Noninvasive and Reversible Cell Adhesion and Detachment via Single-Wavelength Near-Infrared Laser Mediated Photoisomerization

Article abstract of DOI:10.1021/jacs.5b03872

Dynamically regulating cell-molecule interactions is fundamental to a variety of biological and biomedical applications. Herein, for the first time, by utilizing spiropyran conjugated multishell upconversion nanoparticles (UCNPs) as a new generation of single-wavelength near-infrared (NIR)-controlled photoswitch, we report a simple yet versatile strategy for controlling cell adhesion/detachment reversibly and noninvasively. Specifically, the two-way isomerization of the photoswitch was merely dependent on the excitation power density of the 980 nm laser. At high power density, the ring-opening was prominent, whereas its reverse ring-closing process occurred upon irradiation by the same laser but with the lower power density. Such transformations made the interactions between spiropyran and cell surface protein fibronectin switchable, thus leading to reversible cell adhesion and detachment. Moreover, efficient adhesion-and-detachment of cells could be realized even after 10 cycles. Most importantly, the utilization of NIR not only showed little damage toward cells, but also improved penetration depth. Our work showed promising potential for in vivo dynamically manipulating cell-molecule interactions and biological process. (Figure Presented).

Full text of DOI:10.1021/jacs.5b03872

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Article
Noninvasive and Reversible Cell Adhesion and Detachment via
Single-Wavelength Near-Infrared Laser Mediated Photo-isomerization
Wei Li, Zhaowei Chen, Li Zhou, Zhenhua Li, Jinsong Ren, and Xiaogang Qu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b03872 • Publication Date (Web): 28 May 2015
Downloaded from http://pubs.acs.org on June 4, 2015
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Noninvasive and Reversible Cell Adhesion and Detachment
via Single-Wavelength Near-Infrared Laser Mediated Photo-
isomerization
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Wei Li,†‡ Zhaowei Chen,†‡ Li Zhou,†‡ Zhenhua Li,†‡ Jinsong Ren*† and Xiaogang Qu*†
†
State Key Laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of
AppliedChemistry, Chinese Academy of Sciences, Changchun 130022, China
‡Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
KEYWORDS. Upconversion, spiropyran, cell, photoswitch, interaction.
ABSTRACT:Dynamically regulating cellꢀmolecule interactions is fundamental to a variety of biological and biomedical applicaꢀ
tions. Herein, for the first time, by utilizing spiropyran conjugated multiꢀshell upconversion nanoparticles (UCNPs) as a new generꢀ
ation of singleꢀwavelength NIRꢀcontrolled photoswitch, we report a simple yet versatile strategy for controlling cell adheꢀ
sion/detachment reversibly and noninvasively. Specifically, the twoꢀway isomerization of the photoswitch was merely dependent
on the excitation power density of the 980 nm laser. At high power density, the ringꢀopening was prominent, whereas its reverse
ringꢀclosing process occurred upon irradiation by the same laser but with the lower power density. Such transformations made the
interactions between spiropyran and cell surface protein fibronectin switchable, thus leading to reversible cell adhesion and deꢀ
tachment. Moreover,efficient adhesionꢀandꢀdetachment of cells could be realized even after 10 cycles. Most importantly, the utiliꢀ
zation of NIR not only showed little damage towards cells, but also improved penetration depth. Our work showed promising poꢀ
tential for in vivo dynamically manipulating cellꢀmolecule interactions and biological process.
Introduction
ly need to be modified with biomolecules, which requires
complicated synthesis and even causes a distortion of the
compounds.7An intriguing exception is the photochromic spiꢀ
ropyran. Unlike common photochromic systems, spiropyran
itself with two different polar isomerizations have shown to be
able to reversibly intercalate into DNA helix,8 regulate enzyme
activity9 and even specifically interact with cell surface protein
fibronectin.10 However, the need for two wavelength light ꢀ
toxic ultraviolet and low penetrating visible light for ringꢀ
opening (350 nm) and ringꢀclosing (560 nm) interconversion
processes and the difficulties in focusing the two light beams
precisely would complicate the practical process. Moreover,
the inevitable cellular damage and poor tissueꢀpenetration of
the light used would limit their further biomedical applicaꢀ
tions. Recently, lanthanideꢀdoped upconversion nanoparticles
(UCNPs) have attracted much attention in nanobiotechnology
due to their unique photophysical properties.11Generally, they
can emit UV, visible, and/or nearꢀinfrared (NIR) light upon
NIR excitation with a large antiꢀStokes shift, which has been
used to biologically friendly tune photoꢀisomerization procesꢀ
sas reported byour group12 and others.13Specially, Branda and
coꢀworkers reported that the emission of an individual coreꢀ
shellꢀshell UCNP can be modulated between UV and visible
light by changing just the power intensity of one single 980
Dynamically regulating the interactions between specific
molecules and cellꢀsurface molecules is critical for cell bioloꢀ
gy due to its potential to attain unprecedented levels of control
over diverse cell behaviors even cell fate.1 Specially, reversiꢀ
ble control strategies, which enable realꢀtime alternating such
interactions between onꢀoff states, have attracted particular
attention since they can aid in the development of highꢀ
contrast optical bioimaging2 and advanced therapies such as
cellꢀbased diagnostics3 and immunotherapy.4 For instance,
reversible manipulation of polymeric materials and immuneꢀ
cells interactions has been proposed to promote antiꢀtumor
immunity.4aAlthough promising, the further cilnical
application of these reversible methods was limited by the
invasive and destructive implantable procedures.Thus, it still
remains a big challenge to find an appropriate approach to
regulate cellꢀmolecule interactions facilely, reversibly and
noninvasively.
