Photo-triggerable hydrogel-nanoparticle hybrid scaffolds for remotely controlled drug delivery

Article abstract of DOI:10.1039/c4tb01436g

Remotely-triggerable drug delivery systems enable the user to adjust dosing regimens on-demand based on a patient's physiological response and clinical needs. However, currently reported systems are limited by the non-specific leakage of drugs in the absence of triggering and the lack of repeatability over multiple cycles of release. To this end, we have designed a unique hydrogel-nanoparticle hybrid scaffold that provides a chemically-defined, remotely-triggerable and on-demand release of small molecule drugs. Our hybrid platform consists of three distinct components: (1) a photo-triggerable chemical compound, which serves to release a covalently-bound drug upon photo-irradiation, (2) a nanoparticle, which serves to covalently bind the photo-triggerable compound, and (3) a polymeric hydrogel, which serves to hold the drug-conjugated nanoparticle. Upon photo-irradiation, the activation of the photo-triggerable compound is designed to initiate a series of intramolecular chemical rearrangements, which would cleave the covalently-bound drug and release it from the hydrogel. The combination of these distinct components in a single scaffold proved to be an effective drug delivery system, as demonstrated by the delivery of a model drug to a malignant cancer line. Our hybrid scaffold can be easily tuned for practically any biological application of interest, thus offering immense potential for clinical therapies.

Full text of DOI:10.1039/c4tb01436g

Journal of
Materials Chemistry B
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PAPER
Photo-triggerable hydrogel–nanoparticle hybrid
scaffolds for remotely controlled drug delivery†
Cite this: J. Mater. Chem. B, 2014, 2,
7685
*
Shreyas Shah,‡ Pijus K. Sasmal‡ and Ki-Bum Lee
Remotely-triggerable drug delivery systems enable the user to adjust dosing regimens on-demand based
on a patient's physiological response and clinical needs. However, currently reported systems are limited
by the non-specific leakage of drugs in the absence of triggering and the lack of repeatability over
multiple cycles of release. To this end, we have designed a unique hydrogel–nanoparticle hybrid scaffold
that provides a chemically-defined, remotely-triggerable and on-demand release of small molecule
drugs. Our hybrid platform consists of three distinct components: (1) a photo-triggerable chemical
compound, which serves to release a covalently-bound drug upon photo-irradiation, (2) a nanoparticle,
which serves to covalently bind the photo-triggerable compound, and (3) a polymeric hydrogel, which
serves to hold the drug-conjugated nanoparticle. Upon photo-irradiation, the activation of the photo-
triggerable compound is designed to initiate a series of intramolecular chemical rearrangements, which
would cleave the covalently-bound drug and release it from the hydrogel. The combination of these
distinct components in a single scaffold proved to be an effective drug delivery system, as demonstrated
by the delivery of a model drug to a malignant cancer line. Our hybrid scaffold can be easily tuned for
practically any biological application of interest, thus offering immense potential for clinical therapies.
