Synthesis and Physicochemical Properties of Cationic Microgels Based on Poly(N-isopropylmethacrylamide)

Abstract

Surfactant-free, radical precipitation co-polymerization of N-isopropylmethacrylamide (NIPMAm) and the cationic co-monomer N-(3-aminopropyl) methacrylamide hydrochloride (APMH) was carried out to prepare microgels functionalized with primary amines. The morphology and hydrodynamic diameter of the microgels were characterized by atomic force microscopy (AFM) and photon correlation spectroscopy (PCS), with the effect of NaCl concentration and initiator type on the microgel size and yield being investigated. When a V50-initiated reaction was carried out in pure water, relatively small microgels (~160 nm diameter) were obtained in low yield (~20%). However, both the yield and size increased if the reaction was carried out in saline or by using APS as initiator instead of V50. Stable amine-laden microgels in the range from 160 nm to 950 nm in diameter with narrow size distributions were thus produced using reaction media with controlled salinity. Microgel swelling and electrophoretic mobility values as a function of pH, ionic strength and temperature were also studied, illustrating the presence of cationic sidechains and their influence on microgel properties. Finally, the availability of the primary amine groups for post-polymerization modification was confirmed via modification with fluorescein-NHS.

Full text

Synthesis and Physicochemical Properties of Cationic Microgels
Based on Poly(N-isopropylmethacrylamide)
Xiaobo Hua,b, Zhen Tongb, and L. Andrew Lyona,*
a School of Chemistry & Biochemistry and the Petit Institute for Bioengineering & Bioscience,
Georgia Institute of Technology, Atlanta, GA 30332, USA
b Research Institute of Materials Science, South China University of Technology, Guangzhou
510640, P.R. China
Abstract
Surfactant-free, radical precipitation co-polymerization of N-isopropylmethacrylamide (NIPMAm)
and the cationic co-monomer N-(3-aminopropyl) methacrylamide hydrochloride (APMH) was
carried out to prepare microgels functionalized with primary amines. The morphology and
hydrodynamic diameter of the microgels were characterized by atomic force microscopy (AFM)
and photon correlation spectroscopy (PCS), with the effect of NaCl concentration and initiator
type on the microgel size and yield being investigated. When a V50-initiated reaction was carried
out in pure water, relatively small microgels (~160 nm diameter) were obtained in low yield
(~20%). However, both the yield and size increased if the reaction was carried out in saline or by
using APS as initiator instead of V50. Stable amine-laden microgels in the range from 160 nm to
950 nm in diameter with narrow size distributions were thus produced using reaction media with
controlled salinity. Microgel swelling and electrophoretic mobility values as a function of pH,
ionic strength and temperature were also studied, illustrating the presence of cationic sidechains
and their influence on microgel properties. Finally, the availability of the primary amine groups
for post-polymerization modification was confirmed via modification with fluorescein-NHS.
Keywords
Controlled salinity; Microgel; N-isopropylmethacrylamide; cationic polymer
Introduction
Temperature-sensitive poly(N-isopropylacrylamide) (pNIPAm) microgels, which were first
prepared by emulsion polymerization in 1986 [1], have received a significant amount of
attention due to dramatic changes in physicochemical properties observed when temperature
is raised above their volume phase transition temperature (VPTT, ~31 °C). In recent years,
such microgels have been used in drug delivery [2–4], biosensors [5–7], tissue regeneration
[8,9], and chemical separations [10]. In many of these applications, the introduction of
specific chemical functionalities is desired, such that the polymer can be elaborated upon
following synthesis. Toward that end, functionalities such as carboxyl [11], azido [12] and
glycidyl [13] groups have been copolymerized into pNIPAm microgels. However, the
primary amine, a reactive group that is particularly useful for bioconjugation [14], has been
far less studied in microgel synthesis. The utility of this group is associated with the
potential for binding with anionic moieties on oligonucleotides, proteins, or enzymes [14–
*To whom correspondence should be addressed. lyon@gatech.edu.
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Colloid Polym Sci
. 2010 December 4; 289(3): 333–339. doi:10.1007/s00396-010-2347-y.
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16], as well as its use in the covalent attachment to molecules carrying isothiocyanates or
succinimidyl esters.
