Near infrared light triggered release of biomacromolecules from hydrogels loaded with upconversion nanoparticles

Article abstract of DOI:10.1021/ja308876j

Using a photosensitive hybrid hydrogel loaded with upconversion nanoparticles (UCNPs), we show that continuous-wave near-infrared (NIR) light (980 nm) can be used to induce the gel-sol transition and release large, inactive biomacromolecules (protein and

Full text of DOI:10.1021/ja308876j

Communication
pubs.acs.org/JACS
Near Infrared Light Triggered Release of Biomacromolecules from
Hydrogels Loaded with Upconversion Nanoparticles
Bin Yan,† John-Christopher Boyer,‡ Damien Habault,† Neil R. Branda,*,‡ and Yue Zhao*,†
†Dep
́ ́ ́
artement de Chimie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1
‡4D LABS, Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
S
* Supporting Information
characteristics of NIR light takes advantage of NIR-absorbing
nanostructures, such as gold nanoparticles (NPs),10 carbon
ABSTRACT: Using a photosensitive hybrid hydrogel
nanotubes11 and graphene oxide NPs,12 by incorporating them
loaded with upconversion nanoparticles (UCNPs), we
show that continuous-wave near-infrared (NIR) light (980
nm) can be used to induce the gel−sol transition and
release large, inactive biomacromolecules (protein and
enzyme) entrapped in the hydrogel into aqueous solution
“on demand”, where their bioactivity is recovered. This
study is a new demonstration and development in
harnessing the unique multiphoton effect of UCNPs for
photosensitive materials of biomedical interest.
into thermoresponsive hydrogel matrices. In these cases, the
photothermal effect is used to convert NIR light into heat, which
triggers a disruption of the hydrogel that results in the release of
entrapped molecules. However, the photothermal-effect-based
approach has constraints: (1) generally it is applicable only to
thermosensitive polymers having a hydration−dehydration
phase transition temperature and (2) photoinduced disruption
of the hydrogel cannot be retained after the NIR irradiation is
turned off because the heating effect disappears and the initial
solution temperature recovers.
mong the many biomedical applications of polymer
In this report, we demonstrate an alternative use of NPs to
Ahydrogels, their use in encapsulating and releasing large convert NIR light into UV light, enabling UV-light-induced
photoreactions to be used to affect the structure of hydrogels.
Our example is the first of its kind. We use core−shell lanthanide-
doped upconverting NPs (UCNPs) to induce a gel−sol
transition when they are irradiated with 980 nm NIR light,
consequently triggering the release of entrapped biomacromo-
lecules. This is feasible because these particular NPs convert as
many as five NIR photons into a UV photon, which is emitted
back into the system to trigger the photochemical reactions of
active groups within the polymer matrix.13 The UCNPs are
convenient for this application because they are also nontoxic
and do not “blink”.14
We previously demonstrated that UCNPs can be loaded
within block-copolymer micelles and act as internal UV and
visible-light sources upon NIR irradiation, with the emitted UV
or visible-light photons activating a photoreaction leading to
micelle disassembly.15 This and other representative examples
highlight the use of UCNPs in NIR-light-controllable bio-
materials and imaging systems.16 The present study is a novel
demonstration of how this universal and robust strategy allows
photosensitive biomaterials to be activated by continuous-wave
(CW) NIR light using hydrogels. In this report, we show how we
can “hide” large biomacromolecules inside a polymer hydrogel,
effectively “shutting down” their bioactivity, and then release
them “on demand” using NIR light, restoring their bioactivity.
Our approach is illustrated in Figure 1, which shows a
schematic representation of the NIR-light-degradable hydrogel
(black lines for polymer chains and red triangles for photo-
cleavable cross-links) loaded with UCNPs (green spheres) and
biomacromolecules (yellow rods). The figure also shows the
biomacromolecules (e.g., proteins or enzymes) in response to
stimuli such as changes in pH1 or temperature;2 the presence of
redox species,3 biomolecules,4 or ions;5 or electric fields6 or light7
has the great potential to improve how we treat disease and study
complex biochemical processes. Also noticeable are hydrogels
using aptamers and complementary oligonucleotides to achieve
controlled release of multiple drugs.8 In this context, a hydrogel
can act as a “cage” and hide biomacromolecules to prevent them
from interacting with other species until desired. Once the
stimulating signal triggers a structural disruption of the hydrogel
through a process such as a gel−sol transition or gel volume
expansion/contraction, the entrapped biomacromolecules are
released into the bulk solution, where their bioactivity is
recovered.