Among the various control strategies,5photomodulation has
proven to be an ideal option, by which cellꢀmolecule interacꢀ
tions can be readily controlled with noninvasive property and
high spatiotemporal precision.5aꢀd Particularly, photoswitching
systems that undergo photoꢀisomerization reactions have been
used to reversibly guide cellꢀmolecular recognition in interfaꢀ
cial systems.6Despite its usefulness, the photoswitches typicalꢀ
nm
excitation
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SCHEME 1. Schematic illustration of the utilization of spiropyran conjugated multiꢀshell upconversion nanoparticle (SPꢀ
UCNP) as a NIRꢀtriggered photoswitch for noninvasive and reversible control of cell adhesion/detachment by merely alterꢀ
ing the power density of a singleꢀwavelength 980 nm laser. At high power (8 W/cm2) density, the UCNPs can emit UV phoꢀ
tons and activate the isomerization from the spiro (SP) form to the merocyanine (MC) form, resulting in the detachment of
cell. Conversely, when exposed to low power (0.5 W/cm2) density, the same UCNPs can emit visible light to drive the MC
form back to the SP form, leading to cell adhesion again.
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light, which offered a simple wayto regulate photochemical
reactions.14 Accordingly, by taking advantage of this unique
feature, we envision thatmodulating thephotochromic of spiꢀ
ropyran with this singleꢀwavelength excited UCNP would
provide a convenient but novel way to reversibly control the
interactions between spiropyran and cells, which however has
remained unexplored until now.
especially for in vivo tissue engineering and medical implantaꢀ
tion et al.
Results and Discussion
To minimize the energy loss induced by surface quenching
effect,15 we deposited a protecting layer of inert NaYF4 onto
the coreꢀshellꢀshell UCNP14,16(layerꢀbyꢀlayer seedꢀmediated
shell growth process and the enhanced luminescence intensity,
for more details see the Supporting Information, Figure S1, S2
respectively). Transmission electron microscopy (TEM,Figure
1a) demonstrated the asꢀdesigned coreꢀshellꢀshellꢀshell
UCNPs with an average size of 40×60nm (width × length).
The typical dꢀspacing value of 0.52 nm (highꢀresolution TEM,
Figure 1b) and Xꢀray diffraction analysis (Figure S3) illustratꢀ
ed their single hexagonalꢀphase crystals. Subsequently, a 10ꢀ
nmꢀthick (Figure 1c) silica shell was coated on the UCNPs by
microemulsion method (UCNP@SiO2),12a which enabled effiꢀ
cient energy transfer between spiropyran and UCNPs. Furꢀ
thermore, energy dispersive spectroscopic (EDC) element
mapping of Si, Y, Yb, Tm and Er also verified the composiꢀ
tion of the nanoparticles. After functionalized with (3ꢀ
aminopropyl)ꢀtriethoxysilane (APTES), the resulting nanohyꢀ
brids (UCNP@SiO2ꢀNH2) were further covalently conjugated
with carboxylꢀcontaining spiropyran (SPꢀCOOH, Scheme S2
and Figure S4).The surface modification processes (Scheme
S3) were confirmedby Fourier transform infrared (FTIR, Figꢀ
ure S5) spectrum and the typical aryl nitro stretching (1338,
Herein, for the first time, by utilizing spiropyran conjugated
multiꢀshell UCNP (SPꢀUCNP) as a new generation of singleꢀ
wavelength NIRꢀcontrolled photoswitch, we report a simple
yet versatile strategy for controlling cell adhesion/detachment
reversibly and noninvasively. In this system, the isomerization
of spiropyrans was merely dependent on the excitation power
density of the 980 nm NIR laser(Scheme S1). As illustrated in
Scheme 1, at high power density, the UV emissions from
UCNPs were prominent and that, in turn, induced the forꢀ
mation of ringꢀopening merocyanine (MC) isomers. This
isomerization process could be reversed by just reducing the
power density to the point where the visible emission domiꢀ
nated. Such transformations made the interactions between
spiropyran and cell surface protein fibronectin switchable, thus
leading to reversible cell adhesion and detachment. Moreover,
owing to the reversibility and the excellent photostability of
the photoswitch, efficient adhesionꢀandꢀdetachment of cells
could be realized even after 10 cycles. Most importantly, the
utilization of NIR not only showed little damage towards cells,
but also improved penetration depth. We foresee that this new
concept may hold great potential for biomedical application,
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1523 cmꢀ1) and acylamide vibration (1650 cmꢀ1) indicated the
the solution turned back to yellow and the band at 560 nm
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successful preparation of SPꢀUCNPs.
disappeared upon exposing to low power 980 nm irradiation
for 10 min, (Figure S7), revealing the complete photoꢀ
isomerization from MC to SP. The excitationꢀdensityꢀ
dependent UCL emission was attributed to the nonlinearity of
the upconversion mechanisms, where higher excitation power
density was needed to achieve a UVꢀemissionꢀdominated
spectral profile by first saturating other visible transiꢀ
tions.17These results clearly showed that the reversible twoꢀ
way photoswitching of spiropyran could be driven by a singleꢀ
wavelength NIR, and the direction of the photoꢀisomerization
could be modulated by merely increasing (for ringꢀopening)
and decreasing (for ringꢀclosing) the power density. The reꢀ
versibility of the SPꢀUCNP photoswitch was tested by moniꢀ
toring the absorbance at 560 nm while continuously alternatꢀ
ing the power density between high/low (10 cycles). As shown
in Figure 2c and Figure S8, the photoꢀisomerization with
high/low NIR was reversible and the efficiency of the switchꢀ
ing was unaffected, confirming the robustness of SPꢀUCNP
conjugation, which was mainly due to the attenuation of phoꢀ
tofatigue by the utilization of noninvasive NIR.18 Taking toꢀ
gether, these results indicated that a new generation of phoꢀ
toswitch system was successfully built, which would facilitate
the reversible control of cell adhesion/detachment.
