Concentration and pH-modulated dual fluorescence in self-assembled nanoparticles of phototautomerizable biopolymeric amphiphile

Article abstract of DOI:10.1016/j.dyepig.2010.12.014

A dual-emissive biopolymeric amphiphile (GC-HBO) has been prepared by densely conjugating hydrophilic glycol chitosan (GC) with a phototautomerizable hydrophobic dye (a derivative of 2-(2′-hydroxyphenyl)benzoxazole, HBO) showing excited-state intramolecul

Full text of DOI:10.1016/j.dyepig.2010.12.014

Dyes and Pigments 90 (2011) 284e289
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Concentration and pH-modulated dual fluorescence in self-assembled
nanoparticles of phototautomerizable biopolymeric amphiphile
,
a
Chang-Keun Lim a, Jangwon Seo b, Sehoon Kim a , Ick Chan Kwon ,
***
*
,
b,
**
Cheol-Hee Ahn c , Soo Young Park
a Biomedical Science Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea
b Center for Supramolecular Optoelectronic Materials, WCU Program for Hybrid Materials, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea
c Department of Materials Science & Engineering, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A dual-emissive biopolymeric amphiphile (GCeHBO) has been prepared by densely conjugating hydrophilic
glycol chitosan (GC) with a phototautomerizable hydrophobic dye (a derivative of 2-(20-hydroxyphenyl)
benzoxazole, HBO) showing excited-state intramolecular proton transfer (ESIPT). Ratiometric modulation of
phototautomeric (enoleketo) dual emission, governed by the chromophoric aggregation state, was studied
with a low-molecular-weight model compound (GAeHBO) and was well correlated with the nanoparticle-
forming behavior of GCeHBO via amphiphilic self-assembly in water. It has been found that the promoted
chromophoric aggregation at higher concentration or at basic-to-neutral pH assists the nanostructure
formation to generate predominant keto emission while the enol emission is intensified at lower concen-
tration or with decreasing pH by disintegration of self-assembled structure. The assembly-related
advantageous potential of GCeHBO for probing self-concentration and surrounding pH has been
demonstrated with the ratiometric determination of critical particulation concentration (0.04 kg mꢀ3) and
near-physiological pH sensitivity (pKa w 5.1).
Received 27 October 2010
Received in revised form
20 December 2010
Accepted 26 December 2010
Available online 7 January 2011
Keywords:
Excited-state intramolecular
proton transfer (ESIPT)
Ratiometric fluorescence sensor
Fluorescent nanoparticle
pH sensor
Self-assembly
Biopolymeric conjugate
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
of biohybrid nanomaterials that form self-assembled nanoparticles in
aqueous milieu with the interfacial-free energy-minimized structure
Luminescence-based technologies have led to great leaps in
biological, medical, environmental and material sciences. Recently,
molecules capable of fluorescence switching are gaining great
interest for their potential utility from typical polarityeviscosity
probes to more advanced applications including biosensors, logic
gates, and optical recordings [1e5]. Aggregation-triggered fluo-
rescence modulation is a more recent and special example [6e11],
which offers a novel pathway to fluorescence probing of molecular
aggregation-related phenomena.
Aggregation of amphiphiles is a key way of molecular organiza-
tion in biological systems and often employed to develop biomimetic
supramolecular materials. Biopolymeric amphiphiles with an
appropriate hydrophilic/hydrophobic balance are an emerging class
comprising a hydrophilic shell and hydrophobic multicores. Hydro-
phobically modified glycol chitosan is among the representative
examples that hold considerable potential as drug carriers and
bioimaging agents due to high biocompatibility and high tumor
targeting efficiency [11e15]. Basic studies on the nanoparticulate
characteristics of biopolymeric amphiphiles are a research subject of
great importance to understand their advantageous behaviors in
a complex in vivo environment [12,14].
