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Cite this: Mater. Adv., 2022,
3, 5043

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Surfactant-free suspension polymerization ofhydrophilic monomers with an oil-in-water
system for the preparation of microparticles
toward the selective isolation of tumor cells†
Shin-nosuke Nishimura,
Masaru Tanaka*‡ Kei Nishida,*§ Shohei Shiomoto

andCirculating tumor cells (CTCs) are derived from a primary tumor or monastic foci, and are found in the
bloodstream of patients with tumors. We developed polymer droplets of blood-compatible poly(2methoxyethyl acrylate) (PMEA) that selectively accumulate in tumor cells. PMEA microparticles, which
are larger than the size of cells, have potential as a platform for CTC capture devices without the need
to use antibodies. Herein, these microparticles, as well as several, other types of microparticles
composed of hydrophilic monomers, were prepared by surfactant-free suspension polymerization, andtheir selective isolation abilities toward the capture of tumor cells were evaluated via comparative
studies. The microparticles possessed smooth and extremely pure surfaces suitable for evaluating theinteraction force with tumor cells. The number of human platelets adhered to the PMEA microparticles
was clearly lower than the number of platelets adhered to the other polymer microparticles.
Received 6th February 2022,
Accepted 6th May 2022

Interestingly, the PMEA microparticles prepared with a 1% crosslinking ratio showed stronger interactions

DOI: 10.1039/d2ma00129b

eﬃcient recovery of tumor cells from cell suspensions under dynamic conditions in comparison with the

rsc.li/materials-advances

other polymer microparticles. These results provide insights into the possible applicability of PMEA
microparticles as a platform for CTC capture without using antibodies.

with tumor cells than the other polymer microparticles. In addition, the PMEA microparticles enabled theInstitute for Materials Chemistry and Engineering, Kyushu University, 744,
Moto-oka, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail: nishida.k.ak@m.titech.ac.jp,
masaru_tanaka@ms.ifoc.kyushu-u.ac.jp† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d2ma00129b‡ Present address: Faculty of Science and Engineering, Department of Molecular
Chemistry and Biochemistry, Doshisha University, Japan.
§ Present address: Department of Life Science and Technology, School of Life
Science and Technology, Tokyo Institute of Technology, Japan.

antibodies.5,6 However, the utilization of EpCAM-based approaches
is often limited because of the existence of EpCAM-negative CTCs.7
Therefore, there is a need to develop a system for the specificisolation of CTCs that uses technology other than tumor-related
antigens and antibodies. We previously reported that poly(2methoxyethyl acrylate) (PMEA)-coated substrates suppress theadhesion and activation of platelets but enhance the adhesion of
integrin-overexpressed cells, such as non-tumorigenic epithelial
cells and tumor cells.8–10 PMEA-coated substrates showed nocytotoxicity and an improved viability of tumor cells underserum-free conditions.11 Furthermore, we demonstrated thatpolymer droplets composed of PMEA selectively accumulate intumor cells, avoiding normal cells.11–13 Thus, PMEA has thepotential to selectively isolate tumor cells from blood samples
regardless of their EpCAM expression.
Microparticles have been developed as a valuable platform
for microcarrier-based cell culture.14,15 In addition, microparticles conjugated with antibodies or polymers have beenreadily utilized for the isolation of proteins, immune cells, andstem cells.16–18 Microparticles with high surface area-to-volume
ratios can gain an increased contact frequency with cells
by using dynamic systems.19,20 Hence, we hypothesize that© 2022 The Author(s). Published by the Royal Society of Chemistry

Mater. Adv., 2022, 3, 5043–5054 | 5043

Introduction
Circulating tumor cells (CTCs), which invade the bloodstream
from a primary tumor and monastic foci, can act as an eﬀective
biomarker to stipulate a reliable method for diagnosing not
only tumor progression but also tumor metastasis.1 Somemethods for the detection of CTCs have been developed, suchas filtration-based assessment, the DEPArray system, and thesemi-automated CellSearch system.2–4 Because CTCs express
specific membrane antigens, including epithelial cell adhesion
molecules (EpCAMs), they have been routinely captured
through ligand–receptor interactions and by immunomagnetic
separation using microspheres coated with ligands, such as

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PMEA-formed microparticles can provide an effective platform
for the separation of CTCs from blood.
Suspension polymerization via oil-in-water systems has beenused to prepare polymer microparticles.21–23 Conventional
suspension polymerization systems are suitable for preparing
microparticles composed of hydrophobic monomers (e.g., styrene,
methyl methacrylate, and vinyl acetate);21,22 whereas hydrophilic
monomers, including 2-methoxyethyl acrylate (MEA), partially orcompletely dissolve in large amounts of water and are difficult toform oil droplets. The oil droplets are unstable and easily fuse
together, resulting in an indefinite aggregation of polymers.
Stabilizers, including surfactants, are generally used in suspension polymerization processes to inhibit droplet fusion;23
however, the surfactants often remain on the surface of theresulting microparticles, even if the microparticles are carefully
washed with solvents that would dissolve the surfactant. Residual
surfactants often affect the function of the particles, including
their cell adhesion. For water-soluble hydrophilic monomers,
inverse suspension polymerization, which uses water-in-oil
systems, is often employed because these monomers dissolve
in water.24–26 Inverse suspension polymerization requires not only
a surfactant to stabilize the dispersed droplets, but also large
amounts of organic solvents.
As such, suspension polymerization with oil-in-water
systems is less detrimental to the environment as it uses
less organic solvent than inverse suspension polymerization.
Okudaira et al. reported a preparation method for hydrophobicpolystyrene (PSt) microparticles by a surfactant-free suspension
polymerization,27 but there are no such reports on hydrophilicpolymer microparticles, owing to the dissolution of hydrophilic
monomers in water. Thus, it is challenging to establish a
protocol toward hydrophilic polymer microparticles by surfactantfree suspension polymerization with an oil-in-water system.
In the present study, we describe the preparation of PMEA
microparticles by a surfactant-free suspension polymerizationthat enabled the selective adhesion of cells. As controls,hydrophobic poly(2-methoxyethyl methacrylate) (PMEMA) andhydrophilic poly(oligo(ethylene glycol)acrylate) (POEGA),
poly(2-hydroxyethyl acrylate) (PHEA), and poly(2-hydroxyethyl
methacrylate) (PHEMA) were prepared by the developed
protocol, and their aﬃnities for tumor cells were evaluated.

