NIR-triggered drug release from switchable rotaxane-functionalized silica-covered Au nanorods

Article abstract of DOI:10.1039/c4cc02966f

An NIR-triggered drug delivery system was developed by capping photo-switchable azobenzene-based rotaxane onto Au nanorod-mesoporous silica core-shell hybrids. Drug release from the nanocarrier in zebrafish embryo models could be controlled remotely under

Full text of DOI:10.1039/c4cc02966f

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NIR-triggered drug release from switchable
rotaxane-functionalized silica-covered Au
nanorods†
Menghuan Li,ab Hong Yan,a Cathleen Teh,c Vladimir Korzhc and Yanli Zhao*ab
Cite this: Chem. Commun., 2014,
50, 9745
Received 22nd April 2014,
Accepted 17th June 2014
DOI: 10.1039/c4cc02966f
www.rsc.org/chemcomm
An NIR-triggered drug delivery system was developed by capping
photo-switchable azobenzene-based rotaxane onto Au nanorod–
mesoporous silica core–shell hybrids. Drug release from the nano-
carrier in zebrafish embryo models could be controlled remotely
under NIR irradiation, showing significant drug spreading to the
adjacent tissues.
Light stimuli, as a remotely-controllable approach, can easily be
exerted with high precision and malleability at specific loca-
tions.1 Light-responsive drug delivery systems have received
much attention on account of their capability to enhance the
drug delivery efficiency and minimize the side effects. Recently,
several light-responsive drug carriers based on conformational
changes or cleavage of chemical bonds under specific illumination
conditions have been reported, including the photoisomerization
Scheme 1 Illustration of photo-responsive Au@MSN–rotaxane, where
the switch of the rotaxane can be controlled remotely by NIR illumination
to regulate the cargo release.
Herein, we report the development of an NIR-responsive
of azobenzene derivatives,2 self-assembly and disassembly of drug delivery system made by immobilizing azobenzene-based
pseudorotaxanes,3,4 and photo-cleavable linkages.5 For instance, rotaxane onto a Au nanorod–mesoporous silica core–shell
the photoisomerization of azobenzene between the trans and cis hybrid (Au@MSN–rotaxane, Scheme 1 and Scheme S1 in the
geometries has been utilized to achieve the controlled release of ESI†). In this system, Au nanorods, a well-proven photothermal
cargo molecules.6,7 Currently, the majority of these systems have agent,10 serve as the energy converter to activate the isomeriza-
employed UV or visible light as the trigger. However, considering tion of the azobenzene moiety. The intermediate mesoporous
the ‘‘water window’’ (from 700 nm to 1400 nm) of biological silica layer on the surface of the Au nanorods was used as the
tissues for electromagnetic radiation, the tissue penetration of drug storage reservoir11,12 and also as the substrate for post-
UV and visible light is greatly limited, resulting in inefficient deep- modification by the rotaxane.13,14 The rotaxane15 on the silica
tissue drug delivery.8 To overcome this challenge, NIR-responsive layer consists of a-cyclodextrin (a-CD) encapsulating the trans-
drug delivery systems are desired. NIR illumination possesses high azobenzene site, and acts as the capping agent to control the
transmittance and attenuated cytotoxicity in living tissues, and has drug loading and release.16 Initially, the nanocarrier was loaded
already been proven as an efficient technique for noninvasive with cargo molecules via diffusion at 40 1C. The cargo-loaded
cancer therapy.9
nanocarrier was subsequently illuminated with UV light for robust
encapsulation. The cargo release from the nanocarrier was ulti-
mately realized under NIR illumination (Scheme S2 in the ESI†).
The delivery efficacy of the nanocarrier was thoroughly investi-
gated in solution and in vivo in live zebrafish embryo models.
The Au@MSN–rotaxane nanocarrier was firstly prepared
(see ESI† for the experimental procedure) and its physical/
chemical properties were characterized in detail. As shown in
Fig. 1a, the TEM observations confirmed that the Au nanorods
have a uniform and well-defined structure, and this feature was
a Division of Chemistry and Biological Chemistry, School of Physical and
Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link,
Singapore 637371, Singapore. E-mail: zhaoyanli@ntu.edu.sg
b School of Materials Science and Engineering, Nanyang Technological University,
Singapore 639798, Singapore
c Laboratory of Fish Development Biology, Institute of Molecular and Cell Biology,
61 Biopolis Drive, Singapore 138673, Singapore
† Electronic supplementary information (ESI) available: Additional experimental
details. See DOI: 10.1039/c4cc02966f
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which could be assigned to the amide bond, alkyne end group,
and alkyl stretching, respectively. The alkyne-containing Au@MSN
subsequently reacted with azidized b-CD–azobenzene pseudoro-
taxane through a click reaction between the alkyne and azide
groups, resulting in the consumption of the alkyne groups. There-
fore, the characteristic alkyne peak at 2100 cmÀ1 disappeared,
while some new peaks appeared at 760 and 989 cmÀ1, which were
assigned to the benzene ring and the sulfonic group21 on the
azobenzene moiety, respectively. The newly appeared peaks indi-
cate that the a-CD–azobenzene rotaxane was successfully conju-
gated onto the surface of Au@MSN through the click reaction.
