RAFT of sulfobetaine for modifying poly(glycidyl methacrylate) microspheres to reduce nonspecific protein adsorption

Article abstract of DOI:10.1002/pola.27681

The minimization of nonspecific protein adsorption is a crucial step in the development of bioseparation processes, immunoassays, and affinity diagnostics. Among the numerous biomaterials, polyzwitterions are known to effectively suppress protein and cell adhesion. This article describes the formation of monodisperse polymer microspheres coated with polysulfobetaine with the aim to limit nonspecific adsorption of bovine serum albumin (BSA) as a model protein. In this process, 2-μm poly(glycidyl methacrylate) (PGMA) microspheres were prepared by dispersion polymerization. To render the microspheres hydrophilic and biocompatible, [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide (MPDSAH) was grafted from the surface by reversible addition-fragmentation chain transfer (RAFT) polymerization. Elemental analysis of the modified microspheres revealed up to 20 wt % of poly{[3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonimum hydroxide} (PMPDSAH). The microspheres were characterized in terms of particle size, morphology, and zeta potential. The amount of BSA nonspecifically adsorbed on the PMPDSAH-modified microspheres decreased to half of that captured on the unmodified PGMA microspheres.

Full text of DOI:10.1002/pola.27681

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RAFT of Sulfobetaine for Modifying Poly(glycidyl methacrylate)
Microspheres to Reduce Nonspecific Protein Adsorption
1,2
1
1
1
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Jana Koubkova, Hana Mackova, Vladimır Proks, Miroslava Trchova,
1
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Jirı Brus, Daniel Horak
1
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Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Sq. 2, 162 06 Prague 6,
Czech Republic
2Institute of Chemistry and Technology of Macromolecular Materials, Faculty of Chemical Technology, University of Pardubice,
ꢀ
Studentska 95, 532 10 Pardubice, Czech Republic
ꢀ
Correspondence to: D. Horak (E-mail: horak@imc.cas.cz)
Received 3 March 2015; accepted 12 April 2015; published online 00 Month 2015
DOI: 10.1002/pola.27681
ABSTRACT: The minimization of nonspecific protein adsorption
is a crucial step in the development of bioseparation proc-
esses, immunoassays, and affinity diagnostics. Among the
numerous biomaterials, polyzwitterions are known to effec-
tively suppress protein and cell adhesion. This article describes
the formation of monodisperse polymer microspheres coated
with polysulfobetaine with the aim to limit nonspecific adsorp-
tion of bovine serum albumin (BSA) as a model protein. In this
process, 2-lm poly(glycidyl methacrylate) (PGMA) micro-
spheres were prepared by dispersion polymerization. To render
the microspheres hydrophilic and biocompatible, [3-(methacry-
loylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide
(MPDSAH) was grafted from the surface by reversible addition-
fragmentation chain transfer (RAFT) polymerization. Elemental
analysis of the modified microspheres revealed up to 20 wt %
of poly{[3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)-
ammonimum hydroxide} (PMPDSAH). The microspheres were
characterized in terms of particle size, morphology, and zeta
potential. The amount of BSA nonspecifically adsorbed on the
PMPDSAH-modified microspheres decreased to half of that cap-
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tured on the unmodified PGMA microspheres. 2015 Wiley Peri-
odicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015, 00, 000–000
KEYWORDS: adsorption; grafting; hydrophilic polymers; micro-
spheres; polysulfobetaine; reversible addition fragmentation
chain transfer (RAFT); synthesis
persion polymerization is realized with functional monomers,
such as glycidyl methacrylate (GMA)15,17 or 2-(acetoacetoxy)
ethyl methacrylate.18
INTRODUCTION Polymer microspheres became popular in
biological and medical fields in the 1970s, when polystyrene
particles were used as reagents in agglutination tests.1 Since
that time, polymer microspheres have been widely employed
in protein and nucleic acid separations,2–5 immunoassays
and diagnosis,6,7 for the immobilization of polysaccharides8
and enzymes,9,10 and in cell labeling11 and embolization.12,13
Depending on the application, polymer microspheres were
typically 0.1–100 mm in size and contained various func-
tional groups such as aldehyde, carboxyl, hydroxyl, amino,
and oxirane groups.2,7,14,15 Emulsion polymerization was
often used to prepare the polymer particles; however, mono-
disperse microspheres of desirable size and surface proper-
ties can be produced with advantage by dispersion
polymerization, which is a one-step technique that does not
require the emulsification of monomers in the reaction
medium.15,16 On the contrary, the monomer, stabilizer, and
initiator are dissolved in the solvent, from which the poly-
mer precipitates and forms nuclei which continue to grow
into the mature polymer particles.16 Conveniently, the dis-
The most important requirement laid on the polymer par-
ticles in bioapplications is the resistance to nonspecific pro-
tein adsorption and cell adhesion. The uncontrolled
adsorption of proteins may lead to false negative or positive
