Near-infrared upconversion controls photocaged cell adhesion

Article abstract of DOI:10.1021/ja412364m

Dynamic control of cell-surface interactions with near-infrared (NIR) light is particularly attractive for regeneration medicine and cell-based therapy. Herein we successfully achieve NIR-controlled cell adhesion with upconversion nanoparticles (UCNPs) ba

Full text of DOI:10.1021/ja412364m

Communication
pubs.acs.org/JACS
Near-Infrared Upconversion Controls Photocaged Cell Adhesion
Wen Li,†,‡ Jiasi Wang,†,‡ Jinsong Ren,† and Xiaogang Qu*,†
†Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource Utilization, Laboratory of Chemical
Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China
‡University of Chinese Academy of Sciences, Beijing 100039, China
S
* Supporting Information
switch of ring opening−closing of dithienylethene molecule.8
ABSTRACT: Dynamic control of cell-surface interactions
with near-infrared (NIR) light is particularly attractive for
regeneration medicine and cell-based therapy. Herein we
successfully achieve NIR-controlled cell adhesion with
upconversion nanoparticles (UCNPs) based program-
mable substrate. The UCNPs can harvest the biocompat-
ible NIR light and convert it into local UV light, which
results in cleavage of the photocaged linkers and on-
demand release of adhesive cells. The strategy also enables
the feasibility of deep-tissue photocontrol of cell adhesion
on substrate. Our work may open a new avenue for design
of UCNP-based cell scaffolds to dynamically manipulate
cell−matrix and cell−cell interactions.
These works mainly highlight the application of UCNPs in
biosensor,8c imaging,8d photodynamic therapy, drug delivery,8e,f
and activation of DNA/siRNA.8h Inspired by these achieve-
ments, herein we found that UCNPs could also be successfully
used in a dynamic substrate to regulate cell−surface interaction
and cell adhesion with NIR light stimulus. This work may not
only provide a solution to resolve UV light problem in
photocontrolled cell adhesion but also may greatly extend the
growing applications of UCNPs, especially for the fabrication of
dynamic substrate for tissue engineering, cell-based therapy,
and disease-related cell manipulation and analysis.
The general strategy was illustrated in Scheme 1. The
substrate was first modified with UCNPs, which could harvest
Scheme 1. Schematic Illustration of the Using of UCNPs for
NIR Light Controlled Cell Adhesion
a
olecular approaches to regulate cell−material inter-
M
actions are desirable for cell biology and medical
application.1 Among them, dynamic control of cell adhesion
on substrates is the fundamental issue because cell adhesion has
profound effects on the cell fate and diverse cellular responses.2
So far, switchable substrates that are responsible for various
external stimuli have been explored.3 Photoactived cell
adhesion has attracted much attention because it can allow
noninvasively to control cells spatially and temporally.3c−e
However, most of them have achieved limited success for in
vitro study since UV−vis light used in the stimulus process is
not friendly to cells, and it is also quickly attenuated in tissues.
Compared with UV light, NIR light is expected to cause
minimal cell damage and has remarkably deep tissue
penetration.4 Recently, NIR-controlled cell adhesion on a
surface by using photothermal effect of graphene and DNA was
reported.5 However, the thermosensitive DNA was susceptible
to nuclease degradation in body fluid. To bridge the gap
between UV light and NIR stimulus for cell adhesion,
converting NIR light into local UV light can be a good way
to control cell adhesion.
a
Upon absorption of 980 nm light, the UCNPs can emit UV photons
and activate the cleavage of the PR linker, resulting in the
disassociation of RGD and cells from substrate.
Recently, an appealing candidate for the application of NIR
light has emerged, which is based on lanthanide-doped
upconversion nanoparticles (UCNPs).6 These UCNPs are
usually made of host lattices of ceramic materials embedded
with trivalent lanthanide ions. Due to the unique ladder-like
energy level structures of lanthanide ions, UCNPs are able to
absorb NIR light and convert it into high-energy photons in the
UV, visible, and NIR regions.7 Due to this fascinating
photoluminescence property, UCNPs have been successfully
used as NIR-induced mediators for photoisomerization of
azobenzene group, photocleavage of compounds, and photo-
the NIR light and convert it into the necessary UV light locally.
