Nanotentacle-Structured Magnetic Particles for Efﬁ cient...

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Nanotentacle-Structured Magnetic Particles for Effi cient Capture of Circulating Tumor Cells

Seong-Min Jo , Joong-jae Lee , Woosung Heu , and Hak-Sung Kim *

CTCs. A variety of methods for capturing CTCs have been

developed, including microfl uidic devices, [ 5–8 ] immunomag-

netic methods, [ 9–12 ] and other platforms. [ 13–16 ] Such methods

have shown high capture effi ciency, but their capture

purity is signifi cantly low. Conventional immunomagnetic

approaches have been commonly used, offering advantages

such as simple procedure, but resulting in a low capture effi -

ciency and/or purity. [ 17 ] The capture purity of the Cellsearch

system, which is an FDA-approved and commercially avail-

able method based on immunomagnetic particles, has been

also known to be less than 1%, even though high capture effi -

ciency (≈80%) was achieved. [ 18 ] Microfl uidics-based devices

have been attempted to increase the capture effi ciency or

purity, but they have some drawbacks such as a complicated

experimental setup, a long processing time, and low retrieval

rate of captured cancer cells. [ 6–9,17 ] Despite many technolog-

ical advances, the capture effi ciency and purity still remain

challenges that limit clinical practice.

Here, we present the construction of “nanotentacle”-

structured magnetic particles using an M13-bacteiophage

and their use for the capturing of CTCs with high effi ciency

and purity ( Figure 1 a). M13-bacteriophage has a uniform

size of 6.6 nm in diameter and 880 nm in length, expressing

defi ned number of coat proteins on its surface. [ 19 ] Such coat DOI: 10.1002/smll.201402619

Circulating tumor cells (CTCs) have attracted considerable attention as promising markers for diagnosing and monitoring the cancer status. Despite many technological advances in isolating CTCs, the capture effi ciency and purity still remain challenges that limit clinical practice. Here, the construction of “nanotentacle”-structured magnetic particles using M13-bacteriophage and their application for the effi cient capturing of CTCs is demonstrated. The M13-bacteriophage to magnetic particles followed by modifi cation with PEG is conjugated, and further tethered monoclonal antibodies against the epidermal receptor 2 (HER2). The use of nanotentacle-structured magnetic particles results in a high capture purity (>45%) and effi ciency (>90%), even for a smaller number of cancer cells (≈25 cells) in whole blood. Furthermore, the cancer cells captured are shown to maintain a viability of greater than 84%. The approach can be effectively used for capturing CTCs with high effi ciency and purity for the diagnosis and monitoring of cancer status.

Cancer Diagnosis

Dr. S.-M. Jo, J.-j. Lee, W. Heu, Prof. H.-S. Kim Department of Biological Sciences Korea Advanced Institute of Science and Technology (KAIST) Daejeon 305-701 , Republic of Korea E-mail: [email protected]

1. Introduction

Circulating tumor cells (CTCs) are solid tumor-derived epi-

thelial cancer cells in the bloodstream of cancer patients. [ 1 ]

CTCs have been known to play a primary role in cancer

metastasis, providing a crucial biomarker for the diagnosis

of cancer patients. The number of CTCs in the blood is cor-

related with the prognosis of the cancer patient, and moni-

toring the change in the number of CTCs can be useful for

determining the patient's prognosis after treatment or resec-

tion. [ 2 ] Furthermore, molecular analysis of CTCs is expected

to enable a critical decision on the tailored treatment of indi-

vidual patients. [ 3,4 ] However, the extremely rare population

of CTCs in the blood (usually less than hundreds cells per

milliliter), in contrast to a large number of other hemato-

logic cells (≈10 9 cells mL −1 ), has hampered the clinical use of

small 2014, DOI: 10.1002/smll.201402619

http://doi.wiley.com/10.1002/smll.201402619

S.-M. Jo et al.

