NIR light controlled release of caged hydrogen sulfide based on upconversion nanoparticles

Article abstract of DOI:10.1039/c5cc02508g

A NIR light induced H₂S release platform based on UCNPs was constructed. Under NIR light excitation, UCNPs can emit UV light which triggers H₂S release in a spatial and temporal pattern. The platform was also employed to real-time monitor the delivery process in vivo, which may provide a new way for the use of H₂S-based therapeutics for a variety of diseases. This journal is

Full text of DOI:10.1039/c5cc02508g

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NIR light controlled release of caged hydrogen sulfide based on
upconversion nanoparticles
Received 00th January 20xx,
Accepted 00th January 20xx
a#
a#
a
a
a
a
Wansong Chen, Min Chen, Qiguang Zang, Liqiang Wang, Feiying Tang, Yajing Han, Cejun
b
a
a
Yang, Liu Deng * and You-Nian Liu *
DOI: 10.1039/x0xx00000x
www.rsc.org/
The NIR light induced H
2
S release platform based on UCNPs much attention in the fields of biological imaging and targeted
7
was constructed. Under NIR light excitation, UCNPs can emit delivery based on their novel optical properties. UCNPs can absorb
2
UV light which triggers H S release in a spatial and temporal long wavelength NIR light and emit narrow and sharp emissions
8
pattern. The platform was also employed to real-time monitor ranging from UV to visible, which enable them to work as
the delivery process in vivo, which may provide a new way for promising NIR-induced delivery or release cargos for biological
9
the use of H
2
S-based therapeutics for a variety of diseases.
studies. It is well known that NIR light is safer and can penetrates
1
0
several centimetres in tissue. However, to the best of our
knowledge, there is no report in literature of the use of NIR light
In the past decade, hydrogen sulfide (H
2
S), identified to be a novel
gaseous transmitter, as carbon monoxide and nitric oxide, can be act
as a cell-signalling mediator in physiology and pathology. Recently,
2
excitation to emit UV light to spatially and temporally trigger H S
release in vitro and in vivo.
1
the application of H S in medical therapy has drawn much attention.
2
Studies have shown that H S has great therapeutic potential in
2
2
3
4
treatment of cancer, inflammation and neurodegenerative disease.
The rapid growth of the H S biomedical research has led to the
2
concomitant need of the H S donor, since it has great therapeutic
2
5
potentials of exogenous administration of H S. Despite the great
2
progress with respect to H S biomedical application, remote control
2
the release of H S inside cells or tissue in a spatial and temporal
2
precision manner remains a critical challenge.
So far, several intracellular controlled release strategies on the
basis of photolysis, hydrolysis, thiol activation or bicarbonate
5
activation, have been developed to direct endogenous production of
the H S in living cell. Among them, photo-induced release, which
2
Scheme 1. Construction of SP-loaded PEG-UCNPs platform for
can achieve spatial- and temporal-specific control release of H2S NIR-triggered H S release.
2
from donors, has been currently recognized as a new direction for
Herein, we present a simple NIR light reactive nanoparticle carrier
6
their promising biomedical practices in H S release in vivo.
2
system to exert target H S release in living cells on the basis of
2
However, one major problem which exists in current photo-induced
release relies on the short wavelength UV irradiation, which is
suffered from low tissue penetration and biological damage. Thus, a
UCNPs (see Scheme 1). In this system, LiYF :Yb/Tm nanoparticles
4
were chosen as light nanotransducers due to their strong emission at
65 nm with 980 nm irradiation. An amphiphilic compound
polyethylene glycol-octadecylamine (PEG-ODA) was then coated on
3
new way to effectively trigger H S release with minimum side
2
effects in living tissues is highly required.
