In vitro and in vivo uncaging and bioluminescence imaging by using photocaged upconversion nanoparticles

Article abstract of DOI:10.1002/anie.201107919

Trading up: A bioimaging system that is based on caged D-luciferin/ upconversion nanoparticle conjugate has been developed. The nanoparticles upconvert near-infrared light into UV light, which triggers the photorelease of D-luciferin (see scheme) and lead

Full text of DOI:10.1002/anie.201107919

Angewandte
Chemie
DOI: 10.1002/anie.201107919
Bioimaging
In Vitro and In Vivo Uncaging and Bioluminescence Imaging by Using
Photocaged Upconversion Nanoparticles**
Yanmei Yang, Qing Shao, Renren Deng, Chao Wang, Xue Teng, Kai Cheng, Zhen Cheng,
Ling Huang, Zhuang Liu, Xiaogang Liu,* and Bengang Xing*
High temporal and spatial regulation of cellular activities,
biological pathways, and gene expression is critical in complex
biological processes.[1] One remarkable technique that ena-
bles such control is the use of light to manipulate compounds
that are photoactive (or photocaged) in various biological
systems.[2] Previously, this strategy has been used to map
cellular functions, monitor the expression of transgenes, and
image the dynamic processes of cell–cell interactions in vitro
and in vivo.[3,4] Although all of these attempts were successful
in principle, there were significant limitations associated with
the use of high-intensity UV or visible light in the photo-
activation process. Excessive exposure to UV light can cause
photoreactions in nucleic acids and result in cellular damage.
Furthermore, short-wavelength UV or visible light does not
penetrate into tissue very far, which limits its utility for deep-
tissue imaging by photoactivation of the caged compounds.
Alternatively, multiphoton photolysis with long-wavelength
excitation has been used to enable deep-tissue imaging and to
target gene expression[5] Despite its usefulness, the multi-
photon photolytic process typically requires a complex exper-
imental set-up and has low conversion efficiency because of
narrow absorption cross-sections. Therefore, the development
of a simple approach that allows a high depth of penetration
into tissue and precise control of photocaged systems as well
as limiting cellular damage is highly desirable.
Recently, lanthanide-doped upconversion nanoparticles
(UCNPs) have received considerable attention for applica-
tions that range from biolabeling to optical data storage.[6]
These nanoparticles offer high photostability and enable deep
tissue-penetration depths (up to 10 mm) by irradiation with
near-infrared (NIR) light, which makes them particularly
attractive for bioimaging applications.[7] Herein, we demon-
strate a method for uncaging photocaged molecules in vitro
and in vivo and performing bioluminescence imaging studies
by combining versatile photocaged compounds with the
UCNPs.
Scheme 1 illustrates the proof-of-concept design for the
photolysis of caged d-luciferin by using bioconjugated
UCNPs. As a commonly used bioluminescent probe, d-
luciferin can recognize firefly luciferase (fLuc) reporter genes
and produce bioluminescence in the presence of O2, Mg2+
ions, and adenosine triphosphate (ATP). Therefore, d-luci-
ferin provides opportunities for extensive applications in
molecular imaging in vitro and in vivo.[8] In our design, Tm/
Yb co-doped NaYF4 core-shell nanoparticles[9] that have
a reduced surface-quenching effect were chosen as the
platform for the conjugation of d-luciferin. The core-shell
nanoparticles were coated with thiolated silane molecules and
subsequently coupled to d-luciferin that was caged with a 1-
(2-nitrophenyl)ethyl group. As the absorption band of the
photocaged d-luciferin overlaps with the upconverted emis-
sion band of the nanoparticle in the UV region (1I6!
[*] Y. Yang, Q. Shao, Prof. B. Xing
Division of Chemistry and Biological Chemistry
School of Physical & Mathematical Sciences
Nanyang Technological University
Singapore, 637371 (Singapore)
E-mail: bengang@ntu.edu.sg
R. Deng, Prof. X. Liu
Department of Chemistry, National University of Singapore
3 Science Drive 3, Singapore 117543 (Singapore)
E-mail: chmlx@nus.edu.sg
Prof. X. Liu
Institute of Materials Research and Engineering
Agency for Science, Technology and Research (A*STAR)
3 Research Link, Singapore 117602 (Singapore)
3
4F3,1D2! H6 transitions of Tm3+), excitation of the photo-
C. Wang, Prof. Z. Liu
caged UCNPs with NIR light can trigger disassociation of d-
luciferin molecules from the surface of the nanoparticle.
Importantly, the uncaging process can be monitored either by
tracking the luminescence intensity of d-luciferin or by using
an fLuc enzyme reporter, thus providing the possibility of
bioluminescence imaging studies without the need for UV or
visible light.
