A series of poly[N-(2-hydroxypropyl)methacrylamide] copolymers with anthracene-derived fluorophores showing aggregation-induced emission properties for bioimaging

Article abstract of DOI:10.1002/pola.25841

A series of new poly[N-(2-hydroxypropyl)methacrylamide]-based amphiphilic copolymers were synthesized through a radical copolymerization of a monomeric/hydrophobic fluorophore possessing aggregation-induced emission (AIE) properties with N-(2-hydroxypropyl)methacrylamide. Photophysical properties were investigated using UV-Vis absorbance and fluorescence spectrophotometry. Influences of the polymer structures with different molar ratios of the AIE fluorophores on their photophysical properties were studied. Results show that the AIE fluorophores aggregate in the cores of the micelles formed from the amphiphilic random copolymers and polymers with more hydrophobic AIE fluorophores facilitate stronger aggregations of the AIE segments to obtain higher quantum efficiencies. The polymers reported herein have good water solubility, enabling the application of hydrophobic AIE materials in biological conditions. The polymers were endocytosed by two experimental cell lines, human brain glioblastoma U87MG cells and human esophagus premalignant CP-A, with a distribution into the cytoplasm. The polymers are noncytotoxic to the two cell lines at a polymer concentration of 1 mg/mL.

Full text of DOI:10.1002/pola.25841

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A Series of Poly[N-(2-Hydroxypropyl)methacrylamide] Copolymers with
Anthracene-Derived Fluorophores Showing Aggregation-Induced
Emission Properties for Bioimaging
1
,2
1
1
1
1
2
Hongguang Lu, Fengyu Su, Qian Mei, Xianfeng Zhou, Yanqing Tian, Wenjing Tian,
1
1
Roger H. Johnson, Deirdre R. Meldrum
1
Center for Biosignatures Discovery Automation, Biodesign Institute, Arizona State University, Tempe, Arizona 85287
2
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012,
People’s Republic of China
Correspondence to: Y. Tian (E-mail: yanqing.tian@asu.edu)
Received 22 August 2011; accepted 7 November 2011; published online 28 November 2011
DOI: 10.1002/pola.25841
ABSTRACT: A series of new poly[N-(2-hydroxypropyl)methacryla-
mide]-based amphiphilic copolymers were synthesized through a
radical copolymerization of a monomeric/hydrophobic fluoro-
phore possessing aggregation-induced emission (AIE) properties
with N-(2-hydroxypropyl)methacrylamide. Photophysical proper-
ties were investigated using UV–Vis absorbance and fluorescence
spectrophotometry. Influences of the polymer structures with dif-
ferent molar ratios of the AIE fluorophores on their photophysical
properties were studied. Results show that the AIE fluorophores
aggregate in the cores of the micelles formed from the amphi-
philic random copolymers and polymers with more hydrophobic
AIE fluorophores facilitate stronger aggregations of the AIE seg-
ments to obtain higher quantum efficiencies. The polymers
reported herein have good water solubility, enabling the applica-
tion of hydrophobic AIE materials in biological conditions. The
polymers were endocytosed by two experimental cell lines,
human brain glioblastoma U87MG cells and human esophagus
premalignant CP-A, with a distribution into the cytoplasm. The
polymers are noncytotoxic to the two cell lines at a polymer con-
centration of 1 mg/mL. VC 2011 Wiley Periodicals, Inc. J Polym Sci
Part A: Polym Chem 50: 890–899, 2012
KEYWORDS: biopolymers; dyes; fluorescence; imaging; self-
assembly
INTRODUCTION Fluorescence-based optical imaging plays a
crucial role for biological investigation, for example, for the
understanding of biological processes, metabolism and phar-
macokinetics, as well as for the diagnosis/detection of dis-
ease and cancer formation either in vitro or in vivo. Many
fluorescent probes are suitable for optical imaging. Typical
fluorescent probes include but are not limited to fluorescein
been suggested as the possible mechanism of AIE phenom-
enon. The vibrational/torsional motions of the molecules
affect drastically the radiative/nonradiative recombination
processes of the excited state. In biological research fields,
AIE fluorophores have been widely explored as sensors
2
6,27
28
29
30,31
32
33
for DNA,
heparin, ATP, pH,
CO2, and glucose.
Fewer studies were reported about the applications of the
1
1
1
34–39
derivatives, rhodamines, cyanine derivatives, quantum
AIE fluorophores as bioimaging probes
than those about
1
2,3
dots, indocyanine greens (ICGs),
upconversion nanopar-
light emitting diodes and biosensors. This is mainly because
that most of the AIE fluorophores are hydrophobic, limiting
their applications in biological environments. Chemical
modification of the AIE fluorophores with polar functional
groups can improve their solubility/dispersion in aqueous
4
–6
7–14
ticles,
and two-photon absorbing materials.
In general,
these fluorescent probes work well for bioimaging. However,
most of these probes suffer a common problem. If the con-
centration is too high, many of the aforementioned fluores-
cent probes tend to form aggregates, causing aggregation-
3
1,40,41
media.
Nevertheless, most of these chemical modifica-
1
5–
induced quenching (AIQ) to reduce fluorescence intensity.
tions not only require complicated synthetic procedures, but
also sometimes lower the fluorescence quantum efficiencies
due to quenching of their excited states by water and nonra-
1
8
This AIQ effect impedes the performance for bioimaging.
