Aggregation induced emission based fluorescence pH and temperature sensors: Probing polymer interactions in poly(N-isopropyl acrylamide-co-tetra(phenyl)ethene acrylate)/poly(methacrylic acid) interpenetrating polymer networks

Article abstract of DOI:10.1039/c5tc00752f

Aggregation induced emission (AIE) active copolymers P1-P6 with high molecular weights (14 000-17 000) and low polydispersity indices (1.3-1.4) were prepared through copolymerization of N-isopropyl acrylamide (NIPAM) and tetra(phenyl)ethene (TPE)-based acrylate monomers. Copolymers P1-P6 show comparable thermal stability to poly(N-isopropylacrylamide) (PNIPAM), while their glass transition temperatures are higher by 7-9 °C than those of pristine PNIPAM. Copolymers P1-P6 are soluble in common organic solvents as well as in water. They retain a similar thermal sensitivity to PNIPAM, but their lower critical solution temperatures (LCST) are reduced with increase of TPE content. By changing the molar ratio of P1-P6/poly(methacrylic acid) (PMAA) and pH, complexes P1-P6-PMMA were studied by fluorescence spectroscopy and dynamic light scattering (DLS). The complexes are non-emissive in THF, and their fluorescence can be turned on upon addition of water. Moreover, their fluorescence is enhanced with the decrease in pH values due to the formation of interpenetrating polymer networks (IPNs) through inter-polymer hydrogen bonding. Fluorescence spectroscopy and DLS results also reveal that the phase transition behaviour of IPNs upon heating could be significantly modified by pH change. Reduction in the pH value from 7.0 to 4.0 leads to the decrease in LSCT of IPNs by up to 5 °C with respect to PNIPAM. By tuning the pH value to dissociate the formed inter-polymer hydrogen bonds, the formed IPNs would be able to fold cooperatively to a compact structure without a loss of solubility at temperatures below the LCST. Thus, these novel IPNs with AIE active moieties would be used as drug delivery systems, in which the release process could be readily monitored by fluorescence spectroscopy.

Full text of DOI:10.1039/c5tc00752f

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Materials Chemistry C
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Aggregation induced emission based fluorescence
pH and temperature sensors: probing polymer
interactions in poly(N-isopropyl acrylamide-co-
tetra(phenyl)ethene acrylate)/poly(methacrylic
acid) interpenetrating polymer networks†
Cite this:DOI: 10.1039/c5tc00752f
Hui Zhou,a Feng Liu,b Xiaobai Wang,a Hong Yan,a Jing Song,a Qun Ye,a
Ben Zhong Tang*c and Jianwei Xu*a
Aggregation induced emission (AIE) active copolymers P1–P6 with high molecular weights (14 000–
17 000) and low polydispersity indices (1.3–1.4) were prepared through copolymerization of N-isopropyl
acrylamide (NIPAM) and tetra(phenyl)ethene (TPE)-based acrylate monomers. Copolymers P1–P6 show
comparable thermal stability to poly(N-isopropylacrylamide) (PNIPAM), while their glass transition
temperatures are higher by 7–9 1C than those of pristine PNIPAM. Copolymers P1–P6 are soluble in
common organic solvents as well as in water. They retain a similar thermal sensitivity to PNIPAM, but
their lower critical solution temperatures (LCST) are reduced with increase of TPE content. By changing
the molar ratio of P1–P6/poly(methacrylic acid) (PMAA) and pH, complexes P1–P6–PMMA were studied
by fluorescence spectroscopy and dynamic light scattering (DLS). The complexes are non-emissive in
THF, and their fluorescence can be turned on upon addition of water. Moreover, their fluorescence is
enhanced with the decrease in pH values due to the formation of interpenetrating polymer networks
(IPNs) through inter-polymer hydrogen bonding. Fluorescence spectroscopy and DLS results also reveal
that the phase transition behaviour of IPNs upon heating could be significantly modified by pH change.
Reduction in the pH value from 7.0 to 4.0 leads to the decrease in LSCT of IPNs by up to 5 1C with
respect to PNIPAM. By tuning the pH value to dissociate the formed inter-polymer hydrogen bonds, the
formed IPNs would be able to fold cooperatively to a compact structure without a loss of solubility at
temperatures below the LCST. Thus, these novel IPNs with AIE active moieties would be used as drug
delivery systems, in which the release process could be readily monitored by fluorescence spectroscopy.