Light offers many distinct advantages over other stimuli for
controlling this disruption process. It can be tuned (color and
intensity) and used for remote activation of a wide range of
materials at a specific time and location with relatively high
precision. However, the examples of light-responsive hydrogels
developed in recent years suffer from a serious drawback that
hinders their potential use in biomedical applications: typically,
their photochemical reactions all require the use of high-energy
UV or visible light, neither of which can penetrate deeply into
tissues and both of which can cause detrimental side effects to
healthy cells. The most common way to overcome this problem
is the use of photoresponsive systems that can absorb two
photons of near-infrared (NIR) light to induce the same
reactions that are induced by UV or visible light. However, the
usefulness of this approach is limited because the low two-
photon absorption cross-section of typical chromophores9
makes the process very inefficient even when femtosecond-
pulse lasers are used. An alternative strategy to harness the
Received: September 6, 2012
Published: September 26, 2012
© 2012 American Chemical Society
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Communication
Figure 1. (a) Schematic illustration of the NIR-light-triggered
degradation of a photosensitive hydrogel using the UV light generated
by encapsulated UCNPs. The polymeric components are depicted as
black lines, the photocleavable cross-links as red triangles, the UCNPs as
green spheres, and the trapped biomacromolecules as yellow rods. (b)
Chemical structure of the hydrogel containing photocleavable o-
nitrobenzyl moieties in the cross-linker and the NIR-light-induced
photoreaction of the hydrogel via UV light emitted by loaded
NaYF4:TmYb core−shell UCNPs. The number of monomer units
per PEG cross-linker was 104, and the molar ratio between the
acrylamide monomer units and the PEG cross-linker, y/x, was ∼50 (as
Figure 2. (a) Absorption spectrum of the hydrogel dispersed in water
(blue line) and emission spectra of the neat UCNPs in aqueous solution
(red line) and the UCNP-loaded hydrogel (black line) upon 980 nm
excitation. The inset is a transmission electron microscopy image of neat
UCNPs. (b) Photograph of a UCNP-loaded hydrogel under a 980 nm
CW diode laser exposure (3.1 W). (c, d) Photographs of a UCNP-
loaded hydrogel (∼0.08 mL) (c) before and (d) after irradiation with
980 nm light (5 W, 195 min), showing the NIR-light-induced gel−sol
transition of the hydrogel.
1
determined from the H NMR spectrum of a fully UV-degraded gel
sample in CDCl3).
the UCNPs were trapped within the hydrogel. In contrast, the
emission peaks in the visible region (430−500 nm), a spectral
region where the hydrogel has no absorptions, clearly remained.
As the laser beam traveled through the UCNP-loaded hydrogel,
visible photoluminescence (PL) and scattered blue light were
visible (Figure 2b). Figure 2c,d shows photographs of a hydrogel
formed inside a 4.7 mm diameter capillary tube before and after
NIR irradiation using a diode laser, respectively. The NIR-light-
induced degradation (gel−sol transition) is indicated by the flow
of the hydrogel after the NIR irradiation. We note that a relatively
high NIR laser power and a long irradiation time were used to
allow the visualization of the flow of the hydrogel confined in the
tube, which occurred in the late stage of the hydrogel disruption.
Our first demonstration of NIR-light-triggered release of
proteins from the hydrogel used fluorescein isothiocyanate
bovine serum albumin (FITC-BSA) as a general model and
proof-of-concept. The hydrogel-trapped protein was prepared by
adding a phosphate-buffered saline (PBS) solution of the protein
(pH 7.0, 100 mM PBS, protein concentration 1 mg/mL) to an
aqueous solution containing the UCNPs, the acrylamide
monomer, the PEG cross-linker, and an initiator (a redox pair
of ammonium persulfate and N,N,N′,N′-tetramethylethylenedi-
amine). After the polymerization was complete, the hydrogel
loaded with both FITC-BSA and the UCNPs was washed with
fresh PBS solution to remove any nontrapped protein. The
release experiments were carried out by exposing a sample of the
hydrogel (∼100 mg) resting on the bottom of a cuvette to 980
nm light while monitoring the increase in absorption and
emission of the liquid phase above the sample due to the
photoreaction and subsequent release of the nitrobenzaldehyde
cross-linker and the fluorescently labeled protein from the gel.