results and influence the accuracy of the target biomolecule
determination. Minimum nonspecific protein adsorption was
observed on hydrophilic materials.19,20 Such surfaces were
obtained by modification with both natural (e.g., dextran,21
gelatin,22 hyaluronic acid, and alginic acid23) and synthetic
polymers, such as poly(2-hydroxyethyl methacrylate),24,25
poly(N-isopropylacrylamide),24,26 poly[N-(2-hydroxypropyl)-
methacrylamide],27 polyvinylpyrrolidone,28 and poly(ethylene
glycol) (PEG).20 Although the modification with PEG is
widely employed to introduce hydrophilicity to various
surfaces,29,30 the low oxygen and thermal stability of PEG
has prompted the development of other antifouling materi-
als. These materials include zwitterionic compounds, such as
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polyphospho-, polysulfo-, and polycarboxybetaines29–31 that
possess both anionic and cationic groups and support the
formation of a hydration layer, thus hindering nonspecific
protein adsorption.32
trile) (AIBN; Fluka, Buchs, Switzerland) was twice recrystal-
lized from ethanol. As a stabilizer, poly(acrylic acid) (PAA;
Mn 5 50,000 g/mol; Polysciences; Eppelheim, Germany) was
used. Ethanol for UV/Vis spectroscopy (EtOH; 99.98%), ethyl
acetate (EtAc), methanol (MeOH), sulfuric and hydrochloric
acid, ammonium hydroxide, and magnesium sulfate were from
Lach-Ner (Neratovice, Czech Republic). N,N-diisopropylethyl-
amine (DIPEA; 99.5%), a-bromoisobutyryl bromide (BIBB),
1 M phenylmagnesium bromide in tetrahydrofuran (THF), car-
bon disulfide, anhydrous THF, 4,40-azobis(4-cyanopentanoic
acid) (ACVA), and bovine serum albumin fraction V (BSA;
Mw 5 68,000 g/mol) were purchased from Sigma-Aldrich
(Steinheim, Germany). [3-(Methacryloylamino)propyl] dimethyl
(3-sulfopropyl)ammonium hydroxide (MPDSAH) zwitterion
was synthesized from N-[3-(dimethylamino)propyl] methacry-
lamide and 1,3-propanesultone (both from Sigma-Aldrich) in
diethyl ether (Lach-Ner). Acetic buffer and phosphate buffered
saline (PBS) were freshly prepared. Ultrapure Q-water, ultrafil-
tered on a Milli-Q Gradient A10 system (Millipore; Molsheim,
France), was used in the biological experiments.
The effectiveness of antifouling resistance depends on the
thickness of the hydrophilic layer, its molecular structure
and weight and the quality of the surface coverage.33,34 Well-
defined polymer layers have been prepared by surface-
initiated controlled radical reactions, including nitroxide-
mediated polymerization (NMP),35 atom transfer radical
polymerization (ATRP)36,37 and its photo-catalyzed metal-
free alternative,38 or reversible addition-fragmentation chain
transfer (RAFT).39,40 The advantage of the RAFT polymeriza-
tion compared with the other techniques is its versatility,
compatibility with a wide range of monomers and reaction
conditions and absence of metal residues.41,42 The polymers
of controlled molecular weight and narrow molecular weight
distributions were obtained by polymerization in the pre-
sence of thiocarbonyl-thio compounds that acted as an effi-
cient chain transfer agent (CTA).41 The principle of the RAFT
technique is based on the rapid exchange between active
and dormant (thiocarbonyl-thio capped) species, which leads
to equal probabilities for all polymer chains to grow. In gen-
eral, grafting from a substrate can be carried out using (i)
surface-anchored initiator or (ii) surface-anchored CTA.24,42
Attachment of the initiator to the solid surface requires
aggressive reaction conditions, such as strong solvents and
catalysts.43 Grafting from the surface-anchored CTA can be
accomplished using either the Z- or R-group approaches.24,42
In the Z-group technique, CTA is attached to the surface by
its stabilizing group, and the polymer radicals propagate in
the solution before they add back to the surface. This mecha-
nism resembles the “grafting to” approach and thus suffers
from the same drawbacks, such as a relatively low grafting
density caused by the steric hindrance of growing polymer
chains.24,42,44 Conversely, in the R-group approach, CTA is
attached to the surface by its leaving (reinitiating) group,
and the radicals propagate at the terminus of the surface-
grafted polymer. This mechanism resembles the “grafting
from” approach and produces a high grafting density and
complete surface coverage.24,42,45
Preparation of PGMA Microspheres
GMA was polymerized in the presence of PAA in EtOH accord-
ing to a previous report with some modifications.15 Briefly,
PAA (0.32 g; Mw 5 50,000) was dissolved in 50 g of EtOH and
transferred into a 70-mL reaction vessel, and a solution of
AIBN (0.176 g) in GMA (7.57 g) was added. The polymeriza-
tion was started by heating to 70 8C under nitrogen atmos-
phere and continued for 16 h with stirring (400 rpm) using
an anchor-type stirrer. The final microspheres were washed
10 times with water. Oxirane groups on the microspheres
were hydrolyzed in 0.1 M aqueous H2SO4 at 55 8C for 5 h
with stirring (120 rpm) to yield poly(2,3-dihydroxypropyl
methacrylate) (PDHPMA) microspheres. Alternatively, the oxi-
rane groups were ammonolyzed in 26% NH4OH at 20 8C for
24 h with stirring, which resulted in poly(2-hydroxy-3-amino-
propyl methacrylate) (PHAPMA) particles. Both PDHPMA and
PHAPMA particles were washed 10 times with water and
vacuum-dried prior to use.