In this study, we applied the aggregation-induced fluorescence
modulation to probing the concentration and pH-dependant
assembly of biopolymeric amphiphiles in aqueous phase. To this end,
we designed a novel dye-concentrated biohybrid that can self-probe
its own molecular aggregation state with ratiometric fluorescence
modulation. As depicted in Fig. 1, the designed biohybrid (GCeHBO)
is composed of water-soluble glycol chitosan (GC) and a derivative
of 2-(20-hydroxyphenyl)benzoxazole (HBO) as a densely conjugated
hydrophobic pendant. The HBO unit is a well-known chromophore
exhibiting enol-to-keto phototautomerization via the excited-
state intramolecular proton transfer (ESIPT) reaction. Its dual
fluorescence from each phototautomer is independently responsive
* Corresponding author. Tel.: þ82 2 958 5924; fax: þ82 2 958 5909.
** Corresponding author. Tel.: þ82 2 880 8347; fax: þ82 2 886 8331.
*** Corresponding author. Tel.: þ82 2 883 8197; fax: þ82 2 880 5791.
E-mail addresses: sehoonkim@kist.re.kr (S. Kim), chahn@snu.ac.kr (C.-H. Ahn),
parksy@snu.ac.kr (S.Y. Park).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.12.014

C.-K. Lim et al. / Dyes and Pigments 90 (2011) 284e289
285
Fig. 1. Phototautomerizable Amphiphilic Glycol Chitosan (GCeHBO).
to environmental parameters (medium polarityeviscosity, pH, and
temperature, etc.) [16e19]. In particular, it has recently been
reported that in some ESIPT molecules, the weak proton-transferred
keto emission in solution is greatly enhanced over the enol emission
by solidification [19e22], allowing for highly reliable ratiometric
optical probing of molecular aggregation. Ratiometric fluorescence
method offers advantages of increased dynamic range of detection
and built-in correction for environmental effects by eliminating
data distortions caused by instrumental instability, photobleaching,
and probe concentration [23]. Here are reported the synthesis
of GCeHBO as a polymeric model biohybrid and its concentration
and pH dependent assembly behavior, along with a low-molecular-
weight model study on the ratiometric fluorescing characteristics of
nanoaggregated ESIPT system.
added to the mixture and sodium hydroxide was added slowly to
obtain pH 5. The filtered solid was dried and recrystallized from
ethyl acetate to give HBO-COOH (0.45 g, 14%). 1H NMR (DMSO-d6,
ppm) 11.04 (s, 1H), 8.32 (s, 1H), 8.06 (d, 2H, J ¼ 8.1 Hz), 7.93 (d, 1H,
J ¼ 8.1 Hz), 7.58 (t, 1H, J ¼ 7.5 Hz), 7.16 (d, 1H, J ¼ 8.3 Hz), 7.12 (t, 1H,
J ¼ 7.5 Hz).
2.2. Preparation of GCeHBO
Glycol chitosan (GC, MW ¼ 250 kDa; degree of deacetylation ¼
82.7%; Sigma) (0.025 g, 0.122 mmol (unit)) was dissolved in 2 cm3
of distilled water with sonication and a DMSO (10 cm3) solution
containing HBO-COOH (0.0125 g, 0.049 mmol), 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC) (0.014 g,
0.074 mmol) and N-hydroxysuccinimide (NHS) (0.0085 g,
0.074 mmol) was added. Then the reaction mixture was stirred
at ambient temperature for 24 h. To quantify the substitution ratio,
the reaction mixture (0.1 cm3) was mixed with THF (0.9 cm3) and
centrifuged, to settle down precipitates of the GCeHBO conjugate.
The substitution ratio was indirectly determined as 17% through the
measurement of the unreacted free HBO-COOH concentration in
the supernatant by UV absorbance-based quantification. For further
experiments, the remaining reaction mixture was subjected to
the successive dialysis against DMSO (48 h) and water (48 h)
using a Cellu$Sep membrane (Membrane Filtration Products, Inc.,
molecular cutoff ¼ 50 kDa). The resulting dispersion of GCeHBO
was freeze-dried for characterization and imaging experiments.