Then the UV-photocleavable 4-(hydroxymethyl)-3-nitroben-
zoic acid (ONA) molecule9 was covalently attached to the
UCNPs. After that, arginine-glycine-aspartic acid (RGD) was
chosen here as the bioadhesive ligand to immobilize on the
UCNPs modified surface to mediate cell adhesion. The RGD
peptide motif can be specifically recognized by integrins, the
Received: December 5, 2013
Published: January 27, 2014
© 2014 American Chemical Society
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Communication
major receptors of cells for adhesion.10 To ensure more
efficient adhesion, a polyethyleneglycol (PEG) spacer was
introduced between surface and RGD ligand to decrease
nonspecific absorption and steric hindrance.3e,7a In our strategy,
upon irradiation with NIR laser of 980 nm, the UCNPs could
emit photons in the UV regions that, in turn, were absorbed by
photolabile linkers. This resulted in the activation of the
photocleavage reaction in the bioadhesive linker and dynamic
change of cell binding state on surface. In addition, the UCNP-
based system also showed potential in deep tissue activation of
cell adhesion with the NIR stimulus.
After synthesis and characterization of the functionalized
UCNPs, the UCNP-based substrate was fabricated for control
of cell adhesion. First, the UCNPs@SiO2-COOH were
attached to the amino group modified quartz surface after
activating the carboxyl. The topography of the UCNPs@SiO2
decorated substrate was observed with SEM (Figure S5a).
Under the irradiation of NIR laser, the prepared substrate
exhibited purple-blue photoluminescence (Figure S5b). The
photoresponsive linker (PR linker) contained the ONA group,
and the PEG spacer was synthesized (Scheme S1) and then
covalently conjugated on the UCNPs modified surface via ester
bond. The detail modification process was provided in Scheme
S2, and the obtained surface was defined as UCNPs@SiO2-PR
surface. As predicted in Figure 1d, the obtained surface revealed
absorbance in UV region, which confirmed the successful
attachment of o-nitrobenzyl contained linker on surface.
For a starting point of our study, the lanthanide-doped
NaYF4:TmYb nanoparticles were synthesized as reported.11 As
shown in the SEM images (Figure 1a), the UCNPs possessed
To examine whether the UCNPs on surface could be
effective to trigger the photolytic reaction, the substrate was
immersed into certain amount of solution and irradiated with
NIR laser. The absorption spectral change of the surrounding
solution is shown in Figure S6. After exposure to NIR laser,
obvious absorption bands in 300−450 nm were detected, which
were attributed to the formation of photocleaved nitro-
sobenzaldehydes. UCNPs could harvest NIR light and emitted
the UV light to photocleavage the o-nitrobenzyl groups, then
the nitrosobenzaldehyde products were released from the
surface and diffused to the solution. The spectral change after
NIR irradiation had a similar trend as that recorded when the
surface was exposed directly to UV light (360 nm). While after
replacing the UCNPs@SiO2 with SiO2 nanoparticles, the NIR
light exposure did not result in observable absorption change,
thereby confirming the photolysis was, as expected, not
susceptible to NIR light in the absence of the UCNPs. To
further access the ability of photolysis for control the target
molecule release, we used fluorescein isothiocyanate (FITC) as
the model to visualize the detachment process directly. After
immobilization of FITC on the amino terminus of PR linker,
the substrate emitted bright green fluorescence (Figure 2a).
When the substrate was treated with NIR light, the strong
fluorescence dramatically diminished due to the photocleavage
Figure 1. (a) SEM image of the NaYF4:TmYb UCNPs. (b) TEM
image of the UCNPs@SiO2. The inserts showed the SiO2 shell with
red line. (c) Absorption spectrum of the ONA solution (red line) and
the emission spectra of UCNPs@SiO2 (black line). Insert photograph
was the UCNPs@SiO2 solution under NIR laser exposure. (d) UV−
vis absorption spectra of UCNPs@SiO2 modified surface before (black
line) and after immobilization of PR linker (red line) and the changes
in the absorbance after immobilization of PR linker (blue line).
uniform rod shapes with an average length of 1.45 μm and
width of 125 nm (Figure S1). The powder X-ray diffraction
confirmed the UCNPs were hexagonal in phase (Figure S2).11
To increase their water solubility and biocompatibility, the thin
silica shells with the thickness of 18 nm were coated on the
surface of UCNPs (Figure 1b).12 After silica coating, the
UCNPs@SiO2 nanoparticles were well dispersible in water and
easy for surface modification. The nanoparticles functionalized
with a carboxylic group (UCNPs@SiO2-COOH) were
obtained by treatment with 3-aminopropyltriethoxysilane and
succinic anhydride, successively.13 The surface modification
processes were monitored with Fourier transform infrared
spectra (Figure S3)12,13 and ζ potential evaluations (Figure S4).