Received 29th August 2014
Accepted 27th September 2014
DOI: 10.1039/c4tb01436g
www.rsc.org/MaterialsB
reservoir, microchip device which was triggered through a
wirelessly transmitted signal.5 Upon wireless triggering, the
Introduction
electrochemical dissolution of the drug-sealed membrane was
shown to effectively release the drug. While such a device is
effective for delivering multiple doses, the size of the solid-state
device limits the implantation to only certain subcutaneous
compartments in the body. Compared to these microfabricated
solid-state devices, other systems have utilized so materials
(e.g. polymers) which undergo conformational changes due to
microenvironmental conditions. For instance, thermo-respon-
sive polymers or membranes consisting of drugs and gold
nanoparticles (i.e. nanoshells, nanorods) have been shown to
effectively release the embedded drug upon local heating of the
gold nanoparticles via near infrared light irradiation.6,7 In other
variants of such a system, magnetic nanoparticles have been
used in combination with an alternating magnetic eld to
impose local heating.8,9 While local heat induction is one of the
most widely investigated stimuli for such systems, the potential
damage to healthy tissue from thermal injury and the possi-
bility of accidental triggering (e.g. due to fever) can cause
undesired side effects.6
Drug delivery systems are constantly advancing due to the
importance of achieving targeted and controlled release. The
current lack of control over the spatial and temporal adminis-
tration of drugs tends to require larger doses in order to achieve
the desired effect within the therapeutic window, which can
inadvertently cause increased toxicity and other undesirable
side effects.1 Moreover, numerous drug treatment regimens
require frequent or continuous administration based on the
patient's response to the treatment over a period of time, which
can compromise patient comfort and compliance. Addressing
these concerns, recent advances in materials chemistry have led
to the generation of reservoir-based drug delivery systems that
release loaded drugs in response to an externally-applied
stimulus.2 Such systems allow for a highly localized concen-
tration of drugs at or near the target site upon implantation,
while enabling the patient or healthcare professional to control
the dose and timing of drug release.3
Various types of remotely-triggerable drug delivery systems
have been developed, which rely on applying an external stimuli
to release the drug load.4 One type of system employed a multi-
In this regard, light can serve as a superior external stimulus
for drug release since it can be manipulated to attain both
spatial and temporal control with high precision.10 In contrast
to other types of remote triggers for drug delivery, light is the
least invasive, it does not require advanced equipment, and the
drug release can be modulated by simply adjusting the wave-
length of light, power density and time of exposure.11 Various
Department of Chemistry & Chemical Biology, Rutgers, The State University of New
Jersey, Piscataway, NJ 08854, USA. E-mail: kblee@rutgers.edu; Fax: +1-732-445-
5312; Tel: +1-848-445-2081
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4tb01436g
‡ These authors contributed equally to this work.
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types of photo-responsive drug carrier systems have been designed to initiate a series of intramolecular chemical rear-
developed including prodrugs, micellar/liposomal vesicles, rangements, which would cleave the covalently-bound drug
nanoparticles, self-immolative polymers and hydrogels.12–15 Yet, from the nanoparticle surface and ultimately release it from the
there have been several challenges in creating effective photo- hydrogel. In this way, our hybrid scaffold combines the unique
responsive reservoir-based systems which afford controlled intrinsic properties of synthetic chemical compounds, three-
release from a drug-embedded membrane or polymer matrix. dimensional hydrogel scaffolds and multifunctional nano-
Specically, the majority of current approaches rely on simply particles in a single, reservoir-based drug delivery platform. In
encapsulating the drug or biomolecule of interest within a the current work, we demonstrate the utility of our platform to
photodegradable polymer matrix (e.g. hydrogel), whereby the deliver a specic model drug (camptothecin) to a malignant
photo-induced degradation of the matrix releases the cargo.16 brain tumor cell line. However, each of the comprising
However, such systems exhibit a poor ratio of drug release components of our hybrid scaffold (i.e. the small molecule drug,
kinetics between the ‘on’ (light) and ‘off’ (no light) state. As a the stimuli-responsive trigger, the nanoparticle and the hydro-
result, there is unintended leakage of drugs from the polymer gel) can be easily replaced to tune our platform for practically
reservoir in the absence of photo-irradiation due to passive any biological application of interest, thus offering immense
diffusion, thus questioning the long-term stability and clinical- potential for advancing drug delivery systems.
relevance of such systems. Moreover, such designs primarily
allow for only one-time burst releases, compared to the favor-
able continuous or pulsatile release from multiple cycles of
photo-irradiation over a given time frame. Considering the
abovementioned challenges, a reservoir-based system which
Results and discussion
Synthesis of the photo-triggerable chemical adaptor system
A key aspect of our system relies on establishing a stable
chemical linkage to attach small molecule drugs to the nano-
particle surface, while also permitting spontaneous release
upon photo-irradiation (Fig. 1). This requires a synthetic
approach wherein the different functionalities can be linked
together and then fragmented into the individual building
blocks in a controlled manner. Chemical adaptor systems (CAS)
are a unique class of synthetic molecules which have been
designed specically for this purpose.17,18 These unique mole-
cules are based on a core molecule (‘chemical adaptor’) which
provides a chemical linkage between multiple functional
moieties (e.g. stimuli-responsive trigger, small molecule drug,
etc.) that can be triggered for rapid disassembly under pre-
dened conditions. In this study, we designed a photo-
can provide an effective light-triggered, controlled drug release,
with minimal release in the ‘off’ state, would be high benecial.