2. Experimental section
Chemical reagents were purchased from Aldrich and used as
received. 1H NMR measurements were recorded on an Avance
DPX-300 (300 MHz, Bruker, Germany) in CDCl3 and DMSO-d6 solu-
tion. UVevisible absorption and fluorescence spectra were measured
on an 8453 UVeVisible spectrophotometer (Agilent Technology,
USA) and an F-7000 fluorescence spectrophotometer (Hitachi,
Japan), respectively. Transmission electron microscopic (TEM) image
of negatively stained particles (with 2 wt.% uranyl acetate) was
obtained with a CM30 electron microscope (FEI/Philips) operated at
200 kV. Hydrodynamic size distribution of nanoparticles was deter-
mined by dynamic light scattering (DLS) method with a particle sizer
(90Plus, Brookhaven Instruments Corporation). The pH-dependence
measurements were performed in phosphate buffer saline (PBS,
pH 4.0e9.0). Ratios of fluorescence intensities (R) at 510 and 420 nm
as a function of pH were theoretically fitted to extract the pKa value
by using the following equation:
2.3. Synthesis of 2-(2-hydroxyphenyl)-N-((2R,3S,4S,5R)-2,4,5-
trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)benzo[d]
oxazole-6-carboxamide (GAeHBO)
FpH4:0ð510Þ10ꢀpH þ FpH8:0ð510Þ10ꢀpKa
D
-(þ)-Glucosamine hydrochloride (0.084 g, 0.39 mmol, Sigma)
I510
I420
R ¼
¼
(1)
was dissolved in 1 cm3 of distilled water with sonication and
a DMSO (5 cm3) solution containing HBO-COOH (0.1 g, 0.39 mmol),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC) (0.112 g, 0.59 mmol) and N-hydroxysuccinimide (NHS)
(0.068 g, 0.59 mmol) was added. After reaction for 24 h at room
temperature, the mixture was diluted with brine and the product
was extracted three times with ethyl acetate. The dried product was
recrystallized from ethyl acetate to give GAeHBO (0.034 g, 21%). 1H
FpH4:0ð420Þ10ꢀpH þ FpH8:0ð420Þ10ꢀpKa
where FpH
(l) and FpH
(l) designate fluorescence intensities
8.0
4.0
of the endpoint species (at pH 4.0 and 8.0) at the considered
wavelength ( ) [24].
l
2.1. Synthesis of 2-(2-hydroxyphenyl)benzo[d]oxazole-6-carboxylic
acid (HBO-COOH)
NMR (300 MHz, CDCl3:DMSO-d6 ¼ 1:1),
d (ppm): 11.00 (s, 1H), 8.54
A mixture of 4-amino-3-hydroxybenzoic acid (2 g, 13 mmol) and
salicylaldehyde (1.6 g, 13 mmol) in acetic acid (120 cm3) was stirred
for 1 h at room temperature, and then lead acetate (5.8 g, 13 mmol)
was added to the mixture. After 1 h stirring at room temperature,
the mixture was stirred for 24 h at 150 ꢁC. 350 cm3 of water was
(s, 1H), 8.28 (d, 2H, J ¼ 8.1 Hz), 8.06 (d, 1H, J ¼ 8.1 Hz), 7.58 (t, 1H,
J ¼ 7.5 Hz), 7.16 (d, 1H, J ¼ 8.3 Hz), 7.13 (t, 1H, J ¼ 7.5 Hz), 3.31 (s, 4H),
2.93 (s, 3H), 2.87 (s, 1H), 2.60 (s, 3H). 13C NMR (75 MHz, CDCl3),
d
(ppm): 169.82, 166.89, 162.08, 160.03, 149.40, 146.26, 135.55,
128.62, 128.31, 122.52, 120.61, 120.07, 118.44, 113.98, 110.35, 29.25,

286
C.-K. Lim et al. / Dyes and Pigments 90 (2011) 284e289
28.91. MS (MALDI): calcd for C20H20N2O8, m/z ¼ 416.38; found,
m/z ¼ 413.80.
was controlled at a constant concentration (10
water fraction (¼water/[water þ THF] ꢂ 100 by volume) of the
mixed solvent. As shown in Fig. 2a, there exist two
* absorption
mM) by changing
pep
bands at 333 and 345 nm in THF solution (0% water fraction) which
can be assigned to the ground-state syn- and anti-rotational isomers
of the enol tautomer, according to the reported assignments for the
parent HBO molecule [18,19]. The feature of the solution absorption
manifested no change up to 60% water fraction, indicating that
GAeHBO kept being dissolved in a water-rich mixed solvent owing
to the enhanced hydrophilicity by glucosamine substitution. From
80% water fraction upward, however, the main band at 320e360 nm
became structureless along with hypsochromic and hypochromic
effects as well as a remarkable baseline lift by Mie scattering.