Given the utility of amine chemoligation sites, the incorporation of amines into microgels
has been studied in a few cases. Xu et al. prepared primary amine functionalized pNIPAm
microgels by copolymerization of N-isopropylacrylamide (NIPAm) with N-vinylformamide
(NVF) by a semi-batch route, followed by conversion of the formamide moieties to a
primary amine by acid hydrolysis [17]. Unfortunately, this method requires multiple steps
and very strong acid hydrolysis conditions, thereby limiting its broad application. Leung et
al. prepared microgels by graft copolymerization of NIPAm from either PEI or chitosan,
which results in core-shell structures with the amines mainly becoming shell-localized [18].
This approach produces a very thin polymer shell, with the amine concentration being
limited due to the stabilization effect of polymer during the synthesis. Meunier et al.
prepared amine-laden pNIPAm microgels by using 2,2′-azobis (2-amidinopropane)
dihydrochloride (V50) as initiator and 2-aminoethylmethacrylate hydrochloride (AEMH) as
a functional monomer [19]. However, the ester group in the AEMH monomer can be easily
hydrolyzed to carboxyl during the synthesis because of the acid solution and high
temperature, which can cause these microgels to be unstable. In this paper, we discuss
alternate routes for the preparation of amine functionalized microgels based on poly(N-
isopropylmethacrylamide) (pNIPMAm). The use of pNIPMAm, which has a volume phase
transition temperature of ~43 °C was motivated by our recent use of this polymer in drug
delivery applications [4]. Thus, we are very interested in broadening the utility of those
particular microgels by increasing the range of functionalities that can be efficiently
incorporated.
Experimental
Materials
All materials were purchased from Sigma-Aldrich unless otherwise noted. The monomer N-
isopropylmethacrylamide (NIPMAm) was recrystallized from n-hexane (J. T. Baker).
Comonomer N-(3-aminopropyl)methacrylamide hydrochloride (APMH, Polysciences Inc.),
cross-linker N,N′-methylene(bisacrylamide) (BIS), cationic initiator 2,2′-azobis(2-
amidinopropane) dihydrochloride (V50) and anionic initiator ammonium persulfate (APS)
were all used as received. NHS-Fluorescein (Thermo Sci.) and NaCl were used as received.
Pure water was produced by deionization to a resistance of 18 MΩ·cm (Barnstead E-Pure
system), followed by filtration through a 0.2-μm filter to remove particulate matter.
Microgel Synthesis
Microgels were synthesized by surfactant-free, radical precipitation co-polymerization using
methods similar to those previously reported [20]. For all reactions, the molar composition
was 89% NIPMAm, 9% APMH, and 2% BIS, with a total monomer concentration of 140
mM (Table 1). In a typical synthesis, 50 mL of a filtered, aqueous solution of NIPMAm,
APMH, BIS and NaCl was added to the reaction flask, which was then heated to 70 °C. The
solution was purged with N2 gas with continuous stirring until the temperature remained
stable. The reaction was initiated by addition of 2 mL of a V50 or APS solution. The
reaction was allowed to continue for 20 h while being purged by N2 gas with constant
stirring. After synthesis, the solution was filtered through glass wool to remove any
coagulum and then centrifuged/redispersed several times for purification. After purification,
the microgel dispersion was lyophilized for at least 72 hours.
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Atomic Force Microscopy (AFM)
Microgels were imaged using an Asylum Research MFP-3D Instrument (Santa Barbara,
CA). Imaging was performed and processed using the MFP-3D software under the IgorPro
(WaveMetrics Inc., Lake Oswego, OR) environment. Non-contact mode aluminum-coated
silicon nitride cantilevers were purchased from NanoWorld (force constant = 42 N/m,
resonant frequency = 320 kHz). All images were taken in air under ambient conditions.
AFM samples were prepared by centrifugal deposition at a maximum rotor speed of 2250 ×
g for 5 min on glass coverslips [21]. Glass coverslips (22 mm × 22 mm) were cleaned using
an air plasma (Harrick Plasma, Ithaca, NY) for ~ 15 min. Each cleaned slide was
functionalized by exposure to a 1% solution of 3-aminopropyltrimethoxysilane (APTMS) in
absolute ethanol (200 proof) for ~3 h. The glass was then rinsed with ethanol and dried
under a gentle stream of N2. The amine-functionalized coverslips were further modified by
immersion in a 0.1 mg/mL poly(sodium styrenesulfonate) solution before the centrifugal
deposition of microgels. After microgel deposition, the sample was gently rinsed with
deionized water and dried under a gentle stream of N2.