Herein, we report a photo-triggerable, hydrogel–nanoparticle
hybrid scaffold that provides a chemically-dened, remotely-
triggered and on-demand release of small molecule drugs
(Fig. 1). Our hybrid drug delivery platform consists of three
main components: (1) a photo-triggerable chemical compound,
which releases a covalently-bound drug upon photo-irradiation,
(2) a silica nanoparticle, which serves to covalently bind the
photo-triggerable chemical compound, and (3) a poly(ethylene
glycol) (PEG)-based hydrogel matrix, which serves to retain the
drug-conjugated nanoparticle. Upon exposure to UV light, the
activation of the photo-triggerable chemical compound is
Fig. 1 Schematic illustration depicting the fabrication and application of hydrogel–nanoparticle hybrid scaffolds. Photo-triggerable, drug-
conjugated silica nanoparticles were encapsulated within a PEG-based hydrogel. The subsequent photo-irradiation of the hybrid scaffold
initiates a series of chemical rearrangements which ultimately releases the covalently-bound drug to induce cancer cell death.
7686 | J. Mater. Chem. B, 2014, 2, 7685–7693
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triggerable CAS for the release of the covalently-bound model conrmed the release of the covalently-bound CPT molecule
drug camptothecin (CPT), a DNA topoisomerase I inhibitor,19 upon exposure to UV light, with complete release observed
upon exposure to UV light. We utilized the commercially within 24 h (Fig. S1†). Moreover, in the absence of UV light, the
available 4-hydroxymandelic acid as the core chemical adaptor, CAS–CPT was observed to remain intact with no release of CPT
which has three functional end groups available for conjuga- even aer 30 days of incubation, further conrming the
tion: the benzylic hydroxyl group, the phenolic hydroxyl group chemical stability and light-specic sensitivity of this
and the carboxylic acid (Fig. 2). In this case, we used the compound (Fig. S2†).
benzylic hydroxyl group to link the drug molecule, the phenolic
hydroxyl group to attach the photo-reactive trigger molecule and
the carboxylic acid to mediate the conjugation to the
Conjugation of the CAS–CPT to nanoparticles
Aer conrming the effectiveness of the CAS in releasing the
CPT drug, we proceeded to covalently attach the CAS–CPT to a
nanoparticle surface. We utilized silica nanoparticles (silica
NPs) to anchor the CAS–CPT since they are highly inert and
stable, exhibit excellent biocompatibility and allow for facile
surface functionalization.20 Briey, amine-terminated silica NPs
(300 nm; Corpuscular, Inc) were rst activated with N,N0-dis-
uccinimidyl carbonate in the presence of N,N-diisopropylethyl-
amine, followed by reaction with the boc-deprotected CAS–CPT
to obtain the NP–CAS–CPT (Fig. 3a).
The release kinetics of the CPT drug from the photo-trig-
gerable, drug-conjugated NPs was then examined upon photo-
irradiation. The NP–CAS–CPT was dispersed in PBS and
exposed to 365 nm light (2.5 mW cmꢁ2). Prior to each
measurement, the nanoparticles were centrifuged and the
supernatant was collected to measure the emission spectra of
the free CPT (lex ¼ 368 nm). Aer photo-irradiation, a steady
increase in the uorescence intensity at 426 nm (lem of CPT)
was observed (Fig. 3b). The drug release was adjusted by altering
the exposure time to UV light between 0 to 20 min. Measure-
ments acquired at 24 h demonstrate an exposure time-depen-
dent release of CPT from the NP–CAS–CPT construct, wherein
10 min of photo-irradiation was observed to be sufficient to
obtain complete drug release (Fig. 3c). The drug loading from
these measurements was determined to be ꢂ2.13 mmol CPT per
gram of NP. The NP–CAS–CPT was further observed to be stable
in the absence of UV exposure, with a negligible change in the
spectra even aer 21 days of incubation in PBS at 37ꢀ (Fig. 3d).
