Ultrasmall Organic Nanoparticles with Aggregation-Induced Emission and Enhanced Quantum Yield for Fluorescence Cell Imaging

Article abstract of DOI:10.1021/acs.analchem.6b02032

The use of fluorescence probes for biomedical imaging has attracted significant attention over recent years owing to their high resolution at cellular level. The probes are available in many formats including small particle size based imaging agents which

Full text of DOI:10.1021/acs.analchem.6b02032

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Article
Ultrasmall Organic Nanoparticles with Aggregation Induced Emission
and Enhanced Quantum Yield for Fluorescence Cell Imaging
Suying Xu, Xilin Bai, Jingwen Ma, Minmin Xu, Gaofei Hu, Tony D. James, and Leyu Wang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02032 • Publication Date (Web): 28 Jun 2016
Downloaded from http://pubs.acs.org on June 28, 2016
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Analytical Chemistry
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Ultrasmall Organic Nanoparticles with Aggregation Induced
Emission and Enhanced Quantum Yield for Fluorescence Cell
Imaging
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Suying Xu† , Xilin Bai† , Jingwen Ma†, Minmin Xu†, Gaofei Hu†, Tony D. James‡ and Leyu Wang*†
†
State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Environmentally Harmful Chemical
Analysis, Beijing University of Chemical Technology, Beijing 100029, P. R. China, Fax:(86)10ꢀ64427869
Email: lywang@mail.buct.edu.cn
‡Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom
ABSTRACT: Using fluorescence probes for biomedical imaging has attracted significant attention over recent years owing to their
high resolution at cellular level. The probes are available in many formats including small particleꢀsize based imaging agents which
are considered to be promising candidates, due to their excellent stabilities. Yet, concerns over the potential cytotoxicity effects of
inorganic luminescent particles have led to questions about their suitability for imaging applications. Exploration of alternatives
inspired us to use organic fluorophores with aggregationꢀinduced emission (AIE), prepared by functionalizing the amine group on
tetraphenylethene with 3,5ꢀbis(trifluoromethyl)phenyl isocyanate. The asꢀsynthesized novel AIE fluorophore (TPEꢀF) display enꢀ
hanced quantum yield and longer lifetime as compared with its counterparts (TPEꢀAM). Furthermore, the TPEꢀF was encapsulated
into smallꢀsize organic nanoparticles (dynamic light scattering size, ∼10 nm) with polysuccinimide (PSI). The biocompatibility,
excellent stability, bright fluorescence and selective cellꢀtargeting of these NPs enable the asꢀprepared TPEꢀF NPs suitable for speꢀ
cific fluorescence cell imaging.
INTRODUCTION
Optical tags including organic and inorganic labels have
been widely investigated for chemical sensing,
Recently, opposite to the aforementioned ACQ effect, fluoresꢀ
cent molecules with aggregationꢀinduced emission (AIE) feaꢀ
ture, that is, the compound is nonꢀemissive in solution but
highly fluorescent once in its aggregated state, have been
1
ꢀ3
4ꢀ7
8
ꢀ12
studying
crucial biological processes such as proteinꢀprotein interacꢀ
tion, metastasis of cancerous cells and realꢀtime monitoring of
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widely investigated for bioꢀimaging.
One prototypical
1
3ꢀ18
compound with AIE properties is tetraphenylethene (TPE), a
central olefin stator with four peripheral aromatic rotors conꢀ
nected by singleꢀbonds. Owing to the dynamic rotation of the
aromatic rotors as well as twisting motion, the exciton energy
is nonꢀradioactively dissipated when in its free form. While in
an aggregated state, radiationless relaxation pathways are reꢀ
drug/gene delivery.
Among the various optical imaging
5ꢀ7
agents, particleꢀbased imaging agents are of great interest
since organic molecular dyes often suffers from short retention
time and are subject to photoꢀbleaching.
particleꢀbased fluorophores are composed of inorganic speꢀ
cies, even some toxic heavy metal cations, of which the potenꢀ
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9, 20
However, most
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stricted, thus facilitating the fluorescence emission of TPE.
tial cytotoxicity effects have greatly impaired their applicaꢀ
21, 22
By taking advantage of this property, compounds with AIE
properties could accommodate encapsulation in particles while
still maintaining fluorescence emission. Indeed, researchers
have explored the adaptability for constructing luminescent
particles, which show low cytotoxicity and good stability in
tion.