This spectral change is a typical indication for the formation of
nanoaggregates, as evidenced by the particulate size distribution of
the 100% water fraction sample (Fig. 2b): 37.3 ꢃ 4.5 nm by dynamic
light scattering (DLS) and 26.9 ꢃ 7.1 nm by transmission electron
microscopy (TEM). The suspension of self-aggregated GAeHBO
nanoparticles exhibited fairly high colloidal stability, which is
most likely attributed to the amphiphilicity by the water-soluble
glucosamine substitution.
The fluorescence spectra of the ESIPT-active GAeHBO in solution
and nanoaggregate dispersion are shown in Fig. 2c. In the monomer
state in THF solution, the spectrum shows tautomeric dual emission
bands with comparable intensity: a multi-peak band in the region
of 400e450 nm and a broad one centered at 520 nm. ESIPT is
a phototautomerization occurring in the excited state of intramo-
lecularly hydrogen (H)-bonded molecules that exist in the enol (E)
form in the ground state. Upon photoexcitation, the excited E form
undergoes fast tautomerization to the excited keto (K) form through
the intramolecular proton transfer, to generate the K emission that is
far red-shifted compared to the normal E fluorescence. Therefore,
the shorter- and the longer-wavelength bands of the solution spec-
trum in Fig. 2c can be assigned to the emission of rotational E isomers
and the typical proton-transferred K emission with an abnormally
large Stokes’ shift, respectively. In the aggregated state, however,
the spectrum is composed mainly of an intense K band peaking at
500 nm, along with a negligible component of the E emission below
450 nm. It is known that the intensity ratio between E/K emission
2.4. Nanoparticle formation of GCeHBO and GAeHBO
Self-assembled nanoparticles of GCeHBO were prepared by
redispersion of the freeze-dried sample in water at 1 mg cmꢀ3 under
probe sonication at 90 W for 2 min (Sigma Ultrasonic Processor,
GEX-600). GAeHBO nanoparticles were prepared by adding a solu-
tion (0.3 cm3, 1 ꢂ10ꢀ4 M in THF) to the mixed solution of THF/water
with varying water fraction (2.7 cm3/0 cm3, 2.1 cm3/0.6 cm3,1.5 cm3/
1.2 cm3, 0.9 cm3/1.8 cm3, 0.3 cm3/2.4 cm3 and 0 cm3/2.7 cm3) under
bath sonication. The pure water suspension was obtained by solvent
evaporation of GAeHBO solution (0.3 cm3, 1 ꢂ 10ꢀ4 M in THF) and
redispersion of the dried sample in water (3 cm3) under probe
sonication.
3. Results and discussion
Synthetic routes of phototautomerizable bio-amphiphiles are
described in Scheme 1. ESIPT-exhibiting pendant (HBO-COOH) was
obtained by one-pot benzoxazole synthesis comprising in situ Schiff
base formation and oxidative ring closure in the presence of lead
(IV) acetate [25]. GCeHBO was prepared by amidation between
free amines of GC and HBO-COOH (40 mol% fed with respect to
the GC monomeric units) in DMSO/water, by using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
N-hydroxysuccinimide (NHS) as coupling agents. The purification
was conducted by successive dialysis against DMSO and excess pure
water. The mild reaction at ambient temperature for 24 h afforded
a biohybrid polymer with 17 mol% HBO substitution with respect to
the repeating units of GC. To study the aggregation-modulated ESIPT
behavior of the HBO pendant, a model compound (GAeHBO) was
prepared via the same amidation procedure with HBO-COOH and
glucosamine to represent the substituted repeating unit of GCeHBO.