Recently, a new category of fluorophores with exactly the op-
posite characteristic to the AIQ, aggregation-induced emis-
sion (AIE), has been developed. AIE fluorophores have been
shown to have high fluorescence quantum yields in the
4
2
diative decay of the excited dyes in aqueous media. A few
advanced approaches have been used to alleviate these prob-
lems and enable the use of AIE fluorophores for bioimag-
1
9–25
34–39
aggregated states.
Restricted intramolecular motion has
ing.
Nanoparticles such as organically modified silica
V
C
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SCHEME 1 Synthesis of the AIE monomer (AIEM) and its copolymers.
3
4–37
nanoparticles
and amphiphilic block copolymers-based
aminoethyl methacrylate ) (PAEMA) is to enhance the cellu-
lar uptake of the polymers by using cationic amino
3
8
micelles were utilized as nanocarriers to deliver hydropho-
bic AIE probes into cells. Besides the physical incorporation
of hydrophobic AIE fluorophores into nanoparticles, AIE fluo-
rophores were chemically conjugated to silica nanopar-
ticles and biocompatible hydrophilic glycol chitosan for
bioimaging. We have recently reported a class of 9,10-dis-
tyrylanthracene (DSA) derivatives with AIE properties. The
investigation indicates that the restricted intramolecular
rotations between the 9,10-anthylene core and the vinylene
segment are the cause for the AIE phenomenon. We also
used anthracene derived materials as typical AIE fluoro-
phores for pH and DNA sensing. Herein, we report the de-
velopment and bioapplications of a new series of AIE-fluoro-
phore-containing random copolymers, polymerized from an
AIE fluorophore with a styrene moiety as a monomer (AIEM,
Scheme 1), N-(2-hydroxypropyl)methacrylamide (HPMA),
and 2-aminoethyl methacrylate (AEMA). Poly[N-(2-hydroxy-
propyl)methacrylamide] (PHPMA) is a biocompatible poly-
mer with little or non-cytotoxicity, and has been widely
applied in biological research fields of drug delivery, in vivo
4
6–48
groups.
Thus, in light of the development of AIE fluoro-
phores for bioimaging, we will for the first time report the
preparation of biocompatible AIE-fluorophore-containing
random copolymers for bioimaging. Through the tuning of
polymeric structures and the molar ratios of AIE fluoro-
phores in the copolymers, polymer structure-dependent opti-
cal properties were investigated. Bioimaging of the polymers
for two cell lines (human brain glioblastoma U87MG cells
and human esophagus premalignant CP-A) was investigated.
Considering the simple approach of the preparation of ran-
dom copolymers with AIE fluorophores and the abundant
biocompatible polymer structures, it is expected that the
polymer approach will enable the wide applications of
hydrophobic AIE fluorophores for bioimaging.
3
6
3
9
2
5
3
1
EXPERIMENTAL
Materials and Reagents
All chemicals and solvents were analytical grade and used
4
3–45
0
imaging, and photodynamic therapy.
The use of poly(2-
without further purification. Tetrahydrofuran (THF), N,N -
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dimethylformamide (DMF), dimethyl sulfoxide (DMSO), azo-
bisisobutyronitrile (AIBN), anthracene, potassium tert-butox-
ide (KOtBu), n-hexyl bromide, potassium carbonate, 6-chloro-
H NMR (CDCl , 400 MHz), d (TMS, ppm): 8.37-8.39 (m, 4H),
3
7.78 (d, J ¼ 16.5Hz, 2H), 7.60 (d, J ¼ 8.5 Hz, 4H), 7.43-7.46
(m, 4H), 7.13 (d, J ¼ 8.5Hz, 4H), 6.86 (d, J ¼ 16.5Hz, 2H),
5.50 (t, 2H), 3.91-3.97 (m, 2H), 3.61-3.66 (m, 2H), 1.89-2.05
1
-hexanol, potassium iodide, 4-vinylbenzyl chloride, sodium
hydride (NaH), 2-aminoethyl methacrylate hydrochloride
AEMA), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
1
3
(m, 4H), 1.55-1.71 (m, 8H); C NMR (DMSO-d , 100 MHz), d
6
(
(TMS, ppm): 156.97, 136.86, 132.74, 130.98, 129.58, 127.66,
126.49, 125.06, 123.15, 116.78, 96.30, 61.99, 30.30, 25.20,
18.71; HRMS (APCIþ): C H O (MþH), calcd. 583.2848;
acid (HEPES) were commercially available from Sigma-
Aldrich (St. Louis, MO) and were used without further purifi-
cation. Monomer HPMA was prepared according to the
4
0 39 4
found 583.2839.
4
3–45
known procedure.
methylene)phosphonate (compound 1) and 4-tetrahydro-py-
ran-2-yloxy-benzaldehyde (compound 2) were synthesized
Tetraethyl anthracene-9,10-bis
Synthesis of 9,10-bis(4-hydroxystyryl)anthracene (4). 1.75 g
of compound 3 (3.0 mmol) was dissolved into 100 mL of
THF and 5 mL of methanol, and then 10 mL of 2 N HCl
aqueous solution was added into the solution. The reaction
mixture was stirred at room temperature for 12 h. After the
solvent was removed under reduced pressure, the residue
was washed with methanol. The solid was redissolved into 5
mL of CH Cl and reprecipitated into 200 mL of methanol to
(
4
9,50
according to known procedures.
