Research Article

www.acsami.org

Double-Walled Microparticles-Embedded Self-Cross-Linked,

Injectable, and Antibacterial Hydrogel for Controlled and Sustained
Release of Chemotherapeutic Agents
Pooya Davoodi,† Wei Cheng Ng,‡ Wei Cheng Yan,† Madapusi P. Srinivasan,†,§ and Chi-Hwa Wang*,†
†
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585
‡
NUS Environmental Research Institute, National University of Singapore, 1 Create Way, Create Tower #15-02, Singapore 138602
§
Civil, Environmental and Chemical Engineering, RMIT University, GPO Box 2476, Melbourne Victoria 3001, Australia
*
S Supporting Information

ABSTRACT: First-line cancer chemotherapy has been

prescribed for patients suffered from cancers for many years.
However, conventional chemotherapy provides a high
parenteral dosage of anticancer drugs over a short period,
which may cause serious toxicities and detrimental side effects
in healthy tissues. This study aims to develop a new drug
delivery system (DDS) composed of double-walled micro-
particles and an injectable hydrogel for localized dual-agent
drug delivery to tumors. The uniform double-walled micro-
particles loaded with cisplatin (Cis-DDP) and paclitaxel
(PTX) were fabricated via coaxial electrohydrodynamic
atomization (CEHDA) technique and subsequently were embedded into injectable alginate-branched polyethylenimine. The
findings show the uniqueness of CEHDA technique for simply swapping the place of drugs to achieve a parallel or a sequential
release profile. This study also presents the simulation of CEHDA technique using computational fluid dynamics (CFD) that will
help in the optimization of CEHDA’s operating conditions prior to large-scale production of microparticles. The new synthetic
hydrogel provides an additional diffusion barrier against Cis-DDP and confines premature release of drugs. In addition, the
hydrogel can provide a versatile tool for retaining particles in the tumor resected cavity during the injection after debulking
surgery and preventing surgical site infection due to its inherent antibacterial properties. Three-dimensional MDA-MB-231
(breast cancer) spheroid studies demonstrate a superior efficacy and a greater reduction in spheroid growth for drugs released
from the proposed composite formulation over a prolonged period, as compared with free drug treatment. Overall, the new
core−shell microparticles embedded into injectable hydrogel can serve as a flexible controlled release platform for modulating the
release profiles of anticancer drugs and subsequently providing a superior anticancer response.
KEYWORDS: injectable hydrogels, core−shell microparticles, antibacterial surfaces, dual-agent drug delivery, 3D tumor spheroids

1. INTRODUCTION Recently, injectable in situ-gelling hydrogels have gained

Hydrogels are an important class of materials with unique
features for biomedical applications exhibiting an exceptional
biocompatibility given their physiochemical properties and high
capacity for water absorption.1−3 In addition, hydrogels can
provide a 3-dimentional microstructure, which closely mimics
natural extracellular matrix of cells for tissue regeneration.4−6
The mechanical and physiochemical characteristics of hydrogels
can be readily modified for clinical practices. For instance, the
permeability of hydrogels can be readily manipulated through
the control of pore size and degradation rate, where an optimal
porous structure benefits the transport of medications and
nutrients inside the gel network.7 Leveraging these fascinating
properties, researchers have developed numerous hydrogel
systems for various clinical applications ranged from drug
delivery and tissue engineering to diagnostics and coating of
medical implants.8
more attention due their superior advantages compared with
the implantable counterparts.9 The macroscopic dimension of
preformed hydrogels is the main obstacle in the use of hydrogel
implants in a noninvasive surgery. However, injectable
hydrogels can facilitate the administration and protection of
bioactive molecules or cells via simple and noninvasive
approaches.10−12 The use of injectable hydrogels not only
enhances patient compliance but also reduces recovery time,
risk of infection,13,14 and treatment costs.15 Although the highly
porous structure and hydrophilic nature of hydrogels benefit
their widespread applications for tissue engineering, premature
release of entrapped molecules (e.g., anticancer drugs) from
hydrogel confines their utilization as drug delivery vectors. Bulk
Received: March 11, 2016
Accepted: August 17, 2016
Published: August 17, 2016

© 2016 American Chemical Society 22785 DOI: 10.1021/acsami.6b03041

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modification of hydrogels (e.g., increasing cross-link density)
can partially diminish this limitation, while it may have adverse
effects on biocompatibility and mechanical properties of the
hydrogel.16−18 On the other hand, a composite hydrogel system
with the immobilized hydrophobic depots inside a hydrophilic
gel matrix may overcome the physiochemical limitations of
hydrogels for the encapsulation and controlled release of small
molecules.19,20
Over the past two decades, polymeric microparticles loaded
with different medications for local drug delivery have
demonstrated versatile features in the treatment of chronic
disease (e.g., cancer).21−24 Microparticles can locally release
their payload at tumor site, which significantly reduces
detrimental systemic side-effects and improves patients’
satisfaction and compliance. However, the use of single agent
therapy often fails to achieve complete cancer remission due to
rapid development of drug resistance in tumor cells. Recently,
combination chemotherapy has opened a new avenue for the
treatment of drug-resistant tumor cells.25,26 The use of multiple
drugs with different mechanisms of action has exhibited less
cancer adaptation process (i.e., development of drug resistance
through abnormal altered drug transport) and remarkable
therapeutic outcome through synergistic effects against cancer
cells. Therefore, in addition to encapsulating chemotherapeutic
agents, microparticles should be engineered for the controlled
release of multiple agents at target tissues with optimal release
rates.27−29
Microparticle-based local drug delivery is a promising
technique to reduce the risk of recurrence for cancer survivors
after a segmental resection. Drug loaded microparticles can
provide a high local concentration gradient of drugs within 2
cm of a resected tumor and target tumor cells remained after a
surgery. However, retaining the particles (in suspension) in the
surgical cavity during the injection and treatment period is still
a major challenge confining the wide use of such a delivery
system.19,20 Therefore, a 3D biodegradable matrix, which can
efficiently entrap and retain microparticles in the cavity is
strongly needed,30 participate in regulating of release profiles,31
and fill the cavity after tumor resection. An in situ-gelling
hydrogel would be the best candidate that can play all the
aforementioned roles successfully. The pre-encapsulation of
therapeutic medications into microparticulate structures and
the subsequent entrapment of the depots inside a hydrogel
system can elevate drug loading and delivery duration in a
controlled manner, while immobilizing delivery vectors at a
target site.