Table 1

Finally, we demonstrated the capturing of tumor cells using the
PMEA microparticles under dynamic conditions.

Results and discussion
Preparation of the polymer microparticles by surfactant-freesuspension polymerization
We demonstrated the preparation of microparticles composedof PMEA, POEGA, PHEA, PMEMA, and PHEMA by surfactantfree suspension polymerization with an oil-in-water system. To
obtain suitable particles for the cell experiments, three diﬃculties had to be overcome (i.e., removing any remaining
surfactant, selecting the appropriate organic solvents, andensuring low glass transition temperatures (Tg)). Polymers withhigh Tg (e.g., PSt and PMEMA) form hard particles whose
surfaces inhibit their fusion with one another. On the other
hand, polymers with low Tg (e.g., PMEA, POEGA, PHEA, and
PHEMA) form soft particles with sticky surfaces, resulting intheir aggregation. Some research groups have reported
the preparation of PHEMA by suspension polymerization;28,29
however, these protocols used an exorbitant amount of crosslinker, which could give resultant particles that do notcomprise pure PHEMA. Thus, there is a need to establish asurfactant-free suspension polymerization method that uses aminimal amount of crosslinker to prepare pure microparticles
composed of hydrophilic monomers. As such, in this study, we
devised a new system for suspension polymerization thatcombines the salting-out eﬀect of calcium chloride and thestabilization eﬀect of calcium carbonate. Calcium chloride issuitable for this system because it can dissolve in not only water
but also in the lower alcohols (e.g., methanol and ethanol),which are used to remove unreacted monomers from theresultant particles; this is notable as general salts tend to be
insoluble in such alcohols. Calcium carbonate was employed as
the dispersion stabilizer because it can be decomposed byhydrochloric acid into easily removable calcium chloride.
Consequently, the particles prepared by our system can beeasily purified by washing them with a lower alcohol after
treatment with hydrochloric acid. Table 1 summarizes the
preparation of the microparticles used in this study. To clarify

Summary of the surfactant-free suspension polymerization via a two-step heating process

Feed composition

Temp. (1C)

Polymn. time (h)SamplesMonomer(mol)

Initiatora(mmol)

Crosslinkerb(mol%)

CaCl2
(mol)

CaCO3
(mol)

Water
(mL)

Org. solv.c
(mL)

1st

2nd

1st

2nd

Stirring
speed (rpm)

PMEA_1
PMEA_2
POEGA
PHEA
PMEMA
PHEMA

0.19
0.19
0.052
0.22
0.17
0.19

0.61
0.61
0.61
0.65
0.61
0.61

1
2
1
1
1
1

0.54
0.54
1.09
1.02
0.54
1.09

0.10
0.10
0.10
0.10
0.10
0.10

200
200
110
150
220
220

—
—
2.5
10
—
—

60
60
60
40
60
60

85
85
85
60
85
85

0.5
0.5
0.5
0.5
0.5
0.5

1
1
2
1
1
3

300
300
300
300
450
300

a

2,2-Azobis(isobutyronitrile) (AIBN) was used for PMEA, POEGA, PMEMA, and PHEMA. 2,2-Azobis(4-methoxy-2,4-dimethylvaleronitrile) was used
for PHEA. b Ethylene glycol diacrylate was used for PMEA, POEGA, and PHEA. Ethylene glycol dimethacrylate was used for PMEMA and PHEMA.
c
Cyclohexanol and 2,2,2-trifluoroethanol were used for POEGA and PHEA, respectively.

5044 | Mater. Adv., 2022, 3, 5043–5054© 2022 The Author(s). Published by the Royal Society of ChemistryView Article Online

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the eﬀect of the crosslinking ratio (Cr), PMEA microparticles
were prepared with Cr values of 1% and 2%, while the othermicroparticles were prepared using a Cr of 1%. The monomers
suspended in a highly concentrated aqueous calcium chloride
solution were polymerized in the presence of calcium carbonate
with stirring by a propeller agitator. Note that the concentrationand volume of calcium chloride depended on the hydrophilicityof the monomers. To obtain sturdy particles from PMEA,
PMEMA, or PHEMA, no organic solvent was needed, whereasa minimal amount was required to obtain particles fromPOEGA and PHEA. In addition, a two-step heating process
was necessary to accomplish high monomer conversion andobtain sturdy particles. When these conditions suited very well
the properties of the monomers, the microparticles wereformed without aggregation (i.e., the combination of theconcentration of calcium chloride, the condition of a two-stepheating process, and the hydrophilicity of monomers). The
resultant microparticles were treated with hydrochloric acid,followed by Soxhlet extraction with methanol, resulting in pure
microparticles. The purified microparticles were sorted into
two groups based on size via a wet-type classification using asieve (mesh opening: 300 mm) and methanol; they were thenlyophilized after replacement with water to give the pure PMEA
microspheres. The smaller microparticles of the two sizedbased groups of particles were used for the remainder ofthe study.
The chemical structures of the microparticles were confirmed by attenuated total reflectance (ATR)-Fourier transform
infrared (FTIR) spectroscopy (Fig. 1A, solid lines). The spectraof the PMEA and POEGA microparticles showed bands derived
from their respective PMEA and POEGA backbones at 3100–
2730 cm1 (–CH2–, R2CH–, and –O–CH3 C–H stretching),
1731 cm1 (CQO stretching of ester for acylate), 1450 cm1
(C–H bending of –COO–CH2– and –O–CH3), and 1420–800 cm1(fingerprint region). As the PHEA microparticles bear hydroxygroups instead of methyl ether groups like PMEA and POEGA,
they showed –OH stretching vibrations at 3360 cm1 (–OHstretching). Moreover, the PMEMA and PHEMA microparticle
spectra were almost the same as those of PMEA and PHEA,
respectively, except PMEMA and PHEMA exhibited a band at
1728 cm1 corresponding to the CQO stretching of theirmethacrylate ester moieties. Importantly, all the spectra corresponded to those of the homopolymers synthesized by conventional free-radical polymerization (Fig. 1A, dashed lines). Thus,
the surfactant-free suspension polymerizations successfully
proceeded to produce objective microparticles composed of
PMEA, POEGA, PHEA, PMEMA, and PHEMA. Subsequently,
X-ray photoelectron spectroscopy (XPS) measurements of the
microparticles were carried out to clarify the state of the outermost surfaces of the microparticles, which significantly
influences their cell-attachment functions. Fig. 1B shows the
XPS profiles of the microparticles. We did not observe calcium
peaks (ca. 347 eV), indicating calcium chloride and calcium
carbonate did not remain. In addition, the elemental ratios ofcarbon and oxygen obtained from the XPS profiles agreed wellwith the theoretical values (Table S1, ESI†). From these results,© 2022 The Author(s). Published by the Royal Society of Chemistry