Eagle’s minimum essential medium (EMEM) and Keratino-
cyte medium were purchased from Invitrogen (Carlsbad, CA).
EMEM was used for U87 cell culture. Keratinocyte medium
was used for CP-A cell culture. 3-(4,5-Dimethyl thiazol-2-yl)-
2
2
2,5-diphenyltetrazolium bromide (MTT) assay was purchased
give a yellow solid (1.10 g, 88% yield).
from Promega (Madison, WI).
1
H NMR (DMSO-d , 400 MHz), d (TMS, ppm): 9.67 (s, 2H),
6
8
8
.32-8.34 (m, 4H), 7.83 (d, J ¼ 16.5Hz, 2H), 7.60 (d, J ¼
.5Hz, 4H), 7.48-7.51 (m, 4H), 6.82 (d, J ¼ 8.5Hz, 4H), 6.77
Instruments
1
1
3
A Varian liquid-state NMR operated at 400 MHz for H NMR
(
d, J ¼ 16.5Hz, 2H); C NMR (DMSO-d , 100 MHz), d (TMS,
6
1
3
and 100 MHz for C NMR was used for NMR spectra meas-
urements. High resolution mass spectrometry (HRMS) was
performed by the ASU Mass Spectrometry Laboratory. A Shi-
madzu UV-3600 UV-VIS-NIR spectrophotometer (Shimadzu
Scientific Instruments, Columbia, MD) was used for absorb-
ance measurements. A Shimadzu RF-5301 spectrofluoropho-
tometer was used for fluorescence measurements. Waters
ppm): 157.61, 137.03, 132.36, 128.98, 128.09, 128.06,
26.22, 125.31, 121.11, 115.55; HRMS (APCIþ): C30
MþH), calcd. 415.1698; found 415.1699.
1
(
23 2
H O
Synthesis of 4-((E)-2-(10-(4-(hexyloxy)styryl)anthracen-9-
yl)vinyl)phenol (5). Into a 25 mL round-bottomed flask were
added 0.42 g of compound 4 (1.0 mmol) and 0.21 g of
K CO (1.5 mmol) in 10 mL of anhydrous DMF. A solution of
2
3
1515 GPC coupled with a RI detector, in reference to a series
0.15 g of 1-bromohexane (0.9 mmol) in 2 mL of DMF was
of poly(2-vinylpyridine) standards in 5% acetic acid aqueous
solution as the eluent, was used for polymer molecular
weight determination. Dynamic light scattering (DLS) meas-
added to the mixture under stirring. The mixture was stirred
for 12 h at 60 C. After being cooled to room temperature,
the reaction mixture was poured into water and extracted
ꢀ
ꢀ
urements were performed using a 173 back scattering Mal-
with CH Cl2 (3 ꢂ 40 mL). The combined organic extracts
2
vern Nano-ZS instrument. The solution was filtered through
a 0.45 lm Millipore Millex-HN filter to remove dust before
DLS measurements. Atomic force microscopy (AFM, Nano-
Scope III, Digital Instrument) equipped with an integrated
silicon tip/cantilever with a resonance frequency of ꢁ240
kHz in height image model was utilized for the observation
of morphologies. Polymer solutions (4 lL) were dropped on
a mica sample stage and dried at room temperature for the
morphological observation. The AFM topographies showed
no evidence of tip-induced modification during successive
scans.
were washed with brine, dried over MgSO , and concen-
4
trated to dryness under vacuum. The crude product was
purified by silica gel chromatography (CH Cl ) to give a yel-
low solid (0.23 g, 45% yield).
2
2
1
H NMR (CDCl , 400 MHz), d (TMS, ppm): 8.37-8.40 (m, 4H),
3
7.76 (d, J ¼ 16.5Hz, 2H), 7.56-7.61 (m, 4H), 7.43-7.46 (m,
4H), 6.97 (d, J ¼ 8.5Hz, 2H), 6.91 (d, J ¼ 8.5Hz, 2H), 6.83-
6.88 (m, 2H), 4.79 (s, 1H), 4.02 (t, 2H), 1.78-1.85 (m, 2H),
1.34-1.51 (m, 6H), 0.917 (t, 3H); C NMR (CDCl
d (TMS, ppm): 159.15, 155.44, 136.90, 136.69, 132.86,
1
3
, 100 MHz),
3
1
1
6
32.62, 130.45, 129.96, 129.60, 127.99, 127.75, 126.53,
26.46, 125.09, 125.05, 123.01, 122.69, 115.67, 114.81,
8.18, 31.60, 29.23, 25.73, 22.62, 14.06; HRMS (APCIþ):
Synthesis
Synthesis of 9,10-bis(4-(tetrahydro-2H-pyran-2-yloxy)styry-
l)anthracene (3). 2.40 g of compound 1 (5.0 mmol) and 1.68
g of potassium tert-butoxide (15.0 mmol) were dissolved in
C H O (MþH), calcd. 499.2637; found 499.2638.
Synthesis of 6-[4-(2-{10-[2-(4-hexyloxy-phenyl)-vinyl]-anthra-
cen-9-yl}-vinyl)-phenoxy]-hexanol (6). Into a 25 mL round-
bottomed flask were added 1.20 g of compound 5 (2.4 mmol)
and 0.50 g of K CO (3.6 mmol) in 10 mL of anhydrous DMF.