Activation of PDHPMA and PHAPMA Microspheres with
BIBB
The PDHPMA or PHAPMA microspheres (1 g each) were
activated at several BIBB/OH or BIBB/NH2 molar ratios. Tak-
ing the BIBB/OH 5 0.13 mol/mol as an example, 0.628 g of
BIBB (2.73 mmol) and 0.353 g of DIPEA (2.73 mmol) were
added dropwise to the dried particles dispersed in 30 mL of
EtAc at 0 8C. The reaction proceeded at 0 8C for 4 h and
then at room temperature for 20 h under mild shaking
(100 rpm). Both BIBB-activated PDHPMA and PHAPMA
microspheres (denoted as PDHPMA-BIBB and PHAPMA-
BIBB) were washed 10 times with EtAc. The amount of
attached BIBB was calculated from bromine analysis.
The aim of this study was to synthesize zwitterionic poly{[3-
(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammo-
nium hydroxide} (PMPDSAH) brushes from the surface of
poly(glycidyl methacrylate) (PGMA) microspheres via RAFT
polymerization in the presence of dithiobenzoic acid (DTBA)
as the CTA. This zwitterion was selected because of its highly
protein-repellent properties and minimal hydrolysis in aque-
ous media even at high temperatures due to the presence of
hydrolytically stable (meth)acrylamide moieties.46
EXPERIMENTAL
Synthesis of Dithiobenzoic Acid
Materials
Dithiobenzoic acid (DTBA), used as the CTA, was synthesized
according to an earlier report.47 A solution of 1 M phenyl-
magnesium bromide (7.2 g; 39.7 mmol) was added dropwise
€
Glycidyl methacrylate (GMA; Rohm, Darmstadt, Germany) was
vacuum-distilled prior to use. 2,20-Azobis(2-methylpropioni-
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to a solution of CS2 (4.75 mL; 78.8 mmol) in 15 mL THF
over the course of 1 h at 0 8C under nitrogen atmosphere.
The solution was left at room temperature for 16 h before it
was slowly acidified with 10 wt % HCl (70 mL). Water was
removed, the oil phase was dried over MgSO4, which was fil-
tered off, and the solvent was evaporated. Dark purple DTBA
was obtained in 37% yield. The structure of DTBA was con-
firmed by 1H NMR (300 MHz, CDCl3, d, ppm): 7.2–7.9
(5H, Ar H).
and washed once with water and five times with PBS
(0.15 M; pH 7.3). The amount of the grafted PMPDSAH was
calculated from an analysis of nitrogen content.
Protein Sorption on PMPDSAH-Modified Microspheres
To verify the antifouling properties of the PDHPMA-
PMPDSAH microspheres, the adsorption of the BSA was
tested as a model. A solution of BSA (1.8 mL; 2 mg/mL) was
added to the PDHPMA-PMPDSAH particles (18 mg) in PBS
(1.8 mL) and the mixture was kept at 23 8C for 2 h under
shaking. The microspheres were separated by centrifugation
and the supernatant was removed. The BSA concentration in
the supernatant was determined by UV/Vis spectroscopy at
278 nm, and the amount of BSA adsorbed on the micro-
spheres was calculated.50
Attachment of CTA to BIBB-Activated and PGMA
Microspheres
DTBA was bound to PDHPMA-BIBB and PHAPMA-BIBB par-
ticles at DTBA/Br 5 1/1 mol/mol. As an example, 0.094 g of
DTBA (0.61 mmol) and DIPEA (0.079 g; 0.61 mmol) were
added at 0 8C to the suspension of wet PDHPMA-BIBB par-
ticles (1 g; 0.61 mmol Br) in EtAc (20 mL). In the experi-
ments with the PGMA microspheres, the DTBA/oxirane ratio
varied from 0.2 to 2.2 mol/mol. Typically, 0.421 g of DTBA
(2.74 mmol) was added to 1 g of dry PGMA dispersed in
0.05 wt % Tween 20 in EtAc (20 mL) at 0 8C. All reactions
proceeded at 0 8C for 4 h and at room temperature for 7
days under mild shaking (100 rpm). Microspheres with the
bound DTBA were washed 10 times with EtAc and stored at
4 8C. Prior to the RAFT polymerization, the microspheres
were transferred in water or acetic buffer (0.27 mol/L acetic
acid and 0.73 mol/L sodium acetate; pH 5.2) via MeOH or
EtOH. Henceforth in the text, the microspheres are denoted
as PDHPMA-DTBA, PHAPMA-DTBA, and PGMA-DTBA. The
amount of the DTBA was determined from an analysis of sul-
fur content.
Characterization Methods
The oxirane group content was determined from the peak
area at 910 cm21 in the Fourier transform infrared (FT-IR)
spectrum17 recorded using KBr pellets and a Perkin–Elmer
Paragon 1000PC spectrometer (Waltham, MA). The surface
of the microspheres was analyzed in attenuated total reflec-
tion (ATR) mode using a Thermo Nicolet NEXUS 870 spec-
trometer (Madison, WI) equipped with a MKII Golden
GateTM heated diamond top-plate single reflection system
(Specac; Orprington, UK). CHN analysis was performed on a
Perkin–Elmer 2400 CHN apparatus (Waltham, MA). The
bromine and sulfur contents were determined by titration
with mercury and barium perchlorates. 1H NMR spectra
were recorded on a Bruker DPX 300 spectrometer (Biller-
ica, MA). 13C cross-polarization (CP) MAS NMR spectra
were measured on a Bruker Avance 500 WB/US NMR
(Karlsruhe, Germany; B0 5 11.7 T) in 4-mm ZrO2 rotors at
MAS frequency of 11 kHz using a B1(13C) field nutation fre-
quency of 62.5 kHz, a contact time of 1 ms, and a recycle
delay of 5 s.