Paper

Fig. 1 (A) ATR-FTIR and (B) XPS profiles of the polymer microparticles
synthesized in this study. Dashed lines in A show the spectra of thehomopolymers.

we concluded that the obtained microparticles have clean andpure outer surfaces, suggesting that these microparticles are
suitable for evaluating the interaction force with tumor cells.
Characterization of the polymer microparticles
The surface morphology also directly aﬀects cell attachment.
We confirmed the shape of the microparticles using scanningelectron microscopy (SEM) (Fig. 2); the SEM image of thecommercially available PSt microparticles used in this study
as a control are shown in the same figure. All the polymer
microparticles possessed true, spherical shapes with smoothsurfaces, which were appropriate for the cell experiments;
although, the diameters of the PHEA and PHEMA microparticles were smaller than those of the others. There was nodiﬀerence between the sizes of the PMEA_1 and PMEA_2
microparticles, suggesting that the eﬀect of Cr was small forour surfactant-free suspension polymerization system. These

Mater. Adv., 2022, 3, 5043–5054 | 5045

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Fig. 2

Materials Advances

SEM images of the polymer microparticles used in this study.

microparticles were used for the cell experiments under wet
conditions. In the presence of water molecules, the particles
swelled and became larger. We evaluated these changes in size
using optical microscope observations and image analysis
under dry and wet conditions (Fig. S1, ESI†). Note that phosphate buﬀered saline (PBS ()) was used as a solvent for theexperiments under wet conditions to obtain an environment
close to cell culturing conditions, and that the microparticles
were immersed for a suﬃcient amount of time. The results ofthis analysis are summarized in Table 2. The diameters of the
PMEA, POEGA, PHEA, and PHEMA microparticles clearly
increased in size under wet conditions. In contrast, thediameters of the PMEMA and PSt microparticles remained
nearly unchanged. The POEGA microparticles exhibited the
maximum diameter (235.8  33.3 mm), and the PHEA microparticles possessed the minimum diameter (76.2  11.2 mm).
Notably, the diameters of all the microparticles were suﬃciently

Table 2

Summary of the characterization of the microparticles used in this study

Diametera (mm)Samples Dry
PMEA_1
PMEA_2
POEGA
PHEA
PMEMA
PHEMA
PSt

larger than the sizes of the cells (410 mm). The surface areas andvolumes of all the microparticles under wet conditions were
calculated from their diameters and are shown in Table 2. The
swelling ratios of the polymer microparticles were calculatedusing eqn (6). The calculated swelling ratios of the PMEA
microparticles were 125.1% (Cr = 1%) and 114.3% (Cr = 2%),
respectively. These results are reasonable because the large
amount of crosslinker decreases the extent of the polymer chains
and interrupts the diﬀusion of water molecules into the microparticles. The more hydrophilic POEGA, PHEA, and PHEMA
microparticles showed more drastic swelling (340.7% for
POEGA, 284.1% for PHEA, and 241.0% for PHEMA) than the
PMEA microparticles. Furthermore, hydrophobic PMEMA
slightly swelled (102.3%) and PSt shrank (98.9%). Usingeqn (8), the water contents of the polymer microparticles were
defined. The PMEMA and PSt microparticles hardly containedwater, while the POEGA, PHEA, and PHEMA microparticles

172.6  47.3
186.5  38.2
156.7  27.5
53.8  9.9
117.7  30.6
58.1  11.8
112.2  17.5

Wet

Volumed (wet) 
Swelling Water content f Log P valuesg
Surface areab (wet)  Surface areac(103 Å2)
104 (cm2 particles1) (wet) (cm2 g1) 106 (cm3 particles1) ratioe (%) (wt%)

186.0  40.0 10.86
195.0  19.5 11.94
235.8  33.3 17.46
76.2  11.2 1.823
118.6  34.2 4.417
77.9  14.4 1.905
111.8  14.3 3.925

32.26
30.77
25.45
78.74
50.59
77.02
53.67

33.68
38.80
68.61
2.315
8.730
2.474
7.313

125.1
114.3
340.7
284.1
102.3
241.0
98.9

20.1
12.5
70.7
64.8
2.3
58.5
—

0.76
0.76
3.59
5.07
1.11
3.05
24.9

a
The diameter of the microparticles was determined as Feret’s diameter by analysis of the optical microscope images of the microparticles (n =200). b The surface area of the microparticles per particle was calculated from eqn (1). c The surface area of the microparticles per gram wascalculated from eqn (5). d The volume of the microparticles per particle was calculated from eqn (2). e The swelling ratio of the microparticles wascalculated form eqn (6). f The water content of the microparticles was calculated from eqn (8). g The value of the computational octanol–waterpartition coefficients (log P)/Connolly molecular surface area (SA). Log P and SA were calculated by Chem 3D (ver. 19.1) after MM2 minimalizationof the models.13,505046 | Mater. Adv., 2022, 3, 5043–5054© 2022 The Author(s). Published by the Royal Society of ChemistryView Article Online