Particle size characterization
Particle sizes were determined by photon correlation spectroscopy (PCS; Protein Solutions,
Inc.) using an instrument equipped with an integrated Peltier temperature control device,
which provides temperature accuracy within ±0.1 °C. The instrument collects scattering
light at 90° by a single-mode optical fiber coupled to an avalanche photodiode detector. The
samples were thermally equilibrated at each temperature for 30 min before each
measurement. The data presented are the averaged values of 20 measurements, with a 10 s
integration time for each measurement.
Zeta-potential Determination
The ζ-potential was measured with a Zetasizer Nano-ZS (Malvern). Before the
measurements, the 1 mL microgel dispersion in the designated buffer was thermally
equilibrated between parallel electrodes in cuvette for 10 min. The ζ-potential value was the
average of at least three successive measurements.
Fluorescence labeling and imaging
For conjugating the amine reactive fluorescein, 2.5 mg of amine microgels were dispersed in
5 mL of a pH 7.4 PBS buffer, and 1.6 mL of 1 mg/mL DMF solution of fluorescein-NHS
was added. The solution was stirred at 4 °C overnight and then purified by dialysis against
DI water for ~2 weeks with the water being changed twice per day, using Spectra-Pro
10,000 MW cut-off dialysis tubing (VWR). The dyed microgels were imaged by
fluorescence microscopy, which was conducted on an Olympus IX-70 inverted microscope
equipped with a high numerical aperture, oil immersion 100 × objective (NA = 1.30). The
excitation irradiation was a mercury lamp filtered by excitation band-pass filters of 450–490
nm. Images were captured using a color CCD camera (PixelFly, Cooke Corporation).
Results and Discussion
During the preparation of pNIPAm or pNIPMAm microgels, an anionic initiator APS or
cationic initiator V50 is typically degraded thermally to initiate the polymerization, which
results in anionic or cationic microgels, respectively [1,22]. During our experiments, initial
attempts to prepare poly(NIPMAm-co-APMH) microgels (μG) in pure water using a
cationic initiator (V50) resulted in microgels of a very small size with poor yield (Table 1).
A possible reason for the poor yield lies in unfavorable reactivity ratio between the two co-
monomers, which inhibits the formation of copolymer chains that can easily collapse at high
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temperature [23]. Randomly positioned APMH along the copolymer chain creates
sufficiently strong Coulombic repulsion between the cationic groups such that thermo-
induced chain collapse is inhibited. A similar phenomenon has been described previously by
Meunier et al., where high AEMH concentrations accelerated the incorporation rate of BIS
during particle formation, making the capture of polymer formed later in the reaction
unfavorable [19]. As a result, many free polymer chains may be formed instead of collapsed
precursor particles, thereby reducing the yield of microgels, and limiting the subsequent
polymer size due to a decrease in the amount of incorporated polymer/particle. Fortunately,
the reactivity ratio of ionic monomers can be notably influenced by the ionic strength
[23,24]. We therefore investigated the influence of NaCl concentration on microgel
preparation.
From Table 1, we can see that the diameter of μG0 microgels in pH 3 buffer (10 mM) is
~715 nm, with a yield of ~45%. However, the particle diameter decreased to ~157 nm after
the addition of 9 mol% APMH, with the yield of cross-linked microgels dropping to 20%. In
the past, the presence of salt in alkylacrylamide emulsion polymerization media has been
avoided due to the propensity of deswollen particles to aggregate under high salinity
conditions [25]. However, in this study, our results show that increasing the salinity of the
reaction medium increases the microgel size along with a modest increase in microgel yield.
We hypothesize that the salt affects the polymerization in at least two ways. After the
addition of NaCl, the Coulombic repulsion between the positively charged APMH units
becomes sufficiently screened to allow a greater tendency for homopropagation, which will
result in polymers with a longer sequence of APMH units in the copolymer chains. The
block-like copolymer chains will undergo thermally-induced collapse more readily, as has
been observed for other block copolymers containing pNIPAm [26]. Consequently, more
polymer chains will become incorporated into the growing microgels instead of remaining in
solution as uncrosslinked “free” polymer chains. Additionally, it is likely that growing
microgels will undergo aggregation with the other precursor particles under these charge-
shielding conditions, thereby producing a larger average particle size. However, when the
NaCl concentration reached 200 mM, the reaction system became unstable and macroscopic
coagulation of the polymer was observed.