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full papers

proteins can be effectively used for construction of a well-

defi ned nanostructure. In addition, M13-bacteriophage is

long and fl exible, and may easily accessible to the cell sur-

face. We reasoned that magnetic particles functionalized with

M13-bacteriophage will lead to a much higher capture effi -

ciency and purity through a “tentacle-like action” that signifi -

cantly enhances the binding avidity for cancer cells compared

with a conventional fl at-surfaced structure (Figure 1 b,c).

2. Results

2.1. Construction of Nanotentacle-Structured Magnetic Particles Using M13-Bacteriophage

We fi rst constructed the nanotentacle structure derived from

gp3-expressing M13-bacteriophage onto magnetic particles

(2.8 µm in diameter) that had been modifi ed with an anti-gp3

antibody (Figure 1 a and Supporting Information Figure S1).

The resulting nanotentacle-structured magnetic particles

were further modifi ed with 6-arm N -hydroxysuccinimide-

activated polyethylene glycol 15000 (6-arm-PEG 15000 -NHS) to

minimize the nonspecifi c binding of the cells, followed by

conjugation with an anti-HER2 antibody (Figure 1 a). The

6-arm-PEG 15000 -NHS linker reacts with each amine group

of M13-bacteriophage and anti-HER2 antibody. Hydrophilic

property and high mobility of PEG is known to play a crucial

role in reducing nonspecifi c binding of proteins or cells. [ 20 ]

As a marker for cancer cells, we employed HER2, which is

known to be overexpressed in several types of carcinomas

including those of breast, gastric, and ovarian origin. [ 21 ] We

examined the morphology of the constructed magnetic

particles using a transmission electron microscope (TEM)

( Figure 2 a,b). The magnetic particles were shown to be well

modifi ed with the M13-bacteriophage, exhibiting a tentacle-

like structure, whereas no similar structure was observed

when anti-mouse IgG-conjugated magnetic particles (nega-

tive control) were incubated with the M13-bacteriophage

(Supporting Information Figure S2). As expected, one end of

the M13-bacteriophage was conjugated onto magnetic par-

ticles, and the amount of M13-bacteriophages on each mag-

netic particle was estimated to be 1030 ± 141 pfu through a

spectrophotometric analysis at 260 nm. This result indicates

that an M13-bacteriophage can be conjugated onto magnetic

particles in an oriented manner using an antibody specifi c for

the gp3 coat protein of M13-bacteriophage. The number of

anti-HER2 antibody molecules conjugated onto each mag-

netic particle was estimated to be 162 000 using a UV spec-

trometric method, whereas 67 000 antibody molecules were

determined to be conjugated to each conventional magnetic

particle. Analysis of nanotentacle-structured magnetic parti-

cles using dynamic light scattering (DLS) revealed that the

small 2014, DOI: 10.1002/smll.201402619

Figure 1. Nanotentacle-structured magnetic particles for capturing CTCs. a) Schematics of the nanotentacle-structured magnetic particles. One end of the M13-bacteriophage (gp3) was conjugated onto magnetic particles in an oriented manner using an antibody specifi c for the gp3 coat protein of M13-bacteriophage. The particles were further tethered with an anti-HER2 antibody to capture the cancer cells. b) Proposed model for capturing CTCs using nanotentacle-structured magnetic particles. Nanotentacle-structured magnetic particles offer enhanced avidity through a “tentacle-like action”, resulting in enhanced binding avidity. c) Capture of CTCs using conventional magnetic particles directly modifi ed using an anti-HER2 antibody.


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hydrodynamic diameter of the construct increased by around

350 nm compared with bare magnetic particles (Supporting

Information Table S1).

2.2. Cancer Cell-Targeting with the Nanotentacle-Structured Magnetic Particles

We investigated whether the nanotentacle-structured mag-

netic particles will effi ciently interact with the cancer cells.