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contained large amounts of water (ca. 60–70 wt%). The PMEA_1
and PMEA_2 microparticles also swelled, and their water contents were 20.1 wt% and 12.5 wt%, respectively. This trend
almost agreed with the theoretical values of the octanol–waterpartition coefficient (log P), which give the hydrophilic indices ofthe polymers (Table 2). Although the log P value of PHEA (5.07)
was lower than that of POEGA (3.58), a higher amount of water
was captured in the POEGA particles. It seems that the networkof PHEA is restricted from expanding due to the crosslinking via
hydrogen-bonding formation of hydroxy groups on the side
chains. The water content of PMEA_2 was smaller than that of
PMEA_1 for the same reason that their swelling ratios weredifferent: the higher amount of crosslinker constrains the polymer chains and interrupts the permeation of water molecules.
Interestingly, the water contents of these PMEA microparticles
were definitely higher than that of the PMEA homopolymer(9 wt%).9 This indicates that the specific surface area of PMEA,
whose outermost surface swells more easily than that of thepolymer in the bulk state, increased upon forming microparticles
and this promoted hydration.30,31 Subsequently, differential
scanning calorimetry (DSC) measurements of the polymer microparticles were performed under wet conditions. We have previously reported that the PMEA homopolymer and its derivatives
form cold-crystallizable water, called intermediate water (IW), atapproximately 40 1C, which plays a key role in the bloodcompatibility of polymer surfaces.9,32–35 In the case of the PMEA
microparticles synthesized by surfactant-free suspension polymerization, peaks ascribed to the cold crystallization of IW wereobserved (Fig. S2, ESI†). This result suggests that the PMEA
microparticles display excellent blood compatibility similar tothat of the PMEA homopolymer.
Measurement of antithrombogenicity for the polymer
microparticles
Platelets, as components of blood, play a central role in theformation of clots to maintain hemostasis.36 The adhesion andactivation of platelets lead to thrombogenicity on the surface of
biomaterials in contact with blood. Because the formation ofthrombus on tumor-capturing devices interrupts the adhesionof tumor cells on the device surface, antithrombogenic devices
are needed. To clarify the antithrombogenicity of our polymer
microparticles, the adhesion of platelets to their surfaces
were evaluated by the lactate dehydrogenase (LDH) method(Fig. 3).37 The numbers of platelets adhered to the PMEMA and
PSt microparticles were significantly higher than that adhered
to the other microparticles. The PMEA_1, PMEA_2, and PHEMA
microparticles suppressed platelet adhesion on their surfaces.
The number of platelets adhered to PMEA_2 was 0.098 times
that of PMEMA, 0.11 times that of PSt, 0.55 times that of
POEGA, and 0.26 times that of PHEA. The amount of serum
proteins, such as fibronectin, vitronectin, and fibrinogen(in particular), on the surface is related to platelet adhesion andactivation.38 Our previous reports demonstrated that PMEAcoated surfaces repelled fibrinogen adsorption and suppressed
conformational changes induced by fibrinogen adsorbing to
the surface.33,39 Kureha et al. reported that sub-micrometer© 2022 The Author(s). Published by the Royal Society of Chemistry

Paper

Fig. 3 Number of human platelets adhered on the polymer microparticles for 1 h of incubation. *p o 0.05; ***p o 0.005; ****p o 0.001;*****p o0.0005; N.S., not significant.

particles composed of PMEA showed plasma–protein adsorption resistance on their surface.40 Furthermore, fibrinogenadsorption and platelet adhesion on the polymer-coatedsurface were correlated to the amount of IW in thepolymers.41 Specifically, the amount of IW per gram of thepolymer microparticles increased in the order of PS E PMEMAE PHEMA r PHEA o POEGA r PMEA_2 o PMEA_1 (Fig. S2,
ESI†). This suggests that the PMEA microparticles have antithrombogenic properties.
Interaction strength between the polymer microparticles andtumor cells
Next, we investigated the interaction strength between thepolymer microparticles and tumor cells using AFM. Colloidal
cantilevers composed of polymer microparticles were fabricatedby immobilizing the polymer microparticles, i.e., PMEA_1,
PMEA_2, POEGA, PHEA, PMEMA, PHEMA, and PSt, on a tipless cantilever (Fig. S3, ESI†). The colloidal cantilevers interactedwith human cervical adenocarcinoma HeLa cells for 1 s in thepresence of serum proteins (10%). The retraction-force–distancecurve was recorded for each colloidal probe (Fig. 4A–G). The
force curve of PMEA_1 showed a higher force than those of theother probes; the force curves of POEGA, PHEA, and PHEMA
exhibited negligible forces. The value of the minimum force wasquantified as the interaction strength between the colloidal
cantilevers and the cell surface (Fig. 4H). The interaction
strengths of PMEA_2, PMEMA, and PSt were 0.58  0.15,
0.46  0.09, and 0.54  0.24 nN, respectively. Remarkably,
PMEA_1 showed an interaction strength of 1.33  0.47 nN,
resulting in its interaction with HeLa cells being stronger thanthose of the other colloidal cantilevers. Furthermore, the interaction energy was calculated as the area enclosed by the force
curve and the baseline (Fig. 4I). The interaction energies forMater. Adv., 2022, 3, 5043–5054 | 5047