Photo-irradiation aer 21 days of incubation resulted in the
nanoparticle.
The photo-triggerable CAS was synthesized step-by-step
starting with 4-hydroxymandelic acid (1) as shown in Fig. 2.
Briey, 4-hydroxymandelic acid (1) was coupled with monop-
rotected boc-dimethylethylenediamine in the presence of EDC
and HOBt to yield compound (2). Selective alkylation of the
phenolic hydroxyl group with the photo-trigger group (4,5-
dimethoxy-2-nitrobenzyl bromide) resulted in the formation of
an ether linkage (3). The acylation of the benzylic hydroxyl
group was carried out with 4-nitrophenyl chloroformate in the
presence of 4-dimethylaminopyridine and triethylamine at 0 ꢀC
to obtain the p-nitrophenyl ester (4). Finally, compound (4) was
reacted with dimethylethylenediamine-conjugated CPT drug (5)
using triethylamine to yield the CAS–CPT compound (6).
Detailed synthetic procedures and characterization are
provided in the ESI Section 1.†
Prior to attaching the CAS–CPT to the nanoparticle surface,
we determined whether the CPT drug could be released upon
photo-irradiation. As per the CAS–CPT design, exposure to UV
light (365 nm) would trigger the cleavage of the photo-trigger
group and generate an unstable phenol, which would then
undergo a spontaneous rearrangement to release carbon
dioxide via decarboxylation, a dimethyl urea via cyclization and
the active CPT drug (see ESI Section 2† for detailed mechanistic
scheme). The reaction was carefully monitored before and aer
photo-irradiation using mass spectroscopy, which conrmed
the efficient release of CPT from the CAS molecule (see ESI
Section 2† for full characterization). The HPLC assay further
Fig. 2 Synthesis of the chemical adaptor system (CAS) bound with camptothecin (CPT). The core of the CAS (4-hydroxymandelic acid) was
covalently attached with the photo-trigger group (4,5-dimethoxy-2-nitrobenzyl) and the anticancer drug (CPT).
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Fig. 3 The CAS–CPT conjugated nanoparticle constructs show excellent stability and photo-triggered release. (a) Scheme for conjugation of
CAS–CPT on silica NPs. (b) Release of CPT monitored over time after 10 min of UV light exposure (365 nm, 2.5 mW cmꢁ2) using fluorescence
spectroscopy. (c) Release of CPT measured after varying times of UV light exposure (0–20 min). Measurements were acquired 24 h after UV
exposure, using absorption spectroscopy (top) and fluorescence spectroscopy (bottom, lex ¼ 368 nm). (d) Absorbance spectra measured over 21
days in the absence of UV exposure (black & red), followed by 10 min UV irradiation after 21 days (blue).
same level of CPT release observed earlier, indicating that the chemistry reaction. In particular, Click-based hydrogel
CAS–CPT complex was still intact and fully functional (Fig. 3d). networks have been found to produce controlled architecture
This data suggests that the NP–CAS construct can serve as a and have improved mechanical properties due to the controlled
reliable platform to facilitate the photo-triggered release of nature of the Click coupling reaction between orthogonal
small molecule drugs.