In the low-molecular-weight model study, the GAeHBO aggre-
gates were prepared by a nanoprecipitation method in a water/THF
mixed solvent where THF and water are good and poor solvents for
the HBO unit, respectively. The degree of chromophoric aggregation
Scheme 1. Synthetic route of HBO-conjugated bio-amphiphiles.

C.-K. Lim et al. / Dyes and Pigments 90 (2011) 284e289
287
0.4
0.3
0.2
0.1
0.0
a
b
100
0%
60%
80%
90%
100%
80
60
40
20
0
200 nm
200
0
50
100
150
250
300
320
340
360
380
400
Wavelength (nm)
Size (nm)
5000
4000
3000
2000
1000
0
40
30
20
10
0
c
d
400
450
500
550
600
650
0
20
40
60
80
100
Wavelength (nm)
Water Fraction (vol.%)
Fig. 2. Optical and morphological characteristics of GAeHBO in mixed solvents with varying water fraction (¼water/[water þ THF] ꢂ 100 by volume). (a) Absorption spectra of
GAeHBO. Water fraction is indicated. (b) Number-averaged hydrodynamic size (measured by DLS) and TEM image (inset) of nanoaggregates formed in 100% water fraction.
(c) Fluorescence spectra of GAeHBO solution (dotted line, in THF) and nanoaggregates (solid line, in water). (d) Ratiometric plot of fluorescence intensities at 510 nm (keto) and
400 nm (enol), as a function of water fraction.
bands of HBO is dependent on the molecular state: HBO emits strong
E fluorescence with a weak K component when dissolved in H-bond
perturbing solvents [18,19], whereas their K fluorescence can be
greatly enhanced over the E emission in the aggregated state
because the solidification prefers the energetically favored H-
bonded geometry that is prerequisite for the ESIPT reaction [19].
In this context, the exclusive K emission from the GAeHBO nano-
aggregates suggests that the HBO units in the solidified state exist
dominantly in the syn-E conformation, where the phenyl hydroxyl
proton forms an internal H bond with the benzoxazole nitrogen
atom, to maximize the ESIPT efficiency [19,20]. Importantly, it was
aggregated in water due to the interfering motion of the water-
soluble backbone. With lowering concentration, the intensity of K
emission was diminished significantly, while the E component was
intensified. A plot of the intensity ratio between K and E components
(Iketo/Ienol) versus the log of concentration shows a two-phase rela-
tionship, suggesting formation of self-assembled nanoparticles at
higher concentrations (Fig. 3b). The observed inflection point (a
crossing of the two linear fits in the plot) corresponds to a critical
particulation concentration (CPC) of 0.04 kg mꢀ3, above which
GCeHBO forms and retains the self-assembled nanostructure via
chromophoric aggregation. The steady increase in Iketo/Ienol with
increasing concentration below the CPC indicates gradual buildup of
chromophoric aggregation toward self-assembly into particulate
nanostructure.
TEM and DLS data in Fig. 3c directly evidence the formation
of self-assembled GCeHBO nanoparticles above the CPC. The
number-weighted hydrodynamic size distribution of a wet sample
was determined as 87.1 ꢃ1.4 nm by DLS. The TEM image manifests
that the dried GCeHBO nanoparticles are polydisperse in size with
a shrunken diameter of 19.9 ꢃ 11.1 nm (the inset of Fig. 3c). The size
difference between the wet and dried samples suggests that the
dispersed nanoparticles exist in the swollen state and shrink during
the drying process. Below the CPC, no detectable scattering was
observed in the detection limit of the DLS instrument, indicative
of the disintegration of nanostructure. These results indicate that
the chemical conjugation of HBO pendants efficiently introduces
found that the intensity ratio between K and E emission bands (Iketo
/
Ienol) is modulated depending on the degree of aggregation (Fig. 2d),
proving the utility of the ESIPT dual emission for the ratiometric
fluorescence probing of aggregation-related molecular events.