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Figure 1. Structural characterization of the asꢀsynthesized multiꢀ
shell UCNPs. a) TEM image of NaYF4:Tm/Yb@ NaYF4@
NaYF4:Er/Yb@ NaYF4 coreꢀshellꢀshellꢀshell UCNPs. b) Highꢀ
resolution TEM image of a single UCNP. c) Darkꢀfield TEM
image of UCNP@SiO2 and corresponding energy dispersive
spectroscopic (EDC) element mapping of Si Kꢀedge, Y Kꢀedge,
Yb Lꢀedge, Tm Lꢀedge and Er Lꢀedge signals.
Next, we investigated the optical properties of SPꢀUCNP at
high (8 W/cm2) and low (0.5 W/cm2) power densities of 980
nm NIR laser excitations. The maximum absorption peaks of
the ringꢀclosed spiro (SP) form located at 350 nm which overꢀ
laps well with the UV emission of the UCNPs (attributed to 1I6
1
→3F4 (347nm) and D2 →3H6 (365nm) transitions of Tm3+
Figure 2.Optical characterization of the asꢀsynthesized SPꢀ
UCNPs. a) UVꢀVis absorption spectra of the SP form in DMF
solvent and the upconversion luminescence (UCL) emission
spectrum of UCNPs and SPꢀUCNPs under high power density (8
W/cm2) of 980 nm NIR laser. b) UVꢀVis absorption spectra of the
MC form in DMF solvent, and the UCL emission spectrum of
UCNPs and SPꢀUCNPs at lower power density (0.5 W/cm2) of
980 nm NIR laser. c) Irradiation cycles of SPꢀUCNPs; Inset in
(c):photographs ofSPꢀUCNPs in DMF solvent under high power
NIR and low power NIR respectively. d) UVꢀVis absorption
spectra of SPꢀUCNPs modified surface before and
subsequentlyirradiated with high /low power NIR laser.
ions) under high power density excitation (Figure 2a). This
indicated that high power NIR excited UCNPs could be utiꢀ
lized to induce the ringꢀopening reaction of SP to MC through
energy transfer process. The energy transfer process was veriꢀ
fied by the significant quenching of upconversion fluorescence
(UCL) emission around 350 nm, and the energy transfer effiꢀ
ciency was measured to be 75.76%. As illustrated in Figure
S6, exposure of SPꢀUCNPs solution to high power density
excitation resulted in an observable change in color from yelꢀ
low to violet and a new band around 560 nm. The absorption
strength increased over time and reached equilibrium within 6
min. Notably, merely reducing the excitation power density to
0.5 W/cm2 would facilitate the overlap of the characteristic
absorption of MC form at 560 nm and the visible emissions of
In the following, we tested the photoswitching ability of SPꢀ
UCNP for regulating cell adhesion/detachment. Firstly, we
succeeded in fabricating the SPꢀUCNP modified surface by
attaching UCNP@SiO2ꢀNH2 to the amino group functionalꢀ
ized glass surface through glutaraldehyde and conjugatingSPꢀ
COOH to the UCNPs with the grafting amount calculated to
be 7.1 wt % (Scheme S4 and Figure S9). The SEM image reꢀ
vealed the uniform topography of the UCNP@SiO2ꢀNH2 decoꢀ
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the UCNPs generated from H11/2→4I15/2 (520 nm) and S3/2→
4I15/2 (540 nm) transitions of Er3+ ions (Figure 2b), which could
in turn be applied to trigger the reverse photoreactions. The
energy transfer efficiency was calculated to be 68.8%, which
certified the successful interconversion process. Additionally,
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rated surface (Figure S10) and the Xꢀray Photoelectron Specꢀ
to guide cell adhesion. We then examined whether the utilizaꢀ
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troscopy (Figure S11, S12) confirmed the successful proceꢀ
dure. As illustrated in UV/Vis absorption spectra (Figure 2d),
when exposure to 980 nm laser at high power density, the SPꢀ
UCNP modified surface exhibited an obvious enhancement
band around 560 nm, certifying the ringꢀopening process. In
contrast, the characteristic band disappeared after exposure to
low power density of NIR, which ensuring the effective ringꢀ
closing of spiropyran. The distinct change confirmed that the
SPꢀUCNP on surface could be also effectively triggered by
just modulating the power density of NIR laser, thus allowing
the singleꢀwavelength NIR mediated interfacial dynamic proꢀ
cesses.
tion of a singleꢀwavelength NIR could dynamically alter cell
adhesion. After a 4h’s incubation, the surfacewas gently
washed with PBS, stained with calcein (AM) dyes, imagined
and counted under a microscopy. As showed in Figure 3b,
over 95% original adherent cells were released from the SPꢀ
UCNPs based platform after exposure to high power 980 nm
for 15 minutes (2.5 min break after 2.5 min irradiation to
avoid heating effect of 980 nm laser). In contrast, no obvious
detachment from the parallel surface(Figure 3c) was observed.