The conformational transition of the azobenzene-based rotaxane
in Au@MSN–rotaxane was monitored by UV-vis spectroscopy. It has
been reported that UV irradiation can induce the trans-to-cis photo-
isomerization of the azobenzene moiety, and that the cis-azobenzene
can undergo a thermal relaxation process to change back to the trans
state.22 The a-CD ring can effectively encapsulate trans-azobenzene
but not cis-azobenzene, thus leading to UV/heat-controlled a-CD
movement in the rotaxane. When Au@MSN–rotaxane was irradiated
with UV light, the absorption at 380 nm decreased due to the trans-
to-cis photoisomerization of the azobenzene unit. Under NIR illu-
mination, a recovery of the absorption intensity in the same region
was observed (Fig. 3a), indicating that the cis-azobenzene moiety was
transformed back to its trans state. The absorption intensity change
was further plotted against the ‘‘on–off’’ cycle number (Fig. 3b). It
was evident that the reversibility between the two isomerization
states was well maintained even after 5 cycles. These observations
prove that NIR irradiation could efficiently trigger the cis-to-trans
isomerization of the azobenzene moiety for controlled drug release.
Next, a cargo release test from fluorescein isothiocyanate
(FITC)-loaded Au@MSN–rotaxane was carried out, and the
release profiles were monitored using fluorescence spectro-
scopy (Fig. 4). In the control group, where the temperature
was 25 1C, it was observed that the amount of FITC released
from the FITC-loaded Au@MSN–rotaxane was almost negligible
(less than 5%) even after 180 min. In another experiment, when
the temperature was increased to 37 1C, the total release
amount of FITC was less than 20%, indicating that the encap-
sulated cargo molecules could be maintained by the rotaxane
cap when no external trigger was exerted. The release profiles in
the control experiments matched well with the kinetic features
reported in a previous study.23 In the external heat-triggered
Fig. 1 Illustration of the morphological evolution. TEM images of (a) Au
nanorods, (b) Au@MSN, and (c) Au@MSN–rotaxane. Scale bar: 100 nm.
well maintained in the Au@MSN core–shell hybrid (Fig. 1b) and
Au@MSN–rotaxane (Fig. 1c). The length and width of the naked
Au nanorods were 75 Æ 15 nm and 18 Æ 5 nm, respectively. The
thickness of the mesoporous silica layer coated using the
template method was around 10–20 nm. In comparison to
the unmodified Au@MSN, Au@MSN–rotaxane appeared to be
blurry, which might be due to the modification of the rotaxane
on the silica surface.
UV-vis spectra were employed to further characterize the specific
electromagnetic absorption of the nanocarrier. As shown in Fig. S2a
(ESI†), the longitudinal absorption of the naked Au nanorods was
centered at 808 nm. After coating with mesoporous silica, it was
slightly red-shifted to around 830 nm. This phenomenon could be
explained by the Lippert–Mataga equation,17 as increasing the
refractive index of the particle surface from monocrystalline Au
substrate (0.28)18 to silica (1.54)19 would enhance the localized
refraction of the incident light. After the subsequent grafting of
the alkyne groups onto Au@MSN, the longitudinal absorption band
shifted back to a shorter wavelength of 780 nm. It shifted again to a
longer wavelength of 830 nm after the surface immobilization of the
photo-switchable rotaxane. In addition, a new peak at 380 nm was
observed, which was assigned to the characteristic UV absorption of
the azobenzene unit in its trans conformation (Fig. S2 in the ESI†).20
The surface modification process was also monitored by
FTIR spectra. As seen from Fig. 2, the coating of mesoporous
silica onto the Au nanorods led to a strong peak at around
1100 cmÀ1, which was assigned to the Si–O bonds from the silica
layer. After the alkyne grafting, a series of new characteristic
peaks appeared at 1536, 2100 and 2860, as well as 2930 cmÀ1
,
Fig. 3 (a) UV-vis spectra of Au@MSN–rotaxane after treatment with UV
irradiation at 365 nm (‘‘on’’ state) and NIR illumination at 808 nm (‘‘off’’ state).
(b) The changes in the UV absorbance of Au@MSN–rotaxane at 380 nm
upon alternating irradiations with UV light and an NIR laser.