We have reported that double-walled microparticles
consisting of a polymeric core surrounded by a shell layer are
able to concurrently encapsulate hydrophilic and hydrophobic
drugs and release them over prolonged period in a controlled
manner.32,33 In this study, a novel drug delivery system
composed of microparticles and an injectable hydrogel is
introduced to address the shortcomings mentioned above. We
employed coaxial electrohydrodynamic atomization (CEHDA)
for the fabrication of double-walled microparticles loaded with
Cis-DDP and PTX in the core and shell compartments,
respectively. Particles were examined for drug loading,
encapsulation efficiency, and their ability for controlled release
of the therapeutic agents. The interior geometry of the particles
was also examined using laser scanning confocal microscopy.
Next, these particles were entrapped inside an in situ-gelling
carbohydrate-based hydrogel to form a microcomposite
structure. The hydrogel physiochemical properties such as
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degree of swelling and degradation rate, antibacterial effects,
cellular cytotoxicity, and its impact on the release kinetics of
both agents were investigated and compared with the drug
release kinetics achieved by microparticles alone.
2. MATERIALS AND METHODS
2.1. Materials. Poly(lactic-co-glycolic acid) (PLGA 50:50, inherent
viscosity in hexafluoroisopropanol 0.55−0.75 dL g−1) and poly(D,L-
lactide) (PDLLA, inherent viscosity in hexafluoroisopropanol 0.55−
0.75 dL g−1) were obtained from LACTEL Absorbable Polymers
(DURECT Corporation, USA). Sodium alginate (medium viscosity of
3500 cps for a 2% in H2O), branched polyethylenimine (25 kDa),
sodium metaperiodate (NaIO4, ≥99.0%), and picrylsulfonic acid (5%
in H2O) were purchased from Sigma-Aldrich (Singapore). Paclitaxel
was supplied courtesy of Bristol-Meyers Squibb (New Brunswick, NJ)
and cis-diamminedichloroplatinum(II) (Cis-DDP, ≥99.99%) was
obtained from Sigma-Aldrich (Singapore). HPLC-grade dichloro-
methane (DCM) (TEDIA, Fairfield, OH, USA) and phosphate buffer
saline (10 × PBS) were used for the fabrication of microparticles and
in vitro release tests, respectively. Dulbecco’s modified eagle medium
(DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and
trypsin-EDTA (0.25%) were purchased from Life Technology
(Singapore). All other materials and reagents used were of analytical
grade and used without further purification.
2.2. Fabrication of Double-Layered Microparticles. The
fabrication of double-layered microparticles comprising of PDLLA
(core) and PLGA (shell) compartments, and loaded with drugs, were
accomplished using CEHDA technique, as reported earlier.32 Briefly,
PDLLA and PLGA (8 wt.%, in DCM) solutions were prepared
overnight and mixed properly with Cis-DDP drug solution, where Cis-
DDP was dissolved in water or dimethyl sulfoxide (DMSO) or
dimethylformamide (DMF). PTX was directly dissolved in polymer
solution at a predetermined weight ratio. The polymer/drug solutions
were subsequently pumped through a cocentric metal nozzle at
predetermined flow rates for the core (0.5 mL h−1) and shell (2.5 mL
h−1) streams, respectively. The solutions formed a liquid cone with a
thin emerging jet at the tip of the nozzle due to increase of potential
difference between the nozzle and grounded substrate using a high
voltage generator (Glassman High Voltage Inc., NJ, USA). The
polymer jet breakup occurred owing to accumulation of positive
charges at liquid−air interface and formed monodispersed micro-
droplets. Solid polymeric particles were collected on the negatively
charged collectors after quick evaporation of DCM from droplets.
Lastly, microparticles were freeze-dried for 2 days to fully eliminate
residual DCM from samples and stored at −20 °C for characterization
and in vitro experiments.
To have a better understanding of the phenomena that occurred
during particle fabrication, computational fluid dynamic (CFD) study
was performed in the area near the nozzle tip. The equations used in
CFD simulation were introduced in Supporting Information (Table
S1).