Synthesis of Monomer
The MPDSAH zwitterion was synthesized according to an
earlier report.48 N-[3-(dimethylamino)propyl]methacrylamide
(9.02 g; 53 mmol) was dissolved in diethyl ether (60 mL),
and 1,3-propanesultone (6.45 g; 53 mmol) was added. The
solution was left at room temperature for 16 h. Precipitated
monomer was filtered off, washed with acetone and vacuum-
dried (39% yield). The structure of the MPDSAH was con-
firmed by 1H NMR (300 MHz, D2O, d, ppm): 5.71–5.47 (2H,
C@CH2), 3.50–3.44 (2H, N(CH3)2ACH2ACH2ACH2ASO3), 3.35
(4H, NHACH2ACH2ACH2AN(CH3)2), 3.11 (6H, N(CH3)2), 2.97
(2H, CH2ASO3), 2.20 (2H, N(CH3)2CH2ACH2ACH2ASO3), 2.05
(2H, NHACH2ACH2ACH2AN(CH3)2), and 1.93 (3H, C(CH2)
ACH3), which was in agreement with the literature.49
The morphology of initial and surface-modified microspheres
in a dried state was observed with a scanning electron
microscope JEOL JFM 6400 (SEM; Tokyo, Japan). The
P
number-average diameter Dn (Dn 5 Di/N; where N and Di
represent the number and diameter of the microspheres,
P
respectively) and weight-average diameter Dw (Dw5 D4i =
P
D3i ) were calculated using Atlas software (Tescan Digital
Microscopy; Brno, Czech Republic). Particle size distribution
was characterized using the polydispersity index (PDI) and
calculated as a ratio of the weight-average diameter to
number-average diameter. For the determination of particle
size and distribution, at least 800 microspheres were measu-
red. Polymerization kinetics were monitored using a size
exclusion chromatograph (SEC) with a PDA 2800 (Knauer;
Berlin, Germany) and an Alltech 3300 ELSD evaporative
detector (Bannockburn, IL) equipped with Phenomenex
polySep-GFC-P 2000 column with 80% 0.05 M ammonia ace-
tate buffer (pH 6.5)/20% acetonitrile mixture as a mobile
phase. The zeta (f) potential of the particles was measured
on a ZEN3600 Zetasizer Nano-ZS (Malvern Instruments; Mal-
vern, UK). The concentration of BSA in the solution was
Surface-Initiated Polymerization from DTBA-Modified
Microspheres
Wet PDHPMA-DTBA microspheres (200 mg) dispersed in
4 mL of water (optionally acetic buffer, MeOH or EtOH),
MPDSAH (e.g., 0.4 g; 1.37 mmol) and ACVA (e.g., 1 mg; 3.6
mmol) were loaded into a Schlenk flask equipped with a
magnetic stirrer. The reaction mixture was degassed, purged
with argon, and the polymerization was started by heating
to 60 8C (MeOH) or 70 8C (water, acetic buffer, EtOH) and
continued for 20 h with stirring (400 rpm). The resulting
particles (denoted as PDHPMA-PMPDSAH) were separated
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FIGURE 1 SEM micrographs of PGMA (a), PDHPMA (b), PDHPMA-BIBB (c), and PHAPMA-BIBB microspheres (d).
determined using a Specord 250 Plus UV/Vis spectropho-
tometer (Analytic Jena; Jena, Germany).
oxiranes reducing thus the theoretical amount of the amino
groups. Successful hydrolysis and ammonolysis of oxirane
rings was confirmed by the decrease of the characteristic
peak at 907 cm21 and the broadening of the band at
3440 cm21. Both PDHPMA and PHAPMA microspheres pre-
served their regular spherical shape, size and narrow size
distribution [2.2 mm, PDI 5 1.02; Fig. 1(b)].
RESULTS AND DISCUSSION
Monodisperse (PDI 5 1.02) PGMA microspheres 2.2 mm in
size were prepared by the dispersion polymerization of GMA
in EtOH with PAA as a stabilizer. A SEM micrograph of the
particles is shown in Figure 1(a). The regular spherical
shape and monodispersity of the microspheres ensures uni-
form chemical, physical and biological properties. Stabiliza-
tion of the PGMA microspheres with PAA is known to
prevent particle aggregation and to preserve high content of
reactive groups.15 Their concentration, as determined from
FT-IR, reached 5.5 mmol oxirane groups/g.
Activation of PDHPMA and PHAPMA Microspheres
with BIBB
Attachment of the RAFT agent to the particle surface was
investigated using three different approaches: (i) direct reac-
tion of CTA with oxirane groups of PGMA microspheres
[Scheme 1(a)] similarly like in thiol-epoxy chemistry51 and
binding of DTBA to (ii) PDHPMA, or (iii) PHAPMA micro-
spheres via ATRP initiator [Scheme 1(b,c)]. PDHPMA micro-
spheres were activated with BIBB at several BIBB/OH molar
ratios (0.01–1.1), forming an ester bond between the hydroxyl
groups of PDHPMA and acyl groups of BIBB. The reaction was
quantitative and the microspheres contained 3–67 wt % BIBB
(Fig. 2). The size of the PDHPMA-BIBB microspheres with low
BIBB content remained the same as that of the original par-
ticles (2.2 mm), but slightly increased to 2.3 mm at BIBB con-
tents greater than 60 wt %. Nevertheless, the morphology
In the next step, the PGMA microspheres were hydrolyzed in
H2SO4, yielding the PDHPMA particles containing approxi-
mately 10 mmol hydroxyl groups/g, or ammonolyzed in
NH4OH providing the PHAPMA particles with 1.9 mmol
amino groups/g. Whereas the concentration of the hydroxyl
groups was determined from an analysis of carbon and
hydrogen content, the amount of the amino groups was cal-
culated from a nitrogen content analysis. It should be noted
that the ammonolysis was accompanied by the hydrolysis of
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SCHEME 1 Attachment of DTBA to PGMA microspheres by opening of oxirane groups (a) and via hydroxyl (b) and amino groups
(c). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and narrow particle size distribution (PDI 5 1.02) were pre-
served [Fig. 1(c)].