Encapsulation of organic dye molecules into nanoꢀ
particles (NPs) may afford a promising strategy to extend the
circulation lifeꢀtime as particleꢀbased agents and reduce the
potential cytotoxicity. However, the fluorescence intensity of
conventional fluorophores tends to decrease when they are in
in close contact; a common phenomenon known as aggregaꢀ
tionꢀcaused quenching (ACQ), which presents a dilemma for
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23, 30ꢀ38
bioimaging applications.
Recently, it was also found that
the different packing of AIE compounds in the solid state afꢀ
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fects the optical properties. We envisaged that fabrication of
nanoparticle with small particle size would afford a tight packꢀ
ing of AIE compounds, which may increase the quantum
conventional fluorophores used in biomedical imaging.
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yields of the afforded organic particles. In another aspect, naꢀ mation for synthesis of PSIOAm) (38 mg of PSIOAm and 1.0 mL
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noparticleꢀbased imaging agents with hydrodynamic diameters
in the range of 5‒10 nm are considered to be optimal since
larger particles would accumulate in the liver and spleen via
of methanol solution containing TPEꢀAM (0.3 mg) was added
into 10mL NaOH (5 mM) aqueous solution under ultrasoniꢀ
cation (350 W, 6 min). Afterwards, the chloroform and methaꢀ
nol was removed by evaporating at 45 °C for 30 min. Similarꢀ
ly, TPEꢀF was treated with the aboveꢀmentioned method for
TPEꢀF@PSIOAm NPs.
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nonꢀspecific interactions.
Herein, we prepared fluorine
containing AIE compound (TPEꢀF) and encapsulated it in
small size NPs, which were also labeled with a cellꢀtargeting
peptide (ArgꢀGlyꢀAsp, RGD). The acquired luminescent NPs
display enhanced luminescence intensities and good stability
and have been successfully utilized for targeted cellular imagꢀ
ing of HeLa cells.
Cell imaging. HeLa cells were seeded on a sterilized glass
cover slide and cultured in a 12ꢀwell cell culture plate overꢀ
night under recommended conditions at 37 °C in 5% CO2ꢀ
humiditied incubator. Then, the TPEꢀF@PSIOAmꢀRGD stock
solution was added into the cell culture well with a final conꢀ
centration of 30 µg/mL. The HeLa cells were incubated with
the TPEꢀF@PSIOAmꢀRGD for another 6 h. As a control, TPEꢀ
F@PSIOAm without RGD target peptide was incubated with the
HeLa cells under the same condition. Thereafter, the cells on
the glass slide were washed with phosphate buffer saline (PBS,
pH 7.4, 20 mM) and fixed in 4% paraformaldehyde solution
for 15 min. The fluorescence imaging based on the fluoresꢀ
cence of TPEꢀF was conducted on an EVOS FL microscopes
system (Life Technologies) with excitation wavelength at 360
nm and emission at 447 nm.
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EXPERIMENTAL SECTION
Chemicals and Reagents. General chemicals were of the
highest grade available (at least analytical grade) and used as
received without further purification. Absolute ethanol, methꢀ
anol, chloroform, dimethylformamide (DMF), NaOH,
NaH PO ·2H O, and Na HPO ·12H O were supplied by Beiꢀ
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jing Chemical Reagent Company. Polysuccinimide (PSI,
Mw~6000) was obtained from Shijiazhuang Desai Chemical
Company (China). Oleylamine (OAm) was purchased from
SigmaꢀAldrich. 4,4'ꢀDiaminobenzophenone and 3,5ꢀbis (triꢀ
fluoromethyl) phenyl isocyanate were supplied by J&K Scienꢀ
tific LTD. Ethyl dimethylaminopropyl carbodiimide solution
RESULTS AND DISCUSSION
(EDC), NꢀHydroxysuccinimide (NHS), and methyl thiazolyl
tetrazolium (MTT) were purchased from SigmaꢀAldrich. Deꢀ
ionized (DI) water was used throughout all experiments.