2 3
A solution of 0.52 g of 6-chloro-1-hexanol (2.9 mmol) in 2 mL
of DMF was added to the mixture under stirring. The mixture
3
6 35 2
100 mL of anhydrous THF. A solution of 2.47 g of compound
2
(12.0 mmol) in 20 mL of anhydrous THF was added drop-
ꢀ
wisely over a 20 min period at 0 C. The resultant mixture
was allowed to warm to room temperature and then stirred
overnight. After solvent evaporation, the residue was redis-
solved into 5 mL of CH Cl and reprecipitated into 200 mL
ꢀ
was stirred for 12 h at 60 C. After being cooled to room tem-
2
2
of methanol to afford a yellow solid (2.18 g, 75% yield).
perature, the reaction mixture was poured into water and
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extracted with CH Cl (3 ꢂ 50 mL). The combined organic
ACH COA), 1.41-2.01 (broad, backbone protons, A(CH ) A),
1.08 (broad, ACH ).
3
2
2
2
2 5
extracts were washed with brine, dried over MgSO
4
, and con-
centrated to dryness under vacuum. The crude product was
purified by silica gel chromatography (CH Cl ) to give a yel-
low solid (0.75 g, 52% yield).
Synthesis of poly(N-(2-hydroxypropyl) methacrylamide)-co-
poly(2-aminoethyl methacrylate)-co-poly[4-[6-[4-(2-{10-
2-(4-hexyloxy-phenyl)-vinyl]-anthracen-9-yl}-vinyl)-phenoxy]-
2
2
[
1
H NMR (CDCl , 400 MHz), d (TMS, ppm): 8.38-8.40 (m, 4H),
hexyloxymethyl] styrene} (P2) with molar ratios of x:y:z of
100: 3.5: 2. Similar condition as P1, except the weight of
AIEM used for this polymerization was 50 mg. Yield: 80%.
M ¼ 11600, M ¼ 15200, M /M ¼ 1.31.
3
7
6
3
.75-7.81 (m, 2H), 7.59-7.63 (m, 4H), 7.44-7.46 (m, 4H),
.97-7.00 (m, 4H), 6.84-6.90 (m, 2H), 4.01-4.04 (m, 4H),
.66-3.70 (m, 2H), 1.82-1.86 (m, 4H), 1.36-1.49 (m, 12H),
n
w
w
n
1
3
0.92 (t, 3H); C NMR (CDCl
3
, 100 MHz), d (TMS, ppm):
1
H NMR (DMSO-d , 400 MHz), d (TMS, ppm): 7.00-8.39
6
1
1
1
2
59.15, 159.07, 136.88, 136.84, 132.78, 132.75, 130.03,
29.96, 129.60, 127.74, 126.50, 125.04, 122.77, 122.70,
14.79, 68.10, 67.97, 62.92, 32.69, 31.60, 29.23, 25.90,
5.72, 25.55, 22.62, 14.05; HRMS (APCIþ): C H O (MþH),
(
broad, aromatic protons), 4.67 (broad, ACH
2
OCO), 4.32
(
broad, ACH O-Ph), 4.00 (broad, ACH OA), 3.64 (broad,
2
2
ACH NHA), 2.85 (broad, ACH COA), 1.41-2.01 (broad, back-
2
2
4
2 47 3
bone potons, A (CH ) A), 0.98 (broad, ACH ).
2
5
3
calcd. 599.3525; found 599.3528.
Synthesis of poly(N-(2-hydroxypropyl) methacrylamide)-co-
poly(2-aminoethyl methacrylate)-co-poly[4-[6-[4-(2-{10-[2-
Synthesis of 9-(4-(hexyloxy)styryl)-10-(4-(6-(4-vinylbenzylox-
y)hexyloxy)styryl)anthracene (AIEM). 1.20 g of compound 6
(
4-hexyloxy-phenyl)-vinyl]-anthracen-9-yl}-vinyl)-phenoxy]-
hexyloxymethyl] styrene} (P3) with molar ratios of x:y:z of
00: 3.5: 4. Similar condition as P1, except the weight of
AIEM used for this polymerization was 100 mg. Yield:
(
2.0 mmol) was dissolved in 10 mL of anhydrous DMF. 0.46
g of 4-vinylbenzyl chloride (3.0 mmol) and 0.80 g of 60%
NaH in mineral oil (20.0 mmol) were added into the reaction
1
mixture under N atmosphere. The mixture was stirred for
2
6
6%. M ¼ 11800, M ¼ 15700, M /M ¼ 1.33.
n
w
w
n
ꢀ
1
2 h at 60 C. After being cooled to room temperature, the
1
H NMR (DMSO-d , 400 MHz), d (TMS, ppm): 6.97-8.33
reaction mixture was poured into water and extracted with
CH Cl
6
(
broad, aromatic protons), 4.67 (broad, ACH OCO), 4.32
2
2
(3 ꢂ 50 mL). The combined organic extracts were
2
washed with brine, dried over MgSO , and concentrated to
(broad, ACH
ACH NHA), 2.85 (broad, ACH
bone protons, A(CH A), 1.01 (broad, ACH
3
2
OAPh), 4.00 (broad, ACH
COA), 1.21-1.87 (broad, back-
).