These results confirmed that high power density of 980 nm
laser could be applied to induce the photoꢀisomerization in SPꢀ
UCNP photoswitch and further triggered the change of cell
adherent behavior of the surface.Moreover, Live/Dead staining
method (Figure 3d) were used to verify the good viability of
cells released from the surface, which was necessary for pracꢀ
tical cell culture and single cell analysis. In addition, the reꢀ
leased cells remained functional and spread normallyafter reꢀ
cultured on the glass slide (Figure S13), confirming that the
SPꢀUCNPꢀbasedsystemcould be used as an effective nonꢀ
invasive cell adhesion/detachment platform for further biologꢀ
ical investigations.
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Another apparent advantage of the SPꢀUCNPꢀbased system
is the ease of reversibly regulating cell adhesion/detachment
on demand. By merely reducing the power density to 0.5
W/cm2 and kept irradiating for 20 min, the surface could be
reused for multiple cycles study. As demonstrated in Figure
4a, b and S14, even after 10 cycles, the platform still showed
high efficiency to cell detachment, which could be attributed
to the outstanding reversibility and photostability of our NIRꢀ
triggered SPꢀUCNP photoswitch. Furthermore, the spatial
control of cell adhesion was also demonstrated by alternating
the power density between “high” and “low” site (Figure 4c),
which provided an alternative method for cell patterning. Adꢀ
ditionally, we investigated the feasibility of this system for
application in deep tissues by using pork tissues as a model
(Figure 4d, Scheme S5). As expected, only a slightly reduced
efficiency was observed for the NIRꢀtriggered platform. In
stark contrast, the cell release efficiency with UV irradiation
decreased dramatically when came to the tissue study, altꢀ
hough direct exposure to UV showed a much high efficiency
than NIR. Importantly, the SPꢀUCNP decorated surface
showed good reversibility even in tissue study (Figure S15).
Taken together,these results verified that the utilization of NIR
controlled SPꢀUCNP as a new generation of photoswitch not
only greatly enhanced the reversibility of the platform and
enabled spatial control of cell adhesion/detachment, but also
significantly improved penetration depth.
Figure 3.Singleꢀwavelength 980 nm NIR regulates cell
adhesion/detachment using the SPꢀUCNPs. a) After treatment
with different illuminations, cell capture efficiency of UCNPs
modified surface and SPꢀUCNPs modified surface at varied time
intervals. Fluorescence images of high power (8 W/cm2) NIR
induced cell detachment on b) the system we designed and the
system c) without spiropyran. Scale bars are 100 ꢁm. d) Viability
of released cell assessed with a live (green)/dead (red) assay.
Afterwards, we performed cell adhesion/detachment experꢀ
iment via a single wavelength NIR. Before cell adhesion, the
modified surfaces were treated with different illuminations.
Then HeLa cell suspensions (106cells mlꢀ1) in a DMEM mediꢀ
um were incubated with the surfaces and the amount of adꢀ
hered cells was counted by a hemacytometer at varied time
intervals (Figure 3a). Initially, the spiropyran were mainly in
the SP form and the SPꢀUCNP modified surface demonstrated
higher capture efficiency compared with the UCNP@SiO2ꢀ
NH2 modified surface, which could be attributed to the specifꢀ
ic interaction between SP isomerization and cell surface fiꢀ
bronectin protein.10Irradiation at high power density of 980 nm
laser gave rise to the MC form and a decrease in capture effiꢀ
ciency was observed, owing to the weak interaction between
the MC form and fibronectin.12a,19,20Moreover, the lowꢀpower
NIR illustrated surface displayed the highest capture
efficiency among all the surfaces, further confirming that the
SPꢀUCNP could be used as a new generation of photoswitch
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Materials. All reagents and solvents were commercially
3
available and used as received without further purification.
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Synthesis of βꢀNaYF4:Tm/Yb core nanoparticles.Briefly,
1.4 mmol of YCl3•6H2O, 0.6 mmol YbCl3•6H2O and 0.01
mmol of TmCl3•6H2O were added to a 100 ml flask containꢀ
ing12 ml oleic acid and 30 ml octadeceneand heated to 160℃
to form a homogeneous solution.The temperature was then
lowered to 50℃ and the flask was placed under nitrogen gas
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protection. Once the reaction reached 50℃, 10 ml methanol
solution containing NH4F (296 mg, 8.0 mmol) and NaOH (200
mg, 5.0 mmol) were added in and kept stirring for 30 min.
Afterslowly evaporated the methanol and heated the mixture
to 300℃ for 1.5 h under nitrogen protection, the solution was
cooled downed and the resulting nanoparticleswere purified by
addition of ethanol, collected by centrifugation and finally reꢀ
suspended in 10 ml cyclohexane.