Received 17th March 2015,
Accepted 27th April 2015
DOI: 10.1039/c5tc00752f
www.rsc.org/MaterialsC
those hydrogels have been extensively investigated in many
areas, such as controlled drug delivery,2 molecular separation,3
Introduction
Over the last few decades, dual or multi-responsive polymers tissue culture substrates,4 and enzyme activity controlling
have been receiving more attention with a special emphasis on systems.5
the temperature and pH sensitiveness.1 Polymer hydrogels are
Typically, copolymers with multi-responsive characteristics,
water-swollen polymer networks which are cross-linked through which are usually prepared through copolymerization of at least
physical or chemical bonding. Some of the hydrogels exhibit two monomers with respective peculiarities, are employed to
sensitive response to external stimuli, such as pH and tempera- generate interpenetrating polymer networks (IPNs) with multi-
ture. Due to their potential applications in biological systems, responsive properties.6 An alternative method has received
more attention, in which the combined property can be easily
a Institute of Materials Research and Engineering, A*STAR (Agency for Science,
Technology and Research), 3 Research Link, Singapore 117602, Republic of
Singapore. E-mail: jw-xu@imre.a-star.edu.sg
tuned by chain interpenetration of two polymers with respective
properties.7 Each polymer retains its own property while proportion
of each can be varied independently of the other, because there is
no chemical bonding between two polymers.
Poly(methacrylic acid) (PMAA) is an ionizable hydrophilic
polymer due to ionization/deionization of carboxylic acid groups,
b Department of Chemistry, National University of Singapore, 3 Science Drive 3,
Singapore 117543, Republic of Singapore
c Department of Chemistry, The Hong Kong University of Science & Technology,
Clear Water Bay, Kowloon, Hong Kong, China. E-mail: tangbenz@ust.hk
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5tc00752f and thus its swelling behavior is greatly pH-dependent. Under low
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pH conditions, –COOH groups are not ionized and cling to the to monitor the thermal transition of PNIPAM in water.16a While
PMAA network at its collapsed state through hydrogen bonding. the conformation of the PNIPAM chain is very sensitive to its
While under high pH conditions, the –COOH groups are ionized structure, even a subtle modification in the structure can lead
and the charged –COOꢀ groups repel each other, leading to to a huge change.21 In order to minimize these negative effects,
PMAA swelling. Poly(N-isopropyl acrylamide) (PNIPAM) is a we here incorporated TPE with alkyl linkage into PNIPAM chains
temperature-sensitive polymer, which shows drastic swelling through copolymerization of monomers with TPE (M1–M3) and
transition at its lower critical solution temperature (LCST) of N-isopropyl acrylamide (NIPAM). The resultant copolymers could
32 1C.8 At a low T (o32 1C) the gel is swollen, while at a high T function as a fluorescence probe to track the interaction of PMAA
(432 1C) the polymer dehydrates to the collapsed state due to and PNIPAM during IPN formation under different conditions,
breakdown of the delicate hydrophilic/hydrophobic balance in including pH, the component ratio and temperature.
the network structure. IPNs generated by PMAA and PNIPAM
exhibited combined pH- and temperature-sensitivity. Due to the
potential application in the real controlled drug delivery, the
Experimental
formation and interactions of such IPNs were investigated by
several analytical methods, such as attenuated total reflectance
Fourier transform infrared spectroscopy,9 the viscometric method,10
equilibrium swelling and oscillatory swelling studies,11 and
high-sensitivity differential scanning calorimetry.12 However,
those methods did not easily track the status of IPNs and thus
were unable to real-time monitor the controlled drug delivery.
The fluorescence methodology may be ideal for these purposes
due to its ultrahigh sensitivity, superfast response and a con-
venient process.
General
All synthetic manipulations were carried out under an atmosphere
of dry argon gas using standard vacuum-line Schlenk techniques.
All solvents were degassed and purified before use according to
standard literature methods. Diethyl ether, hexanes, tetrahydro-
furan, and toluene were purchased from Aldrich Chemical Co.
Inc. and distilled from sodium/benzophenone ketyl before use.
Materials
NIPAM was purchased from Aldrich and purified by crystallization
before use. 2,20-Azoisobutyronitrile (AIBN) was purchased from
Aldrich and purified by crystallization before use. Other com-
mercially available reagents and solvents were used as received.
PMAA was purchased from Aldrich, and the molecular weight is
9.5 kDa according to GPC.