functional groups.23,24
We exploited these features to form our hydrogel-based
reservoir by the copper(I)-catalyzed cycloaddition of azide-
terminated and alkyne-terminated PEG monomers in the
presence of our NP–CAS–CPT constructs (Fig. 4a). We mixed one
equivalent of four-arm PEG azide (10 kDa) with two equivalents
Nanoparticle encapsulation within the hydrogel matrix
Aer demonstrating the controlled photo-induced release from
the NP–CAS construct, we aimed to create a reservoir-based
drug delivery system by encapsulating the drug-conjugated
nanoparticles within a polymeric hydrogel matrix. Polymeric
hydrogels have emerged as a popular class of materials for
biological applications due to a number of attractive features,
including the close resemblance to native human tissue, high
water content, ease in modication of its physical and chemical
properties, and the facile transport of nutrients and biomole-
cules.21 While hydrogels composed of both natural and
synthetic polymers have been established, poly(ethylene glycol)
(PEG)-based hydrogels have been commonly used since they are
biocompatible, exhibit negligible immunogenicity, prevent
non-specic adhesion and are FDA-approved for various clinical
applications.22 We generated covalently cross-linked PEG
hydrogels using the regioselective and highly efficient Click
ꢀ
of PEG dialkyne (4 kDa) under aqueous conditions at 37 C in
the presence of sodium ascorbate (1 eq.) and copper sulfate (0.4
eq.). The NP–CAS–CPT constructs were embedded within the
hydrogel by simply mixing the nanoparticles from an aqueous
stock solution into the prepolymer solution prior to adding the
copper catalyst. Under these conditions, the hybrid scaffolds
were seen to form within 30 min. The hydrogel–NP scaffolds
were washed with aqueous ethylenediamine tetraacetic acid
(EDTA, 0.1 M) to remove traces of the copper, and then incu-
bated in PBS for at least one day to allow for equilibration.
The incorporation of the NPs within the hydrogel matrix was
rst visualized by using pyrene dye-conjugated NPs, which show
strong blue uorescence under UV irradiation. In addition to
the blue intensity observed throughout the hydrogel,
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Fig. 4 Hydrogel–NP hybrid scaffolds enable the controlled and on-demand release of drugs. (a) Scheme for encapsulation of drug-conjugated
NPs within PEG-based hydrogels formed using copper-catalyzed Click chemistry. (b) Fluorescence and SEM images of NPs distributed
throughout the hydrogel matrix. (c) The cumulative release of CPT from hydrogel–NP hybrid scaffolds (n ¼ 3) after varying times of UV light
exposure (365 nm, 2.5 mW cmꢁ2), measured over 24 h after photo-irradiation. (d) Cumulative CPT release from the hydrogel–NP hybrid scaffolds
(n ¼ 3) with cycles of OFF (48 h) and ON (30 seconds UV light).
uorescence microscopy provides further evidence for the increase in the uorescence intensity (lem ¼ 426 nm) was
uniform distribution of the NPs in the hydrogel matrix (Fig. 4b). observed over time aer photo-irradiation, reaching a satura-
Scanning electron microscopy (SEM) was then used to examine tion point within 24 h (Fig. S4†). By varying the time of photo-
the ne microscopic structure of the hydrogel and the distri- irradiation, the release kinetics of CPT from the hybrid scaffolds
bution of the embedded NP–CAS–CPT constructs. SEM micro- was modulated to achieve varying degrees of maximum drug
graphs indicate that an isotropic gel has formed which has the release (Fig. 4c). For instance, we attained 25%, 60% and 100%
classic, uniform morphology reported for such hydrogels, along cumulative CPT release using 1, 5 and 10 min UV exposure,
with an even distribution of the NPs throughout the hydrogel respectively (Fig. 4c). At the same time, negligible CPT release
matrix (Fig. 4b). Additionally, the hydrogels do not show was observed in the absence of UV exposure. In addition to
signicant changes in the elastic properties upon adding achieving a predened percentage of total release, the amount
increasing amounts of the NP constructs, suggesting that the of drug released with a given time of UV exposure was further
underlying polymer network formed aer gelation is stable and controlled by adjusting the initial NP–CAS–CPT loading within
remains intact (Fig. S3†). This feature is particularly advanta- the hydrogel matrix (Fig. S5†). This type of controlled and trig-
geous from both a drug delivery and clinical standpoint, since it gered drug release, modulated simply by varying the time of
can allow for the versatile loading of the drug-conjugated NP photo-irradiation and the amount of NP loading within the
constructs without altering the mechanical properties of the hydrogel, can afford innumerable types of drug release proles
hybrid scaffold.
that can be dynamically adjusted over time.