Fig. 2 FTIR spectra of Au@MSN before and after modification. (1) Au@MSN,
(2) Au@MSN–alkyne, (3) Au@MSN–rotaxane.
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DOX released from Au@MSN–rotaxane, as DOX itself has strong red
fluorescence that facilitates CLSM detection. As compared to the
unilluminated control groups, the NIR-illuminated specimens
showed greater DOX release. The DOX fluorescence covered a
significantly larger area after NIR irradiation (Fig. 5). Moreover,
the integrated DOX fluorescence intensity ratio of the NIR illumi-
nated embryo to the control group was 3 : 1. It is important to note
that the area injected with DOX-loaded Au@MSN–rotaxane was
extracted from the integration area to avoid the interference of
unreleased DOX. The difference in the fluorescence intensity sug-
gested that Au@MSN–rotaxane could retain the loaded DOX when
no external stimulus was exerted, and was capable of efficiently
releasing the DOX cargo under NIR illumination. The drug-release
behavior of Au@MSN–rotaxane was further investigated by chan-
ging the NIR illumination duration. For those zebrafish embryos
treated with NIR light for 10 min, the distance of DOX fluorescence
spreading was evidently higher than that of the unilluminated
control (Fig. S4a and b in the ESI†). The distance increase indicated
that more of the DOX inside the Au@MSN–rotaxane was released
into the adjacent tissues under NIR irradiation for 10 min. When
the NIR illumination duration was reduced to 5 min, the DOX
fluorescence band still overlapped completely with the optically
opaque Au@MSN–rotaxane in the merged image (Fig. S4c and d,
ESI†). The coincidence indicated that most of the loaded DOX was
still entrapped within the nanocarrier at this stage, similar to the
unilluminated embryo control. It was also consistent with the
kinetic features of the above release profile, where the nanocarrier
showed a release delay following NIR irradiation (Fig. 4d). Addi-
tional in vivo experiments carried out at 37 1C also showed a similar
trend, where the maximum duration of NIR illumination was
extended to 30 min (Fig. S5, ESI†). The observations firmly sup-
ported the hypothesis that DOX release could be effectively con-
trolled by remote NIR illumination.
Fig. 4 Release profiles of FITC-loaded Au@MSN–rotaxane in aqueous
solution (a) at 25 1C, (b) at 37 1C, (c) with external heating at 45 1C, and (d)
under NIR illumination at 808 nm.
group, only a minor increase in FTIC fluorescence was observed in
the initial 5 min, when the temperature was at 25 1C. Interestingly,
the release amount rose rapidly when the incubation temperature
increased to 45 1C, and it ultimately reached around 50% in
180 min, which could be explained as the cis-to-trans relaxation
rate of the azobenzene moiety would speed up under high tem-
perature. When an NIR laser was applied, the loaded FITC was
released rapidly into the medium, and the release amount even-
tually reached approximately 70%. For the NIR-triggered release,
there was a delay of approximate 5 min between the launching of
NIR illumination and the rapid increase of the fluorescence inten-
sity. The possible reason is that during the photothermal treatment,
the Au nanorods may need to first heat up the whole surrounding
environment to evoke the relaxation of cis-azobenzene on the silica
surface, thus causing the delayed drug release in comparison to the
external heating method. Overall, the release profiles demonstrated
well that this drug delivery system could readily respond to remote
NIR irradiation for controlled cargo release.
In conclusion, a novel NIR-responsive nanosystem for anticancer
drug delivery has been developed through the integration of Au
nanorod, mesoporous silica reservoir and azobenzene-based rotax-
ane. The cis-to-trans isomerization of the azobenzene moiety in the
rotaxane could be readily initiated upon photothermal heating with
an NIR laser, leading to remotely controlled cargo release from the
mesoporous silica reservoir. The drug delivery efficacy of the
nanosystem has been demonstrated in solution and in live zebrafish
embryo models under NIR laser stimulation. The current research
presents a successful example of NIR-controlled drug release in vivo.
This research is supported by the National Research Foun-
dation (NRF), Prime Minister’s Office, Singapore under its
NRF Fellowship (NRF2009NRF-RF001-015) and the Campus
for Research Excellence and Technological Enterprise
(CREATE) programme-Singapore Peking University Research
Centre for a Sustainable Low-Carbon Future, and the NTU-
A*Star Centre of Excellence for Silicon Technologies (A*Star
SERC No.: 112 351 0003).
The in vivo release efficiency of Au@MSN–rotaxane loaded with
the anticancer drug doxorubicin (DOX)24 was evaluated in zebrafish
embryo models using confocal laser microscopy (CLSM). The con-
focal images presented a visual demonstration of the distribution of
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Fig. 5 Confocal images showing the DOX release within embryo heads
injected with DOX-loaded Au@MSN–rotaxane without and with NIR
irradiation.
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