In addition to the impact of salinity on microgel formation, the influence of different
initiators was studied, where the anionic initiator APS was used to initiate the
polymerization instead of V50. Despite the obvious potential for forming zwitterionic
microgels when APS is used, no colloidal instability was observed during the syntheses
undertaken in this work. Particles produced under the conditions for μG5 had a diameter of
~500 nm with a yield of 60%, which is much higher than that obtained from the
corresponding V50 synthesis. When the APS concentration was increased to 2 mM, a
particle diameter of 740 nm and 65% yield was obtained (μG6). Whereas these results are
too preliminary to completely explain this phenomenon, we tentatively ascribe these results
to the presence of the oxygen-centered radical associated with APS, which is more effective
in abstracting a hydrogen atom from the carbon next to the amine group in APMH. This
chain transfer reaction will provide additional reactive sites, which will induce continuous
graft polymerization and self cross-linking [27,28], resulting in larger microgels and higher
polymer yields.
The ζ-potentials of the microgels in pure water are listed in Table 1; all microgels display a
positive ζ-potential indicating cationic character. For μG0 and μG1, the ζ-potentials are of
similarly low magnitude. For μG0, the positive charge is due to the amidine end groups
arising from the V50 initiator. The lack of a significant change in this value for μG1, which
was synthesized using 9 mol% APMH, suggests that the degree of APMH incorporation was
very poor. In contrast, there is a large increase in the ζ-potential value for microgels
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synthesized with increasing salinity or with APS as the initiator, which is indicative of
surface charges imparted by more efficient incorporation of APMH. It is also interesting to
note that μG6 displays a slightly higher zeta-potential than the other samples, even though
this particle must also contain some anionic initiator residues. This, coupled with the
observation that these conditions display the highest yield, suggests that APMH
incorporation is maximized for μG6 relative to the other conditions.
Analysis of the microgels via AFM permits visualization of the particle morphology and
polydispersity estimation (Figure 1). It should be noted that in all cases, the strong microgel-
surface interactions, combined with the fact that the particles are dehydrated, result in
severely flattened particles. Of particular interest in these data is the observation that μG3
and μG4 do not appear to be purely spherical particles. Instead, these microgels appear
somewhat irregular and “bumpy”, as if formed via the aggregation of multiple smaller
particles. Such precursor particle aggregation would be predicted to occur more readily in
high salt. Alternatively, the addition of NaCl likely results in the formation of block-like
copolymers during the microgel synthesis [23,24], as described above. If this is indeed the
case, the resultant particles might contain relatively long polyelectrolyte chains at the
microgel surface. This shell-localized polymer will randomly spread on the substrate during
the drying process, resulting in a non-spherical particle appearance. Conversely, microgels
μG5 and μG6 appear spherical, which perhaps suggests a homogeneous particle architecture.
Line profiles taken from these images permit estimation of the dried microgel heights and
diameters. The height/diameter ratio provides a rough, qualitative estimate of microgels
stiffness [29]; this analysis suggests that the APS-initiated microgels (μG5 and μG6) are
somewhat stiffer than those prepared with V50 (Figure 1f), presumably due to increased
cross-linking brought about by the aforementioned chain transfer reactions.
In addition to microgel morphology, it is critical to know how the different synthetic
conditions impact the temperature and pH dependent swelling and charge characteristics.
The physicochemical properties of selected microgels are summarized in Figure 2. As
expected from their pNIPMAm content, all microgels display a volume phase transition
temperature (VPTT) between 40 and 50 °C (Figure 2a). Those lacking cationic co-monomer
(μG0) display a transition very close to the LCST of pure pNIPMAm (~44 °C), with the
VPTT of all other microgels being shifted to higher temperatures, as is typical for microgels
containing hydrophilic or charged groups [30,31]. The swelling ratio is also presumably
influenced by the presence of charged co-monomers (Figure 2b), as the swelling ratios of the
amine containing microgels are all smaller than that of μG0. Amine incorporation is also
evident in the influence of ionic strength on the measured microgel size at pH 3 (Figure 2c).