2
OA), 3.64 (broad,
4
dryness under vacuum. The crude product was purified by
2
2
silica gel chromatography (Hexane: CH Cl ¼ 5:1) to give a
2
)
5
2
2
yellow solid (1.16 g, 81% yield).
Synthesis of poly(N-(2-hydroxypropyl) methacrylamide)-co-
poly(2-aminoethyl methacrylate)-co-poly[4-[6-[4-(2-{10-[2-
(4-hexyloxy-phenyl)-vinyl]-anthracen-9-yl}-vinyl)-phenoxy]-
hexyloxymethyl] styrene} (P4) with molar ratios of x:y:z of
100: 3.5: 8. Similar condition as P1, except the weight of
AIEM used for this polymerization was 200 mg. Yield:
1
H NMR (CDCl , 400 MHz), d (TMS, ppm): 8.38-8.40 (m, 4H),
3
7
.76 (d, J ¼ 16.5Hz, 2H), 7.59 (d, J ¼ 8.5 Hz, 4H), 7.44-7.46
(
m, 4H), 7.38 (d, J ¼ 8.5Hz, 2H), 7.29 (d, J ¼ 8.5Hz, 2H), 6.95-
6
5
2
0
1
1
1
2
.98 (m, 4H), 6.85 (d, J ¼ 16.5Hz, 2H), 6.67-6.74 (m, 1H),
.73 (d, 1H), 5.21 (d, 1H), 4.45 (s, 2H), 4.02 (t, 4H), 3.48 (t,
H), 1.82 (m, 4H), 1.66 (m, 2H) 1.48 (m, 6H), 1.35 (m, 4H),
70%. M
¼ 30300, M
¼ 34800, M
/M
¼ 1.16.
n
w
w
n
1
3
.92 (t, 3H); C NMR (CDCl , 100 MHz), d (TMS, ppm):
1
3
H NMR (DMSO-d , 400 MHz), d (TMS, ppm): 6.84-8.39
6
59.17, 159.12, 136.89, 136.58, 132.79, 130.01, 129.98,
29.62, 127.83, 127.76, 126.53, 126.21, 125.06, 122.75,
22.73, 114.82, 113.70, 72.62, 70.27, 68.18, 68.03, 31.62,
9.72, 29.25, 29.23, 26.03, 25.93, 25.75, 22.64, 14.07; HRMS
(
broad, aromatic protons), 4.67 (broad, ACH
2
OCO), 4.34
(
broad, ACH OAPh), 3.98 (broad, ACH OA), 3.64 (broad,
2
2
ACH NHA), 2.85 (broad, ACH COA), 1.11-1.88 (broad, back-
2
2
bone Hs, A (CH ) A), 0.98 (broad, ACH ).
2
5
3
(
APCIþ): C H O (MþH), calcd. 715.4151; found 715.4144.
5
1 55 3
Synthesis of poly(N-(2-hydroxypropyl) methacrylamide)-co-
poly(2-aminoethyl methacrylate)-co-poly[4-[6-[4-(2-{10-[2-
Synthesis of poly(N-(2-hydroxypropyl) methacrylamide)-co-
poly(2-aminoethyl methacrylate)-co-poly[4-[6-[4-(2-{10-[2-
(
4-hexyloxy-phenyl)-vinyl]-anthracen-9-yl}-vinyl)-phenoxy]-
hexyloxymethyl] styrene} (P5) with molar ratios of x:y:z of
00: 3.5: 16. Similar condition as P1, except the weight of
AIEM used for this polymerization was 400 mg. Yield:
(
4-hexyloxy-phenyl)-vinyl]-anthracen-9-yl}-vinyl)-phenoxy]-
hexyloxymethyl] styrene} (P1) with molar ratios of x:y:z of
00: 3.5: 1. 500 mg of HPMA, 20 mg of AEMA, 25 mg of
1
1
AIEM, and 10 mg AIBN were dissolved in 5 mL of DMF.
The solution was degassed three times through a standard
freeze-thaw process. The monomers were polymerized at
8
3%. M ¼ 28500, M ¼ 33900, M /M ¼ 1.20.
n
w
w
n
1
H NMR (DMSO-d
, 400 MHz), d (TMS, ppm): 6.68-8.30
OCO), 4.32
OA), 3.64 (broad,
COA), 1.21-1.89 (broad, back-
A), 0.98 (broad, ACH ).
6
ꢀ
6
5
C for 20 hours under nitrogen. Polymer was precipi-
(broad, aromatic protons), 4.67 (broad, ACH
(broad, ACH OAPh), 4.00 (broad, ACH
ACH NHA), 2.86 (broad, ACH
bone protons, A (CH
2
tated into 200 mL of ether from the DMF solution. After
filtration, the polymer was redissolved in 10 mL DMF and
reprecipitated into 100 mL of ether. Yield: 410 mg (75%).
M ¼ 11200, M ¼ 15800, M /M ¼ 1.41.
2
2
2
2
)
2
5
3
n
w
w
n
Quantum Efficiency Measurements
1
H NMR (DMSO-d
6
, 400 MHz), d (TMS, ppm): 7.01-8.39
Fluorescence quantum yields for the AIE fluorophores were
obtained by comparing the integrated fluorescence spectra of
the polymers in solutions to the fluorescence spectrum of
(
(
broad, aromatic protons), 4.68 (broad, ACH OCO), 4.00
2
broad, ACH OA), 3.64 (broad, ACH NHA), 2.85 (broad,
2
2
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quinine sulfate in 1.0 N H SO (U ¼ 0.55, excitation wave-
2
4
5
1
length of 365 nm) with a correction of refractive index dif-
5
2
ferences using eq 1.
