A unique aliphatic tertiary amine chromophore: Fluorescence, polymer structure, and application in cell imaging

Article abstract of DOI:10.1021/ja310236m

Although photoluminescence of tertiary aliphatic amines has been extensively studied, the usage of this fundamental chromophore as a fluorescent probe for various applications has unfortunately not been realized because their uncommon fluorescence is easily quenched, and strong fluorescence has been observed only in vapor phase. The objective of this study is how to retain the strong fluorescence of tertiary amines in polymers. Tertiary amines as branching units of the hyperbranched poly(amine-ester) (HypET) display relatively strong fluorescence (Φ = 0.11-0.43). The linear polymers with tertiary amines in the backbone or as the side group are only very weakly fluorescent. The tertiary amine of HypET is easily oxidized under ambient conditions, and red-shifting of fluorescence for the oxidized products has been observed. The galactopyranose-modified HypET exhibits low cytotoxicity and bright cell imaging. Thus, this study opens a new route of synthesizing fluorescent materials for cell imaging, biosensing, and drug delivery.

Full text of DOI:10.1021/ja310236m

Communication
pubs.acs.org/JACS
A Unique Aliphatic Tertiary Amine Chromophore: Fluorescence,
Polymer Structure, and Application in Cell Imaging
Miao Sun, Chun-Yan Hong, and Cai-Yuan Pan*
CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and
Technology of China, Hefei, Anhui, 230026, P. R. China
S
* Supporting Information
polymers containing tertiary amine is relatively easy; with this
ABSTRACT: Although photoluminescence of tertiary
aliphatic amines has been extensively studied, the usage
of this fundamental chromophore as a fluorescent probe
for various applications has unfortunately not been realized
because their uncommon fluorescence is easily quenched,
and strong fluorescence has been observed only in vapor
phase. The objective of this study is how to retain the
strong fluorescence of tertiary amines in polymers. Tertiary
amines as branching units of the hyperbranched poly-
(amine-ester) (HypET) display relatively strong fluores-
cence (Φ = 0.11−0.43). The linear polymers with tertiary
amines in the backbone or as the side group are only very
weakly fluorescent. The tertiary amine of HypET is easily
oxidized under ambient conditions, and red-shifting of
fluorescence for the oxidized products has been observed.
The galactopyranose-modified HypET exhibits low
cytotoxicity and bright cell imaging. Thus, this study
opens a new route of synthesizing fluorescent materials for
cell imaging, biosensing, and drug delivery.
type of polymer, drawbacks of the current chromophores, such as
cytotoxicity and photobleaching,3 might be avoided. Thus,
development of the tertiary amine-based fluorescent polymers is
very valuable.
Although the fluorescence nature of tertiary aliphatic amines is
continuously investigated for a long time,10 successful
fluorescent labeling with this fundamental chromophore has
not been studied. The main problem is how the tertiary amines in
the materials can retain their high fluorescence efficiency. Study
on the TMA and TEA fluorescent behavior demonstrates that
the factors governing the predissociation rate that competes with
emission rate are collisional relaxation and quenching.4 Hyper-
branched and dendritic poly(amido amine)s (PAMAMs), which
are generally prepared from primary and/or secondary amines,
contain different types of amines due to incomplete Michael
addition reaction. Their photoluminescence was observed,11,12
but the chromophore was not investigated,11 except a
speculation based on fluorescence behaviors in our previous
report.12 Plus, the fluorescence of nitrogen vacancy defects in
diamond has been extensively studied, and under excited at 514
nm, a strong fluorescence appears at around 700 nm.13 This
encourages us to study the fluorescence properties of hyper-
branched polymers produced by linking aliphatic tertiary amine
molecules, and this type of polymers is expected to retain high
fluorescence efficiency of the starting amine because they are
fixed in the polymer network, similar to nitrogen in the diamond.
In addition, the microenvironment around the chromophores
can be adjusted in order to reduce the fluorescence quenching by
solvent molecules.8 Thus, the hyperbranched poly(amine
ester)s, HypET11, HypET15, HypET20, and HypET24, were
prepared by Michael addition polymerization of tri(2-
mercaptoethyl)amine (TMEA) and ethylene glycol diacrylate
(EGDA) for 11, 15, 20 and 24 h, respectively, as shown in
Scheme 1.