groups was significantly lower than that of hydroxyl groups
in PDHPMA-BIBB microspheres. At 0.2–3.7 BIBB/NH2 molar
ratios, a rather low amount of BIBB (5–16 wt %) was bound
to the PHAPMA microspheres (Fig. 2), which could be caused
by a reduced concentration of the amino groups on the
PHAPMA microspheres (1.9 mmol/g). Amino groups reacted
with BIBB preferentially and sterically hindered the addi-
tional reaction of BIBB with hydroxyl groups. Consequently,
maximum BIBB content was reached at BIBB/NH2 approxi-
mately 1 mol/mol. The shape, size, and monodispersity of
the PHAPMA-BIBB microspheres were not changed by the
reaction [Fig. 1(d)].
The PHAPMA microspheres were activated with BIBB
through a similar process as the PDHPMA microspheres.
However, substitution of the acyl bromides with the amino
Attachment of CTA to Initial PGMA and BIBB-Activated
Microspheres
Of the various reported CTAs, DTBA is considered to be a
suitable CTA for the polymerization of methacrylamides.42
It can be obtained by several methods, among which the
synthesis with Grignard reagents and CS2 is convenient due
to the commercial availability of both reagents.47,52
Moreover, the synthesis is easy and provides DTBA of rea-
sonable purity.
PGMA-DTBA Microspheres
DTBA was bound to the PGMA microspheres by direct open-
ing of oxirane rings [Scheme 1(a)] at several DTBA/oxirane
ratios (0.2–2.2 mol/mol). With an increasing concentration
of DTBA in the reaction mixture, the amount of CTA attached
to the PGMA-DTBA microspheres increased from 0.05 to 4.7
wt % [Fig. 3(a)]. However, unlike the PDHPMA and PHAPMA
microspheres, the PGMA particles were not sufficiently stable
in EtAc and aggregated [Fig. 4(a)].
FIGURE 2 Dependence of BIBB amount attached to PDHPMA-
BIBB and PHAPMA-BIBB microspheres on BIBB/OH and BIBB/
NH2 ratio, respectively.
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FIGURE 3 Dependence of DTBA amount attached to PGMA microspheres on DTBA/oxirane ratios (a) and dependence of DTBA
amount attached to PHDPMA-DTBA and PHAPMA-DTBA microspheres on Br content (b).
PDHPMA-DTBA and PHAPMA-DTBA Microspheres
was selected because the radical centers formed at the ter-
minus of the polymer chains allowed for growth from the
surface.42,45 After DTBA fragmentation,ꢀC(CH3)2COOR tertiary
radicals of the same structure as those derived from the
monomer were formed. The structural similarity should
ensure efficient chain transfer and control over the
polymerization.42
DTBA was reacted with both BIBB-activated microspheres at
an equimolar ratio relative to bromine [Scheme 1(b,c)]. The
amount of the DTBA attached to the PDHPMA-DTBA micro-
spheres linearly grew with increasing Br content and
reached 36 wt % [Fig. 3(b)]. Up to seven times as much
DTBA was found in the PDHPMA-DTBA microspheres com-
pared with the PGMA-DTBA microspheres. Such a large dif-
ference was likely to be caused by the higher reactivity of
the CABr bond in the PDHPMA-BIBB than that of oxiranes in
the PGMA microspheres. Conversely, the amount of DTBA
attached to the PHAPMA-BIBB microspheres was independ-
ent on the Br content and did not exceed 0.9 wt % [Fig.
3(b)]. This could be caused by relatively low BIBB concentra-
tion and the possible degradation of the DTBA by unreacted
primary amino groups of PHAPMA.42 Morphology, size, and
monodispersity of the PHAPMA-DTBA microspheres
remained unchanged [Fig. 4(b)]. These particles, however,
were not suitable for the RAFT polymerization because of
the low concentration of the surface-anchored DTBA. In con-
trast, the size of the PDHPMA-DTBA microspheres with low
(<17 wt %) and high DTBA content (36 wt %) increased
from 2.2 to 2.3 and up to 2.6 mm. Morphology of the
PDHPMA-DTBA microspheres was preserved, and the PDI
did not exceed 1.04 [Fig. 4(c)]. For further experiments, the
PDHPMA-DTBA microspheres with low DTBA content (<17
wt %; 1140 mmol/g) were chosen, in which the activation of
hydroxyl groups only on the particle surface was expected.