Recently, although fluorescence probes have attracted increasing
interest, the formation and interaction of IPNs formed from
PMAA and PNIPAM have been rarely investigated by fluorescence
spectroscopy mainly due to the lack of suitable luminogens,
which exhibit sensitive response to external stimuli especially in
an aqueous solution.13 Several fluorophores, such as pyrene,
rhodamine and tetraphenylethene (TPE), were covalently attached
to PNIPAM chains to investigate their thermal transitions by
fluorescence methodology. The fluorescence spectrum of the
pyrene containing copolymer in general comprises monomer
and excimer emissions, in which the ratio of these two types of
emissions varies with temperature change.14 The rhodamine
system displays a temperature dependent fluorescence, which
is caused by the PNIPAM dehydration at high temperatures
under acidic conditions.15 However, these common lumino-
gens are not supreme models for the fluorescence probes since
the aggregation-caused quenching effect is undesirable to a
practical application, leading to reduction in efficiencies and
sensitivities of sensors or probes. TPE is an unusual luminogen
system in which aggregation works constructively rather than
destructively in a conventional system, which exhibits high
sensitivity to environmental changes, such as temperature,
hydrophobicity and steric hindrance.16 The emission is induced
by aggregate formation, and thus this phenomenon is termed
as aggregation induced emission (AIE). Due to the uniqueness
of AIE luminogens that are emissive in the solid state, their
applications in optoelectronics,17 fluorescence sensors,18 cell
imaging,19 and explosive chemical detection20 have been paid
increasing attention recently.
Instrumentation
1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker DRX 400 MHz NMR spectrometer in
CDCl3 at room temperature using tetramethylsilane (TMS) as
an internal standard. Operating frequencies of the NMR spec-
trometer were 400.13 MHz (1H) and 100.61 MHz (13C). High
resolution MS (HRMS) were recorded using a Micromass 7034E
mass spectrometer. Elemental analysis was conducted on a
Perkin-Elmer 240C elemental analyzer for C, H, and N determi-
nation. UV-vis and fluorescence spectra were obtained using a
Shimadzu UV3101PC UV-vis-NIR spectrophotometer and a
Perkin-Elmer LS 50B luminescence spectrometer with a Xenon lamp
as a light source, respectively. Thermal analysis was performed on a
Perkin-Elmer thermogravimetric analyzer (TGA 7) in nitrogen or in
air at a heating rate of 20 1C minꢀ1 and on a TA Instruments
Differential Scanning Calorimetry (DSC) 2920 at a heating rate and a
cooling rate of 5 1C minꢀ1 in nitrogen. Dynamic light scattering
experiments were performed on a Brookhaven 90 plus spectrometer
with a temperature controller. An argon ion laser operating at
633 nm was used as a light source.
Preparation of interpolymer complexes
By covalently attaching TPE to the PNIPAN chain through a Stock solutions of copolymers and PMAA in a water or buffer
triazole linkage, PNIPAN displayed high fluorescence sensitivity solution were used for the preparation of inter-polymer complexes.
on the thermally induced chain aggregation of PNIPAM under They were prepared by a drop wise addition of the PMAA stock
neutral conditions, which was used as a fluorescent thermometer solution to copolymer stock solution under continuous stirring.
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The mixtures were incubated at room temperature for 1 day IR (thin film): n = 3440, 3301, 3077, 2975, 2936, 2877, 1652,
prior to the experiment. Polymer composition in the complexes 1549, 1459, 1388, 1368, 1277, 1173, 1131, 701 cmꢀ1
was presented by the polymer molar ratio r, i.e., the ratio of
Copolymer P4. Yield: 88%; 1H NMR (CDCl3): d 3.99 (br, 2H),
molar concentrations of PNIPAM and PMAA. An inter-polymer 2.23–1.40 (m, 3H), 1.14 (s, 6H). Anal. calcd for (C6H11NO)n: C,
composition of the copolymer was kept constant as 0.50 mg mLꢀ1
63.69; H, 9.80; N, 12.38. Found: C, 63.70; H, 9.82; N, 12.38. IR
The pH of the complexes was varied by using appropriate buffer (thin film): n = 3441, 3300, 3077, 2975, 2937, 2877, 1652, 1550,
.
.
solutions: a 10 mM buffer system of sodium phosphate and citric 1459, 1388, 1368, 1278, 1173, 1131, 701 cmꢀ1
.
acid, pH ranging from 2.8 to 7.0.
Copolymer P5. Yield: 79%; 1H NMR (CDCl3): d 3.99–3.88 (m,
2H), 2.26–1.43 (m, 3H), 1.14 (s, 6H). Anal. calcd for (C6H11NO)n:
C, 63.69; H, 9.80; N, 12.38. Found: C, 63.71; H, 9.83; N, 12.37. IR
Synthesis of compounds and polymers
4-(1,2,2-Triphenylvinyl)phenyl acrylate (M1). 4-(1,2,2-Triphenyl- (thin film): n = 3441, 3299, 3080, 2975, 2937, 2877, 1647, 1551,
vinyl)phenol (522.6 mg, 1.5 mmol) was dissolved in dry dichloro- 1460, 1388, 1369, 1280, 1244, 1173, 1132, 701, 668 cmꢀ1
.