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Materials AdvancesFig. 4 Representative force–distance curves of HeLa cells interacting with colloidal cantilevers composed of (A) PMEA_1, (B) PMEA_2, (C) POEGA,(D) PHEA, (E) PMEMA, (F) PHEMA, and (G) PSt; these were recorded with the following parameters: set point = 3 nN, approach rate = 2 mm s1, retraction
rate = 5 mm s1, and holding time = 1 s. Dot plots of (H) interaction strength and (I) energy of HeLa cells interacting with the same colloidal cantilevers (n =8–17, **p o 0.01).

colloidal cantilevers other than PMEA_1 were negligibly low.
These results suggest that PMEA_1 microparticles strongly interact with tumor cells. Although the adsorption and conformationalchanges of fibrinogen were suppressed on the PMEA-coatedsurface, PMEA enhanced the conformational changes inducedby fibronectin adsorbing to the PMEA-coated surface.10 Because
cell binding motifs, including arginine–glycine–aspartic-acid peptide, were exposed to the conformational change of fibronectin,
PMEA could enhance the adhesion of tumor cells on its surface.
In this regard, tumor cells with high expression levels of integrin
were promoted to adhere to PMEA. The expression level ofintegrin in tumor cells, including HeLa cells, was relatively higher
than that in normal cells.42,43 Therefore, PMEA_1 exhibited asignificantly high interaction strength and energy when interacting with tumor cells. Interestingly, PMEA_2 showed a negligible interaction strength with HeLa cells. Because PMEA_2 iscomposed of a higher crosslinker concentration than PMEA_1,
the elasticity of PMEA_2 might be higher than that of PMEA_1.
Many researchers have demonstrated that the elasticity of a
substrate affects the activation and internalization of integrin in

adhering cells, the amount of fibronectin that can adsorb to thecell surfaces, and the conformational changes induced by theadsorbed fibronectin. As such, the elasticity of microparticles may
be a key parameter in modulating their cell adhesion. However,the numbers of platelets adhered to PMEA_1 and PMEA_2 were
negligibly different. The mechanism by which PMEA_1 interactedwith tumor cells with high interaction strength is unclear; hence,
further investigation is necessary in terms of its protein adsorption behavior.

5048 | Mater. Adv., 2022, 3, 5043–5054© 2022 The Author(s). Published by the Royal Society of Chemistry

Capture of tumor cells using PMEA microparticles
The PMEA microparticles showed low adhesion with platelets
and high adhesion with tumor cells, suggesting their potential
use as a CTC capture device. Hence, we investigated whether
PMEA microparticles could capture tumor cells from cellsuspensions. Unfortunately, when capturing CTCs from blood
under static conditions, the large amount of red blood
cells present obstructs the adhesion of the CTCs to the microparticles due to the precipitation of the red blood cells.
However, cell capturing under dynamic conditions has beenView Article Online

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Fig. 5 (A) Diagram of the methodology for capturing tumor cells with the microparticles under dynamic conditions. (B) Phase contrast images of
PMEA_1 and PHEMA microparticles treated with HeLa cells for 24 h under static conditions (Scale bars: 200 mm). (C) Phase contrast images of PMEA_1
and PHEMA microparticles treated with MDA-MB-231 cells for 24 h under static and dynamic conditions (Scale bars: 200 mm). Yellow arrows represent
MDA-MB231 cells. (D) Recovery rate of MDA-MB-231 cells using PMEA_1 and PHEMA microparticles (****p o 0.0001).

developed as a methodology to avoid such precipitation of red
blood cells.44 To verify the ability of the microparticles tocapture tumor cells, PMEA microparticles were shaken intumor-cell-containing suspensions, and the number of tumor
cells adhered to them was calculated (Fig. 5A). The eﬃcacy oftumor-cell capture by the PMEA microparticles was comparedto that of the control PHEMA microparticles, which showed a
low adhesion of both platelets and tumor cells. The PMEA
microparticles exhibited a significant number of adhered HeLa
cells (EpCAM negative) on their surfaces in comparison withthe other polymer microparticles after 24 h of incubation under
static and dynamic conditions (Fig. 5B and Fig. S3, ESI†).45
Additionally, one of our previous reports revealed that MDAMB-231 cells showed higher expression levels of integrin b1
than normal human fibroblasts;46 therefore, the PMEA microparticles developed here should eﬃciently recognize andcapture MDA-MB-231 cells. In the present study, human breast
tumor cells (EpCAM-negative MDA-MB-231 cell line) wereused as one of model cells for testing our tumor capture

technology.45,47 After incubation for 24 h, the MDA-MB-231
cells adhered to the PMEA microparticles, but only negligibly
adhered to the PHEMA microparticles (Fig. 5C). The recovery
rate of MDA-MB-231 cells from the cell suspensions was 12.2fold higher when using the PMEA microparticles than whenusing the PHEMA microparticles (Fig. 5D).
PMEA microparticles captured tumor cells under both staticand dynamic condition. Our previous reports demonstrated
that PMEA-coated substrates did not cause substantial cytotoxicity
under serum-free conditions, but rather improved cell viability.11
PMEA microparticles would not cause insignificant damage tocaptured tumor cells. On the other hand, the advantage ofdynamic conditions using microparticles would be demonstrated
in the presence of multiple cells, including tumor cells, platelets,
and white and red blood cells. Therefore, the result that tumor
cells could adhere on to the microparticles under dynamic aswell as static conditions was an important consideration in
the present study. The utilization of PMEA microparticles
under dynamic conditions shows promise as a methodology for© 2022 The Author(s). Published by the Royal Society of Chemistry

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Papercapturing EpCAM-positive and -negative tumor cells from whole
blood without using antibodies.
CTCs are detected at a very low level in the blood fromcancer patients, and form clusters to suppress anoikis andreduce shear stress in blood.48,49 In addition, blood with a
specific viscosity and components aﬀects the recovery rate oftumor cells. PMEA microparticles with a dynamic system maycontribute to the capture of CTC clusters in blood with a
specific viscosity because of their size (4190 mm), which was
suﬃciently larger than that of the cells (410 mm) and the
dynamic system was capable of changing the shaking rate. For
our tumor capture microparticles to be used in clinical applications for accurately capturing rare CTCs from blood, theyrequire further investigation; specifically, their performancein tumor patient-derived blood should be evaluated, and theirtumor-cell capture eﬃciency should be improved.