SK-Br3, which is known to overexpress HER2, was employed

as a model cancer cell. The nanotentacle-structured magnetic

particles were incubated with the SK-Br3 cells, followed by

observation using an optical microscope. Interestingly, many

nanotentacle-structured magnetic particles were revealed to

bind on a single cancer cell (Figure 2 c), compared with the

conventional magnetic particles that were directly modifi ed

with the anti-HER2 antibodies (Figure 2 d). The number of

nanotentacle-structured magnetic nanoparticles attached

onto each cancer cell was estimated to be 45 ± 15 ( n = 12),

whereas the number of conventional magnetic particles

attached onto each cancer cell was 13 ± 4 ( n = 12). In addi-

tion, each captured cell was placed on a disposable chip, and

it was observed by fl uorescence microscope. The number

of captured cells by the nanotentacle-structured magnetic

particles was larger than the number of captured cells by

conventional particles (Figure 2 e,f). These results strongly

imply that the nanotentacle-structured magnetic particles

had much better cancer cell-targeting ability, mainly owing

to a signifi cantly enhanced avidity of the nanotentacle-

structured magnetic particles for cancer cells. The long and

fl exible structure of an M13-bacteriophage is likely to offer

high accessibility to cancer cells, leading to a multivalent

binding of a nanotentacle-structured magnetic particle to

the cells. In addition, topographical interactions between

the cancer cells and nanotentacle-structured magnetic parti-

cles will be much more favorable and effective than conven-

tional fl at-surfaced particles owing to the structural feature

small 2014, DOI: 10.1002/smll.201402619

Figure 2. TEM and optical images of nanotentacle-structured magnetic particles. a) Image at 10 000-fold magnifi cation. b) Image at 40 000-fold magnifi cation. Black spheres indicate the magnetic particles, and long objects show an M13-bacteriophage. The M13-bactriophages were negatively stained using a 0.7% uranyl acetate solution at pH 4.5. Image contrast was slightly adjusted to highlight the M13-bacteriophages using the ImageJ program. c) Optical microscopic image of SK-Br3 cells targeted by the nanotentacle-structured magnetic particles (500-fold magnifi cation). d) Optical microscopic image of SK-Br3 cells targeted by conventional magnetic particles (500-fold magnifi cation). e) Representative image of captured SK-Br3 cells by the nanotentacle-structured magnetic particles (100-fold magnifi cation). f) Representative image of captured SK-Br3 cells by the conventional magnetic particles (100-fold magnifi cation).

S.-M. Jo et al.


full papersof an M13-bacteriophage as well as the high surface area

(18 700 nm 2 per M13-bacteriophage). [ 19 ] Considering the

large number of M13-bacteriophages conjugated onto a mag-

netic particle, the surface area of a nanotentacle-structured

magnetic particle is presumed to be much larger than a con-

ventional particle.

2.3. Capture Performance of the Nanotentacle-Structured Magnetic Particles

We evaluated the capture effi ciency using the nanotentacle-

structured magnetic particles for SK-Br3 cells at different

cell numbers ranging from 2.0 × 10 4 to 3.0 × 10 1 cells mL –1

in 1 mL of a serum-free medium without negative cells. The

number of cells captured by the nanotentacle-structured

magnetic particles was linearly correlated with the number

of initially spiked cells (Supporting Information Figure S3a),

and the capture effi ciency was higher than 95% ( Table 1 ). In

contrast, the number of cells captured by conventional mag-

netic particles was much smaller than the number of initially

spiked cells (Supporting Information Figure S3a), and conse-

quently the capture effi ciency was lower than 40% (Table 1 ).

This result also supports the idea that the nanotentacle-struc-

tured magnetic particles are highly effi cient for capturing

cancer cells. In the experiments using 100% fetal bovine

serum (FBS), similar results were obtained in terms of the

capture effi ciency for the nanotentacle-structured magnetic

particles (Supporting Information Figure S3b). However, in

the case of conventional magnetic particles, the capture effi -

ciency was much lower (<20%) than those in a serum-free

medium (Supporting Information Figure S3b). This result

implies that some serum proteins generated severe interfer-

ence with the capturing of the cancer cells by the conven-

tional magnetic particles.