The results of these experiments are summarized in Figure 3. The
experimental setup is illustrated in Figure 3a. The changes in the
UV−vis absorption spectrum of a hydrogel sample containing
the UCNPs but no protein (Figure 3b) demonstrated the success
of the multiphoton effect and the conversion of the photo-
sensitive o-nitrobenzyl groups to their nitrosobenzaldehyde
products, which diffused from the gel into the aqueous phase
only when NIR light was used. Prior to NIR light exposure, only
very minor changes in the absorption spectra were detected even
chemical structure of the hydrogel, which has a cross-linked
hybrid polyacrylamide−poly(ethylene glycol) (PEG) structure
held together by photoresponsive o-nitrobenzyl groups. NIR
irradiation of the UCNPs within this cross-linked system
generates the UV light needed to cleave the o-nitrobenzyl
groups in their typical photooxidation process. This results in the
breakdown of the entire gel (gel−sol transition), releasing the
trapped components. Details of materials synthesis and
characterization as well as experimental procedures for loading
of UCNPs in the hydrogel, simultaneous loading of proteins or
enzymes, and NIR-light-triggered release are provided in the
Supporting Information (SI). The photocleavable PEG cross-
linker was synthesized using a literature method7a−d with some
modifications. The core−shell NaYF4:TmYb NPs (core =
NaYF4:0.5 mol % Tm3+:30 mol % Yb3+; shell = NaYF4), which
had a uniform hexagonal prism shape with an average length of
36.0 1.1 nm and width of 32.0 1.5 nm, were synthesized
using a literature method.17 For incorporation of the UCNPs
into the hydrogel, a water-compatible system is required (the
UCNPs are prepared in an organic-soluble form coated with
oleate ligands). This was achieved by a ligand exchange process
that replaced the hydrophobic oleate ligands with water-
compatible polyvinylpyrrolidone.18 The UCNPs and biomacro-
molecules were conveniently loaded into the photosensitive
hydrogel by polymerizing an aqueous mixture of the acrylamide
monomer and the photocleavable PEG cross-linker by a redox-
initiated radical polymerization process.
As designed, the emission spectrum of the UCNPs changes
when the NPs are trapped within the hydrogel as a result of the
overlap of specific bands with the absorption band of the
photoreactive component holding the hydrogel together (Figure
2a). When irradiated with 980 nm light, the NPs emit at several
wavelengths within the UV−vis region of the spectrum. The ones
relevant to the current study are those appearing between 250
and 400 nm, since these wavelengths are absorbed by the
hydrogel and induce photoreactions of the o-nitrobenzyl groups.
This phenomenon was clearly revealed by the fact that the UV
emissions from the UCNPs could no longer be observed when
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Journal of the American Chemical Society
Communication
because the protein molecules remained entrapped in the
hydrogel until the release was triggered again by turning on the
laser.
Control over the bioactivity due to NIR-triggered release from
our hybrid hydrogel was readily demonstrated by monitoring the
activity of trypsin (an enzyme that cleaves peptide bonds in
proteins) on a fluorogenic substrate, (CBZ-Arg)2-R110. The
results are shown in Figure 4. The enzyme was loaded into our
Figure 3. (a) Setup used to detect molecular species diffusing from the
hydrogel into the aqueous solution as a result of 980 nm excitation. (b)
Changes in the UV−vis absorption spectrum of an aqueous solution
containing a hydrogel exposed to NIR light (5 W) for different times
(0−3 h). For comparison, the absorption spectrum of the hydrogel
immersed in the solution for 12 h without NIR irradiation is shown in
red. (c) Plots of fluorescence emission intensity (λem = 514 nm, λex = 494
nm) detected from FITC-BSA loaded in the hydrogel vs NIR irradiation
time, showing NIR-light-triggered release of the protein from the
hydrogel. The results of two control tests are also shown for comparison.
(d) Temporal control of the protein release by turning the NIR diode
laser on (5 min) and off.
Figure 4. Increase in the emission intensity revealing the enzymatic
activity of trypsin on a fluorogenic substrate (λex = 498 nm) recorded
with a hydrogel loaded with the enzyme and UCNPs in aqueous solution
(using the setup shown in Figure 3a): (a) in the dark (black curves) and
upon 5 W 980 nm NIR irradiation from 0 to 57 min (red curves); (b)
plots of fluorescence emission intensity at the 521 nm peak vs time with
□
●
( ) and without ( ) NIR light irradiation.
hydrogel as described above. After washing and removal of
nontrapped enzyme molecules, the hydrogel was immersed in
water containing (CBZ-Arg)2-R110. There was little increase in
fluorescence due to the enzymatic conversion of the substrate
into its emissive product (rhodamine 110) in the absence of NIR
light even after 57 min, indicating that the enzyme was effectively
trapped within the hydrogel, which suppressed its activity. The
observed weak activity was likely caused by a very small amount
of trypsin that diffused from the gel (as was shown previously for
BSA) and/or diffusion of (CBZ-Arg)2-R110 into the hydrogel,
where it could be converted into its fluorescent product within
the gel matrix before diffusing into the whole solution. Exposing
the hydrogel to 980 nm light produced an immediate increase in
the fluorescence corresponding to rhodamine 110, indicating
successful release of trypsin and recovery of its enzymatic activity.