Preparation of TPE-AM. The 4,4',4'',4'''ꢀ(etheneꢀ1,1,2,2ꢀ
tetrayl)tetraaniline (TPEꢀAM) was prepared by following preꢀ
25
viously reported procedures with slight modification. In brief,
,4'ꢀdiaminobenzophenone (0.4 g, 1.8 mmol) was dissolved in
4
concentrated hydrochloric acid (18 mL) at 60 °C under stirring,
followed by the addition of Tin powder (1.2 g, 10 mmol) and
the reaction mixture was stirred at 75 °C for 5 h. After cooling,
the mixture was vacuum filtrated and washed by NaOH (1 M)
and H O. After drying overnight at room temperature, TPEꢀ
2
AM was obtained as a yellowish powder (228 mg) in 65%
1
yield. H NMR (400 MHz, d ꢀDMSO) δ (TMS, ppm): 6.59 (d,
6
J = 8.72 Hz, 8H), 6.27 (d, J = 8.36 Hz, 8H), 4.85 (s, 8H)
Scheme 1. Reaction procedures for preparation of TPEꢀF.
Preparation of TPE-F. Compound 1,1',1'',1'''ꢀ(etheneꢀ
1
,1,2,2ꢀtetrayltetrakis(benzeneꢀ4,1ꢀdiyl))tetrakis(3ꢀ(3,5ꢀ
Compound TPEꢀAM was synthesized using the previousꢀ
42
bis(trifluoromethyl)phenyl)urea) (TPEꢀF) was readily syntheꢀ
sized by reacting TPEꢀAM with 1ꢀisocyanatoꢀ3,5ꢀ
bis(trifluoromethyl)benzene. Specifically, 80 mg (0.2 mmol)
of TPEꢀAM was dissolved in 60 mL of dichloromethane, then
3,5ꢀbis (trifluoromethyl) phenyl isocyanate (160 µL, 0.9 mmol)
in 5 mL of dichloromethane was added dropwise and stirred
for 30 min in iceꢀbath. The reaction mixture was then refluxed
for 3 h. Thereafter, the organic phase was concentrated under
reduced pressure and washed by dichloromethane (DCM).
Finally, the product was dried at room temperature to afford a
ly reported method. The TPEꢀF compound was prepared by
facile reaction of TPEꢀAM with 1ꢀisocyanatoꢀ3,5ꢀ
bis(trifluoromethyl)benzene (Scheme 1). The compounds were
confirmed using nuclear magnetic resonance (NMR) and mass
spectrometry (MS), and given in the Supporting Information
Figure S1ꢀS4. Both TPEꢀAM and TPEꢀF display weak emisꢀ
sion in pure methanol solution, but their optical properties
follow the typical behavior of AIE probes when varying water
fractions (Figure 1 and 2). More specifically, for TPEꢀAM, the
emission intensity remained unchanged with water fraction
less than 80 vol%, but significantly enhanced with further
increase of the water fraction. The emission intensity inꢀ
creased up to ~125 fold when compared to that in pure methaꢀ
nol, when the water fraction reaches 90 vol%. As for TPEꢀF,
the emission intensity increased about 143 fold when comꢀ
pared to that for pure methanol. The conjugation of TPEꢀAM
with 3,5ꢀbis(trifluoromethyl) phenyl isocyanate further exꢀ
tends the conjugation system and also increases the rigidity of
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yellowish powder (125 mg) in 44% yield. H NMR (400 MHz,
d6ꢀDMSO) δ(TMS, ppm): 9.38 (s, 4H), 8.96 (s, 4H), 8.12 (s,
8
8
H), 7.64 (s, 4H), 7.28 (d, J = 8.48Hz, 8H), 6.93 (d, J = 8.4Hz,
19
H); F NMR(376 MHz, d ꢀDMSO) δ(CFCl , ppm): ꢀ61.68
6
3
+
(C H ꢀCF ); HRꢀMS (m/z ): M , calcd. for C H F N O ,
6 5 3 62 37 24 8 4
1
413.2549; found, 1413.2489
Preparation of hydrophilic TPE-AM@PSIOAM NPs. 1.0 mL
of chloroform colloidal solution containing oleylamine funcꢀ
41
tionalized polysuccinimide (PSIOAm, see Supporting Inforꢀ
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the molecule, thus producing significantly enhanced emission
intensities.
Interestingly, the TPEꢀF compound displays its maximal
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emission intensities with the water fraction at 40% (Figure 2).