To further assess the specifi c capture effi ciency, we spiked

SK-Br3 or SK-Ov3 cells (HER2-positive cells) into 1 mL of

a serum-free medium at 2.0 × 10 5 cells mL –1 , and added the

5 × 10 6 magnetic particles followed by incubation for 15 min.

As a result, the capture effi ciency was estimated to be 80%

for both SK-Br3 and SK-Ov3 ( Figure 3 a), whereas the cap-

ture effi ciency using conventional magnetic particles was less

than 50% (Figure 3 b). We tested three kinds of HER2-nega-

tive cells, i.e., HL-60, MDA-MB-231, and MCF-7, and found

a negligible capturing of the cells by both the nanotentacle-

structured and conventional magnetic particles (insets of

Figure 3 a,b). These results indicate that the nanotentacle-

structured magnetic particles are effective for specifi cally

capturing cancer cells expressing a target marker, leading to

high capture effi ciency. The expression status of HER2 in the

cell lines is shown in Supporting Information Figure S4 and

Table S2.

We tested the capture effi ciency and purity for the

reduced number of target cancer cells with cell mixture.

Approximately 250 and 25 cells of SK-Br3 (HER2-positive

cells) were mixed with HL-60 cells of 4.0 × 10 6 (HER2-neg-

ative cells) in 1 mL of a serum-free medium, and the cap-

ture effi ciency and purity were analyzed. These experimental

conditions are presumed to closely mimic the real number of

CTCs and leukocytes in the blood. As shown in Figure 4 a (left

part), the capture effi ciency and purity were estimated to be

90% and 80%, respectively, when 250 cells of SK-Br3 were

small 2014, DOI: 10.1002/smll.201402619

Table 1. Capture effi ciency (%) for SK-Br3 cells at different cell numbers ranging from 2.0 × 10 4 to 3.0 × 10 1 cells mL –1 in 1 mL of a serum-free medium and 100% FBS. Data represent the mean and standard deviations of six independent experiments.

Number of cells spiked in serum free media

Nanotentacle-structured magnetic particles

Conventional magnetic particles

2.0 × 10 4 95.7 ± 2.1% 59.3 ± 3.1%

2.0 × 10 3 87.3 ± 3.2% 32.4 ± 5.7%

2.0 × 10 2 89.9 ± 5.6% 26.4 ± 3.5%

3.0 × 10 1 85.4 ± 9.1% 36.2 ± 2.3%

Number of cells spiked in 100% FBS

Nanotentacle-structured magnetic particles

Conventional magnetic particles

2.0 × 10 4 92.3 ± 2.5% 17.7 ± 1.6%

2.0 × 10 3 90.7 ± 5.0% 10.4 ± 3.5%

2.0 × 10 2 91.0 ± 13.2% 25.6 ± 6.7%

3.0 × 10 1 86.4 ± 8.2% 14.0 ± 6.1%

Figure 3. Capture effi ciency of cancer cells. Capture effi ciency by nanotentacle-structured magnetic particles (a) and conventional magnetic particles (b) for positive cancer cells (SK-Br3 and SK-Ov3). The inset shows the capture effi ciency for negative cells (HL-60, MDA-MB-231, and MCF-7). The number of cells spiked into the medium was 2.0 × 10 5 . Error bars represent the standard deviations ( n = 6).



mixed with 4.0 × 10 6 cells of HL-60. In contrast, conventional

magnetic particles showed about 30% capture effi ciency and

40% purity (Figure 4 a). When the number of SK-Br3 cells

was reduced to 25 in a medium containing 4.0 × 10 6 cells of

HL-60, the capture effi ciency and purity were estimated to

be 80% and 38%, respectively (Figure 4 b), whereas conven-

tional magnetic particles resulted in about 40% capture effi -

ciency and purity of lower than 10%. It is noteworthy that the

capture effi ciency and purity of the nanotentacle-structured

magnetic particles remained high, even though the number

of SK-Br3 cells was reduced from 250 to 25 cells mL –1 .