2.3. Aldehyde Functionalized Sodium Alginate. The aldehyde
functionalization of sodium alginate was performed as previously
described elsewhere.34 Briefly, sodium alginate (5.0 g) was dispersed in
25 mL of ethanol. Sodium metaperiodate (2.5 g) was dissolved in 25
mL of ultrapure water and was added dropwise to the suspension of
alginate/ethanol. The reaction mixture was then stirred magnetically
for 6, 12, and 18 h at room temperature in dark. The solution was then
transferred into tubular dialysis membrane (MWCO = 1000 Da,
Spectrum Laboratories, Inc.), where it was extensively dialyzed against
deionized-water (5 L). A minimum of five dialysis cycles were
performed prior to lyophilization of the solution.
2.4. Synthesis of in Situ Gelling Hydrogels and the
Microcomposite. To prepare Alg-Ald (3 w/v %) and bPEI (1%,
2.5%, 5%, and 10 w/v %) solutions, the chemicals were separately
dissolved in ultrapure water and the pH of the solutions was adjusted
at ∼7.5 using hydrochloric acid (HCl, 32.5%, VWR International).
Hydrogels were formed by mixing an equal volume (150 μL) of Alg-
Ald and bPEI solutions. The hydrogel was allowed to reach to stable
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form for 30 min at 37 °C. For microparticles loaded hydrogels,
microparticles at the final 25%, 50%, and 75% of dried-gel weight were
suspended in Alg-Ald solution and quickly mixed with bPEI solution
to form hydrogel composites. The hydrogel is formed upon mixing
bPEI (1%) and Alg-Ald (3%) solutions (short gelation time, ∼2−3 s).
However, the gelation time becomes longer as bPEI concentration
increases, where mixing bPEI (10%) and Alg-Ald (3%) solutions does
not produce obvious hydrogel pellet (the viscosity of the mixture
increases, but no obvious hydrogel is formed).
2.5. Physicochemical Characterization. 2.5.1. Morphology,
Particle Size, and Uniformity. The surface morphology and size
distribution of microparticles were examined via Scanning Electron
Microscopy (SEM) (JEOL JSM 5600LV, Tokyo, Japan) with an
accelerating voltage of 10 kV. The microparticles were collected on the
surface of a double-side adhesive carbon tape and subsequently coated
with a thin layer of platinum using a JFC-1300 Platinum Coater
(JEOL, Tokyo, Japan) at 30 mA for 50 s prior to analysis.
The inner morphology of microparticles was observed using
Confocal Laser Scanning Microscopy (CLSM) (Olympus FluoView
FV1000, Olympus America Inc., USA), where coumarin 6 (Sigma-
Aldrich, Singapore), a green fluorescence dye, was mixed with the
polymer solution of the inner nozzle prior to fabrication of
microparticles.
2.5.2. Drug Loading and Encapsulation Efficiency. The drug
loading and encapsulation efficiency were determined by dissolving 15
mg of microparticles in 2 mL DCM and were allowed to stand until
complete disappearance of the microparticles. Then, 5 mL 1 × PBS
was added to the previous solution and the mixture was incubated at
37 °C overnight. The concentrations of Cis-DDP and PTX were
determined using inductively coupled plasma optical emission
spectrometry (ICP-OES) and high-performance liquid chromatog-
raphy (HPLC), respectively. The HPLC apparatus was equipped with
a C18 reverse-phase (RP) column and UV-2487 UV-detector where
acetonitrile: H2PO4 buffer (50:50 v/v) at 1.0 mL min−1 was utilized as
mobile phase.
2.5.3. Determination of the Degree of Cross-Linking. The cross-
linking degree of the hydrogel (Ald-Alg: bPEI of 3:1 w/w %) at
different pH was determined using picrylsulfonic acid (2,4,6-
trinitrobenzenesulfonic acid (TNBSA)) reagent, known as a rapid
and sensitive technique for quantitating unreacted primary amines.
After the formation of hydrogels, the samples were centrifuged and 10
μL of each supernatant was diluted with 90 μL of 0.1 M sodium
bicarbonate (pH ∼8.5) before being transferred (25 μL) into 96-well
plate. Next, 50 μL of freshly prepared TNBSA (0.01 w/v %) reagent
was introduced to the diluted samples and incubated at 37 °C for 2 h,
required for the completion of the reaction. The reaction was
terminated after adding 25 μL of 10% sodium dodecyl sulfate (SDS)
and 12.5 μL of 1 N HCl to each sample. The absorbance of the
mixtures was measured at a wavelength of 335 nm using Infinite 200
PRO microplate reader (TeCAN, Switzerland). The amount of
unreacted bPEI was calculated after comparing the results with a
standard curve of serially diluted pure bPEI solutions.
2.5.4. Fourier Transform Infrared Spectroscopy (FTIR) and Solid-
State 13C NMR. Fourier transform infrared spectrum of Alg-Alds and
final hydrogel products were recorded using Bio-Rad FTS-3500ARX at
transmittance mode in the range of 400 cm−1 to 4000 cm−1. Potassium
bromide (KBr) pellets were used for the preparation of samples. A
bare KBr pellet was considered as background, where its
corresponding spectrum was subtracted from that of samples. All
samples were purged by nitrogen atmosphere for 15 min before
recording their absorbance. The chemical structure of the hydrogel
and the formation of Schiff’s base chemical bonds were determined
using solid-state 13C-nuclear magnetic resonance (13C NMR)
spectroscopy (BRUKER 400 MHz, equipped with a 4 mm MAS
BB-BB probe) at room temperature (296.1 K). The frequency and the
number of scans were set at 100 MHz and 16380, respectively.