Upon excited by a 980 nm CW laser, UCNPs@SiO2-COOH
displayed emission bands in the UV−vis region that were
attributed to the transitions from the emitting energy levels of
Tm3+ (Figure 1c). When the laser beam traveled through the
UCNPs colloidal solutions, the purple-blue photoluminescence
was also readily observed (Figure 1c inset). For current study,
the UV light emitted from the UCNPs was appealing, since
these wavelengths overlapped with the absorption band of
ONA linker.
Figure 2. (a) Fluorescence images of substrate immobilized with FITC
before laser exposure (1) and after 980 nm laser exposure (2). (b)
Viability of cells on the substrate without (blue) and with (red)
UCNPs exposed to various doses of 980 nm CW NIR laser for 30 min.
(c) Fluorescence images of cells incubated on the substrate. Insert was
the corresponding bright-field image of cells in the blue square. Scale
bars are 100 μm.
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Communication
of linkers. And the photocleavage efficiency was affected by the
distance between ONA molecule and UCNPs (Figure S7),
which suggested the effect of local UV light converted from
UCNPs. To certify that the attenuation of fluorescence was not
caused by the photobleaching, the control substrates without
PR linker were fabricated. Irradiation with same NIR light did
not cause obvious fluorescence change (Figure S8).
Prior to examining the NIR-controlled cell adhesion, we
checked the biocompatibility of UCNPs as well as the safety of
NIR light and especially the unconverted UV light from
UCNPs. The NIH 3T3 fibroblast cells were cultured on
UCNPs modified surface and then were irradiated with a
different dose of 980 nm laser (2.5 min break after 2.5 min
irradiation). Cells incubated on the surface without UCNPs
were also exposed to NIR light and used as a control to study
the particular toxicity of NIR light. The cell viability was
assessed with MTT assay. As shown in Figure 2b, no obvious
reduction in cell viability was detected in any of the cell samples
treated with UCNPs, NIR laser, or the UCNPs + NIR laser.
These results were consistent with previous reports, which have
proved that the UCNPs have low toxicity and even can be used
in vivo studies.6 Additionally, the above results also showed that
under our experimental conditions, not only the NIR light but
also the upconverted UV had little damage to cells. The
biocompatibility of upconverted UV light might be attributed
to the localized UV radiation produced by the UCNPs
compared to conventional whole-cell irradiation with direct
UV light.8h Furthermore, the live/dead staining also exhibited
that the cells were almost alive after exposure to NIR-to-UV
UCNPs (Figure S9). Although the 980 nm laser can increase
the water temperature, the short interval irradiation condition
in our experiment could avoid this problem, and no obvious
temperature increase (<3 °C) was observed. In addition,
recently a new class of UCNPs with the excitation wavelength
of ∼800 nm has been successfully synthesized,14 and it also
provided an efficient solution to avoid the heating effect of 980
nm laser. Taken together, the UCNPs provided a safe approach
for photoactivation of cell adhesion.
Figure 3. Fluorescence images of NIR light-induced cells release on
(a) the substrate we designed and (b) substrate without UCNPs. Scale
bars are 100 μm. (c) Percent of cell release after irradiation with NIR
laser with 2 (red line), 4 (blue line), and 6 W/cm2 (green line). 2.5
min break after 2.5 min irradiation. Inset was the detailed release
process with laser on−off exposure for 0−10 min. (d) Viability of
released cells assessed with a live (green)/dead (red) assay. Scale bars
are 100 μm. (e) Normalized cell numbers of the released cells (black
bar) and the controlled sample (blue bar) after proliferation of
different times. (f) Illustration of in-deep tissue irradiation by covering
the substrate with pork tissue. (g) Cell release induced by directed UV
light irradiation (blue bar) and 980 nm NIR laser irradiation (orange
bar) through 0, 2, or 4 mm of pork tissue.