To further demonstrate the robustness of these hydrogel–NP
hybrid scaffolds, CPT release was monitored using an alter-
nating ON/OFF exposure to light. The hybrid scaffolds were rst
incubated for 24 h (OFF), followed by UV light exposure for 30
Controlled drug release from hydrogel–NP hybrid scaffolds
The release kinetics of CPT from the hydrogel–NP hybrid scaf-
folds was next analyzed upon photo-irradiation. A steady
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seconds (ON). The gels were then kept incubated for 48 h (OFF), which are derived from an extremely aggressive form of a
followed by UV light exposure for 30 seconds (ON). This cycle primary brain tumor known as glioblastoma multiforme. The
was repeated four times, over a span of nine days, to evaluate brain cancer cells were rst seeded into cell culture well plates
the repeatability and triggered release capabilities of the (3 ꢃ 104 cells per mL) and allowed to attach for one day.
hydrogel–NP scaffold. The cumulative release during the ON– Hydrogel–NP scaffolds were made, as described above, in the
OFF cycling shows a stepwise drug prole, in which photo- TransWell® inserts, rinsed with EDTA (0.1 M) and incubated in
irradiation (ON) induced a steep increase in CPT release and the PBS for one day to allow for equilibration. The hybrid scaffolds
absence of photo-irradiation (OFF) showed minimal release were then irradiated with 365 nm light (10 min, 2.5 mW cmꢁ2),
(Fig. 4d). This stepwise, photo-triggered release over an followed by deposition of the inserts into the culture dish
extended period of time, along with the negligible release in the containing the cancer cell monolayers (Fig. 5a).
OFF state, highlights the effectiveness of our hydrogel–NP
Compared to the cells in the untreated control condition, the
hybrid scaffold as a promising remotely-triggerable, reservoir- cells in the hydrogel only and non-irradiated hybrid scaffold
based drug delivery system.
conditions exhibit similar cell densities and morphology, indi-
cating negligible toxicity from the hydrogel scaffolds and
negligible non-specic release of the CPT drug from the hybrid
scaffold (Fig. 5c). In contrast, the condition with the photo-
Hydrogel–NP hybrid scaffolds for drug delivery to cancer cells
We further evaluated the efficacy of our system in a cancer cell irradiated hybrid scaffolds shows a signicantly reduced
model. We utilized permeable support cell culture inserts number of viable cells, which suggests the successful triggered
(TransWell®) as depicted in Fig. 5. The hybrid scaffold was release and delivery of the CPT drug (Fig. 5c). Aer four days of
made directly within the TransWell® insert, which contains a incubation, the MTS assay was performed to further quantify
microporous membrane that would allow for the facile trans- the cellular viability. A dose-dependent trend was observed in
port of the released drug into the underlying culture well con- cellular viability for the photo-irradiated conditions, wherein
taining the cellular monolayer (Fig. 5a). While this experimental the cells incubated with hybrid scaffolds containing greater
setup allows for the facile testing of drug release, the insertion amounts of the drug-conjugated nanoparticles showed a greater
of the culture insert containing the hydrogel–NP scaffold into degree of cell death (Fig. 5d). At same time, there was a negli-
the culture wells containing the cancer cells further mimics an gible effect on cellular viability observed for the non-irradiated
implantable reservoir-based drug delivery platform for an in scaffolds, which further affirms the lack of non-specic drug
vitro culture system (Fig. 5b). As a proof-of-concept, we utilized release in the absence of photo-irradiation.
human U87 glioblastoma cells expressing the mutant onco-
genic epidermal growth factor receptor vIII (U87-EGFRvIII),
Fig. 5 Cancer cells exhibit dose-dependent sensitivity to the CPT drug photo-released from hydrogel–NP hybrid scaffolds. (a) Cell culture setup
for assessing CPT drug delivery to U87 glioblastoma brain cancer cells. (b) Images of hydrogel–NP hybrid scaffolds generated in cell culture
inserts. (c) Phase images of brain cancer cells after four days in varying conditions: control (untreated cells), hydrogel without NPs, hydrogel–NP
without UV irradiation and hydrogel–NP with 10 min UV irradiation (365 nm, 2.5 mW cmꢁ2). Scale bars ¼ 50 mm. (d) Cellular viability after four
days in the presence of varying NP-loaded hydrogels (n ¼ 3), with and without UV irradiation (normalized to control untreated cells).