Jiangsu Key Laboratory for Carbon-Based Functional Materials &
Devices, Institute of Functional Nano & Soft Materials, Soochow
University (P. R. China)
K. Cheng, Prof. Z. Cheng
Molecular Imaging Program at Stanford (MIPS)
Department of Radiology, Stanford University Medical Center (USA)
X. Teng, Prof. L. Huang
Division of Biomedical Engineering
School of Chemical and Biomedical Engineering
Nanyang Technological University (Singapore)
In a typical experiment, silica-coated UCNPs with reac-
tive terminal thiol groups were synthesized by using a water-
in-oil microemulsion method (see the Supporting Information
for details).[7d–f] TEM images demonstrate that the silica-
modified nanoparticles have a narrow size distribution of
about 50 nm (Figure S1 in the Supporting Information). Both
solutions of unmodified and silica-coated UCNPs have
similar emissions in the UV, visible, and NIR spectral regions
[**] This work was supported in part by NTU Start-Up Grant (SUG),
Tier 1 (RG 64/10), the Singapore-Perking-Oxford Research Enter-
prise (SPORE), and the Singapore Ministry of Education
(MOE2010-T2-083).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107919.
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luciferin derivative and the d-luciferin/
nanoparticle conjugate upon exposure
to UV light (Figure S4 in the Supporting
Information).
The photolysis of the caged d-luci-
ferin/nanoparticle conjugate was further
confirmed by analysis of the biolumines-
cence mediated by the fLuc enzyme in
a solution of phosphate-buffered saline
(PBS, 10 mm, pH 7.2). Prior to photo-
activation with NIR light, there were no
detectable bioluminescence signals from
either the caged d-luciferin derivative or
the d-luciferin/nanoparticle conjugate
in the presence of the fLuc enzyme,
Mg2+, and ATP. Similarly, biolumines-
cence signals were not detected from the
caged d-luciferin derivative after exci-
tation with NIR light for 2 h. In stark
contrast, upon excitation of the d-luci-
ferin/nanoparticle conjugate with NIR
light, a significant increase in the biolu-
minescence signal at 560 nm was
detected (Figure 1b), which can be
attributed to the light emission from
the enzymatic oxidation of the photo-
released d-luciferin substrate.
Scheme 1. Experimental design for uncaging d-luciferin and subsequent bioluminescence
through the use of photocaged core-shell upconversion nanoparticles.
Encouraged by the results of the
in vitro uncaging experiments and the
under irradiation with NIR light (Figure S1 in the Supporting
Information), which demonstrates that coating the UCNPs
with a silica shell did not significantly affect the upconversion
properties. To ensure a high-efficiency coupling of the
photocaged molecules to the particle surface, d-luciferin
that was photocaged with a 1-(2-nitrophenyl)ethyl group was
modified with a poly(ethylene glycol) linker that contained
a terminal maleimide group, which is reactive with thiols.[10]
The loading density of photocaged d-luciferin on the surface
of a nanoparticle after the cross-coupling reaction was
approximately 490 molecules, as determined by UV/Vis
absorption spectroscopy (Figure S3 in the Supporting Infor-
mation).
bioluminescence studies, we investigated the intracellular
We examined the feasibility of uncaging d-luciferin by
using UCNPs. The poly(ethylene glycol)-modified d-luciferin
derivative was chosen as a control. Without irradiation with
NIR light, both the caged d-luciferin derivative and the d-
luciferin/nanoparticle conjugate did not exhibit d-luciferin
fluorescence, which indicated that the d-luciferin molecules
were effectively blocked in the photocaged systems. However,
upon excitation with laser light (980 nm), the fluorescence of
the solution of the d-luciferin/nanoparticle conjugate
increased significantly, which can be attributed to the photo-
deprotection of the 1-(2-nitrophenyl)ethyl group by upcon-
verted UV light (Figure 1a). Laser irradiation of the caged d-
luciferin derivative that was not conjugated to the nano-
particles did not lead to a significant increase in fluorescence
from d-luciferin (Figure 1a), which indicated that the pho-
tocaged molecule itself was not sensitive to NIR light. Similar
increases in fluorescence were also detected for the caged d-
Figure 1. a) Time-dependent fluorescence increase from d-luciferin
released from photocaged UCNPs (0.5 mm) under irradiation with NIR
light (0–3 h). Black line=d-luciferin/UCNP conjugate; red line=pho-
tocaged d-luciferin only b) Time-dependent bioluminescence generated
by adding the fLuc enzyme to uncaged d-luciferin under irradiation
with NIR light (0–3 h). Purple bars=d-luciferin/UCNP conjugate; blue
bars=photocaged d-luciferin only
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uptake and uncaging of the d-luci-
ferin/nanoparticle conjugate in living
cells. In a typical cell imaging study,
the d-luciferin/nanoparticle conjugate
([d-luciferin]: 0.5 mm) was incubated
with C6 glioma cells in Dulbecco’s
Modified Eagle Medium (DMEM) at
378C for 2 h. As a control, the caged
d-luciferin derivative was incubated
with the same type of cells under the
same conditions. These two samples
were then irradiated with NIR light
for intracellular uncaging studies (Fig-
ures 2b and d). Before exposure to
NIR light, the fluorescence signals
within the cells in both of the samples
were weak (Figures 2a and c), which
indicated the possible quenching of
the fluorescence from d-luciferin by
the 1-(2-nitrophenyl)ethyl group or by
the silica shell of the UCNPs.[7f–g] The
NIR-activated uncaging of the d-luci-
ferin/nanoparticle conjugate was fur-
ther confirmed by bioluminescence
studies in MCF-7 cells that were
expressing the fLuc enzyme. Upon
irradiation with NIR light, strong
bioluminescent emission was detected
at 560 nm (Figure 2 f and Figure S9 in
the Supporting Information). The
emission signal from the cells that
were transfected with fLuc was pro-
portion to the number of cells used
Figure 2. Fluorescence imaging of C6 glioma cells incubated with photocaged d-luciferin (0.5 mm) and the
photocaged d-luciferin/UCNP conjugate (0.5 mm) in the absence (panels a and c) and presence (panels b
and d) of NIR light. Excitation filter: 460/40 nm; emission filter: 535/50 nm (exposure time to NIR light:
2 h). e) Bar graph of the fluorescence intensity of uncaged d-luciferin in cell lysate shown in panels a)–d).