Synthesis of NaYF4:Tm/Yb@NaYF4 coreꢀshell nanoparꢀ
ticles. 1.8 mmol of YCl3•6H2O was added to a 100 ml flask
containing12 ml oleic acid and 30 ml octadeceneand heated to
120℃. The temperature was lowered to 80℃ and the disperꢀ
sion of NaYF4:Tm/Yb core nanoparticles in hexanes were
added. Afterslowly evaporated the hexanes and heated to
110℃, the mixture was cooled down to 50℃. Once the temꢀ
perature reached 50℃, 10 ml methanol solution containing
NH4F (259 mg, 7.0 mmol) and NaOH (175 mg, 4.4 mmol) was
added in and kept stirring for 30 min. Afterslowly evaporated
the methanol and heated the mixture to 300℃ for 1.5 h under
nitrogen protection, the solution was cooled downed and the
resulting nanoparticleswere purified by addition of ethanol,
collected by centrifugation and finally reꢀsuspended in 10 ml
cyclohexane.
Figure 4.Singleꢀwavelength 980nm NIR regulates reversible
Synthesis
of
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control of cell adhesion and detachment. a), b) Reversible
adhesion and detachment of cells on SPꢀUCNPs modified system.
Scale bars are 100 ꢁm. c) NIRꢀinduced detachment of cells with
pattern light irradiation. Scale bars are 100 ꢁm. d) Cell release
efficiency induced by UV and NIR through 0, 2 or 4 mm of pork
tissue.
NaYF4:Tm/Yb@NaYF4@NaYF4:Er/Ybcoreꢀshellꢀshell
nanoparticles. The same procedure outlined above for the
synthesis of coreꢀshell NaYF4: Tm/Yb@NaYF4 was employed
except that the coreꢀshell nanoparticle was used as core and
YCl3•6H2O was used as precursor for epitaxial shell growth.
Synthesis of NaYF4:Tm/Yb@NaYF4@NaYF4:Er/Yb@
NaYF4coreꢀshellꢀshellꢀshell nanoparticles. The same proceꢀ
dure outlined above for the synthesis of coreꢀshell NaYF4:
Tm/Yb@NaYF4 was employed except that the coreꢀshellꢀshell
nanoparticle was used as core and YCl3•6H2O was used as
precursor for epitaxial shell growth.
Conclusion
In summary, for the first time, noninvasive and reversible
regulation of cellꢀmolecules interaction was successfully realꢀ
ized by utilizing spiropyran conjugated multiꢀshell UCNP as a
singleꢀwavelength NIRꢀcontrolled photoswitch. Notably, cell
adhesion/detachment can be switched conveniently by merely
decreasing/increasing the excitation power density of the same
980 nm laser, which greatly simplified the treatment process.
Moreover, efficient adhesionꢀandꢀdetachment of cells was
achieved even after 10 cycles based on the reversiblephotoꢀ
isomerization reactionsand the excellent photostabilityof the
photoswitch. Most importantly, the utilization of NIR not only
showed little damage towards cells, but also significantly imꢀ
proved penetration depth, which is superior for further appliꢀ
cation. We anticipate that this singleꢀwavelength NIR conꢀ
trolled approach would be further developed for in vivo dyꢀ
namic control of biological process, tissue engineering and
medical implantation.
Synthesis of SPꢀCOOH. During the synthesis, all the reacꢀ
tion vessels were wrapped in aluminum foil to ensure the reacꢀ
tion was performed in the dark.The preparation of the carꢀ
boxyꢀcontaining spiropyran 1’ꢀ(βꢀcarboxyethyl)ꢀ3’, 3’ꢀ
dimethylꢀ6ꢀnitrospiro [indolineꢀ2’, 2ꢀchromane] was perꢀ
formed as our reported paper.12In brief, 0.06 mol of 2, 3, 3ꢀ
trimethylindoleꢀnine, 0.06 mol of 3ꢀiodopropanoic acid and 5
ml of ethyl methyl ketone were refluxed under nitrogen at 100℃
for 3 h. The resulting mixture was dissolved in water, and the
solution was washed with chloroform.After theevaporation of
water, 1ꢀ(βꢀcarboxyethyl)ꢀ2, 3, 3ꢀtriꢀmethylindolenine iodide
was achieved. Next, 0.04 mol of the resulting iodide, 0.04 mol
of 5ꢀnitrosaliꢀcylaldehyde and 3.8 ml (0.04 mol) piperidine
were dissolved in ethyl methyl ketoneand refluxed for 3 h. On
standing overnight, a yellow crystalline powder was precipiꢀ
tated. The product was filtered and washed with methanol to
Experimental Section
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yield SPꢀCOOH (70% yield).The product was characterized
Cell Adhesion.The UCNP modified surface and SPꢀUCNP
by 1HNMR spectroscopy as shown in Figure S1.1HNMR (400
MHz, CDCl3, 25℃, TMS): δ = 1.0ꢀ1.3 (6H; 2CH3), 2.6 (2H;
CH2COO), 3.4ꢀ3.5 (2H; CH2N), 5.9ꢀ6.0 (2H; olefinic protons),
6.6ꢀ8.2 (aromatic protons).
modified surface were preꢀtreated with different illumination.
Afterwards, they were placed into a sizeꢀmatched 24ꢀwell
LabꢀTekTM Chamber Slide. Hela cells were added to each
culture flask to obtain a cell density of 1×106 cells per flask.