If the water fraction was further increased, the emission intenꢀ
sity was reduced, which can probably be ascribed to the preꢀ
cipitation of the TPEꢀF aggregates due to poor solubility of
the TPEꢀF compound, as shown by the photographic images
under UV light (Figure S5). These issue could be solved by
encapsulating the TPEꢀF into a hydrophobic particle core usꢀ
ing hydrophilic and biocompatible PSIOAM, which in one asꢀ
pect, increases the packing density, meanwhile avoiding any
undesired precipitation.
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Figure 3. TEM images (A, B) and DLS (C, D) analysis of TPEꢀ
AM@PSIOAm (A, C) and TPEꢀF@PSIOAm (B, D), respectively.
To this end, fabrication of luminescent AIE nanoaggreꢀ
gates was achieved by using oleylamine functionalized amꢀ
phiphilic polysuccinimide (PSIOAm). The hydrophobic AIE
compounds were encapsulated in the hydrophobic cores of the
particles after being subjected to ultrasonication under alkali
conditions. The transmission electron microscopy (TEM) imꢀ
ages and dynamic light scattering (DLS) results (Figure 3)
indicated that the afforded TPEꢀAM@PSIOAm and TPEꢀ
F@PSIOAm have average sizes of 16.5±3.2 and 9.9±1.8 nm,
respectively, which are smaller than most of the reported AIE
32ꢀ35
organic particles.
With respect to the optical properties, the
Figure 1. (A) Fluorescent spectra of compound TPEꢀAM at difꢀ
TPEꢀF@PSIOAm displayed stronger emission intensities when
compared with TPEꢀAM@PSIOAm (Figure 4) at the same conꢀ
centration, similar to the phenomenon observed in solution.
Additionally, increased amounts of AIE compounds would
result in much more enhanced emission intensities. The fluoꢀ
rescence quantum yields for TPEꢀAM@PSIOAm and TPEꢀ
F@PSIOAm were measured to be 0.05 and 0.52, respectively
when using quinine sulfate as a reference. The observed high
quantum yield of TPEꢀF@PSIOAm originates from the dense
packing of the TPEꢀF in the hydrophobic cores as well as the
increased rigidity of the molecule itself. Moreover, the emisꢀ
sion intensities of TPEꢀF@PSIOAm NPs were inert to changes
of pH, while the emission of TPEꢀAM@PSIOAm has a strong
pH dependency (Figure S6). This is reasonable given that the
amine group is prone to protonation under acidic condition,
thus leading to a decrease of electron density for the comꢀ
pounds, this may in turn affect the emission intensity of TPEꢀ
AM. In another aspect, fabrication of TPEꢀF@PSIOAm NPs
also offers an effective way to avoid unwanted precipitation
observed with isolated TPEꢀF aggregates in mixed solvents,
since the carboxylic acid groups on the amphiphilic PSIOAM
ferent water fraction (f ); (B) Photographs of TPEꢀAM solution
w
with different water fraction under UV light. [TPEꢀAM] = 50
ꢁg/mL; λex = 365 nm.
allow for good dispersion of the asꢀprepared TPEꢀF@PSIOAm
NPs (Figure S5).
Figure 2. A) Fluorescent spectra of compound TPEꢀF at different
water fraction; (B) Photographs of TPEꢀF solution with different
water fractions under UV light. [TPEꢀF] = 50 ꢁg/mL; λex = 365
nm.
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NPs remains constant when left in both phosphate buffer saline
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(PBS) and DMEM culture media for over one week (Figure S8),
indicating their excellent stability. To further demonstrate their
performance in bioimaging, HeLa cells were incubated with TPEꢀ
F@PSIOAm and TPEꢀF@PSIOAMꢀRGD NPs, respectively. As indiꢀ
cated by Figure 6, HeLa cells incubated with TPEꢀF@PSIOAm
ꢀ
RGD display bright typical AIE emission while those incubated
with TPEꢀF@PSIOAM, no emission was observed, implying the
specific targeting ability of the TPEꢀF@PSIOAMꢀRGD NPs.