Again, according to a control test using the same amount of
trypsin dissolved in solution, ∼72% of the enzyme was released
after 57 min of NIR irradiation at a laser power of 5 W (Figure
S6).
Before concluding, we mention that several in-depth studies
have examined the long-term toxicity of NaYF4-based UCNPs
using both cell and small-animal models;19 the results suggest
that these UCNPs possess little or no toxicity over extended
exposure times. For future studies of NIR-light-sensitive UCNP-
loaded hydrogels, it would be of interest to apply the same design
to other biocompatible hydrogels and use photocleavable
moieties that may be more suitable for biomaterials (e.g.,
coumarin).20 In view of the on-demand release activated by NIR
light, this type of hydrogel would be better suited for applications
where a temporally controllable, stepwise release is desired,
rather than applications requiring prolonged release (e.g., drug
release from implants).
12 h after immersion of the hydrogel in the solution. Irradiation
of the same hydrogel with 980 nm light (∼0.1 mm diameter
beam size) resulted in the appearance of absorption bands at
300−400 nm characteristic of the nitrosobenzaldehydes
produced from the photocleavage reaction. The intensity of
these absorption bands increased as long as the gel was exposed
to NIR light, indicating a continuous photoreaction.
Figure 3c illustrates release of trapped protein from our
hydrogel using NIR light. In this case, the amount of FITC-BSA
released into the solution was monitored by the increase in the
PL intensity at the emission maximum wavelength. Only a small
observable amount of the protein was released from the hydrogel
containing the NPs in the absence of NIR light, as expected on
the basis of several cases reported in the literature.1a,3a,b Similar
amounts of released protein were observed for samples of the
hydrogel containing only the protein upon exposure to 980 nm
light (5 W). The amount of protein released from the hybrid
system increased significantly when the hydrogel loaded with
both UCNPs and protein was exposed to the NIR laser (3−5 W),
clearly demonstrating the NIR-light-triggered release of the large
biomacromolecules as a result of hydrogel degradation. As
expected, the release was power-dependent, slowing when the
laser intensity was reduced. On the basis of the emission intensity
of the same amount of FITC-BSA in solution without the
hydrogel, the release of the protein entrapped in the UCNP-
loaded hydrogel after 50 min of NIR irradiation was ∼67% at a
laser power of 5 W and ∼38% at 3.1 W (Figure S6 in the SI). The
latter experiment was repeated twice, and the release of protein
was found to be ∼40 and ∼33%, respectively, suggesting quite
good reproducibility. The on-demand release of protein from the
hybrid hydrogel was best demonstrated by alternating between
“light” and “dark” conditions (Figure 3d). As designed, release
events occurred only when the NIR light was on (3−5 W), as
illustrated by the increase in fluorescence intensity. Each time the
light was turned off, there was no change in the emission intensity
In conclusion, our hybrid UCNP−hydrogel system represents
the first demonstration of the use of the multiphoton effect of
UCNPs to trigger structural changes in photosensitive hydrogels.
It allows the use of CW NIR light to induce the gel−sol transition
and release large, inactive biomacromolecules on demand, where
their bioactivity can be recovered. This study is an important new
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ASSOCIATED CONTENT
* Supporting Information
■
S
Details of materials synthesis and characterization and
experimental procedures for loading of UCNPs in the hydrogel,
simultaneous loading with proteins or enzymes, and NIR-light-
triggered release. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
nbranda@sfu.ca; yue.zhao@usherbrooke.ca
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We acknowledge the financial support from the Natural Sciences
and Engineering Research Council of Canada (NSERC), le
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■
Fonds Queb
Technologies of Queb
Chairs Program, and Simon Fraser University through the
Community Trust Endowment Fund. Y.Z. is a member of
FQRNT-funded CSACS, CQMF, and FRQS-funded Centre de
́
ec
́
ois de la Recherche sur la Nature et les
ec (FQRNT), the Canada Research
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Mater. 2012, DOI: 10.1002/adfm.201201020.
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Smith Foundation for Health Research for support. B.Y. thanks
the China Scholarship Council for scholarship support.
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