This prevented growth of the graft polymer from the bulk of
the particles, thus avoiding the coexistence of core PGMA
and graft PMPDSAH polymers in the boundary layer.53
Preliminary experiments with RAFT polymerizations of
MPDSAH from the PDHPMA-DTBA microspheres in water
and acetic buffer (pH 5.2) resulted in a rather low amount of
grafted PMPDSAH (1.2–1.9 and 0.6–0.9 wt %, respectively).
To improve CTA fragmentation and to enhance the amount
of grafted polymer,54 increasing volumes of polar organic sol-
vents (MeOH, EtOH, and N,N-dimethylformamide) were
added to the aqueous medium. However, only the polymeri-
zation reactions in pure MeOH and EtOH provided micro-
spheres grafted with significantly higher amounts of
PMPDSAH (2–14 wt %; Table 1). The successful grafting can
be ascribed to the poorer solubility of the PMPDSAH in
MeOH or EtOH compared with aqueous media.49,55 Polymer
chains thus remained at the particle surface and continued
to grow with a minimal tendency toward solution polymeri-
zation. Conversely, aqueous salt solution was considered a
thermodynamically good solvent for PMPDSAH, thus sup-
porting the polymerization and expansion of polymer coils in
the solution.56 This was in accordance with the time depend-
ence of ln([M]0/[M]) (Fig. 5), where [M]0 and [M] are the ini-
tial and instant monomer concentrations in the solution,
respectively. While in MeOH/water, the dependence was li-
near and MPDSAH consumption was fast; the dependence in
MeOH slightly differed from the linearity, and the polymeri-
zation was significantly retarded. Nevertheless, depending on
the polymerization time, 6–17 wt % PMPDSAH was attached
on the PDHPMA microspheres in MeOH, compared with
only 3 wt % PMPDSAH on the same microspheres in a
Surface-Initiated RAFT Polymerization from
PDHPMA-DTBA Microspheres
For the RAFT polymerization of MPDSAH, an analog of the R-
group approach (CTA attached via the fragmenting bond)
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chain transfer reactions.45,57 PDHPMA-DTBA microspheres
with both high (1140 mmol/g) and low DTBA concentrations
(134 mmol/g) were used in the surface-initiated RAFT poly-
merization. The PMPDSAH amount grew with increasing
monomer concentration, from 6.2 to 12 wt % on micro-
spheres with high DTBA content and up to 20 wt %
PMPDSAH on microspheres activated with a low DTBA
amount (Table 2). This result is in agreement with earlier
published results,57 where a high concentration of surface-
anchored CTA induced degenerative chain transfer between
the radicals localized on the surface instead of propagation
in the reaction medium. The degenerative chain transfer
retarded the polymerization and, as a consequence, resulted
in a smaller amount of grafted polymer compared with
microspheres that contained a low CTA concentration.57
The size of the PDHPMA-PMPDSAH microspheres increased
from 2.2 to 2.6 mm, depending on the amount of grafted
PMPDSAH; the narrow particle size distribution (PDI 5 1.02–
1.06), regular shape, and dispersibility in aqueous medium
were preserved (Fig. 6).
Characterization of PDHPMA-PMPDSAH Microspheres by
ATR FT-IR and 13C CP/MAS NMR Spectroscopy
The modification of PDHPMA microspheres with PMPDSAH
was confirmed by ATR FT-IR and 13C CP/MAS NMR spectros-
copy. ATR FT-IR spectra of starting and modified microspheres
exhibited minimal differences [Fig. 7(a)]. More information
was obtained from differential spectra [Fig. 7(b)], where
intensity of the peaks situated at 3475, 1272, and 1148 cm21
increased after reaction with BIBB [Fig. 7(b), spectrum 1].
They were assigned to the stretching vibrations of OAH, CAO
and CAOH bonds.58 After DTBA attachment, a sharp peak at
1044 cm21 corresponding to C(@S)S vibrations appeared in
the ATR FT-IR spectrum59 [Fig. 7(a), spectrum 3]. In the dif-
ferential spectrum, the increase in intensity of the broad band
at 3475 cm21 was attributed to the OH vibrations [Fig. 7(b),
spectrum 2]. The newly formed peaks at 1089 and
1048 cm21 were ascribed to skeletal CAC vibrations of the
aromatic rings and C(@S)S vibrations of the thiocarbonyl
groups of DTBA, respectively.58,59 After surface modification
with PMPDSAH, two new peaks at 1645 and 1533 cm21
appeared in the differential spectrum [Fig. 7(b), spectrum 3],
corresponding to the C@O (amide I) and NAH (amide II)
vibrations.59 The presence of amide bonds originating from
the MPDSAH zwitterion was also confirmed by a broad band
at 3392 cm21 belonging to the NAH stretching vibrations.59
The increased number of CH2 groups58 was connected to the
bands in the region of 3034–2883 cm21. The positive peak at
1464 cm21 corresponded to the vibrations of quaternary
ammonium groups originating from PMPDSAH.46,58 The bands
situated at 1203 and 1048 cm21 belonged to the asymmetric
and symmetric stretching vibrations of sulfonate groups,
respectively. The latter peak could be partly overlapped by
vibrations of DTBA thiocarbonyl groups.46,58
FIGURE 4 SEM micrographs of PGMA-DTBA (a), PHAPMA-
DTBA (b), and PDHPMA-DTBA microspheres (c) containing 4.7,
0.7, and 25 wt % DTBA, respectively.
MeOH/water mixture (data not shown). It can thus be noted
that the solution polymerization proceeded preferentially in
the MeOH/water mixture compared with MeOH.