1
methane (10.0 mL). After adding 3.0 equiv. of triethylamine
Copolymer P6. Yield: 86%; H NMR (CDCl3): d 3.98 (s, 1H),
(607.0 mL, 4.5 mmol) to the solution, 2.0 equiv. of acryloyl 3.56 (s, 2H) 2.26–1.43 (m, 2H), 1.14 (s, 6H). Anal. calcd for
chloride (235.0 mL, 3.0 mmol) were added drop wise at 0 1C. (C6H11NO)n: C, 63.69; H, 9.80; N, 12.38. Found: C, 63.70; H,
The mixture was then stirred at room temperature for 4 h. The 9.81; N, 12.36. IR (thin film): n = 3438, 3300, 3081, 2975, 2937,
ammonia salts were filtered off and the solvent was evaporated. 2878, 1650, 1550, 1459, 1389, 1369, 1336, 1280, 1245, 1173,
The crude product was purified on a silica gel using column 1132, 701, 668 cmꢀ1
chromatography with a dichloromethane and hexane (1: 2)
mixture as the eluent, giving the desired product as white solids
of 567.8 mg (yield: 94.0%). 1H NMR (CDCl3): d 7.14–7.06 (m,
.
Results and discussion
17H), 6.91 (d, 2H, J = 8.0 Hz), 6.58 (d, 1H, J = 16.8 Hz), 6.30 (dd,
1H, J = 6.8, 10.5 Hz), 5.99 (d, 1H, J = 10.5 Hz). 13C NMR (CDCl3):
d 164.2, 149.1, 143.7, 143.6, 141.5, 141.4, 140.1, 132.5, 132.4,
131.5, 131.4, 128.1, 127.9, 127.8, 126.7, 126.6, 120.8. IR (thin film):
n = 3075, 3049, 3120, 2924, 1952, 1903, 1805, 1745, 1739, 1638,
1598, 1503, 1492, 1443, 1297, 1248, 1199, 1170, 1147, 1022, 997,
902, 764, 753, 701, 614 cmꢀ1. HRMS (ESI†): [M + Na+] calcd for
C29H22O2Na, m/z 425.1517; found, m/z 425.1534.
Synthesis and characterization of monomers and related
copolymers
The synthetic routes leading to copolymers P1–P6 are shown in
Scheme 1. M1 could be prepared in a yield of 94% by the
reaction of 4-(1,2,2-triphenylvinyl)phenol with acryloyl chloride
in the presence of triethylamine in dry dichloromethane.
Monomers M2 and M3 were prepared according to our previous
work (Scheme S1, ESI†).20a,b Poly(N-isopropyl acrylamide-co-
tetra(phenyl)ethene acrylate) P1–P6 were prepared from a mix-
ture of monomers M1–M3 and NIPAM in different ratios by
radical polymerization using AIBN as a radical initiator. P1–P6
were purified by re-precipitation twice in diethyl ether to give
white solids with 79–85% isolated yields.
P1–P6 are not only soluble in many common organic solvents,
such as tetrahydrofuran (THF), dichloromethane, chloroform,
methanol and dimethyl sulfoxide (DMSO), but also in water.
P1–P6 were structurally characterized by various spectroscopic
methods and elemental analysis. Using P1 as an example, the
General procedure for copolymerization
All polymers were prepared by free radical polymerization of
the corresponding monomers in the presence of AIBN. A
solution of 0.50 mmol of monomers and 1.5 mg of AIBN
dissolved in 1.0 mL of dry THF was degassed by three freeze/
thaw cycles. The solutions were stirred for 48 h at 70 1C in
sealed vessels. The reaction mixture was added dropwise into
diethyl ether with stirring, and then the precipitate was collected by
filtration. This purification process was repeated several times to
ensure that all unreacted starting materials were removed. The
polymer was re-dissolved in 5.0 mL of THF and re-precipitated with
Et2O to afford a white solid. The polymer was then filtered and
dried under vacuum at 40 1C for 72 hours for further analysis.