The flasks were cultured in DMEM containing 10% FBS at
37℃ in an atmosphere of 5% CO2. After incubating for 30
min and 1 h, the surfacewas gently washed with PBS for 3
times, and cell numbers in supernatant was counted by a heꢀ
Preparation of silica coated UCNP. Firstly, 7.2 ml triton,
28 ml cyclohexane, 7.2 ml hexanol, 680ꢁl water and 50 mg of
UCNPs were mixed in a 100 ml flask by sonication and agitaꢀ
tion for 40 min.Then,100 ꢁl of TEOS was added drop wise
into the mixture. Afterward, 160ꢁl NH3•H2O was added into
the flask and stirred for 24 h. The resulting nanoparticles were
precipitated out by the addition of ethanol to yield
UCNPs@SiO2.
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macytometer again. The number of adhesion cells (n
)
adhesion
on the surfaces and the capture efficiency (e) can be calculatꢀ
ed by the following equations respectively:
n
adhesion =n total ꢀ n supernatant
Preparation of amino functionalized UCNP@SiO2. Briefꢀ
ly, 20 mg of UCNPs@SiO2 and 30 ml toluene were firstly
mixed in a 100 ml flask and heated to 110℃. Next, 30ꢁl of (3ꢀ
1minopropyl) trierhoxysilane (APTES) was added drop wise
into the mixture. After stirring for 24 h, the precipitates were
separated by centrifugation, washed with ethanol three times,
and dried at 60℃ for 12 h to obtain the UCNPs@SiO2ꢀNH2.
e= n adhesion / n total
NIR controlled Cell Detachment.After incubated with
cells for 4 h, the platform was examined for NIR triggered
release. Irradiation NIR laser at the powers density of 8 W/cm2
for 15min (2.5 min break after 2.5 min irradiation to avoid
heating effect of 980 nm laser), the surface was gently washꢀ
ing with PBS in 15 s to remove the release cells. Fluorescent
microscopic imaging was used to monitor the cell release. For
live/dead cell staining, the released cell was collected from the
washing buffer by centrifugation at 1000 rpm for 5 min, and
then propidium iodide (PI) and calcein (AM) dyes were added
into the released cell. After 15 min incubation, the cells were
imaged by the microscope. For repeatability study, the subꢀ
strates firstly were digested by trypsin and washed with PBS
for 3 times. Then the substrates were exposed to NIR at the
powers of 0.5 W/cm2 for 20 min and reused for cell adhesion
and detachment.Furthermore, to control cell detachment reꢀ
gionally, we firstly treated the SPꢀUCNP modified surfaces
with a dose of 20 min lower power NIR illuminations and then
incubated them with cells. Afterwards, we shield the surfaces
with a lightꢀproof photomask on one half and put the surfaces
under high power density NIR for 15 min. As the light source
and photomask were removed, the surfaces were washed genꢀ
tly with PBS and recorded with microscope. For deep tissue
irradiation, pork tissues (muscle tissue) with 0 mm, 2 mm, 4
mm thickness was placed above the cell adhesive surface with
a distance of 1 mm. The laser was irradiation from the top of
tissue.
Preparation of spiropyran conjugated UCNP@SiO2ꢀ
NH2.During the synthesis, all the reaction vessels were
wrapped in aluminum foil to ensure the reaction was perꢀ
formed in the dark. 0.526 mmol of SPꢀCOOH (200 mg) was
firstly dissolved in 30 ml dimethylformamide (DMF). 4 ml
MES buffer (pH 6.0, 20 mM) containing EDC (500 mg) and
NHS (400 mg) was added into the solution and agitated for 1.5
h. Afterwards, 100 mg UCNPs@SiO2ꢀNH2in 4 ml MES buffer
was added in and stirred for 48 h. The resulting nanoparticles
were washed by water and DMF.
Preparation of the UCNPs Modified Substrate.The glass
slide of 1×1cm were cleaned and immersed into 10 ml 0.5%
APTES solution for 1 h to introduce amino group. Then the
amine terminated substrates were reacted with the solution of
UCNPs@SiO2ꢀNH2 (10 ml/ml) for 24 h at room temperature.
Preparation of the SPꢀUCNPs Modified Surface.Firstly,
SPꢀCOOH (200mg, 0.526mmol) was dissolved in 30 ml DMF.
Then EDC (500 mg) and NHS (400 mg) in 4mml MES buffer
(pH 6.0, 20 nM) was added to the solution. After agitating the
solution overnight in the dark, the UCNPs modified surfaces
were immersed into the solution for 24 h to conjugate with SPꢀ
COOH through EDC/NHS chemistry. The grafting amount of
spiropyran on the UCNPs was calculated by UV/Vis spectra.
Fluorescent Microscopic Imaging.For the imaging test, the
surfacewas placed into a 24ꢀwell plates and incubated with a
density of 1×106 cell/well for 4 h at 37℃ in an atmosphere of
5% CO2. AM dyes were added into the flask for 15 min. Then
the substrates were gently washed with PBS and finally imꢀ
aged under an Olympus BXꢀ51 optical equipped with a CCD
camera. After NIR irradiation, the substrates were gently
washed with PBS, stained with AM for 15 min and viewed
under the microscope.
NIR triggeredPhotoꢀisomerization of the SPꢀUCNPs on
modified surface.Before exposure to NIR illumination, the
absorption spectra of the SPꢀUCNPs modified surfaces were
recorded on JASCO Vꢀ550 UVꢀVis spectrophotomeꢀ
ter.Subsequently, the surface was immersed in 500ul of DMF
and exposed to high power NIR irradiation for 5 min. The
surfaces were dried out quickly before being measured. Afꢀ
terwards, the surface was immersed in 500ul of DMF, exposed
to low NIR irradiation for another 15 min and measured again.