The ability of UCNPs for dynamic control of cell adhesion
was then examined. For this purpose, the bioadhesive RGD
ligand was covalently conjugated on the photosensitive
substrate. Then, the cells were seeded on the substrate and
monitored with fluorescence microscopy. After 5 h, the cells
efficiently attached and spread on the surface with fully
extended morphology (Figure 2c). After the cell adhesion, we
studied whether the substrate could dynamically stimulate cell
release with NIR laser. As shown in Figure 3a, after exposure to
4 W/cm2 NIR light for 30 min, ∼70% of the original adherent
cells were released from the surface. The residual cells on the
surface were mainly due to the photoresponsive linkers that
were not completely cleaved by NIR laser. And clearly, the rate
and amount of the cell release could be easily adjusted by the
laser power intensity and exposure duration (Figure 3c). The
on-demand release of cells from the responsive surface was
demonstrated by alternating the NIR laser between “light” and
“dark” conditions (Figure 3c inset). In addition, instead of the
whole exposure to NIR light, the substrate was selectively
irradiated with a laser through a mask. As designed, few cells
were left on the irradiation site, while most of the cells on the
nonirradiation region were still attached well (Figure S10). This
showed that the NIR-responsive UCNPs could control of cell
adhesion with high spatial resolution. For control, the cells on
the substrate without UCNPs were almost not affected by laser
irradiation (Figure 3b), indicating that the cell release was
mediated by the upconverted UV light not the direct NIR laser.
Additionally, to further confirm the cell detachment was caused
by the cleavage of PR linker, the substrate without photo-
sensitive ONA molecule was prepared. There was little
reduction in cell numbers for it with the NIR light treatment
(Figure S11). From the live/dead cell staining assay, most
released cells were still alive (Figures 3d and S12). MTT assay
was also performed which showed that our releasing condition
did not affect the proliferation ability of cells (Figure 3e). In
addition, after reincubated on the glass slide, the released cells
remained functional and spread normally (Figure S13). These
results confirmed that the UNCP-based photoactivation
provided a biologically friendly platform for light control of
cell adhesion.
Furthermore, we studied whether this system could be used
for regulating cell adhesion in deep tissue, which was an
important factor for further in vivo applications. For this
purpose, pork tissues (muscle tissue) with different thickness
were used as the tissue models plated on the top of the
photoresponsive surface15 (Figure 3f). The release of cells
would occur only if the laser penetrated the tissue and reached
the surface. Upon irradiation with NIR light, the cell release
reduced with the increase of tissue thickness, but ∼50%
activation was still observed even with 4 mm tissue. When it
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came to UV light, direct exposure to the light sources without
the pork tissue resulted in a more efficient cell release than
using NIR laser. However, the cell release was dramatically
reduced to 35% when the UV light was blocked by 2 mm tissue,
and only 10% was detectable once the light was blocked by 4
mm tissue (Figure 3g). The result was consistent with the
absorbance difference of the tissue in UV and NIR region
(Figure S14) and showed that the UCNP-based NIR light
system offers significantly improved penetration depth and is
preferred for the control of cell adhesion in deep tissue.
In summary, a new paradigm for NIR-controlled cell
adhesion has been presented by using UCNPs acting as the
nanotransducers. UCNPs can harvest NIR light and convert it
into local UV light for cleavage of photocaged linker, thereby
realizing on-demand cell release. This has been demonstrated
in deep tissue penetration. To the best of our knowledge, this is
the first example of using UCNPs to control cell adhesion, and
rapid developments for UCNPs will further improve the
performance of this platform, such as the use of a new kind of
UCNP which can be irradiated with more suitable NIR light.
Therefore, our work will facilitate the design of UCNP-based
multifunctional cell scaffold for dynamic study of biological
process, regeneration medicine, and disease-related cell
isolation and analysis.
ASSOCIATED CONTENT
* Supporting Information
Additional experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
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S
AUTHOR INFORMATION
Corresponding Author
xqu@ciac.ac.cn
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by 973 Project (2011CB936004,
2012CB720602) and NSFC (21210002, 91213302).
■
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