Recently, upconversion nanoparticles (UCNPs) have attracted
the surface of LiYF :Yb/Tm nanoparticles to convert hydrophobic
4
nanoparticles into aqueous phase. At the same time, propane-2,2-
diylbis((1-(4,5-dimethoxy-2-nitrophenyl)ethyl)sulfane) (SP), as a
2
new H S donor (see Scheme S1 in ESI), was synthesized for the first
time and loaded at the surface of UCNPs through hydrophobic
interactions. Upon 980 nm laser irradiation, the upconverted UV
light emitted from UCNPs is supposed to photo-cleavage SP to gem-
dithiols through luminescence resonance energy transfer (LRET)
effect. As we know, gem-dithiols are unstable and apt to hydrolysis
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to release H
2
S (showed in Scheme 1). Upon the nanocarriers arrive inner filter effect. These results confirmed that there is efficient
at the specific physiological targets, H
80 nm laser, thus giving a remotely, non-invasive and sustainable the short distance between UCNPs and SP and the perfect match
stimulus to trigger spatially and temporally controllable H S release. between UCNPs emission and SP absorption.
The Yb /Tm co-doped UCNPs were prepared according to the
2
S release can be trigged by LRET effect between PEG-UCNPs and SP, which is probably due to
9
2
3
+
3+
1
1
protocol from the literature. The morphologies of the UCNPs were
characterized by transmission electron microscopy (TEM). As
shown in Figure 1a, the UCNPs are monodisperse rhombus with the
long axis around 100 nm. From the high resolution transmission
electron microscopy (HRTEM) image (Figure S1), it also can be
seen that the interplanar spacing for all lattice fringes is 0.41 nm,
which corresponds to the (101) planes of tetragonal-phase of
11
LiYF4. To improve the dispersion stability of nanoparticles in
aqueous solutions, amphiphilic PEG-ODA synthesized from mPEG
and ODA, was then bound with UCNPs through the lipophilic
interaction between octadecyl chain and oleic acid at the surface of
UCNPs. After coated with PEG-ODA the nanoparticles disperse Figure 1. (a) TEM images of the as-prepared UCNPs and (b) PEG-
-1
UCNPs; (c) photos of the PEG-UCNPs in water (~3.5 mg mL )
well in water (see Figure 1c). In the TEM image of PEG-ODA
modified UCNPs (PEG-UCNPs), a very thin layer was observed at
the edge of UCNPs which mainly attributes to the high hydrophilic
nature of PEG (see Figure 1b). The successful PEG-lyation was also
-
2
under 980 nm laser irradiation (20 W cm ). (d) UV-vis absorption
spectrum (blue line) of SP; emission spectra of UCNPs (black line)
and SP-loaded PEG-UCNPs (red line). (e) Fluorescence spectra of
DNS-Az in the presence of SP-loaded PEG-UCNPs with 365 nm
confirmed by IR spectroscopy (Figure S2). The IR spectrum of PEG- UV light irradiation for 5 min or 980 nm laser irradiation for 2 h.
-
1
UCNPs shows a band at 1,100 cm , which is the characteristic
stretching vibration of C-O-C bonds of PEG. In contrast, the PEG-
modification did not affect the optical properties of UCNPs (see
We next studied the light-induced release of H
platform, where dansylazide (DNS-Az) was used as a fluorescence
probe to monitor the generation of H S (Scheme S2 and Figure S10).
Firstly, the controlled release properties of the developed platform
under UV light irradiation was carried out. As shown in Figure 1e,
upon irradiation with UV light for 5 min, the fluorescence emission
peak of DNS-Az shifts from 480 nm to 520 nm and the fluorescence
2
S from the
2
Figure S3). H S donor SP was loaded on PEG-UCNPs through the
2
lipophilic interaction. The size distribution of PEG-UCNPs and SP
loaded PEG-UCNPs were analysed by dynamic light scattering
(
DLS) measurement (Figure S2). The average particle size of PEG-
UCNPs are around 156 nm in water, after SP loading the size of
PEG-UCNPs has a little increase. The loading capacity for SP on the
surface of PEG-UCNPs was determined by UV-Vis method (see
Figure S4-S6), and the loading amount was estimated to be about
2
intensity is significantly increased, indicating H S release caused by
1
3
UV irradiation achieved. Then, SP-loaded PEG-UCNPs were
exposed to NIR light, the intensity around 520 nm is also increased
and significantly much higher than that without light irradiation,
3
4,000 SP molecules loaded on one particle of UCNP. To
which indicates the successful H
from the platform. NIR-controlled H
in vitro with varying NIR light intensity and irradiation time. As
shown in Figure 2, the concentration of H S is increased when
prolonging the irradiation time or increasing the irradiation intensity.