ASSOCIATED CONTENT
Supporting Information.Detailed experimental procedures and
supplementary figures and tables.This material is available free of
charge via the Internet at http://pubs.acs.org.
Cell Culture. The Hela cells were cultured at 37℃ in an
atmosphere of 5% (v/v) CO2 in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine seꢀ
rum(FBS). The media was changed every three days, and the
cells were digested by trypsin and reꢀsuspended in fresh comꢀ
plete medium before plating.
AUTHOR INFORMATION
Corresponding Author
jren@ciac.ac.cn; xqu@ciac.ac.cn
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(10)(a) Higuchi, A.; Hamamura, A.; Shindo, Y.; Kitamura, H.;
ACKNOWLEDGMENT
Yoon, B. O.; Mori, T.; Uyama, T.; Umezawa, A. Biomacromole-
cules2004,5, 1770;(b) Wang, N.; Li, Y.; Zhang, Y.; Liao, Y.; Liu, W.
Langmuir 2014,30, 11823;(c) Edahiro, J.ꢀi.; Sumaru, K.; Tada, Y.;
Ohi, K.; Takagi, T.; Kameda, M.; Shinbo, T.; Kanamori, T.; Yoshimi,
Y. Biomacromolecules2005,6, 970.
(11)(a) Cheng, L.; Yang, K.; Li, Y.; Chen, J.; Wang, C.; Shao, M.;
Lee, S.ꢀT.; Liu, Z. Angew. Chem. Int. Ed. 2011,50, 7385;(b) Huang,
P.; Zheng, W.; Zhou, S.; Tu, D.; Chen, Z.; Zhu, H.; Li, R.; Ma, E.;
Huang, M.; Chen, X. Angew. Chem. Int. Ed. 2014,53, 1252;(c) Liu, J.;
Liu, Y.; Liu, Q.; Li, C.; Sun, L.; Li, F. J. Am. Chem. Soc. 2011,133,
15276;(d) Liu, Y.; Chen, M.; Cao, T.; Sun, Y.; Li, C.; Liu, Q.; Yang,
T.; Yao, L.; Feng, W.; Li, F. J. Am. Chem. Soc. 2013,135, 9869;(e)
Yang, Y.; Shao, Q.; Deng, R.; Wang, C.; Teng, X.; Cheng, K.; Cheng,
Z.; Huang, L.; Liu, Z.; Liu, X.; Xing, B. Angew. Chem. Int. Ed.
2012,51, 3125; (f) Liu, J.; Liu, Y.; Bu, W.; Sun, Y.; Sun, Y.; Du, J.;
Shi, J. J. Am. Chem. Soc. 2014,136, 9701.
(12)(a) Chen, Z.; Zhou, L.; Bing, W.; Zhang, Z.; Li, Z.; Ren, J.;
Qu, X. J. Am. Chem. Soc. 2014,136, 7498;(b) Zhou, L.; Chen, Z.;
Dong, K.; Yin, M.; Ren, J.; Qu, X. Adv. Mater. 2014,26, 2424.
(13)(a) Lai, J.; Zhang, Y.; Pasquale, N.; Lee, K.ꢀB. Angew.Chem.
Int. Ed. 2014,53, 14419;(b) Min, Y.; Li, J.; Liu, F.; Yeow, E. K. L.;
Xing, B. Angew. Chem. Int. Ed. 2014,53, 1012;(c) Wang, L.; Dong,
H.; Li, Y.; Xue, C.; Sun, L.ꢀD.; Yan, C.ꢀH.; Li, Q. J. Am. Chem. Soc.
2014,136, 4480;(d) Zhang, B. F.; Frigoli, M.; Angiuli, F.; Vetrone, F.;
Capobianco, J. A. Chem.Commun.2012,48, 7244;(e) Zhang, C.; Xu,
C.ꢀH.; Sun, L.ꢀD.; Yan, C.ꢀH.Chem. Asian J.2012,7, 2225; (f) Liu, J.;
Bu, W.; Shi, J. Angew. Chem. Int. Ed. 2013,52, 4375; (g) Yang, T.;
Liu, Q.; Li, J.; Pu, S.; Yang, P.; Li, F., RSC Adv.2014,4, 15613; (h)
Boyer, J.ꢀC.; Carling, C.ꢀJ.; Chua, S. Y.; Wilson, D.; Johnsen, B.;
Baillie, D.; Branda, N. R., Chem. Eur. J.2012,18, 3122.
(14)Boyer, J.ꢀC.; Carling, C.ꢀJ.; Gates, B. D.; Branda, N. R. J. Am.
Chem. Soc. 2010,132, 15766.
(15)(a) Han, S.; Deng, R.; Xie, X.; Liu, X. Angew. Chem. Int. Ed.
2014,53, 11702;(b) Peng, J.; Xu, W.; Teoh, C. L.; Han, S.; Kim, B.;
Samanta, A.; Er, J. C.; Wang, L.; Yuan, L.; Liu, X.; Chang, Y.ꢀT. J.
Am. Chem. Soc. 2015,137, 2336.
(16)(a) Wang, F.; Deng, R.; Wang, J.; Wang, Q.; Han, Y.; Zhu, H.;
Chen, X.; Liu, X. Nat. Mater.2011,10, 968;(b) Wen, H.; Zhu, H.;
Chen, X.; Hung, T. F.; Wang, B.; Zhu, G.; Yu, S. F.; Wang, F. An-
gew. Chem. Int. Ed. 2013,52, 13419.