ꢀ
ꢁꢀ ꢁꢀ
ꢁ
2
Ar
As
Is
n
n
s
gs ¼ gr
(1)
2
Ir
r
where (g ) and (g ) are the fluorescence quantum yield of
r
s
r s
standards and the samples, respectively. A and A are the
absorbance of the standards and the measured samples at
the excitation wavelength, respectively. I and I are the inte-
r
s
grated emission intensities of standards and the samples. nr
and n are the refractive indices of the corresponding sol-
s
vents of the solutions, respectively (the refractive indexes of
H O and DMSO are 1.333 and 1.479, respectively). The final
2
value of quantum yield was obtained from the average of
four measurements with different absorbance in the range
between 0.03 and 0.09. The standard deviation is less than
10%.
Culture of U87MG and CP-A Cells for Bioimaging
U87MG cells (American Type Culture Collection, ATCC, Mana-
ssas, VA) were cultured in EMEM supplemented with 10%
fetal bovine serum, 5% penicillin, 2 mM L-glutamine (Sigma-
ꢀ
Aldrich), and incubated at 37 C in a 5% CO2 atmosphere.
CP-A cells (kindly provided by Dr. Brian J. Reid at Fred
Hutchison Cancer Research Center, Seattle, WA) were cul-
tured in Keratinocyte-serum free medium (Invitrogen, Carls-
bad, CA) supplemented with Bovine Pituitary Extract (BPE)
and human recombinant Epidermal Growth Factor (rEGF,
ꢀ
Invitrogen) at 37 C in a 5% CO2 atmosphere. CP-A (also
identified as KR-42421 or QhTERT) was derived from an en-
doscopic biopsy specimen obtained from a region of nondys-
plastic metaplasia and transduced with the retroviral expres-
sion vector, pLXSN-hTERT, to create an immortalized cell
line. Cells were seeded onto 96-well plates at 10,000 cells
per well, and incubated for 1 day. Polymer P4 dissolved in
10 mM HEPES buffers were added to the medium to make
the AIE fluorophore concentration of 5 lM, corresponding to
the polymer concentration of 0.013 mg/mL. After incubation
ꢀ
at 37 C for 24 hours, cells were washed using fresh medium
and then used for bioimaging. Under Nikon Eclipse TE2000E
confocal fluorescence microscope (Melville, NY), the poly-
mers were excited at 402 nm and their green emissions
were collected using a 515/30 nm filter set.
FIGURE 1 (A) Absorbance and emission spectra of a typical
polymer P3 in DMSO and 2% DMSO aqueous solution. (B)
Emission intensity change of P3 in the mixture of DMSO and
water. The spectra were excited at 405 nm. (C) Quantum yields
of polymers P1 to P5 in DMSO and water mixtures.
MTT Assay
The assay was performed by an in vitro MTT-based toxicol-
ogy assay kit (Promega). U87MG cells were cultured in
EMEM supplemented with 10% fetal bovine serum, 5% peni-
ꢀ
cillin, and 2 mM L-glutamine (Sigma-Aldrich) at 37 C in a
were removed and the cells were washed with PBS buffer
5
% CO2 atmosphere. CP-A cells were cultured in Keratino-
and then incubated in fresh medium (100 lL) and 15 lL of
ꢀ
ꢀ
cyte-serum free medium at 37 C in a 5% CO atmosphere.
MTT solution (5 mg/mL) in 5% CO at 37 C for another 3
2
2
U87MG and CP-A cells were seeded onto 96-well plates at
h. One hundred microliters of stabilizer (Promega) was
added to each well to dissolve the internalized purple forma-
zan crystals by gentle pipetting up and down. The absorb-
ance was measured at a wavelength of 570 nm using a Spec-
traMax 190 from Molecular Devices (Downingtown, PA).
Each experiment was conducted twice in triplicate. The
10,000 cells per well, and incubated for 1 day. The polymers
in 10 mM HEPES buffers were added into the cell culture
media to a final polymer concentration of 0.1 to 1.0 mg/mL
and the cells with the polymers were incubated in the 96
well plates. Twenty-four hours later, the medium in the wells
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monomer was used to copolymerize with HPMA and AEMA
ꢀ
under the normal radical polymerization conditions at 65 C
for 20 hours using AIBN as a thermal initiator to form a se-
ries of copolymers of P1 to P5. The molar ratios of HPMA to
AEMA were kept constant of 100:3.5 (Scheme 1), while the
AIEM molar ratio to the sum of HPMA and AEMA was
increased from 1.0:103.5 for P1 to 16.0:103.5 for P5. Thus,
the influence of AIEM molar fractions on the photophysical
properties could be investigated. The polymers have very
good solubility in DMSO and DMF with a concentration of at
least 50 mg/mL. After dispersing the DMSO solution into
water or HEPES buffer and removing the DMSO using dialy-
sis, the polymers can be dissolved in aqueous solutions with
a concentration of at least 10 mg/mL.
Photophysical Properties and Possible Nanostructures
Figure 1(A) shows the typical absorbance and emission spec-
tra of P3 in DMSO and in DMSO/H O (2:98 by volume). The
2
polymer in DMSO has almost no emission with quantum effi-
ciency of 0.08%. At the same polymer concentration, with
the increase of water fraction in DMSO, the absorbance
decreases and the fluorescence intensity increases [Fig. 1(B)].