The cell lines without sample incubation were used as controls. f) Bioluminescence visualization of different
concentrations of photocaged UCNPs in living cells (MCF-7/fLuc). g) Cell viability in the presence of silica–
UCNPs or d-luciferin/UCNP conjugates that were exposed to NIR light for 2 h (purple bars), UV light for
5 min (blue bars), or no light (gray bars). As a control, cells that were not incubated with UCNPs or
d-luciferin/UCNP conjugates were also exposed to UV and NIR light.
(Figure S10 in the Supporting Information). As a negative
control, no bioluminescence was detected in MCF-7 cells that
were incubated with the d-luciferin/nanoparticle conjugate
without NIR irradiation, which clearly suggests that the use of
the cell-penetrating nanoparticles enables the effective iden-
tification of fLuc enzymatic activity in living cells. More
importantly, MTT assays showed that no significant cytotox-
icity was observed in C6 glioma cells that were treated with
the d-luciferin/nanoparticle conjugate after two hours of
irradiation with NIR light. This was in stark contrast to
a parallel experiment with UV irradiation, which showed
significant cellular damage after a short exposure time (5 min,
Figure 2g).
Another unique advantage of using photocaged silica-
UCNPs is the ability to carry out bioimaging studies at
substantial tissue depths. As a proof-of-concept experiment,
the d-luciferin/nanoparticle conjugate and the fLuc enzyme
were subcutaneously injected into living mice at a tissue depth
of 2 mm, and then the bioluminescence was measured by
using a modified Evolve in vivo imaging system. In contrast to
the bioluminescence that was detected in the left thigh of
a mouse that was directly injected with d-luciferin, there was
no detectable bioluminescence signal at the point of injection
in the right thigh of the mouse without irradiation with NIR
light (Figure 3a). In comparison, strong bioluminescence
signals were detected in the mouse that was injected with
Figure 3. Bioluminescent images of fLuc activity in living mice that
were treated with d-luciferin. a) Left: injection with d-luciferin (20 mm,
20 mL); right: injection with photocaged nanoparticles without NIR
light irradiation. b) Left: injection with photocaged nanoparticles and
irradiation with UV light for 10 min; right: injection with photocaged
nanoparticles and irradiation with NIR light for 1 h.
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NIR light (Figure 3b). It should be noted that under UV
irradiation, no notable bioluminescence was detected (Fig-
ure 3b and Figure S11 in the Supporting Information). Taken
together, these data show the versatility of using photocaged
UCNPs for bioluminescence studies at substantial tissue
depths.
In conclusion, we have reported a system for the
controlled uncaging of d-luciferin and bioluminescence
imaging that is based on photocaged upconversion nano-
particles. This method takes advantage of photon upconver-
sion of NIR light to UV light to trigger the uncaging of d-
luciferin from d-luciferin-conjugated UCNPs. The released
d-luciferin effectively conferred enhanced fluorescence and
bioluminescence signals in vitro and in vivo with deep light
penetration and low cellular damage. With compositional or
structural tuning of the UCNPs and the availability of diverse
photocaged functional groups, this UCNP-based photolysis
could be readily extended to other biomedical studies. These
results may also offer new possibilities for non-invasively
monitoring the dynamic functions of cells and selectively
delivering drug molecules to targeted areas in vitro and
in vivo on the basis of photocaged techniques.
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Received: November 10, 2011
Revised: December 13, 2011
Published online: January 12, 2012
Keywords: bioluminescence · d-luciferin · nanoparticles ·
.
photochemistry · upconversion
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