(17)(a) Pollnau, M.; Gamelin, D. R.; Lüthi, S. R.; Güdel, H. U.;
Hehlen, M. P. Phys. Rev. B 2000,61, 3337;(b) Shen, J.; Chen, G.;
Ohulchanskyy, T. Y.; Kesseli, S. J.; Buchholz, S.; Li, Z.; Prasad, P.
N.; Han, G. Small 2013,9, 3213.
We thank Wen Li from Graduate School of the Chinese Academy
of Sciences for the helpful advice on the manuscript. Financial
support was provided National BasicResearch Program of China
(Grant 2012CB720602,2011CB936004) and the National Natural
Science Foundationof China (Grants 91213302, 21210002,
21431007, 91413111).
REFERENCES
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(1)(a) Hynes, R. O. Science2009, 326, 1216; (b)Herbert, S. P.;
Stainier, D. Y. R. Nat. Rev. Mol. Cell Biol.2011, 12, 551; (c) Robꢀ
ertus, J.; Browne, W. R.; Feringa, B. L. Chem. Soc. Rev.2010, 39,
354;(d) Stevens, M. M.; George, J. H. Science2005, 310, 1135.
(2)(a) Kim, Y.; Jung, H.ꢀy.; Choe, Y. H.; Lee, C.; Ko, S.ꢀK.; Koun,
S.; Choi, Y.; Chung, B. H.; Park, B. C.; Huh, T.ꢀL.; Shin, I.; Kim, E.
Angew. Chem. Int. Ed. 2012,51, 2878;(b) Takahashi, S.; Piao, W.;
Matsumura, Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kamachi, T.;
Kohno, M.; Nagano, T.; Hanaoka, K. J. Am. Chem. Soc. 2012,134,
19588.
(3)Liu, Q.; Wu, C.; Cai, H.; Hu, N.; Zhou, J.; Wang, P. Chem. Rev.
2014,114, 6423.
(4)(a) Ali, O. A.; Huebsch, N.; Cao, L.; Dranoff, G.; Mooney, D. J.
Nat. Mater.2009,8, 151;(b) Kim, J.; Li, W. A.; Choi, Y.; Lewin, S. A.;
Verbeke, C. S.; Dranoff, G.; Mooney, D. J. Nat.Biotechnol.2015,33,
64;(c) Okano, F.; Merad, M.; Furumoto, K.; Engleman, E. G.
J.Immunol.2005,174, 2645.
(5)(a) Lee, T. T.; García, J. R.; Paez, J. I.; Singh, A.; Phelps, E. A.;
Weis, S.; Shafiq, Z.; Shekaran, A.; del Campo, A.; García, A. J. Nat.
Mater.2015,14, 352;(b) Li, W.; Wang, J.; Ren, J.; Qu, X. Angew.
Chem. Int. Ed. 2013,52, 6726;(c) Li, W.; Wang, J.; Ren, J.; Qu, X. J.
Am. Chem. Soc. 2014,136, 2248;(d) Liu, D.; Xie, Y.; Shao H.; Jiang
X. Angew. Chem. Int. Ed. 2009,48, 4406;(e) Hou, S.; Zhao, H.; Zhao,
L.; Shen, Q.; Wei, K. S.; Suh, D. Y.; Nakao, A.; Garcia, M. A.; Song,
M.; Lee, T.; Xiong, B.; Luo, S.ꢀC.; Tseng, H.ꢀR.; Yu, H.ꢀh. Adv. Ma-
ter. 2013,25, 1547;(f) Liu, H.; Li, Y.; Sun, K.; Fan, J.; Zhang, P.;
Meng, J.; Wang, S.; Jiang, L. J. Am. Chem. Soc. 2013,135, 7603;(g)
Zhang, P.; Chen, L.; Xu, T.; Liu, H.; Liu, X.; Meng, J.; Yang, G.;
Jiang, L.; Wang, S. Adv. Mater. 2013,25, 3566;(h) Zhang, Z.; Chen,
N.; Li, S.; Battig, M. R.; Wang, Y. J. Am. Chem. Soc. 2012,134,
15716.
(6)Weber, T.; Chandrasekaran, V.; Stamer, I.; Thygesen, M.
B.;Terfort, A.; Lindhorst, T. K. Angew.Chem. Int. Ed. 2014,53,
14583.
(7) (a) Schönberger, M.; Trauner, D. Angew. Chem. Int. Ed.
2014,53, 3264;(b) Zhang, J.; Zou, Q.; Tian, H. Adv. Mater.2013,25,
378.
(18)Tong, R.; Hemmati, H. D.; Langer, R.; Kohane, D. S. J. Am.
Chem. Soc. 2012,134, 8848.
(19)Gelmi, A.; Zanoni, M.; Higgins, M. J.; Gambhir, S.; Officer,
D. L.; Diamond, D.; Wallace, G. G. J. Mater.Chem. B 2013,1, 2162.
(20)Bergkvist, M.; Carlsson, J.; Oscarsson, S. J. Biomed.Mater.
Res. Part A2003,64A, 349.
(8) Andersson, J.; Li, S.; Lincoln, P.; Andréasson, J. J. Am. Chem.
Soc. 2008,130, 11836.
(9) Song, Y.; Xu, C.; Wei, W.; Ren, J.; Qu, X. Chem.Commun.
2011,47, 9083.
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