FIGURE 2 (A) Typical DLS of P1 and P4 in 2% DMSO aqueous
solutions and the AFM image of P4 (inserted, 0.75 lm ꢂ 0.75
lm). (B) A possible schematic drawing of the micelles formed
from the polymers using a flower-micelle model.
result was expressed as a percentage of the absorbance of
the blank control.
Flow Cytometric Analysis
To demonstrate the time and temperature dependent cellular
uptake of the polymers, fluorescence measurements were
carried out by using a FACS caliber cytometer (Becton Dick-
inson Immunocytometry Systems, San Jose, CA). One hun-
dred twenty five microliter of P4 with AIE fluorophore con-
centration of 200 lM (polymer concentration was of 0.52
mg/mL) in HEPES buffer was added to about 1 million cells
in 5 mL medium in a flask. The mixture was incubated at 37
ꢀ
ꢀ
C or 4 C in a cell incubator for various time periods. After
incubation, the cells were centrifuged, washed twice with
fresh medium, and then suspended in fresh medium again.
Fluorescence was determined by counting 10,000 events.
RESULTS AND DISCUSSION
Synthesis of AIE Monomer and Polymers
The AIE fluorophore was constructed from an anthracene
derivative (compound 1, Scheme 1). Through the condensa-
tion of 1 and 2, compound 3 was obtained. Compound 4
was synthesized through the deprotection of the tetrahydro-
pranyl units on 3 under mild acidic condition in methanol
and THF. 4 was then reacted with 1-bromohexane to get a
mono-hexyloxy-substituted 4, that is, compound 5. Com-
pound 6 was obtained by a reaction of 5 with 6-chloro-1-
hexanol. Through a reaction of 6 with 4-vinylbenzyl chloride,
the monomeric AIE fluorophore (AIEM) was obtained. This
FIGURE 3 Polymer concentration dependent fluorescence for
CMC determination of P1 (A) and P4 (B) in aqueous solutions.
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FIGURE 4 Confocal fluorescence images of P4 for CP-A (A–C) and U87MG (D–F). A and D are fluorescence images from the poly-
mer. B and E are bright field images. C is the overlay of A and B. F is the overlay of D and E.
These phenomena were observed in other AIE materi-
quantum efficiency change is not significant with the further
increase of AIE fractions.
2
6,27,31
als,
suggesting the typical AIE characteristics. The quan-
tum efficiency of P3 in DMSO/H O (2:98 by volume)
increased to 7.9%, which is 98-fold greater than that in pure
DMSO. Figure 1(C) shows the typical quantum efficiencies of
2
It should be noted here, the organic small molecule-based
anthracene AIE dyes formed J-aggregations (31), which was
observed by the red-shift of absorption maximum at the
aggregated states while without obvious emission maximum
changes. In the AIE-containing polymers, the J-aggregations
were not obvious. Most likely, the stoichiometry of the poly-
mer main and side chains interfered the fluorophores’ inter-
actions and aggregations.
the polymers P1-P5 at the DMSO/H O mixtures, indicating
2
materials dependent photophysical properties. DMSO is a
strong solvent for all the polymer segments, PHPMA, PAEMA,
and PAIE. The AIE fluorophores can rotate freely in the strong
solvent of DMSO to lose their energies to result in their
extremely weak emission intensities. With the addition of
water into the polymer solutions in DMSO, the hydrophobic
AIE fluorophores started to reassemble with the hydrophilic
PHEMA and PAEMA polymer chains, possibly through the for-
mation of micellar nanostructures (Fig. 2). The hydrophobic
PAIE segments aggregate together as the hydrophobic cores,
limiting or restricting the intramolecular rotations of the AIE
fluorophores to increase the fluorescence quantum efficien-
cies. With more water fractions, the aggregations become
stronger. From Figure 1(C), it can be seen that, for the poly-
mers with more hydrophobic PAIE segments, the aggregations
of the PAIE segments start earlier with lower water fractions.
To determine whether these polymers can form nanostruc-
tures, two typical polymer solutions of P1 and P4 were
measured using dynamic light scattering (DLS). The absence
of reliable scattering data of these polymers in DMSO indi-
cated their unimer structures. Small particles with diameters
of 11.0 6 3 nm for P1 and 10.4 6 4 nm for P4 in DMSO/
H O (2:98 by volume) were observed [Fig. 2(A)], suggesting
2
the polymers’ aggregated nanostructures [Fig. 2(B)]. Consid-
ering the AIE fluorophore has a longer molecular length than
the HPMA (the fully extended lengths of AIE monomer and
HPMA are 3.6 nm and 0.6 nm, respectively, calculated using
ChemDraw 3D software), we proposed the micelle structures
using the ‘‘flower micelle model’’ reported by Tominage
2
At the same DMSO/H O fraction, the polymers with higher
AIE fractions exhibit higher quantum efficiencies of the PAIE
fluorophores. When the AIE fluorophore’s fraction reaches a
certain level, for example, over the molar fraction of P4, the
5
3
et al. Such micelles can be formed through an aggregation
of the AIE probes as the micelle cores and the entangled
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PHPMA/PAEMA chains as the hydrophilic loop-like shells
Fig. 2(B)]. According to this model, if the polymers are
[
packed tightly, the diameters are about 8.4 nm, which is quite
close to the diameters (10 to 11 nm) measured using DLS.