Copolymer P1. Yield: 85%; 1H NMR (CDCl3): d 3.99–3.88 (m,
2H), 2.23–1.40 (m, 3H), 1.14 (s, 6H). Anal. calcd for (C6H11NO)n:
C, 63.69; H, 9.80; N, 12.38. Found: C, 63.74; H, 9.68; N, 12.37. IR
(thin film): n = 3436, 3300, 3077, 2974, 2936, 2877, 1651, 1548,
1459, 1388, 1368, 1276, 1172, 1131, 701 cmꢀ1
.
Copolymer P2. Yield: 87%; 1H NMR (CDCl3): d 3.99–3.73 (m,
2H), 2.23–1.40 (m, 3H), 1.14 (s, 6H). Anal. calcd for (C6H11NO)n:
C, 63.69; H, 9.80; N, 12.38. Found: C, 63.73; H, 9.82; N, 12.36. IR
(thin film): n = 3437, 3299, 3077, 2974, 2936, 2877, 1651, 1549,
1459, 1388, 1368, 1277, 1173, 1131, 702 cmꢀ1
.
Copolymer P3. Yield: 84%; 1H NMR (CDCl3): d 3.99 (br, 2H),
2.23–1.40 (m, 3H), 1.14 (s, 6H). Anal. calcd for (C6H11NO)n: C,
63.69; H, 9.80; N, 12.38. Found: C, 63.72; H, 9.81; N, 12.37.
Scheme 1 Synthetic route of monomer copolymers (P1–P6).
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Table
1
Molecular weight, polydispersity (PDI) and thermal data of
PNIPAM and P1–P6a
Feed
Experimental
Mw (104) Mn (104) PDI Td Tg
b
Polymers ratio x : y m : n
PNIPAM
P1
P2
P3
P4
P5
P6
—
—
1.48
1.62
1.46
1.49
1.60
1.63
1.61
1.01
1.23
1.13
1.15
1.18
1.19
1.19
1.4 357 130
1.3 361 138
1.3 360 137
1.3 357 138
1.4 359 139
1.4 360 138
1.4 361 140
100 : 1
200 : 1
300 : 1
400 : 1
400 : 1
400 : 1
96 : 1
211 : 1
357 : 1
438 : 1
411 : 1
419 : 1
a
Mw: the weight-average molecular weight. Mn: the number-average
b
molecular weight. Td: the decomposition temperature at which 5%
weight loss occurs.
Fig. 1 Fluorescence spectra of P4/PMAA in THF/H2O; lex = 318 nm;
[P4] = 0.5 mg mLꢀ1, 5.0 equiv. of PMAA, 20 1C, inserts are photographs
of P4/PMAA solutions taken under UV illumination (365 nm).
1H NMR spectrum of P1 is illustrated in Fig. S1 (ESI†). The
presence of signals at d 7.00–6.52 corresponding to protons of
TPE indicated that the TPE residues remained intact during the
radical polymerization and purification process. The molecular
weights of these copolymers against polystyrene standards were
determined by gel permeation chromatography (GPC) and the
data are summarized in Table 1. P1–P6 have high molecular
weights of 14 600–16 300 with a relatively narrow polydispersity
index of 1.3–1.4. Those polymers exhibit high thermal stability
with decomposition temperatures ranging from 357 to 361 1C
under nitrogen, which are comparable to 357 1C of PNIPAM
(Fig. S2a, ESI†). The DSC study indicates that P1–P6 exhibit a
entangling of polymer chains has no effect on the fluorescence
enhancement (ESI,† Fig. S6).
Considering the good fluorescence response of TPE to the phase
change of the polymer chain, TPE could be used as a fluorescence
probe to investigate the formation of hydrogen bonds in PMAA and
PNIPAM interpenetrating polymer networks.
The thermally induced fluorescence of copolymers
glass transition temperature of 138–140 1C under nitrogen, Fig. 2a shows the effect of temperature on P1–P6 fluorescence
which is slightly higher than that of PNIPAM (DTg 4 7 1C, in an aqueous solution. Taking P4 for example, a small change
ESI,† Fig. S2b). The ratio of TPE to NIPAM was determined by in the emission intensity was observed when P4 was heated
UV-vis absorption spectra (ESI,† Fig. S3).
from 14.0 to 25.0 1C, and a continuous increase in fluorescence
intensity was recorded in the range of 25.0 to 30.0 1C, finally the
fluorescence intensity reached the maximum at 32.5 1C.