2.5.5. Gel Permeation Chromatography (GPC). Relative molecular
weight and weight distribution of the native and oxidized alginates
were measured by gel permeation chromatography system equipped
with a PL aquagel−OH Mixed 300 column (300 × 7.5 mm, 8u) and
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Waters 2414 Reflective Index detector at constant temperature (35
°C). Water was used as mobile phase at a flow rate of 1.0 mL min−1.
Sample injection was set at 20 μL. A set of five polyethylene glycol
(PEG) solution with standard molecular weights was used for
molecular weight calculation.
2.5.6. Elemental Analysis. The percentage estimation of three key
elements (i.e., carbon (C), hydrogen (H), and nitrogen (N)) in the
hydrogel samples were measured using Elementar vario MICRO cube
analyzer. All samples were freeze-dried for 48 h prior to analysis.
2.5.7. Thermogravimetric Analysis (TGA). TGA was performed to
investigate the thermal profile of intermediates and final hydrogel
products, where bare sodium alginate was used as control sample.
Samples of 5−15 mg were prepared and freeze-dried overnight and
subsequently loaded in thermogravimetric analyzer (DTG-60 AH,
Simultaneous DTA-TG apparatus, Shimadzu) furnace equipped with
TA-60WS thermal analyzer device. The samples were heated up to 600
°C under dry nitrogen gas at a flow rate of 10 mL min−1 and the
measurements were conducted at the scanning speed of 10 °C min−1.
The weight loss pattern of each sample was recorded simultaneously
during the analysis.
2.5.8. Differential Scanning Calorimetry. The thermal properties
of intermediates Alg-Ald and final hydrogel products and the physical
state of the drug inside microparticles were determined using
METTLER TOLEDO 822e (Greifensee, Switzerland) equipped with
a gas controller (TSO800GC1) and controlled by STARe Default DB
V9.10-STARe software. Approximately 5−10 mg of the microparticles
were sealed in covered aluminum pans. A standard empty pan was
used as a reference for all analyses. Samples were tested over the
temperature range from −10 to 110 °C at a heating rate of 10 °C
min−1. The equipment was purged with nitrogen gas at a rate of 50
mL·min−1 and a liquid nitrogen stream was employed as coolant.
Blank microparticles were used as control in all thermal analysis tests.
2.5.9. Determination of Swelling Ratio. The samples were
prepared as described above and were freeze-dried overnight. A
known weight of dry gels was soaked in ultrapure water at room
temperature. At predetermined intervals, the excessive surface water
was removed using filter paper. After weighting the samples, the
swelling ratio was calculated using the equation
SR(%) = (Wi , st /Wi , s0) × 100 (1)
where Wi,st, and Wi,s0 were the weight of sample i at time t and the dry
sample, respectively.
2.5.10. Bulk Degradation of Hydrogels. The hydrogels were
freshly prepared and placed in Eppendorf test-tubes filled with 1.5 mL
1 × PBS solution. The samples were incubated at 37 °C and 240 rpm
and the degradation rates were determined at predetermined time
intervals. The samples were lyophilized at indicated time-points and
accurately weighted to calculate the weight loss for each sample.
2.5.11. Rheological Measurement. The rheological properties of
bPEI (1%)/Alg-Ald (3%) hydrogel was investigated using AR-G2
Stress Controlled Rheometer (TA Instruments, U.S.A.). Stress sweep
and frequency sweep measurements were carried out at 37 °C using a
parallel plate configuration (diameter, 20 mm) with a gap of 500 μm.
The storage (elastic) modulus (G′) and the loss of modulus (G″) of
the hydrogel were measured as functions of oscillatory pressure (0.5−
1000 Pa, 1.0 Hz) and angular frequency (∼0.6−600 rad s−1, 0.1 kPa).
All samples were tested ∼1 h after preparation.
2.6. Bacteria Culture. The antibacterial property of the synthetic
hydrogels was examined against Escherichia coli (E. coli, Gram
negative) and Bacillus pumilus (B. pumilus, Gram positive) bacteria.
Prior to the experiments, the bacteria were cultivated in a streaked
agar-plate to form distinct colonies overnight. A single colony was
harvested and subsequently inoculated in autoclaved tryptic soy broth
(TSB). The bacteria were allowed to grow overnight at 37 °C, shaking
at 120 rpm to ensure that they grew at the exponential growth-phase.
The bacteria concentration was monitored photometrically 600 nm
using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific
Inc., U.S.A). The initial optical density (O.D.) of the bacteria solution
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Figure 1. SEM micrographs of electrosprayed microparticles depicting the surface morphology and the size distribution of core-sell microparticles:
core flow rate = 0.5 mL h−1and shell flow rate = (a, a′, a″) 1.0 mL h−1, (b, b′, b″) 1.5 mL h−1, (c, c′, c″) 2.0 mL h−1, (d, d′, d″) 2.5 mL h−1.
was adjusted to ∼0.1, corresponded to concentrations ∼108 CFU
ml−1.
2.6.1. Concentration-Dependent Antibacterial Activity. Hydrogels
used for the antibacterial experiments were freshly prepared by mixing
an equal volume of bPEI solutions (100 μL) at different concentration
with 100 μL of Alg-Ald (3 wt %, MMw, 6 h oxidation) solution. The
hydrogels were then transferred to a 96-well plate and incubated at 37
°C for 1 h before washing gently with sterilized 1 × PBS. Next, the
stock bacterial samples were further diluted to 104 CFU ml−1 in 1 ×
PBS, followed by introducing 100 μL to the surface of the gels.