Below the VPTT, the diameter of μG3 is observed to decrease as the ionic strength increased
from 2 mM to 100 mM. Furthermore, the VPTT is shifted to lower temperatures with a
concomitant sharpening of the transition. These effects arise largely from charge screening,
competitive solvation, and osmotic effects [25,32,33]. By measuring the microgel swelling
ratio as a function of pH (defined as the ratio of the microgel diameter at a particular pH
relative to that at pH 11.5), we observe the expected result that the swelling ratio gradually
decreases at pH values higher than 7.4 and plateaus above the pKa (~10) of APMH (Figure
2d). The pH dependence of the ζ-potential closely mirrors the swelling data (Figure 2e),
where in the absence of APMH (μG0), no pH dependence is observed, but a strong pH effect
is observed in the presence of APMH (μG3 and μG6). For the APMH containing microgels,
the ζ-potential decreases as the pH increases. For example, the ζ-potential of μG6 suddenly
decreases with a pH change from 9.5 to 10.5, with this pH corresponding to the pKa of
primary amine group. However, for μG3, the ζ-potential gradually decreases once the pH
increases above 7.4. This difference in pKa between the polyelectrolyte and the parent
monomer is common due to the decreased propensity for deprotonation in the presence of
the neighboring ionic groups [34]. For example, poly(2-aminoethyl methacrylate), which has
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a similar structure to pAPMH, has a pKa of approximately 7.6. This pKa is significantly
lower than that of its monomer (pKa =10) [35]. This result is therefore also suggests that the
addition of NaCl results in the formation of block-like copolymers. Due to the cationic
initiator fragments, the ζ-potential is always positive for the μG0 and μG3 microgels over
the pH range studied here. However, due to the neutralization of the amine sidechains and
the presence of anionic initiator groups, the ζ-potential of μG6 microgels is negative at pH
11.5. Finally, to illustrate that the amine groups are accessible to small molecule coupling
reactions, fluorescein-NHS was used as a coupling probe for microgels μG3 and μG6; a
representative image of μG6 is shown (Figure 2f). Studies investigating the accessibility of
those groups in a wider range of bioconjugation reactions are currently underway.
Conclusions
In this investigation, we have shown that primary amine containing pNIPMAm microgels
can be produced by a surfactant free radical precipitation copolymerization. By controlling
the reaction salinity or using an anionic initiator APS, low polydispersity, primary amine
containing pNIPMAm microgels can be obtained in high yield. The increase of particle size
and yield by the salt addition originates from the screening of Coulombic repulsion between
APMH units, which results in more favorable polymer incorporation. The resultant amine-
laden microgels show the expected swelling properties of thermoresponsive cationic
microgels as a function of temperature, pH and ionic strength, as well as reactivity in
standard amide bond-forming reactions. Studies of these microgels in the context of
biomolecule conjugation and thin film self-assembly are currently underway.
Acknowledgments
This work was partially supported by the National Institutes of Health (1 R01 GM088291-01). XH thanks China
Scholarship Council (CSC) for fellowship support. We thank the Kröger group at GT for the use of their zeta
potential equipment.
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Figure 1.
AFM height images and line profiles of (a) μG0, (b) μG3, (c) μG4, (d) μG5, and (e) μG6. (f)
Histogram of calculated height/diameter ratios.
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Figure 2.
(a) Hydrodynamic diameter and (b) swelling ratio vs. T of the indicated microgels in pH 3
buffer (I = 10 mM). (c) Diameter vs. T for μG3 microgels in pH 3 buffers with different
ionic strengths. μG0 microgels in pH 3 (10 mM) is shown for comparison. The dependence
of: (d) swelling ratio and (e) ζ-potential of the indicated microgels as a function of pH with
the ionic strength held constant at 2 mM. (f) Fluorescence microscopy image of fluorescein-
labeled μG6 microgels.
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NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Hu et al. Page 11
Table 1
Synthesis conditions, yields, and microgel properties.
CodeaAPMH/mol % V50/mM APS/mM [NaCl]/mM Dh/nmbζ-potential in DI water/mV Yield/%
μG0 0 2 - 0 714 13.4 45
μG1 9 2 - 0 157 12.9 20
μG2 9 2 - 50 318 33.8 25
μG3 9 2 - 100 508 37.8 35
μG4 9 2 - 150 950 38.9 38
μG5 9 - 1 0 498 33.8 60
μG6 9 - 2 0 744 41.7 65
aReaction conditions: total monomer concentration = 140 mM, temperature = 70 °C, reaction time: 20 h.
bSolution conditions: pH 3, 10 mM ionic strength.
Colloid Polym Sci. Author manuscript; available in PMC 2011 March 16.