AIE properties of copolymers
P1–P6 were non-emissive when dissolved in THF, which is a Further heating to 50.0 1C led to no change in the fluorescence
good solvent for both TPE and NIPAM. P1–P6 with different intensity. When cooled down from 50.0 to 14.0 1C, a reversible
degrees of TPE labelling also showed similar AIE properties. temperature-dependent fluorescence curve was obtained with the
Taking P4 solution in THF as an example (Fig. S4, ESI†), the same trend in the same temperature regions (ESI,† Fig. S6a). This
fluorescence started to become obvious when a large amount of phenomenon is supported by dynamic light scattering (DLS) and
H2O (470%) was added, indicating that TPE maintained its AIE solution turbidity measurements (Fig. 2b and Fig. S6b, ESI†), which
activity after being incorporated into the polymer NIPAM, show the reversible temperature-dependent particle size and
which was further evidenced by the strong emission of films solution turbidity curves, implying that P4 chains swell well in
fabricated from P1–P6 and M1–M3 (ESI,† Fig. S5). When water at the beginning and start to collapse at 22.5 1C due to
compared to P1–P6 films, obvious red-shifts (4–6 nm) in emission dehydration, and the particle reaches the smallest size at 32.5 1C.
spectra were found for the M1–M3 films, implying their better
As shown in Fig. 2, P1–P3 with a higher degree of labeled
stacking in the solid state. The AIE property of the P4–PMAA TPE display down-shift LSCT and easily precipitate out when
complex in a THF/H2O mixture was studied. As shown in Fig. 1, heating. It is well known that the conformation of the PNIPAM
the mixture showed very good solubility in pure THF and was non- chain is very sensitive to its structure, and even a subtle
emissive. While increasing water content, the fluorescence could be modification in the structure can result in a huge change.21
turned on when water content is 60%, and finally reached the Thus, by tuning the degree of labelled TPE, the conformation of
maximum fluorescence intensity in 100% water. The P4–PMAA the PNIPAM chain could be modified well, and P4–P6 with a
complex showed similar AIE properties to P4, but emitted 1.5-fold very low degree of labeled TPE keep the similar conformation of
stronger fluorescence than that of neat P4 with the same concen- PNIPAM.
tration, because the polymer interaction suppressed the molecular
The starting point of fluorescence enhancement for P4
motions of TPE.16a Another possibility to restrict the TPE motion is relates slightly to its concentration, e.g. from 0.1 to 1.0 mg mLꢀ1
that the entangling of polymer chains may lead to the enhance- (ESI,† Fig. S7). However, the fluorescence intensity of polymers with
ment in fluorescence. No obvious increase in fluorescence intensity a higher concentration, such as 1.0 mg mLꢀ1 solution, decreased
in a non-hydrogen-bonded system P4/polystyrene indicates that swiftly on heating from 37.0 to 50.0 1C, due to their precipitation at
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Scheme 2 The illustration of PMAA, PNIPAM and the hydrogen-bond
formation between the two networks in the IPNs.
Fig. 2 (a) Plot of I/I0 vs. temperature of P1–P6 in H2O. (b) The particle
size vs. temperature of P1–P6 in H2O. The concentration of the polymer is
0.50 mg mLꢀ1, I0 = intensity at 469 nm at 14 1C, excited at 318 nm.
a higher temperature than LCST. Moreover, as shown in Fig. 2,
P1–P3 with higher TPE labeling degrees showed lower LSCT of
30.0, 28.5 and 18.0 1C, respectively. As the hydrophobic inter-
action between the TPE residues may enable the aggregation of
TPE, this likely promotes the hydrophobic backbones of PNIPAM
chains to conglobulate.16a While P5 and P6 functioned similarly
as P4, and the LSCTs were not changed, although TPE was
attached to the PNIPAM chain through an alkyl linkage. Thus,
P4–P6 with a lowest degree of TPE maintained the same LSCT as
that of PNIPAM, and the LSCT of copolymers could be tuned by
adjusting the degree of TPE residues in copolymers.
Formation of interpenetrating polymer networks
Scheme 2 illustrates the hydrogen bond formation in IPNs
formed by P4 and PMAA. The formation and interaction of
hydrogen bonds between PMAA and P4 was investigated by
fluorescence spectroscopy in the pH range of 2.8–7.0, and the
concentration of P4 was set as 0.50 mg mLꢀ1. Fig. 3a sum-
marizes the fluorescence spectra of the PMAA–P4 complex with
different compositions at pH 2.8, 3.4, 4.0, 4.6, 5.2 and 7.0,
respectively. Pronounced changes in the fluorescence intensity
were observed upon PMAA addition at pH values less than 5.2,
Fig. 3 (a) Plot of I/I0 vs. ratio of PMMA and P4 at different pH, r = 0–5.0,
pH = 2.8–7.0. (b) Plot of I/I0 vs. pH at r = 3.0. (c) Plot of I/I0 vs. ratio of
PMMA and P4–P6 at pH = 4.0. [Copolymer] = 0.5 mg mLꢀ1, 10.0 mM
Na2HPO4–citric acid buffer, I0 = intensity at 469 nm at 20 1C, lex = 318 nm.