Bacteria samples without hydrogels were employed as control. The
samples were then incubated at 37 °C with constant shaking at 120
rpm for 18 h, after which 10 μL of the supernatant was serially diluted.
The number of colony-forming units (CFUs) (on agar-plate) was
determined and compared with control after incubation at 37 °C for
12 h.
To investigate the effect of bacteria concentration on the
antibacterial behavior of the hydrogel, sample 1 (S1) was incubated
with a 90 μL of bacteria solution prepared after 1:10 serial dilutions of
a concentrated stock of bacteria (108 CFU ml−1) with TSB. The O.D.
and the number of colony-forming units (CFUs) (on agar-plate) were
determined and compared with control as previously explained.
2.6.2. Time-Dependent Antibacterial Activity. The killing kinetic
of the hydrogel was determined via monitoring the concentration of
bacteria solutions at indicated intervals. Briefly, the freshly prepared
hydrogels were incubated with 100 μL of bacteria solution (104 CFU
mL−1) at 37 °C, shaking at 120 rpm. At the specific time-points, 10 μL
of the supernatant was removed, serially diluted in 1 × PBS, and
placed on an agar plate surface at 37 °C. The number CFUs was
counted after 12 h of incubation.
2.6.3. Assessment of Bacteria-Hydrogel Contact on Antibacterial
Behavior. Fresh hydrogels (400 μL of bPEI 1.0 wt. % + 400 μL of Alg-
Ald 3 wt. %) were washed with 1 × PBS once, and transferred into
transwell cell culture (Corning Transwell) inserted in a 24-well plate.
A volume of 400 μL of 104 CFU ml−1 bacteria (E. coli) solution in TSB
was loaded to each well and an additional 100 μL of bacteria-free TSB
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was introduced on top of the gels in the transwell inserts. A transwell
insert and bacteria solution without hydrogel were employed as
negative controls. All samples were incubated at 37 °C with
continuous shaking at 120 rpm for 18 h. The number of colony-
forming units (CFUs) was determined and compared with control
after 12 h incubation at 37 °C.
2.6.4. Fluorescent Imaging of Bacteria-Hydrogel Contact on
Agar Gel. Luria broth (LB) agar gels were prepared in six-well plates
according to the manufacturer’s instructions. A circular piece of the
agar gel was removed by 1 mL blue pipet tip to make a cylindrical
cavity inside the gel. A freshly prepared hydrogel was transferred into
the cavity and incubated at 37 °C for 30 min, after which the surface of
sample and agar gel were washed with 1 × PBS twice. A volume of
diluted bacteria solution (400 μL) in 1 × PBS (E. coli, 104 CFU ml−1)
was added to each well and rocked to uniformly cover the surfaces. An
agar gel with cavity and without hydrogel was used as control. The
plate was incubated at 37 °C for 18 h, followed by staining bacteria
with green fluorescent SYTO 9 dye (Life Technologies, Singapore).
After 15 min, the plate was imaged using Confocal Laser Scanning
Microscopy.
2.7. In Vitro Cytotoxicity of Hydrogels. The cytotoxicity of the
hydrogels was evaluated using hepatocellular carcinoma (HepG2) liver
cancer and MDA-MB-231 triple negative breast cancer cell lines, Hela,
MRC5, NIH3T3 fibroblast, and smooth muscle cell. The cells were
harvested from 25 cm2 T-flask culture dish and seeded at a density of
∼4 × 105 cell per well onto a 24-well plate and allowed to attach
overnight. The cells were incubated with freshly prepared hydrogels in
400 μL of DMEM supplemented with 10% FBS and 1% antibiotics at
37 °C and 5% CO2. After 24 h, the cells were rinsed with 1 × PBS
twice and resuspended in 400 μL fresh growth medium supplemented
with 80 μL of CellTiter 96 AQueous One Solution Cell Proliferation
Assay (Promega Corporation, U.S.A.). After 4 h, the absorbance of the
solution was measured at 492 nm using TeCAN microplate reader.
Cells without hydrogels were used as control.
To examine the long-term cytotoxic effect of hydrogels, cells
(MDA-MB-231) were incubated with hydrogels (bPEI(1%)/Alg-Ald
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Figure 2. Confocal micrographs illustrating coumarin 6 (green) loaded core−shell microparticles. The radially averaged fluorescence intensity
profiles depicting the spatial location of the core compartment inside microparticles (scanning intervals = 32).
(3%)) for 1, 2, and 3 days. Then, cells were washed with 1× PBS and
stained with fluorescence-based LIVE/DEAD Cell Viability Assays
according to manufacturer’s instructions. The samples were imaged
using confocal laser scanning microscopy.
2.8. Three-Dimensional Spheroids for Cell Viability Assay.
The MDA-MB-231 tumor spheroids were prepared according to
method reported by Friedrich et al.35 For homospheroid formation,
cell suspension was diluted to a density of 5000 cells mL−1 in high-
glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS). Two hundred microliters of
diluted cells was added to wells of 96-well plate precoated with 50 μL
agarose gel (1 wt. %). The spheroids formed after 5 days and
subsequently were incubated with freshly prepared microparticles/
hydrogel delivery systems (day 0). The size and morphology of the 3D
tumor spheroids were monitored at predetermined intervals and
compared with control groups.