In contrast, there’s no significant H S release from SP solution in the
absence of UCNPs at the same experimental condition (Figure S11).
It indicates that the H S release is mainly triggered by NIR light in
2
S release triggered by NIR light
investigate the H2S release mechanism, reversed-phase high-
performance liquid chromatography (RP-HPLC) and mass
spectrometry (MS) measurements were performed. As displayed in
Figure S7, the retention time for SP is 13.8 min. By contrast, a main
peak at 4.4 min was observed after UV irradiation for 10 min. The
eluates at 4.4 min were collected and then analysed by HRMS. The
molecular ion peak located at m/z 210.0759 (Figure S8), is assigned
2
S release was further measured
2
2
+
2
to C H NO , which further demonstrates the formation of
10
12
4
the presence of UCNPs, and can be controlled by either NIR
irradiation time or NIR irradiation intensity. As a proof of concept,
compound 1. Thus, the results confirmed the SP photo-cleavage
mechanism shown in Scheme 1 and SP could be employed as a H2S
donor.
The LRET efficiency between the UCNPs and SP is of great
2
importance for the NIR-induced H S release. Hence, the LRET
in our experiment the highest H
loaded PEG-UCNPs is 31 µM with the 1 mg mL concentration of
2
S concentration released from SP-
-
1
-
2
SP-loaded PEG-UCNPs under 20 W cm of irradiation intensity for
h. In the further biological application, the H S concentration can
2
2
effect between UCNPs and SP was assessed. Figure 1d shows the
emission spectra of the UCNPs dispersed in aqueous solution before
and after SP loading under excitation at 980 nm. SP shows an
absorption band between 300–400 nm, and the emission intensity of
UCNPs at 350–370 nm is significantly decreased after SP loading,
be tuned by varying the concentration of the SP-loaded PEG-
UCNPs, NIR irradiation time or irradiation intensity. It should be
2
noted that H S could affect the biological process even when the
concentration is in the range of 1–10 pmol per second per milligram
1
4
1
2
2
protein. Thus, our H S delivery platform will be able to satisfy the
which could be ascribed to the LRET effect or inner filter effect.
widely requirement of further application.
We also tested the fluorescence spectrum of the mixture of UCNPs
and SP at the same concentration in the SP-loaded PEG-UCNPs, and
little change in fluorescence was observed (Figure S9), suggesting
that the fluorescence quenching around 365 nm is not caused by
The cytotoxicity of SP-loaded PEG-UCNPs to both normal cells
1
5
and cancer cells were also measured with MTT method. For either
L929 fibrosarcoma cells or MCF-7 breast cancer cells, the cell
2
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survival rates are above 80% (see Figure S12) when incubated with
COMMUNICATION
To further explore the potential of H
2
S release from SP-loaded
the SP-loaded PEG-UCNPs. In addition, SP and PEG-UCNPs as PEG-UCNPs in deep tissue triggered by NIR light, we performed
well as NIR light irradiation also show low cytotoxicity (see Figure ex-vivo experiment. Pork skin was used as the tissue model plated
S12). These results suggest that the SP-loaded PEG-UCNPs are low on the top of SP-loaded PEG-UCNPs solution because pork skin is
1
6
cytotoxicity and have potential to be further used in biological thought to be very similar to human’s (see Figure 4a). As shown in
system.
Figure 4b after NIR irradiation for 0.5 h and 1 h the H
concentration was up to 3.7 µM and 5.6 µM, respectively. In
contrast, when irradiated with UV light only a little H S was released
under the same condition. It should be noted that without pork skin
S release triggered by UV irradiation was much higher than that
2
S
2
H
2
by NIR light (see Figure 1e). Therefore, compared with UV light
NIR light significantly improves tissue penetration and is preferred
for the controlled release of H S in deep tissue.
2
Figure 2. (a) H S release as a function of irradiation time from SP-
2
-
1
-2
loaded PEG-UCNPs (1 mg mL ) under 980 nm laser (20 W cm ).
b) H S release as a function of irradiation intensity from the SP-
loaded PEG-UCNPs (1 mg mL ) under 980 nm laser for 2 h. Error
bars mean S.D. (n =3).