The decrease in the capture purity seems to be due to the

fact that the total number of captured positive cells (SK-Br3)

decreased as the initial number of spiked cells was reduced

from 250 to 25 cells, whereas the number of nonspecifi cally

captured negative cells (HL-60) remained almost constant.

We investigated the effect of the PEG shape using a six-arm

PEG 15000 (star-shaped) and linear-PEG 3400 . Interestingly, no

signifi cant differences were observed in terms of non-specifi c

binding (Supporting Information Figure S5). However, the

use of the six-arm PEG allowed a simpler conjugation of

antibodies than the hetero-bifunctional linear-PEG, and we

therefore used the six-arm PEG in our study. We tested the

PEGylated magnetic particles in terms of capture effi ciency

and purity. For this, PEG 3400 was used for conjugating anti-

HER2 antibody to magnetic particles. The PEGylated parti-

cles were shown to result in much lower capture effi ciency

and purity compared with nanotentacle-structured magnetic

particles (Supporting Information Figure S6). This result

strongly implies that the nanotentacle-structured particles

constructed by M13-bacteriophage are effective for capturing

cancer cells with high capture effi ciency and purity through

enhanced avidity toward cancer cells.

2.4. Performance of the Nanotentacle-Structured Magnetic Particles in Fresh Whole Blood

Next, we examined the performance of the nanotentacle-

structured magnetic particles for capturing cancer cells from

whole blood. We spiked about 25 and 250 cells of dye-stained

SK-Br3 into 0.5 mL of fresh whole mouse blood, and iso-

lated the cells using the nanotentacle-structured magnetic

particles. The isolated cells were then stained with an anti-

mouse CD45-antibody and DAPI for counting the negative

cells. CD45 is a representative leukocyte membrane marker,

and this method is a gold standard for detecting white blood

cells. [ 7 ] As shown in Figure 5 , the capture effi ciencies were

shown to be higher than 90% for both 25 and 250 cells. The

capture purities were estimated to be 45% and 85% for

25 and 250 cells, respectively. This result indicates that the

small 2014, DOI: 10.1002/smll.201402619

Figure 4. Performance using nanotentacle-structured magnetic particles and conventional particles for different numbers of cancer cells. a) Capture effi ciency and purity for 250 SK-Br3 cells in 4.0 × 10 6 HL-60 cells. About 250 SK-Br3 cells were spiked in 4.0 × 10 6 HL-60 cells, and the capture effi ciency and purity were determined. b) Capture effi ciency and purity for 25 SK-Br3 cells in 4.0 × 10 6 HL-60 cells. About 25 SK-Br3 cells were spiked in 4.0 × 10 6 HL-60 cells, and the capture effi ciency and purity were determined. The cells were prestained by a cell-tracking dye with a different color (SK-Br3, green; HL-60, orange), and were spiked into 1 mL of a serum-free medium. All captured cells were directly counted using a fl uorescence microscope. Error bars represent the standard deviations ( n = 6).

Figure 5. Capture effi ciency and purity for capturing the cancer cells from whole mouse blood. The SK-Br3 cells (25 and 250 cells) were spiked into 0.5 mL of whole mouse blood. The Sk-Br3 cells were prestained using an orange dye. The captured cells were further stained using an anti-CD45 mouse antibody and DAPI to distinguish them from leukocytes, followed by counting. All captured cells were directly counted using a fl uorescence microscope. Error bars represent the standard deviations ( n = 9).

S.-M. Jo et al.


full papersnanotentacle-structured magnetic particles can be effectively

used for capturing cancer cells from the whole blood without

any pretreatment, leading to much higher capture purity and

effi ciency than previously reported methods. [ 5–16 ] Further-

more, no aggregation or blood-particle clots were observed in

the experiment, suggesting that the nanotentacle-structured

magnetic particles have high blood compatibility.