2.9. Statistical Analysis. All experiments have been conducted in
triplicate or more per condition. The statistical analysis (student t-test)
was performed, where p < 0.05 and p < 0.01 were considered
statistically significant.
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3. RESULTS AND DISCUSSION
3.1. Particle Fabrication and Characterization. Coaxial
electrohydrodynamic atomization (CEHDA) technique was
employed in this study for the fabrication of core−shell
microparticulate structures. As seen in Figure 1, the size of
microparticles increased with the increase of shell flow rate,
while the morphology of the particles slightly changed from
irregular-shaped microparticles to well-formed microspheres.
Our previous studies demonstrated that dichloromethane
(DCM) was a suitable candidate for dissolution of PLGA and
PDLLA and fabrication of microparticles using CEHDA
technique. In this study, a mixture of DCM and dimethyl
sulfoxide (DMSO) (5:1 v/v) was used for the core solution due
to the higher solubility of Cis-DDP in DMSO compared to
aqueous solution. The presence of DMSO in the core solution
led to the formation of microparticles with smoother surfaces.
However, it adversely affected the morphology of the particles,
particularly at a lower shell flow rate (1.0 mL h−1).
DCM has a higher vapor pressure compared with common
organic solvents such as DMSO and dimethylformamide
(DMF). Therefore, microdroplets can quickly form and solidify
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Figure 3. CFD simulation results: (a) Taylor cone-jet and droplet formation processes (core, PLA solution; shell, PLGA solution; surrounding fluid,
air), (b) electric potential distribution, electric field strength distribution, charge density distribution, and (c) velocity vector. Operating conditions:
Voltage (5600 V), shell:core flow rate (2.5:0.5 mL h−1).

Figure 4. Schematic representation of the preparation of injectable hydrogel composed of alginate aldehyde (Alg-Ald) and PEI-25k.

after breaking-up of the polymer jet at the tip of the nozzle and
flying toward the collecting substrate. Although the fast
evaporation of organic solvents is crucial to fabricate micro-
particles with uniform size distribution, it can affect the surface
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morphology of particles produced. Solvent evaporation rate
affects polymer diffusion, which plays a determinant role in
morphology of final products. A greater particle size and higher
surface roughness are the major impacts of using a highly
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volatile solvent. The fast solvent evaporation usually confines
the interaction and rearrangement of polymer chains inside a
droplet flying toward the oppositely charged substrate. In
addition, the fast evaporation of the solvent may cause a highly
porous surface, which can influence the release rate at a later
stage. The presence of DMSO reduces the volatility of DCM
and allows polymer chains to rearrange and form a less porous
surface during the solidification step. However, the micro-
particles may reach the substrate before being fully dried, where
the particle’s shape may further change at this stage.
As seen in Figure 1, the size of the particles increased as the
shell flow rate changed from 1.0 to 2.5 mL h−1. However, the
formation of secondary droplets at high flow rate (i.e., 2.5 mL
h−1) adversely affected the size distribution of the particles. The
size of microparticles is correlated with the flow rates of the
core and shell solutions. Therefore, the flow rates must be
carefully chosen to allow complete solvent evaporation from
droplets before reaching the collector.
The core−shell structure of microparticles was confirmed
using laser scanning confocal images, where the fluorescent dye
(coumarin 6) was mixed with the core polymer solution prior
to fabrication process. From the confocal image, the core
compartment was completely surrounded by the shell matrix,
where the fluorescence intensity profiles showed that the shell
layer was uniformly distributed around the core (Figure 2).
However, due to the diffusion and dissolution of coumarin 6
into the shell solution containing DCM, the estimation of shell
thickness (∼8−10 μm) using the confocal image may not be so
accurate.
3.2. CFD Simulation of CEHDA Process. To observe the
Taylor cone-jet mode and droplet formation, which dominate
the formation of core−shell microparticles, a CFD simulation
was carried out near the tip of nozzle. Figure 3 illustrates the
CFD simulation results involving Taylor cone-jet and droplet
formation processes, electric potential distribution, electric field
strength distributions, charge density, and velocity vector. Two
fluid meniscus were set at the tip outlets of the nozzle as initial
conditions for both streams. As time proceeded, the fluids were
elongated along the axis and formed a cone-jet due to the force
balance among the gravity, surface tension, electric stress, and
viscous stress. Finally, the jet broke into droplets and formed
core−shell droplets with the size of ∼30 μm. The final particle
size (∼13 μm) can be predicted from droplet size by eq S8,
which is correlated well with the experimental result. From the
velocity vector distributions, the motions of fluids inside the
Taylor-cone can be obtained. The liquids moved down along
two vortex edges located near liquid−gas interface. At the liquid
cone apex, the fluids moved into the jet flow, followed by jet
breaking up and droplet formation. The results also showed
that the highest potential appeared at the tip and liquid cone,
while the highest electric field strength was located at the gas−
liquid interface near the cone apex. This phenomenon can be
attributed to the presence of induced charges at the surface of
liquid cone, especially near the cone apex as confirmed by the
results of charge density distribution.
3.3. Alginate-Aldehyde and Hydrogel Formation. The
synthetic procedure of injectable hydrogel was illustrated in
Figure 4. Three kinds of alginate-aldehyde of different
molecular weight were synthesized using sodium metaperiodate
for various duration. Periodate molecules target the α-glycol
groups of polysaccharides and form their dialdehyde derivatives.