Fluorescent biomaterials have become a focus of research in
biology due to their broad applications in cellular imaging,
biosensing, and drug delivery,1 and the fluorescent materials in
the studies include fluorescent organic dyes, fluorescent proteins,
and quantum dots.2 Some aliphatic biodegradable synthetic
polymers, which show intriguing photoluminescence phenom-
ena, have developed.3 However, usage of tertiary amines as a
fluorescent probe has never been reported, although the
fluorescence of tertiary amine has been extensively studied.4,5
Tertiary aliphatic amines of different structures display different
fluorescence behaviors due to the formation of different
excimers, such as α,ω-diaminoalkanes form various intra-
molecular excimers.5−7 Very high fluorescence quantum yields
(around 98%) of aliphatic tertiary amines including TMA and
TEA were obtained only in the vapor phase.7 However, the
fluorescence of primary and secondary amines is not observed
even in the vapor phase, owing to easy loss of the hydrogen atom
via predissociation,4 and the quantum yields of tertiary amines
measured in various solvents are very low generally due to
quenching of the solvents, which is explained by possible energy
transfer and electron (or charge) transfer.8,9 So, the photo-
luminescence behaviors of tertiary amine chromophores are
obviously different from the current fluorescent materials, such as
fluorescent organic dyes and quantum dots, and synthesis of the
biodegradable, biocompatible, and water-soluble fluorescent
Since there are two types of terminal groups, mercapto and
acryloyl, in the resultant HypETs, cross-linking reactions occur
during storage. All HypETs were treated with mercaptoethanol;
both reactions produced the same terminal hydroxyl group. This
1
is confirmed by the H NMR spectra (Figure S1) with two
methylene group proton signals next to OH group, one at δ =
3.91 ppm, terminal SH reaction with mercaptoethanol forming
the linear unit, and the other at δ = 3.75 ppm, acyloyl reaction
with mercaptoethanol, indicating existence of linear and terminal
Received: October 16, 2012
© XXXX American Chemical Society
A
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Communication
Figure 1b shows that vibrational relaxation in the excited state
occurs prior to emission.4 Using Williams’ method,14,15 the
quantum yield (Φ) of HypET24 in CHCl3 was measured and is
0.43. All these data support that the tertiary amine in the
hyperbranched polymers can retain high fluorescence efficiency.
Ester linkages of EGDA units are easily hydrolyzed in alkaline
solution; theoretically the completely hydrolyzed product of
HypETs is tri(5-carboxylate-ethylene-3-sulfo-ethylene)amine
(TCESEA). Fluorescence of the hydrolyzed products, obtained
by hydrolysis of the HypET24 in NaOH solution of THF/H2O
(1:1, v/v), was measured, and a significant decrease of the
fluorescence was observed (Figure 1c). The fluorescence is
stronger than the pure TCESEA obtained from reaction of
TMEA and methyl acrylate and subsequent hydrolysis (Figure
1c), which is ascribed to small amount of branched oligomers in
the hydrolyzate. Results demonstrate again the importance of the
hyperbranched structure for retaining high fluorescence
efficiency of the tertiary amine.
The emission spectra of different molecular weight HypETs
have similar profiles, but the relative fluorescence intensity
increases with increasing the molecular weight (Figure 1b),
which was also observed in the study of PAMAM dendrimers16
and hyperbranched PAMAM (HPAMAM).12 Quantum yields of
HypETs have the same trend as the fluorescence intensity
(Figure 2b) are presumed to be mainly due to decreased interior
mobility in the high molecular weight HypETs, because solution
NMR studies demonstrated that the interior mobility of the
dendrimer decreased with increasing generation.17 The high
molecular weight HypETs also contain a high tertiary amine
content in the branching units than the lower molecular weight
HypETs, which is supported by the DBs increase of the HypETs
with increasing molecular weights (Figure 2b).
Scheme 1. Synthesis and Hydrolysis of the Hyperbranched
Polymer HypET
a
a
Structures of the Linear Polymers with Tertiary Amine in the
Backbone [L-poly(EGDA-BMEA)] and as Side Group [L-poly(PPT-
APAMA)].
units in the HypETs. 1H NMR spectrum in Figure S1 supports
the HypETs formation. Based on integral values of signals at
2.5−3.0 and 4.32 ppm, the degree of branching (DBs) was
calculated, and the results are shown in Figure 2. The molecular
weights and molecular weight distributions were estimated by
size exclusion chromatography (SEC) measurements with multi-
angle laser light scattering (MALLS) detector, and the results are
listed in Table S1 and Figure S2.
Examining hyperbranched structure effects on fluorescence,
the fluorescence spectra of starting materials TMEA and EGDA
were measured. As shown in Figure 1a,a′, EGDA is non-
Figure 2. Influence of the polymer structures on fluorescence behaviors.