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mg) and N,N-diisopropylethylamine (DIPEA, 25 mL) to activate
the amine group. The reaction was carried out for 4 h. In order
to functionalize maximal free amine groups on the silica NP
surface as possible, the reaction was repeated a total of two
times. Thereaer, the silica NPs were washed with DMF,
resuspended in DMF (0.5 mL) and kept at room temperature.
The CAS–CPT compound was then dissolved in 0.4 mL CH2Cl2
Conclusion
Overall, we have demonstrated a unique hydrogel–nanoparticle
hybrid scaffold which allowed for the remotely-triggered,
controlled and on-demand release of drug molecules. Our
methodology involved incorporating the distinct properties that
are intrinsic to hydrogels (e.g. three-dimensional, biocompat-
ible, in vivo tissue-mimics) and nanoparticles (e.g. large surface
area-to-volume, facile surface functionalization, multifunc-
tional) within a single hybrid platform. Such depot formula-
tions have recently gained attention for modulating drug
release.25,26 However, compared to previous reports, we
employed a robust synthetic strategy to stably link chemother-
apeutic drugs to the nanoparticle surface, while simultaneously
allowing for controlled drug release from the hydrogel upon
photo-irradiation. The combination of these discrete compo-
nents in a single hybrid system provided a number of advan-
tages compared to conventional approaches, including
chemical control over drug loading and release, negligible
release in the absence of photo-irradiation, ease in adjusting
drug release kinetics and high stability for long-term studies.
While we have established our platform using a specic model
drug and cancer cell line, the versatile design of our system
allows for the replacement of any of the comprising elements,
including the small molecule drug (e.g. anticancer drugs,
steroids, anti-inammatories), the stimulus-responsive trigger
(e.g. light, enzyme, pH), the nanoparticle (e.g. silica, magnetic
and gold nanoparticles) and the hydrogel (e.g. PEG, collagen,
gelatin). In this way, our hydrogel–nanoparticle platform is
potentially amendable to any type of biological application that
requires the controlled release of drugs or biomolecules. We
envision that our hybrid biomaterial approach will be essential
for the development of advanced therapies in biomedicine.
ꢀ
and cooled to 0 C. Triuoroacetic acid (TFA, 25 mL) was then
added dropwise and the reaction was continued in the dark at
0
ꢀC for 45 min. The TFA with CH2Cl2 were removed under
reduced pressure and the residue was dissolved in 750 mL DMF.
The above boc-deprotected compound was reacted with amine-
activated silica NPs in the presence of Et3N (0.25 mL). The
reaction was continued in the dark at room temperature over-
night. The NP–CAS–CPT was puried by washing several times
with DMF, followed by MeOH and nally, the product was iso-
lated and dried.
Characterization of CPT release from NP–CAS–CPT
The solid NP–CAS–CPT powder was suspended in phosphate
buffer saline (1ꢃ PBS, pH 7.4). The samples were exposed to UV
light (365 nm, 2.5 mW cmꢁ2) for different times intervals (0–20
ꢀ
min), followed by incubation at 37 C. Prior to each measure-
ment, the samples were centrifuged (6000 rpm) and the CPT
release was monitored from the supernatant solution using
absorbance and uorescence spectroscopy.