As the aqueous solution of alginate forms a highly viscous
solution even at low concentrations, the oxidation of alginate
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was preferably preformed in a heterogeneous medium
composed of ethanol:water (1:1 v/v). As reported by
Balakrishnan and Jayakrishnan,34 the oxidation degree of
alginate is associated with the quantity of periodate in the
reaction medium and the duration of the reaction. It means that
a longer reaction produces more aldehyde groups ready for
reacting with cross-linkers at a later stage. However, an
extensive depolymerization happens during the oxidation
reaction (Table 1), which may adversely affect the formation
Table 1. Gel Permeation Chromatography (GPC) Results of
Sodium Alginate and Alg-Ald
Mw (g mol−1)
item Mn PDI
sodium alginate (MMw) 207 200 223 000 1.08
sodium alginate (HMw) 259 500 270 300 1.04
MMw Alg-Ald −6h 134 000 162 600 1.21
HMw Alg-Ald −12 h 141 100 166 300 1.18
HMw Alg-Ald −18 h 126 100 156 600 1.24
and stability of hydrogels. Depolymerization reaction can be
attributed to the formation of 1-hydroxyethyl radicals produced
as a side product during the desired reaction.36
The hydrogel was formed upon mixing of Alg-Ald solution
with PEI-25k solution in deionized water pH ∼7.5. PEI-25k
chains act as a cross-linker where their ε-amino groups reacted
with aldehyde groups of oxidized alginate via Schiff’s base
formation at physiological temperature. The concentration of
Alg-Ald solution was kept constant at 3 wt. % throughout the
experiments, while the PEI concentration was changed from 1
to 10 wt. %. The time required for hydrogel formation showed
a PEI concentration dependent trend. The time was quite short
when the PEI concentration was 1 wt. %, while no obvious gel
network was formed at PEI concentrations above 5 wt. %, even
after 15 min. This was due to the change of primary amines
concentrations available to react with aldehyde (RCHO)
groups. At low PEI concentrations (>5 wt. %), primary amines
of one PEI molecule react with a number of RCHO groups
from different alginate chains. Therefore, it can make a dense
cross-linked network. However, as PEI concentration increases,
the probability of the reactions between amines of one PEI
molecule and RCHO decreases. It can be attributed to the
physical mixing ability of the high concentration of bPEI with
Alg-Ald solution. These solutions cannot be mixed homoge-
neously, where the access of RCHO groups to primary amines
suddenly reduces. Thus, a weak polymer network with a
nonporous morphology is formed (Figure S1).
3.4. Chemical Characterization of Hydrogel. FTIR
spectra of sodium alginate, oxidized alginate, and composite
hydrogel are shown in Figure 5a. The spectrum of sodium
alginate showed characteristic absorption bands around 945
(C−O stretching), 1029 (C−O−C stretching), 1130 (C−C
stretching), and 1321 cm−1 (C−O stretching). The strong
bands at 1612 and 1413 cm−1 are associated with asymmetric
and symmetric stretching bands of the carboxylate salt groups.
The appearance of aldehyde symmetric band at 1724 cm−1
confirmed the formation of alginate-aldehyde due to reaction
with sodium meta-periodate. However, hemiacetal formation of
free aldehydes, in some cases, may reduce the intensity of this
band. As seen in Figure 5b, the aldehyde symmetric band at
1724 cm−1 disappeared upon the mixing of alginate-aldehyde
and PEI and formation of hydrogel. Therefore, the absence of
the band in hydrogel spectrum clearly indicated the formation
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Figure 5. Chemical characterization of synthetic hydrogels. (a and b) FTIR spectra of the hydrogel formed after the mixing of alginate-aldehyde with
PEI at different concentrations. (c) Solid-state 13C NMR spectrum of hydrogel. (d) Thermogravimetric analysis of bare sodium alginate, oxidized
alginate, and synthetic hydrogel. (e) DSC curves of bare sodium alginate, oxidized alginate, and synthetic hydrogel.
of Schiff’s base (i.e., CN chemical bond) between −CHO
and −NH2 groups. Figure 5c presents the solid-state 13C NMR
spectrum of the hydrogel upon mixing alginate aldehyde and
PEI-25k solutions at room temperature. Compared to bare
sodium alginate spectrum shown in Figure S2, the peaks
appeared at ∼35−60 ppm and 160−163 ppm are assigned to
PEI carbons and Schiff base carbon (−HCNH−),
respectively. The presence of −HCNH− peak in the
hydrogel 13C NMR spectrum is in a good agreement with the
results of FTIR analysis, where the aldehyde peak appeared and
disappeared upon the oxidation of alginate molecules with
NaIO4 and formation of the hydrogel, respectively.
The elemental analysis (EA) results are presented in Table
S2, where the molar ratio of carbon/nitrogen (nC/nN) is
calculated from the weight percentage of both components.
Because of constant carbon:nitrogen ratio in each PEI unit
(2C:1N) and alginate unit (6C:0N), the grafting rate can
subsequently be calculated using eq S9. Using this equation, α
= 0.003 for PEI (1%)/Alg-Ald (3%) is the maximum grafting
ratio.