and fluorescence was further enhanced until the ratio of PMAA (ca. 10-fold), suggesting that no strong interaction takes place
and polymer (r) is 3.0. As shown in Fig. 3b, the fluorescence between P4 and PMAA. In other words, no inter-polymer com-
enhancement fold reached a maximum of 1.93 at pH 2.8. When plexes are formed between P4 and PMAA although mixed in the
the pH was increased to 7.0, fluorescence enhancement was solution. This phenomenon could be related to the high
not observed even with the addition of high excess of PMAA ionization degree of PMAA at pH 7.0 that it is not able to form
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Fig. 4 (a) FTIR spectra of solid P4 and IPNs4 under different pH condi-
tions. IPNs4 solid samples were prepared from solutions by freeze drying
to remove water. (b) 1H NMR spectra of P4, PMAA and IPNs4 in D2O at
various temperatures, [P4] = 0.50 mg mLꢀ1, r = 3.0.
Fig. 5 (a) Plot of I/I0 vs. temperature of IPNs4 during a heating and
cooling cycle at pH = 4 and r = 3. (b) The particle size and solution
turbidity (kcps) vs. temperature of IPNs4. [P4] = 0.50 mg mLꢀ1, r = 3.0,
pH = 4.0, 10.0 mM Na2HPO4–citric acid buffer. I0 = intensity at 469 nm at
14 1C, lex = 318 nm.
hydrogen bonds with PNIPAM, because PMAA has a related
low pKint of 4.85.22 All fluorescence changes occurred in a While further heating to 50 1C, a continuous decrease in
narrow range of r from 1.0 to 3.0, indicative of the cooperative fluorescence density was observed due to thermally activated
character of IPNs4 formation. However, P5 and P6 labeled with molecular motions of TPE,16a which was supported by the DLS
TPE units through a longer alkyl chain displayed much lower results that particle sizes became smaller but no precipitation
fluorescence enhancement upon PMAA addition (Fig. 3c). FTIR was produced during this heating process (Fig. 5b). When
spectra was widely used to verify the formation of hydrogen cooled down from 50.0 to 14.0 1C, a coincided fluorescence
bonds.23 Using P4 and its IPNs as examples, FTIR spectra of P4, intensity–temperature curve and a particle size curve were
PMAA and IPNs4 at different pH values were studied. As shown obtained, implying its good thermal reversibility. The fluores-
in Fig. 4a, the peak at 1707 cmꢀ1 represents the carbonyl cence and DLS measurements showed the LCST of IPNs4 at
1
(–CQO) stretching vibration in the carboxylic acid groups of 27.5 1C, which was 5.0 1C lower than that of P4. The H NMR
PMAA. The peaks at 1655 and 1551 cmꢀ1 belong to –CQO spectra of IPNs4 are measured in D2O at various temperatures
stretching vibration and –N–H deformation in the amide groups to give insight into its thermal transitions (Fig. 4b). IPNs4 start
in P4, respectively. Their mixture, for example, IPNs4 at pH 4.0, to dehydrate at 25 1C but still swell well in H2O. The protons
exhibits two signals at 1641 and 1574 cmꢀ1, corresponding to the chains could be identified in the 1H NMR spectra when the
red-shifted –CQO stretching vibration of carboxylic acid and the temperature is at 25 1C. However, when IPNs4 solution is
amide group of P4, and blue-shifted –N–H deformation in the heated up to 35 1C or higher, IPNs4 collapse to form compact
amide group, indicating the formation of hydrogen bonds between particles and thus lead to a gradual decrease in intensity of
P4 and PMAA.
NMR peaks.