Effect of CTA Concentration
The surface coverage and content of grafted polymers is
influenced by the amount of the attached CTA, which deter-
mines the concentration of free radicals and the frequency of
Surface modification of the microspheres with the PMPDSAH
was also well demonstrated in the 13C CP/MAS NMR spectra
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TABLE 1 Influence of Reaction Medium and Monomer Concen-
tration on the Amount of the PMPDSAH Grafted on PDHPMA-
DTBA Microspheres Containing 437 mmol DTBA per g
Surface Charge of PDHPMA-PMPDSAH Microspheres
To determine the resistance of polyzwitterions to nonspecific
protein adsorption, electrostatic forces must be considered.
The interactions depend not only on protein charge and the
ionic strength of the solution but also on the surface
charge.56,60,61 The overall net charge of the polymer particles
was expressed as zeta (f) potential.
a
d
Polymerization cMPDSAH
Nb
(wt %) (wt %)
PMPDSAHc Dn
Medium
(g)
(lm) PDIe
MeOH
0.4
0.8
1.2
0.4
0.8
1.2
0.18
0.59
0.79
0.77
1.33
1.08
1.88
6.16
8.25
8.04
13.89
11.28
2.2
2.2
2.3
2.3
2.3
2.3
1.02
1.02
1.03
1.03
1.03
1.03
The f potential of starting PGMA microspheres decreased
with increasing pH of the media, which was ascribed to
intense deprotonation of the carboxyl groups of the PAA sta-
bilizer at higher pH (Fig. 9). The microspheres grafted with
19.8 wt % of PMPDSAH had a negative charge over the
whole pH range [Fig. 9(a)]. This behavior was explained by
the acid–base equilibrium involving tetra-alkylammonium
and sulfonate ions.61 Because of the strong acidity of sulfonic
acid, the sulfonate groups were in the form of anions. More-
over, some tetra-alkylammonium ions associated with the
hydroxyl ions in water, thus forming uncharged tetra-
alkylammonium hydroxide.61 Due to the excess of negative
sulfonate over the positively charged ammonium groups, the
PMPDSAH, and consequently PMPDSAH-modified micro-
spheres displayed an overall negative charge. To reach the
charge neutrality of PMPDSAH, the pH has to be further low-
ered to favor of the formation of positive ammonium ions.
This was observed on microspheres modified with a low
amount of PMPDSAH (2.8 wt %), where the reduced concen-
tration of sulfonate groups shifted the acid–base equilibrium
such that a neutral charge occurred at a pH value of approxi-
mately 4.5 [Fig. 9(a)].
EtOH
a
Amount of MPDSAH in polymerization feed.
b
c
Nitrogen content in PDHPMA-PMPDSAH microspheres.
Amount of grafted PMPDSAH calculated from nitrogen content.
Number-average diameter.
Index of polydispersity.
d
e
(Fig. 8). A prominent decrease in the signal intensity of
CHAO units at 49 ppm that was accompanied by the corre-
sponding increase of the signal at 67.4 ppm clearly reflected
cleavage of oxirane rings. Whereas partial oxirane cleavage
was found in PDHPMA-DTBA microspheres, complete oxirane
opening and extensive formation of hydroxyl groups was
achieved in PDHPMA-PMPDSAH microspheres. This fact was
proven by the overlay of 13C CP/MAS NMR spectra [Fig.
8(b)]. The weak signal at 129 ppm in both the PDHPMA-
DTBA and PDHPMA-PMPDSAH spectra demonstrated the
incorporation of aromatic units [Fig. 8(a)]. The considerably
increased signal at 18 ppm and the weak signal at 38 ppm
reflected the incorporation of building units containing
methyl groups and formation of ACH2AS fragments, respec-
tively [Fig. 8(b)]. This confirmed the successful grafting of
PMPDSAH on the PDHPMA microspheres.
As aforementioned, the
f potential of the PDHPMA–
PMPDSAH microspheres was also influenced by the ionic
strength of the solution. With the increasing salt concentra-
tion, the absolute f potential value of the microspheres with
both high and low PMPDSAH content decreased [Fig. 9(b)].
This was ascribed to the preferential interaction of sulfonate
anions with Na1 counterions, compared with that of tetra-
alkylammonium and Cl2 ions.62 Moreover, due to the
TABLE 2 Influence of Monomer Concentration in MeOH on
the Amount of the PMPDSAH Grafted on PDHPMA-DTBA
Microspheres with Different DTBA Content
Nb
PMPDSAHc
(wt %)
Dn
a
d
cDTBA
cMPDSAH
(mmol/g)
(g)
(wt %)
(lm)
PDIe
1140
0.4
0.8
1.2
0.4
0.8
1.2
0.78
1.14
0.60
0.27
1.31
1.90
8.15
2.3
2.4
2.3
2.2
2.5
2.6
1.02
1.02
1.02
1.02
1.04
1.06
11.90
6.27
134
2.82
13.68
19.84
a
Amount of MPDSAH in polymerization feed.
b
c
Nitrogen content in PDHPMA-PMPDSAH microspheres.
FIGURE 5 Dependence of ln([M]0/[M]) ratio in MeOH/water and
MeOH on the polymerization time t. [M]0 and [M] are initial
and instant monomer concentrations.
Amount of grafted PMPDSAH calculated from nitrogen content.
Number-average diameter.