Conclusions
In this study, we prepared microparticles composed of hydrophilic
monomers via a surfactant-free suspension polymerization with anoil-in-water system, and demonstrated their eﬃcient recovery oftumor cells through comparative studies with several other typesof polymer microparticles. All the polymer microparticles types,
except for commercially available PSt microparticles, were successfully prepared by the polymerization system. Among them, the
PMEA microparticles showed IW formation, which suppressed theadhesion of human platelets. In particular, the PMEA microparticles with a 1% crosslinking ratio strongly interacted with thetumor cells, resulting in a high tumor-cell recovery ratio. Thus,
our suspension polymerization system provides an opportunity todesign and synthesize various microparticles, composed of hydrophilic monomers, whose surfaces are extremely clean. Conventional methods, such as the filtration-based system, DEPArraysystem, and semi-automated CellSearch system, rely on the sizeof the CTCs and the antibody to antigens expressed on the CTCs.
Meanwhile, the developed PMEA microparticles with a dynamic
system have great potential to selectively capture tumor cells fromblood without using antibodies, regardless of their size. We believethat they can be applied in the diagnosis of tumor progression.

Experimental
Materials
Solvents of analytical grade were used unless otherwise stated.
Methanol and hydrochloric acid (12 M HCl) were purchasedfrom Kanto Chemicals Co., Ltd, (Japan). Calcium chloride
dihydrate, calcium carbonate, and 2,2 0 -azobis(4-methoxy-2,4dimethylvaleronitrile) (V-70) were purchased from FUJIFILM
Wako Pure Chemical Co., Ltd (Japan). Oligo(ethylene glycol)
methyl ether acrylate (OEGA) (Mn = 480 g mol1) was purchasedfrom Merck/Sigma-Aldrich (Germany). Cyclohexanol was purchasedfrom Nacalai Tesque Inc. (Japan). 2-Methoxyethyl acrylate (MEA),
2-hydroxyethyl acylate (HEA), 2-methoxyethyl methacrylate (MEMA),
2-hydroxyethyl methacrylate (HEMA), diethylene glycol diacrylate

5050 | Mater. Adv., 2022, 3, 5043–5054

Materials Advances(DEGDA), diethylene glycol dimethacrylate (DEGDMA), 2,20 azobis(isobutyronitrile) (AIBN), 2,2,2-tirfluoroethanol (TFE), andpolystyrene (PSt) resin (1% divinylbenzene, 100–200 mesh) werepurchased from Tokyo Chemical Industry Co., Ltd (Japan). PSt
resin was purified by Soxhlet extraction with methanol for 3 days
prior to use.
Measurements
The FTIR spectra were acquired by an FT/IR-6600 system(JASCO Co., Japan) using a deuterated l-alanine triglycine
sulfate detector fitted with an attenuated total reflection(ATR) accessory that used a germanium internal reflection
element (resolution: 4 cm1, 16 scans). The XPS analyses werecarried out with an ULVAC-PHI APEX ESCA system (ULVAC-PHI
inc., Japan) at 2.0  107 Pa using a monochromatic Al-Ka X-ray
source at 150 W. The SEM images were obtained using a RealSurface View VE-7800 system (KEYENCE Co., Japan). The optical microscope images were collected with a VHX-900F system(KEYENCE Co., Japan).
Preparation of poly(2-methoxyethyl acrylate) (PMEA)
microparticles
MEA (25 g, 0.19 mol), DEGDA (0.42 g, 1 mol% or 0.84 g,
2 mol%), and AIBN (0.10 g, 0.61 mmol) were mixed. Calcium
chloride dihydrate (80 g, 0.54 mol) was dissolved in water(200 mL), and calcium carbonate (10 g, 0.10 mol) was added
as a dispersion stabilizer. These solutions were combined andheated to 60 1C with stirring at 300 rpm using a propelleragitator under an argon atmosphere. After 30 min, the suspension solution was allowed to react for 1 h at 85 1C. Hydrochloric
acid (12 M, 20 mL) was slowly added to the reaction mixture todecompose calcium carbonate into calcium chloride and carbon dioxide. The resultant PMEA microparticles were purified
by Soxhlet extraction with methanol over 2 d. The purified
PMEA microparticles were sorted into two size-based groups by
wet-type classification using a sieve (mesh opening: 300 mm)
with methanol as a solvent, and then lyophilized after replacement with water to give the pure PMEA microparticles. The
chemical structures of the particles were evaluated by FTIR and
XPS analyses.
[Crosslinking ratio = 1%] yield: 16.4 g (64.5%); 11.2 g(smaller-size group), 5.2 g (larger-size group).
[Crosslinking ratio 2%] yield: 19.1 g (73.9%); 15.6 g (smallersize group), 3.5 g (larger-size group).
Preparation of poly(oligo(ethylene glycol)methyl ether acrylate)(POEGA) microparticles
OEGA (25 g, 0.052 mol), DEGDA (0.11 g, 1 mol%), and AIBN
(0.10 g, 0.61 mmol) were mixed with cyclohexanol (2.5 mL).
Calcium chloride dihydrate (190 g, 1.09 mol) was dissolved inwater (110 mL), and then calcium carbonate (10 g, 0.10 mol)
was added as a dispersion stabilizer. These solutions werecombined and heated to 60 1C with stirring at 300 rpm using
a propeller agitator under an argon atmosphere. After 30 min,the suspension solution was allowed to react for 2 h at 85 1C.
Hydrochloric acid (12 M, 20 mL) was slowly added to the© 2022 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Materials Advances
reaction mixture to decompose calcium carbonate into calcium
chloride and carbon dioxide. The resultant POEGA microparticles were purified by Soxhlet extraction with methanol for 2 d.
The purified POEGA microparticles were sorted into two sizebased groups by wet-type classification using a sieve (meshopening: 300 mm) with methanol as a solvent, and thenlyophilized after replacement with water to give the pure
POEGA microspheres. The chemical structures of the particles
were evaluated by FTIR and XPS analyses.
Yield: 12.6 g (50.2%); 4.3 g (smaller-size group), 8.3 g (largersize group).
Preparation of poly(2-hydroxyethyl acrylate) (PHEA)
microparticles
HEA (25 g, 0.22 mol), DEGDA (0.52 g, 1 mol%), and V-70 (0.20 g,
0.65 mmol) were mixed with TFE (10 mL). Calcium chloride
dihydrate (150 g, 1.02 mol) was dissolved in water (150 mL), andthen calcium carbonate (10 g, 0.10 mol) was added as adispersion stabilizer. These solutions were combined andheated to 40 1C with stirring at 300 rpm using a propelleragitator under an argon atmosphere. After 30 min, the suspension solution was allowed to react for 1 h at 60 1C. Hydrochloric
acid (12 M, 20 mL) was slowly added to the reaction mixture
to decompose calcium carbonate into calcium chloride andcarbon dioxide. The resultant PHEA microparticles were purified by Soxhlet extraction with methanol for 2 d. The purified
PHEA microparticles were sorted into two size-based groups by
wet-type classification using a sieve (mesh opening: 300 mm)
with methanol as a solvent, and then lyophilized after replacement with water to give the pure PHEA microspheres. The
chemical structures of the particles were evaluated by FTIR and
XPS analyses.
Yield: 15.6 g (61.1%); 13.2 g (smaller-size group), 2.4 g(larger-size group).
Preparation of poly(2-methoxyethyl methacrylate) (PMEMA)
microparticles
MEMA (25 g, 0.17 mol), DEGDMA (0.42 g, 1 mol%), and AIBN
(0.10 g, 0.61 mmol) were mixed. Calcium chloride dihydrate(80 g, 0.54 mol) was dissolved in water (220 mL), and thencalcium carbonate (10 g, 0.10 mol) was added as a dispersion
stabilizer. These solutions were combined and heated to 60 1C
with stirring at 450 rpm using a propeller agitator under anargon atmosphere. After 30 min, the suspension solution wasallowed to react for 1 h at 85 1C. Hydrochloric acid (12 M,
20 mL) was slowly added to the reaction mixture to decompose
calcium carbonate into calcium chloride and carbon dioxide.
The resultant PMEMA microparticles were purified by Soxhlet
extraction with methanol for 2 d. The purified PMEMA microparticles were sorted into two size-based groups by wet-typeclassification using a sieve (mesh opening: 300 mm) withmethanol as a solvent, and then lyophilized after replacementwith water to give the pure PMEMA microspheres. The
chemical structures of the particles were evaluated by FTIR
and XPS analyses.