(a) Fluorescence spectra of the HypET20 with Mn = 6000 g/mol and
Mw/Mn = 3.50, the linear polymer with the tertiary amine in the
backbone (L-poly(EGDA-BMEA), Mn = 6570 g/mol, Mw/Mn = 1.74)
and the linear polymer with tertiary amine as side group (L-poly(PPT-
APAMA), Mn = 4300 g/mol, Mw/Mn = 1.34). (a′) Fluorescence spectra
of linear polymers. Conditions: 1 mg/mL in CHCl3; and λex = 375 nm at
rt. (b) Relationship of degree of branching and quantum yield with
weight-average molecular weight.
Figure 1. TMEA and EGDA fluorescence used in the preparation of
HypETs (a, a′); fluorescence (b) and UV/vis spectra (b′) of the
HypET11, HypET15, HypET20, and HypET24 using Michael addition
polymerization of TMEA and EGDA with feed molar ratio of 1:2 at 50
°C for 11, 15, 20, and 24 h, respectively; and fluorescence of the
hydrolyzed products of HypET24, N(CH2CH2SCH2CH2COOH)3 (c).
Conditions: 1 mg/mL in CHCl3; λex = 375 nm at rt. TMEA and EGDA
solution (da); and HypET24 solution in CHCl3 (db).
To verify the contribution of branching structure to tertiary
amine fluorescence, we synthesized a linear polymer with tertiary
amine in its backbone by Michael addition polymerization of
EGDA and bis(2-mercaptoethyl)methylamine with equal molar
ratio (Figure S3). The resultant L-poly(EGDA-BMEA),
Mn(GPC) = 6750 and Mw/Mn = 1.74 (Figure S4), emits weak
fluorescence (Φ = 0.025), as shown in Figure 2a. Since collisional
relaxation is very important factor influencing the fluorescence
efficiency,4 the tertiary amine in the linear polymer backbone has
relatively high mobility in comparison with the tertiary amine in
the branching units of HypEts, decreasing fluorescence via
fluorescent, and TMEA emits very weak fluorescence. When
EGDA and TMEA solution in chloroform was irradiated at λex =
375 nm, no fluorescence was observed (Figure 1da). But
HypET24, from polymerization of TMEA and EGDA for 24 h,
displays very strong fluorescence (Figure 1b), and under
irradiation at λex = 375 nm, a blue solution of the HypET24 in
CHCl3 is clearly seen (Figure 1db). Distribution of intensity in
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collisional relaxation. If this is correct, then a polymer with a
tertiary amine side group should display more weak fluorescence
because the side groups have higher mobility than the groups in
the polymer backbone. Thus a polymer with a tertiary amine side
group was prepared by polymerization of 1,3-propanedithiol and
1-acryloyl-1-di(isopropyl)amineethyl-2-methacryloyl-glycol
(Figure S5) and the resulting L-poly(PPT-APAMA) with
Mn(GPC) = 4300, Mw/Mn = 1.34 (Figure S6) display weaker
fluorescence (Φ = 0.006) than L-poly(EGDA-BMEA) (Figure
2a). However, their fluorescence spectra are almost the same
with HypET in both appearance and position (Figure 2a′). We
reasonably infer that the hyperbranched structure with the
tertiary amine as branching unit is a key for reserving
fluorescence efficiency of the tertiary amine. Based on the this
discussion, terminal and linear units emit very weak fluorescence,
and branching units situated in the interior of HypETs have the
biggest contribution to the fluorescence, which is consistent with
the previous fluorescence-quenching study of the PAMAM
dendrimers where the fluorescence originates from the interior
region, and the fluorescence of surface region was not
observed.16 To further understand the fluorescence, a similar
method reported16 was applied to measure the lifetimes of
fluorescence for HypETs, and the results are listed in Table S1.
Different from the results of PAMAM dendrimers that have two
lifetimes,16 only one lifetime (around 2.5 ns) for the HypETs was
obtained. This suggests that only a single fluorescent moiety,
TMEA, exists in the HypETs.
absorption between 250 and 400 nm (Figure 3a′), but for the
HypET24-O7, a new absorption band at 320 nm is clearly seen
and is strengthened significantly in the case of HypET24-O20.
Fluorescence behaviors of the HypET24 and its oxidized
products are quite different (Figure 3c,d); HypET24 emits
fluorescence at 440 nm, altering the excited wavelength from 305
to 390 nm does not change the fluorescence both in appearance
and in position. For the ring films formed by evaporation of the
HypET24 solution in CHCl3, only blue rings were observed by
optical fluorescence microscope under excitation at 330−385 nm
(Figure 3ba), and the color was not changed with altering excited
wavelengths, which is different from the HPAMAMs that exhibit
color change from blue to green to red with increasing the
excitation wavelength.12,15 However, the oxidized products of
HypET24 show two fluorescence peaks centered at 440 and
∼560 nm, respectively (Figure 3c,d), thus, blue and red ring films
of the HypET24-O20 were observed by optical fluorescence
microscope under excitation of 330−385 and 510−550 nm,
respectively (Figure 3bb,bc). Compared to the fluorescence of
HypET24, the fluorescence intensities of the oxidized HypET24s
at 440 nm decrease greatly, indicating continuous decrease of the
unoxidized tertiary amine content with increasing oxidization
time from 7 to 20 days (Figure 3c), which is contrary to the
oxidation of PAMAM dendrimers.18 But the fluorescence at 560
nm is strengthened significantly with increasing oxidization time
(Figure 3d) due to the content increase of a new chromophore.