Index of polydispersity.
d
e
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FIGURE 6 SEM micrographs of PDHPMA-PMPDSAH micro-
spheres containing 11.9 (a; prepared in MeOH) and 16.1 wt %
PMPDSAH (b; prepared in EtOH).
FIGURE 8 13C CP/MAS NMR spectra of PGMA, PDHPMA-DBTA,
and PDHPMA-PMPDSAH microspheres in full (a) and expanded
range scaled on the main chain ACH2A signal (b).
FIGURE 7 ATR FT-IR spectra (a) of PDHPMA (1), PDHPMA-BIBB (2), PDHPMA-DTBA (3), PDHPMA-PMPDSAH microspheres (4) and
MPDSAH (5). Differential spectra (b) of PDHPMA-BIBB minus PDHPMA (1), PDHPMA-DTBA minus PDPHMA-BIBB (2) and PDHPMA-
PMPDSAH minus PDHPMA-DTBA (3).
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FIGURE 9 Dependence of n potential on the pH of dispersions of initial PGMA and PDHPMA-PMPDSAH microspheres with low
and high amount of PMPDSAH in 0.01 M (a) and 0.1 M NaCl solution (b).
CONCLUSIONS
expansion of PMPDSAH chains at high salt concentrations,
the electrophoretic mobility and, consequently, the absolute f
potential value of the microspheres slightly decreased.63
New polyzwitterion-modified PGMA microspheres with a
narrow size distribution and enhanced antifouling properties
were successfully developed using the grafting from
approach. To initiate the controlled radical polymerization
from the surface, the microspheres were functionalized with
CTA attached through several synthetic routes. Both the
direct reaction of oxiranes with DTBA and the activation
with the ATRP initiator via amino groups resulted in either
extensive particle aggregation or a small amount of surface-
bound DTBA. Only the hydrolysis of oxirane groups followed
by activation with the ATRP initiator and attachment of
Antifouling Properties of PMPDSAH-Grafted
Microspheres
Polyzwitterions are known to suppress protein and cell
adhesion.29,31,32,46,61 The influence of PMPDSAH coating on
protein adsorption was studied using BSA as a model. BSA
belongs among the most abundant blood proteins having iso-
electric point at pH 4.7, that is, it possesses an overall nega-
tive charge in PBS (pH 7.3). As a result, BSA should not be
attracted to the PDHPMA–PMPDSAH particle surface.61
The unmodified PGMA microspheres exhibited rather low
nonspecific BSA adsorption (4 wt %), which could be
ascribed to the presence of hydrophilic carboxyls from PAA
and hydroxyls from hydrolyzed oxiranes. Moreover, in PBS,
the negative charges of both PGMA microspheres and BSA
(231 and 228 mV, respectively) prevented their mutual
contact. After the attachment of DTBA, that is, after hydro-
phobization of the particle surface, the nonspecific protein
adsorption was doubled (Fig. 10). Hydrophobic dithioben-
zoate groups probably induced protein denaturation and
conformation changes, negatively influencing protein–surface
interactions.64 Nevertheless, the nonspecific BSA adsorption
gradually decreased with an increasing amount of PMPDSAH
grafted on the PDHPMA microspheres, up to half of that on
the unmodified PGMA microspheres (Fig. 10). This result
was explained by the increased concentration of hydrophilic
tetra-alkylammonium and sulfonate groups that improved
surface hydration, thus excluding the BSA from the particle
surface and simultaneously preserving protein conforma-
tion.61 Microspheres grafted with ꢁ13.5 wt % PMPDSAH
thus reduced the nonspecific protein adsorption more effi-
ciently than the unmodified PAA-stabilized PGMA particles.
FIGURE 10 Amount of BSA adsorbed on PDHPMA-PMPDSAH
microspheres relative to BSA amount on the unmodified
PGMA microspheres.
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9 G. Peng, C. Zhao, B. Liu, F. Ye, H. Jiang, Appl. Surf. Sci.
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DTBA allowed the introduction of dithiobenzoate moieties in
high yields (up to 36 wt %). At the same time, the morpho-
logy, particle size, distribution, and dispersibility in aqueous
media were preserved. The successful PMPDSAH grafting (up
to 20 wt %) was accomplished only in polar organic sol-
vents. This was ascribed to limited PMPDSAH solubility and
the tendency of its chains to remain at the particle surface.
The amount of grafted polymer was also affected by the con-
centration of surface-anchored CTA. The highest PMPDSAH
content was found on the microspheres with the smallest
concentration of attached DTBA. Surface properties, in partic-
ular the f potential of the microspheres, were primarily
determined by the amount of grafted PMPDSAH. The micro-
spheres with high PMPDSAH concentration (19.8 wt %)
were negatively charged in both 0.1 and 0.01 M salt solu-
tions within the whole pH range. In contrast, the f potential
of the microspheres with low zwitterion grafting (2.8 wt %)
was shifted from the positive to negative values in the solu-
tion containing low NaCl concentration. This was ascribed to
a lower concentration of sulfonate groups on such particles
and thus their easy protonation compared with that of
PDHPMA–PMPDSAH microspheres containing large amounts
of zwitterions. However, highly efficient reduction of nonspe-
cific protein adsorption was reached only on the micro-
spheres with PMPDSAH content ꢁ13.5 wt %, ensuring
excellent hydration of the particle surface and thus hindering
protein–surface interactions. These properties make the
PMPDSAH–PDHPMA microspheres suitable for biomedical
applications in which antibodies or peptides are conjugated
to the surface; this work is in progress.
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