(
2
-
1
Figure 4. (a) Illustration the pork skin model used for in-deep tissue
irradiation. (b) The ex-vivo H S release from SP-loaded PEG-
2
UCNPs with UV (365 nm) or 980 nm laser irradiation for different
time; (c) In vivo imaging of SP-loaded PEG-UCNPs.
UCNPs are supposed to be trackable in vivo because of their NIR
fluorescence emission. To demonstrate the feasibility of in vivo
imaging for SP-loaded PEG-UCNPs, we subcutaneously injected
SP-loaded PEG-UCNP solutions into the back of Kunming mice,
and then imaged it using the IVIS imaging system with the 980 nm
laser as the excitation light. As shown in Figure 4c, SP-loaded PEG-
UCNPs revealed strong NIR fluorescence emission under 980 nm
irradiation, which can be used to track the SP-loaded PEG-UCNPs in
Figure 3. CLSM images of L929 cells (a-c) before and (d-f) after
9
80 nm laser irradiation. The cells were treated with SP-loaded
-
1
vivo and facilitate to tune the
2
H S release at the specific
PEG-UCNPs (2 mg mL ) and DNS-Az (50 µM) in CTAB (100µM)
solution. The merged image represents the overlay of UCNP channel physiological targets.
and bright field channel. The scale bars are 20 µm.
In summary, we have developed an NIR light induced H S release
2
platform based on UCNPs. Being exposed to 980 nm NIR laser, the
functionalized SP-loaded PEG-UCNPs can emit UV light which is
Encouraged by the promising results of H S release with NIR
light irradiation in vitro, we further examined the possibility of NIR
2
able to trigger H
controlled manner. Compared to the existing protocols for UV-
controlled H S release, the use of unique NIR-to-UV properties of
UCNPs would result in negligible photo-toxicity and guarantee high
tissue penetration by NIR irradiation. Furthermore, H S delivery
2
S release in both spatially and temporally
light controlled H S release from SP-loaded PEG-UCNPs in living
2
cells. DNS-Az was used as the fluorescence probe to determine the
2
intracellular H S generation. As shown in Figure 3b and 3e the
2
fluorescence of UCNPs distributing around both cell surface and
cytosol indicates that PEG-UCNPs were uptaken by cells. The
fluorescence emission of DNS-Az were shown in Figure 3a and 3d,
from which we can see that both of the cell samples showed green
fluorescence signals. Obviously, the cells which were irradiated with
2
process in vivo can also be tracked in real-time with the NIR
fluorescence emission of UCNPs, which is highly beneficial to the
practical application of the H
diseases, such as cancer, inflammation as well as neurodegenerative
diseases. The multiple advantages of PEG-UCNPs in H S release,
2
S-based therapeutics in a variety of
9
80 nm light exhibit much stronger fluorescence intensity than the
2
one without light exposure (Figure 3a and 3d). The fluorescence
intensity within cells after NIR irradiation is about 1.7 times as high
as that before NIR irradiation (analysed by ImageJ software). In
contrast, in the absence of SP-loaded PEG-UCNPs, there is little
difference in the intracellular fluorescence intensity before and after
NIR irradiation (data not shown). Thus, the increase of intracellular
H S concentration can be ascribed to H S release from SP-loaded
NIR imaging and potential therapeutic applications could make it
serve as an outstanding template in future biomedical research and
therapeutic applications.
Improvements to this platform are still possible. The produced
H
2
S or the gem-dithiols as nucleophile can react with compound 1
before H S diffused into solution, thus affecting H S release
efficiency, more efficient photoinduced H S donors should be
2
2
2
2
2
PEG-UCNPs under NIR irradiation.
developed. Because the excitation of UCNPs at 980 nm overlaps
with water absorption peak at 970 nm, long time irradiation can lead
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to overheating issue. However, this problem could be ameliorated by
short interval irradiation in our experiment. In addition, novel
UCNPs with other excitation wavelength such as 800 nm or 1490
nm are in development in our lab, which will avoid the overheating
issue in another way.
This work was supported by the National Natural Science
Foundation of China (Nos. 21476266 and 21276285). The Advanced
Research Center of CSU (Changsha, China) is acknowledged for
providing access to NMR instrumentation.
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