The 8.4 nm was calculated by using a double of a radius of
4
.2 nm (3.6 nm þ 0.6 nm) of the fully extended AIE mono-
mer and HPMA. Because the hydrophilic shells formed from
PHEMA and PAEMA chains may contain significant amount of
water to extend the thickness of shells, the hydrophilic chains
may entangle in the shell, and also the polymers are usually
not packed tightly in solutions, the calculated sizes may usu-
FIGURE 6 Cytotoxicity of polymers P1 and P4 to U87MG (A)
and CP-A (B) cells. Cellular internalization time is 24 hours at
ꢀ
3
7 C.
ally be smaller than those measured by DLS. The flower mi-
celle model can also explain that the diameters of the
micelles are neither significantly affected by the molar frac-
tions ratios of the AIE fluorophores in the polymers, nor by
the polymeric molecular weights. Further confirmation of the
micelles was visualized under AFM using P4 as an example
at its dry state. Small spherical micelles [Fig. 2(A), inserted
figure] with an average diameter of 35 6 15 nm were
observed. The size is larger than the average diameter meas-
ured using DLS, which is probably due to the flattening of the
micelles on the mica surface during the drying process.
Some random copolymers with both the hydrophilic and
hydrophobic units were reported to form micelles having av-
erage diameters ranging from a few nanometers to hundreds
nanometers with the hydrophobic segments as the micellar
4
8,53–56
cores and hydrophobic chains as the shells.
Critical
micellar concentration (CMC) is an important factor for
determination of micelles formation. For the fluorescent
polymers, CMCs can be determined by the measurement of
the concentration dependent fluorescence in aqueous solu-
4
8
FIGURE 5 (A) Time dependent flow cytometry of P4 by U87MG
tions. For the AIE polymers, before the micelle formation,
the fluorophores have no aggregations and very weak fluo-
rescence will be observed. Once the micelle is formed, the
hydrophobic AIE fluorophores will aggregate, resulting in the
ꢀ
cells at 37 C. (B) Time dependent flow cytometry of P4 by CP-
ꢀ
A cells at 37 C. (C) Time dependent flow cytometry of P4 by
ꢀ
CP-A cells at 4 C.
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observation of fluorescence. Meanwhile the fluorescence
intensities after CMC will increase with the increase of con-
centrations. Fluorescence intensities at 539 nm were plotted
against the polymer concentrations in aqueous solutions
CONCLUSIONS
A new series of PHPMA based random copolymers with
hydrophobic AIE fluorophores were prepared. The increase of
the molar fractions of the hydrophobic AIE fluorophores
results in the higher quantum efficiencies of the AIE-contain-
ing copolymers. These polymers had almost no emissions in
their strong solvents of DMSO, but showed fluorescence in the
DMSO/water mixtures. These polymers were confirmed to
form small micelles with average diameters of around 10 nm
in their aqueous solutions. The polymers were cell permeable
and were located in the cytoplasma area of U87MG and CP-A
cells. The polymers did not show obvious cytotoxicity to the
two experimental cell lines after cellular internalization for 24
hours with a polymer concentration up to 1 mg/mL. The cel-
lular uptake of the polymer is time and energy dependent,
indicating the endocytosis cellular internalization mechanism.
Although the AIE fluorophore is water insoluble, its chemical
conjugation with biocompatible polymers such as the PHPMA
enables its application in biological condition for bioimaging
and endows the noncytotoxicity to cells.
(
Fig. 3). It was found the CMCs of P1 and P4 are 0.031 mg/
mL and 0.011 mg/mL, respectively. P4 has more hydropho-
bic AIE segments than P1, exhibiting a lower CMC value
than P1. It has been known that the CMC values depend on
polymer structures, molecular weights, and the fractions of
hydrophobic segments to hydrophilic segments. Some ran-
dom copolymers were reported to have high CMCs of 2.0
5
5
mg/mL. Some other random copolymers were reported to
ꢃ4
56
have much lower CMCs of around 5.3 ꢂ 10 mg/mL. Our
polymers’ CMCs are within the reported ranges.
Bioimaging
Figure 4 shows the confocal fluorescence microscopy images
of one typical polymer (P4) for two cell lines (U87MG and
CP-A) after 24 hour internalization with cells at 37 C. Green
emission was observed, which is randomly distributed in the
cytoplasma area of the cells, showing that the polymer in the
form of micelles was successfully taken up by the cells and
the AIE fluorophores were in the aggregated states. Similar
results were observed for other polymers.
ꢀ
This work was supported by NIH National Human Genome
Research Institute, Centers of Excellence in Genomic Science,
Grant Number 5 P50 HG002360, Deirdre Meldrum, PI, Director.
Brian J. Reid and Tom Paulson at Fred Hutchinson Cancer
Research Center (Seattle, WA) were acknowledged for provid-
ing the CP-A cell line. Hongguang Lu thank China Scholarship
Council scholarship program for overseas graduate students.
Time and Temperature Dependent Flow Cytometry
To understand the possible cellular uptake mechanism, flow
cytometry was used to measure the fluorescence of the cells
after internalization with the polymers. P4 was used as a
typical polymer. Figure 5(A,B) gave the time dependent fluo-
ꢀ
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