© 2022 The Author(s). Published by the Royal Society of Chemistry

Paper
Yield: 46.4 g (61.1%); 10.2 g (smaller-size group), 1.6 g(larger-size group).
Preparation of poly(2-hydroxyethyl methacrylate) (PHEMA)
microparticles
HEMA (25 g, 0.19 mol), DEGDMA (0.47 g, 1 mol%), and AIBN
(0.10 g, 0.61 mmol) were mixed. Calcium chloride dihydrate(190 g, 0.109 mol) was dissolved in water (220 mL), and thencalcium carbonate (10 g, 0.10 mol) was added as a dispersion
stabilizer. These solutions were combined and heated to 60 1C
with stirring at 300 rpm using a propeller agitator under anargon atmosphere. After 30 min, the suspension solution wasallowed to react for 3 h at 85 1C. Hydrochloric acid (12 M,
20 mL) was slowly added to the reaction mixture to decompose
calcium carbonate into calcium chloride and carbon dioxide.
The resultant PHEMA microparticles were purified by Soxhlet
extraction with methanol for 2 d. The purified PHEMA microparticles were sorted into two size-based groups by wet-typeclassification using a sieve (mesh opening: 300 mm) withmethanol as a solvent, and then lyophilized after replacementwith water to give the pure PHEMA microspheres. The chemical
structures of the particles were evaluated by FTIR and XPS
analyses.
Yield: 13.5 g (53.0%; smaller-size group only).
Calculation of the surface areas, volumes, swelling ratios, andwater contents of the polymer microparticles
The surface areas and volumes of the polymer microparticles
under wet conditions (per particle) are given by the following
formulae:
Swet = 4prwet2(1)

Vwet = 4/3  prwet3

(2)

and

1

2

where Swet (cm particles ) is the surface area of the polymer
microparticles per particle under wet conditions, Vwet (cm3particles1) is the volume of polymer microparticles per particle
under wet conditions, and rwet (cm) is the radius of the polymer
microparticles estimated from the optical microscope images.
Assuming that all the densities are the same (i.e., 1 g cm3), therelationship between the volume and weight is given by thefollowing formulae:
Vdry = Wdry

(3)

Vwet = Wwet

(4)

and

where Vdry (cm3 particles1) is the volume of the microparticles
per particle under dry conditions; Wdry and Wwet (g particles1)
are the weights of the microparticles per particle under dry andwet conditions, respectively. From eqn (1)–(3), the surface area
per particle can be translated to the surface area per gram.
0

Swet ¼ Swet =Vwet ¼ Swet =Wwet(5)

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0

where Swet (cm2 g1) is the surface area of the microparticles
per gram under wet conditions. In addition, the swelling ratioof the microparticles is expressed below, derived from eqn (3)and (4).
Vwet/Vdry  100%

(6)