We tried to completely oxidize the tertiary amine in the HypETs
using a strong oxidation agent, such as H2O2, to further
understand the influence of oxidation on fluorescence but was
unsuccessful, owing to HypET degradation during the oxidation
reaction.
What is the new chromophore formed during the oxidation?
Imae assumed a chromophore of oxygen-doped tertiary amine
formed in oxidation of the PAMAM dendrimers.18 Oxygen-
amine “contact” donor−acceptor complex formed in the
oxidation of aliphatic tertiary amine was presumed as a
chromophore.8 We suggest that the N→O may be formed
based on IR (Figure S8) and 1H NMR spectra (Figure S7) of the
HypET24 and its oxidized products. The 1H NMR spectrum of
HypET24-O20 in Figure S7 reveals that all proton signals remain
unchanged, except the proton signals (d and e) of three nitrogen-
attached methylene groups at 2.54−2.72 ppm are shifted to
downfield, and the unshifted tail of d and e signals represents
unoxidized tertiary amine. With increasing oxidization time, tail
strength decreases gradually, but weak proton signals still remain,
demonstrating the oxidation reaction is incomplete. To explore
the resulting oxidized product, we measured the IR spectra of
HypET24 and its oxidized products (Figure S8). Although the IR
spectrum of HypET-O7 has no big difference with that of the
HypET24, the spectrum of HypET24-O20 clearly shows
characteristic N→O absorption bands at υ = 1184, 1362, and
1460 cm−1 (dimer),19 but the NO absorption band in the
region of 1585−1539 cm−119 was not found. The characteristic
absorption bands for the unconjugated C−N of tertiary amine in
the region of 1250−1100 cm−119 decreased significantly but did
not disappear completely, which supports the result of 1H NMR
analysis. The N→O formed may be responsible for the
fluorescence.
HypETs fluorescence is centered at 440 nm, which is much
lower than the fluorescence of nitrogen-vacancy defects in
diamond.13 To explore the red-shifting possibility of the
fluorescence, the HypET24 dissolved in THF was stirred
under ambient conditions at ∼35 °C for 7 and 20 days,
respectively, and oxidized products, HypET24-O7 and
HypET24-O20, were obtained after removal of THF and
subsequently dried. UV/vis and fluorescence spectra were
measured in chloroform. Figure 3a shows the UV/vis spectra
of HypET24 and its oxidation products. HypET24 has
Figure 3. UV/vis absorption (a) and fluorescence spectra (c,d) of
HypET24, HypET24-O7, and HpyET24-O20 solutions in CHCl3 (1
mg/mL) measured at rt; excited wavelength for (c) λεx = 365 and (d) λex
= 510 nm. (b) Optical fluorescence microscope photos of the ring-shape
films prepared, respectively, by evaporating a drop of HypET24 (ba) and
HypET24-O20 solutions in CHCl3 on the clean glass slides under
excited wavelength of 330−385 nm (WU) filter (bb) and 510−550 nm
(WG) filters (bc).
Similar to current fluorescent materials studied, this
fluorescent polymer may have various applications. We applied
this polymer in the cell imaging, which requires high water
solubility of the HypET. Considering special interaction of
HepG2 cells with galactose,20 a water-soluble galactopyranose-
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modified HypET (HypET-AlpGP) was prepared by hydrolysis of
1,2:3,4-di-O-isopropylidene-α-D-galactopyranose-grafted
HypET with quantum yield = 0.10 in acidic solution (pH = 3) of
THF/H2O (Figures S9 and S10). First we tested cytotoxicity of
the HypET-AlpGP by MTT assay because toxicity is very
important for biological applications. Branched PEI with
molecular weight of 25k (PEI25k) was used as the control. In
Figure 4a, the PEI25k exhibits high cytotoxicity to HepG2 cells
applications including cell imaging, biosensing, and drug
delivery.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
pcy@ustc.edu.cn
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Work is supported by the National Natural Science Foundation
of China under contract no. 20974103, 21074121, and
21090354. We thank Prof. A.-D. Xia from Institute of Chemistry,
Sinica for helpful discussion on fluorescence of polymers.
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a new route of designing fluorescent materials for various
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