The aggregation-probing potential of ESIPT dual fluorescence
in an actual biopolymeric amphiphile system was evaluated with
the self-assembly and ratiometric optical modulation behaviors of
GCeHBO. It was found that the lyophilized GCeHBO solid after
purification by dialysis forms a clear dispersion in water by sonica-
tion. Fig. 3a shows representative fluorescence spectra of the
GCeHBO dispersion at a high concentration of 1 kg mꢀ3, the spec-
trum shows tautomeric dual bands. As discussed above in the model
study, they can be assigned as E (400 nm) and K (w500 nm) emission
of the HBO pendant. On considering the predominant K emission
observed from the GAeHBO nanoaggregates (Fig. 2c), the E/K
dual fluorescence from the GCeHBO dispersion indicates that the
chemically conjugated hydrophobic pendants are not fully
the hydrophobic and chromophoric
pep interactions to the free
motion of the water-soluble GC backbone in aqueous environment,

288
C.-K. Lim et al. / Dyes and Pigments 90 (2011) 284e289
a
a
pH 4.0
pH 7.0
1.0
0.5
formic acid
pH 9.0
450
0.0
400
500
550
600
Wavelength (nm)
b
b
3.5
1.5
1.0
0.5
0.0
3.0
2.5
2.0
1.5
1.0
-3
-2
-1
0
0.5
Concentration (Log(mg/mL))
3
4
5
6
7
8
9
10
pH
c
100
80
60
40
20
0
Fig. 4. pH response of GCeHBO dual emission. (a) Normalized fluorescence spectra in
PBS with varying pH and formic acid. (b) Ratiometric plot of fluorescence intensities at
510 nm (keto) and 420 nm (enol and protonated enol), as a function of pH. The solid
curve is the fitting result obtained by using Eqn. (1).
saline (PBS, pH 4.0e9.0), the K emission is the most dominant over the
E component at the highest pH (pH 9.0) and the ratio (Iketo/Ienol) drops
gradually with decreasing pH. This optical pH response is attributable
to the basicity of GCeHBO arising from its chemical structure.
As shown in Fig. 2, GCeHBO is composed of two basic constituents, i.e.,
unsubstituted repeating units of the GC backbone and benzoxazoles
in the pendant. On the basis of the reported pKa values of the
protonated GC (pKa w 6.5) [15] and the protonated HBO (pKa w 1.3)
[26], it is anticipated that protonation takes place at free amines in the
GC backbone near neutral pH and then occurs at the benzoxazole
nitrogen at lower pH. Partial protonation of the GC backbone at neutral
pH may raise the hydrophilicity of amphiphilic GCeHBO and thus
cause its formation of hydrogel-like swollen nanostructure in water,
as discussed in Fig. 3c. The enhanced K emission at pH 9.0 (Fig. 4a)
suggests that the GC backbone changes from the partially protonated
state at neutral pH into a deprotonated free amine form by basification,
which makes GCeHBO less hydrophilic to promote chromophoric
aggregation of the ESIPT pendants via hydrophobically shifted
amphiphilic balance. In this context, the opposite modulation of the
dual emission at pH 4.0e5.0, i.e., intensified E emission over the K
band, indicates that the degree of pendant aggregation, preferring
the generation of K emission, is reduced at lower pH. One can expect
that the hydrophilicity of GCeHBO will be maximized under the given
acidic conditionviadual protonation of thewhole backboneandpartof
the HBO pendants. The occurrence of partial protonation at the
pendants at lower pH is evidenced by the fluorescence spectrum
200 nm
50
100
150
200
250
Size (nm)
Fig. 3. Optical and morphological characteristics of GCeHBO in water with varying the
amphiphilic polymer concentration. (a) Normalized fluorescence spectra at 1.0 kg mꢀ3
(solid line) and 0.001 kg mꢀ3 (dotted line). (b) Ratiometric plot of fluorescence
intensities at 510 nm (keto) and 400 nm (enol), as a function of the log of concen-
tration. The solid lines are the linear fits for experimental data in 0.001e0.05 kg mꢀ3
and 0.05e1.0 kg mꢀ3, respectively. (c) Number-averaged hydrodynamic size (measured
by DLS) and TEM image (inset) of self-assembled nanoparticles at 1.0 kg mꢀ3
.
to induce the amphiphilic self-assembly into swollen nanoparticles.
