scientic, 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 humidied
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,
RSC Adv., 2017, 7, 22468–22478 | 22469
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RSC Advances
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
mL 1. 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 specic chemical groups in
the modied 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, Gareld, NJ). The spectra of the samples were then processed by Bruker OPUS soware. 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. Aer magnetic separation, the precipitate was imaged under a confocal microscope and ow cytometry. The number of cells before and aer 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 mL 1) 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 mL 1) 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 mL 1
MTT reagent into each well and incubated for additional 4
hours. Aer 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 mL 1) 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 mL 1) 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 aer 3 minutes the target
cells were attached to the tube wall while the supernatant was
collected carefully using a pipet. Aer 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 specicity 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 mL 1 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%). Aer 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. Aer 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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Paper
For potassium thiocyanate method, 1 106 cells from Ramos
lymphoma cell line, hybridoma or CHO cells were incubated with
30 mg mL 1 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
disulde 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
inte …
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