Preparation of nanoparticle-encapsulated hydrogels by
copper-catalyzed click chemistry
The PEG monomers were prepared as described in ESI Section
3.† To form the hydrogel, PEG-tetraazide (MW ¼ 10 kD) was
reacted with two equivalents of PEG-dialkyne (MW ¼ 4 kDa) at
37 ꢀC under aqueous conditions in the presence of copper
sulfate and sodium ascorbate as a reducing agent. Based on
previously reported conditions,24 a prepolymer solution con-
taining a [dialkyne] : [tetraazide] : [copper] : [ascorbate] ratio of
2 : 1 : 0.4 : 1 was made. To encapsulate the drug-conjugated
nanoparticles (NP–CAS–CPT) within the hydrogel matrix, the
dried nanoparticle powder was dispersed in DI water to make a
stock solution from which a given volume was transferred
directly into the prepolymer solution. Varying amounts of NP–
CAS–CPT were dispersed within the hydrogel matrix (0–3.75%
w/v). Aer thorough vortexing and mixing, gelation was
Experimental
General synthesis of CAS–CPT & hydrogel PEG monomers
All reactions were carried out under nitrogen or argon atmo-
sphere. All light sensitive reactions were carried out in the dark.
All reagents were purchased from Acros, Sigma-Aldrich, or Alfa
Aesar and used without further purication. Analytical thin
layer chromatography was performed on EM Reagent 0.25 mm
silica gel 60 F254 plates. Visualization was accomplished with
UV light and potassium permanganate stain followed by heat-
ing. Purication of reaction products was carried out by ash
column chromatography using Sorbent Technologies Standard
ꢀ
observed to occur within 30 min at 37 C. The gels were then
washed with Versene (Gibco®; 0.48 mM EDTA) to remove traces
of copper and then washed extensively with 1ꢃ PBS.
˚
Grade silica gel (60 A, 230–400 mesh). The ESI† outlines the
detailed synthesis and characterization of the CAS–CPT
compound (ESI Section 1†), CPT release from CAS–CPT (ESI
Section 2†) and the hydrogel PEG monomers (ESI Section 3†).
Characterization of CPT release from hybrid scaffolds using
uorescence spectroscopy
The hydrogel–nanoparticle hybrid scaffolds were immersed in
1ꢃ PBS (pH 7.4) and exposed to UV light (365 nm, 2.5 mW cmꢁ2
)
Conjugation of CAS–CPT to silica nanoparticles
for different time intervals (1, 5 and 10 min). The hybrid scaf-
To conjugate the CAS–CPT compound to nanoparticles, amine- folds were then incubated at 37 ꢀC, and CPT release was
terminated silica nanoparticles (silica NPs; corpuscular, Inc; monitored using uorescence spectroscopy (lex ¼ 368 nm, lem
Cat. no. 140360-10) were rst suspended in DMF (0.5 mL). The ¼ 426 nm). For control, similar conditions were carried out
silica NPs were reacted with N,N0-disuccinimidyl carbonate (25 without exposure to UV light.
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SEM preparation
Acknowledgements
The hydrogel samples were equilibrated in 1ꢃ PBS at room
temperature and subsequently frozen at ꢁ80 ꢀC for 1 h. The
samples were then freeze-dried overnight, and the fractured
cross-sections were mounted on aluminum studs. Aer sputter-
coating with gold, the Zeiss Sigma eld emission scanning
electron microscope (FE-SEM) was used to image the samples.
K.B.L. acknowledges nancial support from the NIH Director's
New Innovator Award [1DP20D006462-01], National Institute of
Biomedical Imaging and Bioengineering of the NIH
[1R21NS085569-01], N.J. Commission on Spinal Cord grant
[CSCR13ERG005] and the Rutgers IAMDN. S.S. acknowledges
NSF DGE 0801620, Integrative Graduate Education and
Research Traineeship (IGERT) on the Integrated Science and
Engineering of Stem Cells. We acknowledge Prof. David
Mechanical testing using rheology
Rheology experiments were performed to measure the elastic Shreiber and Kathryn Drzewiecki for assistance with the
modulus (G0) and viscous modulus (G00) of the gel samples. Gels rheometer. We acknowledge Dr. Dipti Barman and Dr. Jinping
were rst prepared as described above and equilibrated in 1ꢃ Lai for their valuable input during the synthesis process.
PBS for at least one day. The Malvern Kinexus rheometer with a
parallel-plate geometry was used, with a parallel plate diameter
of 8 mm and gap distance of about 0.45 mm. An amplitude
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J. Mater. Chem. B, 2014, 2, 7685–7693 | 7693