2.5. Viability of the Captured Cancer Cells

To determine the viability of the captured cells, we isolated

5.0 × 10 3 SK-Br3 cells using the nanotentacle-structured

magnetic particles, and tested the viability of the cells. High

viability is crucial to further analysis of captured CTCs. The

viability of the SK-Br3 cells captured by the nanotentacle-

structured magnetic particles was 84%, whereas the viability

of intact SK-Br3 was estimated to be 95% (Supporting Infor-

mation Figure S7). When conventional magnetic particles

were used, the viability of the captured SK-Br3 was 88%. In

addition, the captured SK-Br3 cells can be cultivated under

normal culture conditions. This result suggests that the con-

structed magnetic particles have a low toxicity against the

cells. Moreover, our approach led to a high viability of the

captured cancer cells (≈84%), which is also valuable for a

further analysis of the captured CTCs. The high viability

strongly supports the idea that the nanotentacle-structured

magnetic particles give rise to a negligibly toxic or harmful

effect on the cells.

3. Discussion

We have shown that the nanotentacle-structured magnetic

particles lead to a high capture effi ciency (>90%) and purity

(45%–85%) even though the number of cancer cells reduced

to 25 in whole blood, which seems to closely mimic real con-

ditions. Based on these results, the high effi ciency and speci-

fi city seem to come from a signifi cantly enhanced binding

avidity of the nanotentacle structures due to long and fl ex-

ible properties of M13-bacteriophage that allows multiva-

lent binding, high accessibility, and increased topographical

interactions with cancer cells. In clinical aspects, high capture

purity is crucial to the molecular diagnosis and monitoring

of the cancer status such as metastasis and drug response

through the current standard methods, including direct DNA

sequencing. Many capture methods are diffi cult to apply for

molecular analysis of CTCs, since the sample purity required

for direct DNA sequencing is known to be about 25%. [ 22 ]

In contrast, our method can provide a suffi cient purity for

molecular diagnosis of CTCs. The high capture effi ciency of

the nanotentacle-structured magnetic particles is useful for

predicting the cancer statues based on the number of CTCs

found in the blood. The high capture effi ciency ensures an

accurate monitoring the change in the number of CTCs that

can be used for determining the prognostic value of cancer

patients after treatment or resection.

A variety of methods for capturing CTCs have been devel-

oped, including microfl uidic devices, [ 5–8 ] immunomagnetic

methods, [ 9–12 ] and other platforms. [ 12–16 ] Such methods have

shown high capture effi ciency, but their capture purity is sig-

nifi cantly low. [ 18 ] Other approaches have shown high capture

effi ciencies (60%–90%) or purity (>10%), but few study to

improve both effi ciency and purity have been reported. [ 17,18 ]

It has been shown that an increase in capture purity usu-

ally causes a decrease in capture effi ciency, whereas, efforts

to increase capture effi ciency may induce signifi cant non-

specifi c cell capture, causing low purity. [ 8 ] In contrast, our

study provides high capture effi ciency as well as high cap-

ture purity. Another distinct advantage of our immunomag-

netic approach is a simple and rapid procedure enabling

the isolation of CTCs within a 20-min period, and no com-

plicated experimental setup is necessary. Microfl uidic-based

approaches usually require excessively elaborated capture

devices, long processing times (up to several hours), the

device fabrication time-consuming, and complex experi-

mental setup for sample handling. [ 23 ]

Our results confi rmed the utility of HER2 as an effective

marker for capturing cancer cells. HER2 has been known to

be useful marker in breast cancer to monitor patients though

analysis of CTCs. [ 24,25 ] Hence, our study thought has a great

potential for applying breast cancer diagnosis. But, epithelial

cell adhesion molecules (EpCAM; CD326) have been exten-

sively used as a marker for capturing CTCs, [ 5–16 ] because of

expressing in wide range of epithelial-origin tumor cells. [ 26,27 ]

Thus, more extensive application of capturing CTCs might be

fulfi lled through the replacement of targeting ligands, such as

EpCAM.

4. Conclusion

We have shown that the nanotentacle-structured magnetic

particles lead to high purity (45%–85%) and effi ciency

(>85%), offering a great potential for use in clinical fi elds.