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Figure 4. (A) Fluorescent spectra of TPEꢀF@PSIOAm (a,b,c) and
TPEꢀAM@PSIOAm (d,e,f) at different concentrations: 12.5 (c, f),
2
5.0 (b,d) and 50.0 ꢁg/mL(a,e), respectively; (B) Photographs of
TPEꢀAM@PSIOAm and TPEꢀF@PSIOAm solutions at different
concentrations: 12.5 (c, f), 25.0 (b, d) and 50.0 ꢁg/mL (a, e), reꢀ
spectively
Figure 6. Fluorescence images of HeLa cells cultured with TPEꢀ
F@PSIOAm (A,B,C) and TPEꢀF@PSIOAmꢀRGD (D, E, F) nanoparꢀ
ticle suspension for 6 hours, respectively.
CONCLUSION
In summary, ultrasmall fluorescent organic NPs with aggreꢀ
gated induced emission and high quantum yields were develꢀ
oped by encapsulating compound AIEꢀF as the hydrophobic
core with an amphiphilic polymer (PSIOAm). The asꢀprepared
NPs display good biocompatibility when compared to convenꢀ
tional inorganic quantum dots, and display excellent stability
even in biological medium. Additionally, modification with
cell targeting RGD peptide allowed for specific cancer cell
imaging, clearly demonstrating the bioꢀimaging potential of
these particles. Currently we are also exploring the potential of
19
using TPEꢀF for multimodal imaging coupled with F magꢀ
netic resonance imaging (MRI) by using the fluorine atom on
the compound of TPEꢀF.
ASSOCIATED CONTENT
Supporting Information
Figure 5. Cell viabilities after treatment with various concentraꢀ
tions of TPEꢀF@PSIOAmꢀRGD NPs suspension for 24 and 48 h,
respectively.
Instrumentation, synthesis of PSIOAM, RGD grafting, cell viability
tests, NMR and MS results of compound TPEꢀAM and TPEꢀF
(
Figure S1ꢀS4), photographs of TPEꢀAM and TPEꢀF solution
under UV light (Figure S5), pH influence on the fluorescence
Figure S6), cell viability treated with different concentration of
Furthermore, the RGD moiety was attached to phosphatidyl
ethanolamine, which was further employed as coꢀligands to preꢀ
pare TPEꢀF@PSIOAMꢀRGD NPs. The cytotoxicity of the TPEꢀ
F@PSIOAMꢀRGD NPs was evaluated via the methyl thiazolylteꢀ
trazolium (MTT) assay, where HeLa cells were incubated with
different amounts of TPEꢀF@PSIOAMꢀRGD NPs for 24 and 48 h,
respectively (Figure 5). As a control, TPEꢀF@PSIOAM NPs were
treated under identical experimental conditions (Figure S7). As
shown in Figure 5 and Figure S7, over 95% cell viability was
observed for both types of NPs, showing an ultrahigh biocompatiꢀ
bility. In addition, the emission intensity of TPEꢀF@PSIOAMꢀRGD
(
TPEꢀF@PSIOAm (Figure S7), DLS results and fluorescent intensiꢀ
ties changes of TPEꢀF@PSIOAmꢀRGD (Figure S8).
AUTHOR INFORMATION
Corresponding Author
lywang@mail.buct.edu.cn (Leyu Wang)
Notes
1
these two authors contributed equally to this work.
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The authors declare no competing financial interest.
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(
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ACKNOWLEDGMENT
This research was supported in part by the National Natural Sciꢀ
ence Foundation of China (Grant Nos. 21475007, 21275015 and
(31) Zhao, Q. L.; Li, K.; Chen, S. J.; Qin, A. J.; Ding, D.; Zhang, S.; Liu,
2
1505003), and the Fundamental Research Funds for the Central
Y.; Liu, B.; Sun, J. Z.; Tang, B., J. Mater. Chem. 2012, 22, 15128ꢀ
1
5135.
Universities (YS1406, buctrc201507 and buctrc201608). We also
thank the support from the “Innovation and Promotion Project of
Beijing University of Chemical Technology”, the “Public Hatchꢀ
ing Platform for Recruited Talents of Beijing University of Chemꢀ
ical Technology, the HighꢀLevel Faculty Program of Beijing Uniꢀ
versity of Chemical Technology (buctrc201325), and BUCT Fund
for Disciplines Construction and Development (Project No.
XK1526)”.
(32) Li, K.; Qin, W.; Ding, D.; Tomczak, N.; Geng, J. L.; Liu, R. R.; Liu, J.
Z.; Zhang, X. H.; Liu, H. W.; Liu, B.; Tang, B. Z., Sci Rep 2013, 3, 10.
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