The temperature dependent fluorescence transition of IPNs4
IPNs4 particles under different pH and temperatures were
at a constant mixture composition (r = 3.0, Fig. 5a) was examined (Fig. 6). IPNs4 particles with a bright blue emission,
examined. At pH 4.0, a noticeable difference in the fluorescence which resemble the stars in the galaxy, could be clearly
trend was observed, which could be split into three stages. A big observed upon excitation with UV light (excitation wavelength:
bump in the emission intensity of IPNs4 was found when it is 378 nm). Fig. 6c, g and h display IPNs4 particles at 20, 35 and
heated from 20.0 to 27.5 1C, and the fluorescence intensity 50 1C, respectively, which is consistent with observations in
reached the maximum at 27.5 1C. In the range from 27.5 to Fig. 5a. Under low pH (2.8, 3.4 and 4.0), IPNs4 formed crystal-
35.0 1C, a stable fluorescence intensity region was recorded, like particles with strong fluorescence emission due to the
indicating its thermal tolerance during this temperature range. more efficient hydrogen bonding at a lower pH. While at a
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Fig. 6 Optical microscopy images and fluorescence microscopy images
(inserted photos) of IPNs4 particles under different pH and temperatures:
(a) 2.8, 20 1C; (b) 3.4, 20 1C; (c) 4.0, 20 1C; (d) 4.6, 20 1C; (e) 5.2, 20 1C;
(f) 7.0, 20 1C; (g) 4.0, 35 1C; and (h) 4.0, 50 1C. The fluorescence
microscopy images of particles were taken using a fluorescence micro-
Fig. 8 Schematic illustration of the structures of P4–PMAA complexes at
different pH and temperatures.
scope with a 337 nm excitation; scale bar is 50 mm. [P4] = 0.50 mg mLꢀ1
r = 3.0, 10.0 mM Na2HPO4–citric acid buffer.
,
Considering the down-shift LSCT together with the fluores-
cence transition, the effect of mixture composition and the pH
value on the structure of IPNs4 is illustrated in Fig. 8. When pH
(7.0 and 5.2) 4 pKint, less hydrogen bonds between PMAA and
PNIPAM were formed because most of the carboxylic groups in
PMAA were ionized. Even when the PMAA content in a mixture
was very high (for example, r = 10.0), LCST of PNIPAM changed
only slightly. In this case, the length of the hydrophobic
PNIPAM blocks in the complexes was long enough to provide
high cooperativity of the phase transition of PNIPAM, and thus
fluorescence transitions of mixtures show similar trends to P4.
At pH (4.6, 4.0, 3.4 and 2.8) o pKint, due to the fact that more
carboxylic groups in PMAA were protonated, more hydrogen
bonds between PNIPAM were generated to form IPNs4. Under
this condition, the phase transition of PNIPAM will be affected
even though the PMAA content is low. In the IPNs4 complex, the
hydrophobic blocks are much shorter, consequently resulting in
the low transition cooperativity. Thus, the down shifts of LSCT
for IPNs4 were observed with an increase in pH values.
higher pH than 4.0, IPNs4 become amorphous and emitted less
fluorescence.
At higher pH values than pKint (4.85) as shown in Fig. 7a, IPNs4
tend to display similar fluorescence behavior to P4 because of the
weaker hydrogen bonding interaction caused by ionization in
different degrees of PMAA. On the other hand, a decrease in pH
from 7.0 to 4.6 led to a gentle reduction in LSCT from 32.5 to
29.0 1C. For pH 3.4 and 2.8, IPNs4 showed a slight opalescence and
started to precipitate out on heating up to 28.0 1C (Fig. 7b).
Overall, the ionized PMAA chains introduced more changes to
the mixture of PMAA–P4, which improved the solubility of IPNs4 and
maintained the cooperativity of phase transition of P4. In contrast,
PNIPAM chains could be hydrophilized by conjugation with more
hydrophilic PMAA to generate IPNs4 through the formation of
hydrogen bonding, leading to a decrease in LSCT and P4 lost its
original features as that of PNIPAM.
Conclusions
In summary, a new type of IPNs was formed through the inter-
molecular hydrogen bonding interaction between PMAA and
PNIPAM containing AIE active TPE. The TPE moieties that were
covalently linked to the PNIPAM chain displayed highly sensi-
tive response to the phase transition of IPNs, which could be
used as a fluorescence probe to track the IPNs’ structural
changes caused by external pH and temperature. By adjusting
pH, the formed IPNs would be able to fold cooperatively to a
compact structure without a loss of solubility at temperatures
below the LCST. Thus, these IPNs would be used as convenient
drug delivery systems, in which the release process can be
monitored by fluorescence spectroscopy in situ.
Fig. 7 (a) Plot of I/I0 vs. temperature of IPNs4. (b) Plot of the particle
size of IPNs4 vs. temperature. [P4] = 0.50 g mLꢀ1, r = 3.0, pH = 2.8–7.0,
10.0 mM Na2HPO4–citric acid buffer. I0 = intensity at 469 nm at 14 1C,
lex = 318 nm.
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Acknowledgements
The authors would like to acknowledge the financial support
(Grant No: 1321760011) from the Institute of Materials Research
and Engineering (IMRE), Agency for Science, Technology and
Research (A*STAR).
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