High capture purity is crucial to the molecular diagnosis and

monitoring of the cancer status such as metastasis and drug

response through the current standard methods, including

direct DNA sequencing. The sample purity required for direct

DNA sequencing is known to be about 25%. [ 22 ] The high cap-

ture effi ciency of the nanotentacle-structured magnetic par-

ticles is useful for predicting the cancer statues based on the

number of CTCs found in the blood. Monitoring the change

in the number of CTCs can be used for determining the prog-

nostic value of cancer patients after treatment or resection.

Furthermore, the concept of “enhanced binding avidity”

opens up a novel possibility for particle-based biosensors

to elevate their sensitivity. This concept can be applied to

other sensing purposes, such as the detection and isolation of

microorganisms and bio-macromolecules. [ 19 ]

5. Experimental Section

Materials : Epoxy-activated magnetic particles (2.8 µm in diameter), cell tracker dye (green CMEDA and orange CMRA), and sulfo-SMCC were purchased from Invitrogen (Carlsbad, CA, USA). Anti-gp3 antibody was obtained from Abcam (Cambridge,

small 2014, DOI: 10.1002/smll.201402619



England). M13-bactriophage (M13KO7) was purchased from New England Biolabs (Ipswich, Massachusetts, USA). Herceptin was kindly gifted by Asan Medical Center (Seoul, Republic of Korea). Uranyl acetate, propidium iodide, and anti-mouse IgG were pro-vided by Sigma–Aldrich (St. Louis, MO, USA). NHS-6-arm PEG 15000 was purchased from Sunbio (Anyang, Republic of Korea). Dispos-able hemocytometer chip was purchased from iNCYTO (Chonan, Republic of Korea).

Cell Culture : SK-Br3 (breast cancer cells), SK-Ov3 (ovarian cancer cells), MDA-MB-231 (breast cancer cells), MCF-7 (breast cancer cells), and HL-60 (human promyelocytic leukemia cells) were provided by Korean Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) under 5% CO 2 condition at 37 ºC in CO 2 incubator (MCO-5AC, Sanyo, Japan). BALB/c female mice (6 weeks) were obtained from Orient Bio (Sungnam, Republic of Korea), and were fed for 12 weeks.

Construction of Nanotentacle-Structured Magnetic Particles Using M13-Bacteriophage : M13-bacteriophage was prepared by following described elsewhere. [ 28 ] To construct the nanotentacle-structured magnetic particles, 7.5 mg (5.0 × 10 8 in number) of epoxy-activated magnetic particles (2.8 µm in diameter, Invit-rogen, Carlsbad, CA, USA) was reacted with 0.1 mg of an anti-gp3 antibody (Abcam, Cambridge, England) for overnight. The resulting particles were washed three times, followed by incu-bation with 2.0 × 10 12 pfu M13-bacteriophages overnight. After washing three times, the particles were further conjugated with 80 µg of N -hydroxysuccinimide-activated six-arm PEG 15000 (Sunbio, Republic of Korea) for 20 min. The resulting magnetic parti-cles were washed three times, and subjected to incubation with 375 mg of anti-HER2 antibodies (Herceptin, Roche, Switzerland) for 8 h, followed by three washings. The fi nal concentration of the magnetic particles was adjusted to 10 8 /100 µL.

Construction of Herceptin-Conjugated Magnetic Particles : 7.5 mg (5.0 × 10 8 ) of epoxy-activated magnetic particles (2.8 µm in diameter) was directly reacted with 375 mg herceptin for overnight at room temperature (50 × 10 −3 M borate buffer, pH 8.6). Following a reaction, the particles were washed three times using same buffer by magnetic separator (CS15000, Invitrogen). The fi nal con-centration of magnetic particles was adjusted to 10 8 /100 µL.