The concentration-dependence of Iketo/Ienol provides information
on the concentration and self-assembly status of GCeHBO itself, to
make it useful as a self-probing imaging agent.
In addition to the concentration-probing capability, the dual
emission of GCeHBO nanoparticles exhibit pH-responsive spectral
evolution, as observed in Fig. 4. When examined in phosphate buffer

C.-K. Lim et al. / Dyes and Pigments 90 (2011) 284e289
289
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obtained at pH 4.0. As shown in Fig. 4a, the E emission band at pH 4.0
has a longer-wavelength shoulder that is well matched with the fully
protonated emission from a GCeHBOsolutioninformicacid. Itisnoted
that protonation of the benzoxazole amine interferes the internal H
bond and thus blocks a pathway for ESIPT, to generate protonated enol
fluorescence at the cost of phototautomeric (ketoeenol) dual emission
[5]. Accordingly, it is concluded that the partial pendant protonation
decreases the K/E emission ratio of GCeHBO by disturbing the
chromophoric aggregation as well as by intensifying the E emission
range due to the spectral similarity between E and protonated bands.
The obtained titration plot of Iketo/Ienol enables ratiometric pH probing
in the range of pH 4e8 (Fig. 4b). This result suggests that the pendant
protonation-assisted hydrophilicity enhancement of GCeHBO and
the cooperative dual emission modulation thereby lead to the nano-
scopically improved pH sensitivity in spite of a low pKa value of the
pendant unit [23]. From the nonlinear fitting of the pH response
(the solid line in Fig. 4b), the resulting pKa value (determined by the
overall self-assembly behavior of GCeHBO) was extracted as w5.1,
which is useful for pH measurement in the cell’s acidic organelles such
as endosomes or lysosomes [27].
[12] Lee KY, Kwon IC, Kim Y-H, Jo WH, Jeong SY. Preparation of chitosan self-aggregates
as a gene delivery system. Journal of Controlled Release 1998;51:213e20.
[13] Kim K, Kwon S, Park JH, Chung H, Jeong SY, Kwon IC. Characterizations of
size-controlled self-aggregates based on glycol chitosans modified with
deoxycholic acid. Biomacromolecules 2005;6:1154e8.
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4. Conclusions
We have designed and prepared a new biopolymeric hybrid
(GCeHBO) that is composed of a hydrophilic GC backbone and
densely conjugated hydrophobic ESIPT pendants, to take advantage
of the resulting correlation between the amphiphilic assembly
behavior and the dual emission modulation thereby. It has been
demonstrated that GCeHBO can self-assemble into nanoparticles
in aqueous phase by chromophoric aggregation and generate
signal modulation of dual emission responsive to the changes in
self-concentration and surrounding pH. We anticipate that the
ratiometric fluorescence response of GCeHBO will offer a possible
way to study the molecular aggregation behavior of self-assembled
biopolymeric nanoparticles in an in vivo system with complex
biodistribution and varying pH.
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a preexisting hydrogen bond in semirigid polyquinoline. Macromolecules
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Acknowledgment
This work was supported by the Real-Time Molecular Imaging
Project and the Pioneer Research Program (2009-0081523) funded
by the Korea Ministry of Education, Science and Technology
(MEST). The work at Seoul National University was supported by
the Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by MEST (CRI; RIAMIAM0209
(0417-20090011)).
[22] Kim H-J, Lee J, Kim T-H, Lee TS, Kim J. Highly emissive self-assembled organic
nanoparticles having dual color capacity for targeted immunofluorescence
labeling. Advanced Materials 2008;20:1117e21.
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nanoparticles as a ratiometric fluorescent pH probe by one- and two-photon
excitation. Chemical Communications; 2006:2071e3.
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An efficient fluorescent probe for ratiometric pH measurements in aqueous
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monomers and polymers containing 2-phenylbenzoxazole moiety in meso-
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