As mentioned above, if the volume is equal to the weight, theamount of water in the microparticles is determined as follows:
Vwet  Vdry = Vwater = Wwater(7)

where Vwater (cm2 particles1) is the volume of water in the
microparticles, and Wwater (g particles1) is the weight of thewater in the microparticles. Therefore, the water content of the
microparticles (wt%) can be defined by the following equation:
Vwater/Vwet  100% = Wwater/Vwet  100%(8)

Hydration state analysis for the polymer microparticles using
DSC measurements
The hydration state in the hydrated polymer microparticles
under wet conditions (in PBS()) was analyzed by diﬀerential
scanning calorimetry (DSC) (X-DSC7000, Seiko Instruments,
Japan) with reference to a previously reported method.35 Drypolymer samples were prepared by storing them under vacuum
conditions for more than 7 d. Saturated hydration polymer
samples were prepared by immersing the dry samples inPBS() for more than 3 d. The samples were cooled to100 1C at a rate of 5 1C min1, held at 100 1C for 5 min,and then heated to 50 1C at the same rate under a nitrogenpurge flow. The amount of IW per gram of the polymer
microparticles (WIW (mg g1)) was calculated using the following
equation:
WIW = (DHcc/DHfus)/Wpolymer  1000,ULTRA TAYOTO SU Premium SOFT, Konishi Co., Ltd) was
placed on the edge of the cantilever. The glue-treated tip-lesscantilever interacted with the polymer microparticles, whichwere spread on a glass slide with cellophane tape. The cantilever approached one microparticle and the particle waspressed for 10 min. The microparticle-attached cantilevers weredried for 1 d at ambient pressure and room temperature toobtain colloidal cantilevers.

(9)

where DHcc (J) is the enthalpy change during the cold crystallization of water estimated from the DCS thermogram, andDHfus (J g1) is the heat of fusion of ice (334 J g1).
Platelet adhesion assay
The polymer microparticles (50 mg) were incubated with PBS
for 1 h at 37 1C. Platelets derived from human whole blood(Tennessee Blood Services, USA; density = 4  107 cells)
were treated with the polymer microparticles for 1 h at 37 1C.
The solution was then centrifuged at 1000 rpm for 1 min. The
supernatant was removed and the microparticles were washedtwice with PBS. The platelet-adhered microparticles were treated with an LDH assay kit (Dojindo, Kumamoto, Japan). After
1 h of incubation, the absorbance of the cells was measured at
490 nm using an Infinite 200PRO M Plex microplate reader(Tecan, Zürich, Switzerland).

Analysis of the interaction strength between the polymer
microparticles and cells
The colloidal cantilevers were incubated with DMEM (Wako) containing 10% FBS for 24 h at 37 1C. HeLa cells (1.0  104 cells cm2)(RIKEN BRC, Japan) were seeded on a 35 mm dish andincubated for 24 h. The force curves between the polymer
microparticles and cells were recorded using an AFM system(CellHesion 200, JPK) equipped with a colloidal cantilever (setpoint = 3 nN, approach rate = 2.0 mm s1, holding time = 1 s,
retraction rate = 5 mm s1). The interaction force was defined as
the maximum force for the detachment of the colloidal cantilever from the cell, corresponding to the force at the minimum
point of the retraction curve. The interaction energy wasestimated as the amount of work required to detach the cells
from the substrate, corresponding to the area enclosed by thebaseline and retraction curve.
Capture of tumor cells from cell suspensions using the polymer
microparticles
The polymer microparticles (50 mg) were incubated with PBS
for 1 h at 37 1C. The glass bottom area of the cell culture
container (IWAKI) was coated with poly(2-methacryloyoxyethyl
phosphorylcholine-co-n-butyl methacrylate) (PMPC; 30 : 70 mol%,
Lipidure-CM5206, NOF Corporation, Tokyo, Japan). HeLa
(5.0  104 cells) or MDA-MB-231 (ATCC) cells (2.0  104 cells)
were treated with the polymer microparticles (50 mg) in thecontainer. After 24 h of incubation under shaking conditions(10 rpm), the polymer microparticles were washed with PBS
twice and treated with the Cell Courting kit-8 (Dojindo). The
absorbance of the cells was measured at 560 nm using an
Infinite 200PRO M Plex microplate reader (Tecan, Zürich,
Switzerland). The recovery rate of tumor cells was calculated
from the ratio of the number of seeded tumor cells to thenumber of polymer microparticles. HeLa cells were stainedwith Tracker Green (Thermo Fisher Scientific).

Author contributions

A tip-less cantilever (TL-CONT, spring constant = 0.2 N m1,
NANOSENSORS) was equipped on an atomic force microscope(CellHesion 200, Bruker). A small amount of glue (BOND

S. N. and K. N. conceived the research and designed theexperiments. S. N., S. S., and K. N. performed the experiments.
M. T. and K. N., and S. N. obtained founding for the project.
M. T. and K. N. oversaw the research. S. N., S. S., K. N., and
M. T. co-wrote the manuscript. All authors discussed the results
and commented on the manuscript.

5052 | Mater. Adv., 2022, 3, 5043–5054© 2022 The Author(s). Published by the Royal Society of Chemistry

Fabrication of the colloidal cantileverView Article Online

Materials Advances

Conflicts of interest

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Mater. Adv., 2022, 3, 5043–5054 | 5053

There are no conflicts to declare.
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Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research(KAKENHI) (JP19H05720 to M. T., JP20J00282 and JP20K20201 to
S. N., JP19J00686 and 21K18066 to K. N.) from the Japan Society
for the Promotion of Science (JSPS), ‘‘Dynamic Alliance Open
Innovation Bridging Human, Environment and Materials’’ from
the Ministry of Education, Culture, Sports, and Technology ofJapan (MEXT). This research was also supported by Grant-in-Aid
for the Nakayama Foundation for Human Science (to S. N.), theJapan Prize Foundation (to S. N.) and Fukuoka Public Health
Promotion Organization Cancer Research Fund (to K. N.).

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5054 | Mater. Adv., 2022, 3, 5043–5054© 2022 The Author(s). Published by the Royal Society of Chemistry