Staining of M13-Bacteriophage for TEM Image : M13-bacte-riophage was stained for TEM image according to the previously reported method with slightly modifi cation. [ 29 ] Briefl y, sample solution was dropped onto carbon/formvar grid, followed by drying under ambient condition for 3 h. The grid was placed on fi lter paper, and uranyl acetate solution (0.7%, pH 4.5) was dropped. The resulting grid was further dried under ambient condi-tion, and subjected to TEM analysis at 200K fi eld emission (JEM-2100F, Japan).

Hydrodynamic Diameter : The hydrodynamic diameter of nano-tentacle-structured magnetic particles was determined by the dynamic light scattering (DLS) method (ELSZ-1000, Otsuka Elec-tronics, Japan) at pH 7.4 HEPES buffer solution.

Flow Cytometry : For the analysis of the expression level of HER2, the cells were trypsinized and washed twice using DPBS. FITC-labeled anti-HER2 antibody (ebioscience, San Diego, CA, USA) was added to the cell suspension of 0.5 mL (DPBS with 1% bovine serum albumin), and incubated for 15 min with mild shaking. The cells were washed twice and analyzed by BD FACSAria III fl ow

cytometer (BDbiosciences, Franklin Lakes, NJ, USA). To test the viability, the cells were trypsinized and washed using 1% BSA-con-tained DPBS (if needed, cells went through a capture procedure), suspended in 0.45 mL solution. Propidium iodide (10 µg mL −1 ) of 0.05 mL was added to the cell suspension, and further incubated for 30 min. The resulting cells were analyzed by FACSAria III fl ow cytometer at PerCP channel. Control (dead cells as a PI-stained) cells were prepared by exposing 60 °C for 3 min.

Capture and Enumeration of Cancer Cells : Prior to the capture experiments, individual cells were prestained using a cell-tracking dye (Invitrogen). Positive cells were stained by a green cell tracker, and negative cells by a red one. The cells were spiked into 1 mL of a serum-free medium, FBS, or whole mouse blood, followed by incubation with magnetic particles of 5.0 × 10 6 for 15 min at room temperature. A neodymium magnet (CS15000, Invitrogen) was applied to the mixture to separate the magnetic particles, and any non-captured cells were discarded by a washing. The separated magnetic particles were washed twice using DPBS, and fi nally resuspended in 20 µL of DPBS for separating the captured cells. The number of captured cells was counted using a fl uorescence microscope (Olympus, Japan). When counting the smaller number of cells (25 cells and 250 cells), the number of spiked cells was counted ten times, and then averaged. The capture effi ciency per-centage was calculated through the following Equation ( 1)

= ×Capture efficiency (%)

Number of captured positive cellsNumber of spiked positive cells

100

(1)

The capture purity percentage was determined using the fol-lowing Equation ( 2)

Capture purity (%)

Number of captured positive cellsNumber of all captured cells

100= ×

(2)

To test the performance of the magnetic particles for isolating the cancer cells from whole mouse blood, healthy 12-week-old female mice (BALB/c) were sacrifi ced, and about 0.5 mL of whole blood was obtained for the capturing experiments. All experi-mental plastic wares were pretreated with a heparin solution (2000 units mL –1 ). Mouse white blood cells were stained by anti CD45 antibody (R&D systems, Minneapolis, MN, USA). Captured cells (prestained by cell tracker dye) were dispersed in 20 µL DPBS, then placed onto a disposable cell chip. All cells in a channel were counted by fl orescence microscope (Olympus, Japan) and confocal microscope (LSM780, Carl Zeiss, Germany).

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

This research was supported by the Bio & Medical Technology Development Program (NRF-2013M3A9D6076530) and Mid-career Researcher Program (NRF-2014R1A2A1A01004198) of the National

small 2014, DOI: 10.1002/smll.201402619

S.-M. Jo et al.


full papers

small 2014, DOI: 10.1002/smll.201402619

Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning. The authors also thank Yoonha Hwang, Howon Seo, and Pilhan Kim for supplying the mouse blood.

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Received: September 1, 2014 Revised: October 2, 2014 Published online:

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