The data provided by TGA analysis of sodium alginate,
alginate aldehyde, and the hydrogel were presented in Figure
5d and Figure S3. The thermograms showed different thermal
behavior of the samples. Sodium alginate exhibited three
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degradation steps, where the first one occurred ∼100 °C. The
first step of mass loss corresponded to the elimination of free
water and bound water from the sample. The second step
occurred ∼220 °C that can be attributed to the decomposition
of alginate at C−H and C−O−C bonds in the main
polysaccharide chain and vaporization and elimination of
volatile products. The third step was associated with the
formation of Na2CO3 and other carbonized materials. The
thermogram of alginate-aldehyde, however, showed a few
differences in the peak area and position as compared with bare
alginate. The first degradation step of alginate-aldehyde was
faster than that of alginate. The variation on the peak area and
position during the first step of mass loss was corresponded to
water molecule interactions with polymer chains and the lower
capacity of alginate-aldehyde for holding of water. In addition,
the second degradation step of alginate-aldehyde exhibited a
maximum peak at ∼200 °C, which was slightly lower than the
maximum peak (∼220 °C) of alginate thermogram. As the
stability of the samples is directly correlated with their
decomposition behavior, the results demonstrated a significant
decrease in the thermal stability of alginate-aldehyde compared
to pure alginate. In the case of hydrogel, the results
demonstrated a two-step mass loss similar to that of alginate-
aldehyde, followed by a gradual mass loss from ∼220 °C
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Figure 6. Swelling ratios of different hydrogels as a function of time: (a) hydrogels prepared at different Ald-Alg: PEI weight ratios and (b) hydrogel
prepared at Ald-Alg: PEI of 3:1 w/w %, where high molecular weight alginate was oxidized by NaIO4 for 12 and 18 h. Medium molecular weight
alginate was oxidized for 6 h using the same process. (c) The swelling ratios of hydrogels at different pH values. (d) PEI release percentage during 5
days at different pH values (hydrogel: Ald-Alg: bPEI of 3:1 w/w %). All data are expressed as the average ± standard deviation of three independent
experiments. (The deswelling behavior of the hydrogel is reported in Supporting Information).

onward. The new peak appeared at ∼440 °C might be due to
the decomposition of the materials and was in agreement with
results (i.e., a less thermal stability for biopolymeric Schiff based
materials) of previous study.37
The data provided by DSC curves (Figure 5e) were in
agreement with TGA results. The DSC curves showed that
dehydration process of pure alginate occurred at a lower
temperature as compared with other samples. The exothermic
peaks ∼200 and 220 °C can be attributed to decomposition of
the samples, where a broad peak in this area was associated with
a gradual decomposition of hydrogel due to temperature
increase.
3.5. Preparation of Microparticles Embedded Hydro-
gel. Figure S4 shows the morphological images of core−shell
microparticles embedded in Alg-Ald/PEI hydrogel networks.
Microparticles were dispersed in Alg-Ald (3 wt. %) solution and
PEI-25k (1 wt. %) solution was subsequently added to form the
hydrogel matrix. Figure S1a shows the morphology of the
hydrogel prior to incorporation of microparticles. The hydrogel
had a highly porous structure, which can significantly help to
absorb large amount of water and increase its biocompatibility.
The addition of microparticles (25, 50, 75 w/w %) did not
change the morphology of hydrogel and microparticles (Figure
S4b−d). However, because of hydrophobicity of microparticles,
the homogeneity of final product is affected by the increase of
particle weight percentage. This conclusion was further proved
using higher magnification SEM micrographs for the particles
embedded hydrogels at 50 and 75 w/w % (Figure S4e and f).
The dispersion of microparticles (Figure S4g) within the
hydrogel was further examined using fluorescence dye-loaded
microparticles and corresponding fluorescence intensity profiles
(Figure S4h). Although the intensity profiles correlated well
with the concentration of microparticles, a low light intensity
was observed near the gel surface in tube (iv), which means that
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the particles could not be uniformly dispersed at high (∼75 w/
w %) concentration.
3.6. Physical Characterization of Hydrogel. The
swelling ratio (SR) of hydrogels with different compositions
was measured in 1× PBS buffer at different pH values and 37
°C (Figure 6). The hydrogels generally reached to a state of
swelling equilibrium within half an hour, although average
quantities were slightly fluctuating around the equilibrium
ratios. As illustrated in Figure 6a, the SR of the hydrogels
increased significantly with the decrease of PEI concentration
used for hydrogel preparation. However, the SR of hydrogels
cross-linked by PEI at concentrations of 1.0 and 5.0 wt. %
fluctuated around 1800% of their initial dry weights and
remained almost constant for 12 h.
The water absorption in the hydrogels is a function many of
variables such as cross-link density, hydrogel microstructure
(porous or poreless), network parameters, nature of the
materials, etc. For instance, the cross-link density determines
the distance between two cross-link nodes on a single polymer
chain. Therefore, a longer distance results in a lower cross-link
density. In addition, the swelling process can be controlled by
diffusion and relaxation processes. The diffusion process,
identified as the rate-determining step at the beginning of the
swelling process, depends on solvent molecular weight, porosity
of hydrogel, and temperature. The chain relaxation step occurs
slower than diffusion process at low cross-link density.
Therefore, as explained by Omidian and Park,38 the chain
relaxation step is considerably slower than diffusion process at a
high cross-link density, which results in a sharp increase in SR,
followed by an equilibrium step.
The effect of alginate molecular weight on SR was
investigated as shown in Figure 6b. The concentration of PEI
solutions was kept constant at 1 wt. % for all experiments. As
can be seen, the SR quickly reached an equilibrium condition
for hydrogel prepared using medium molecular weight alginate
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(MMw 6), while it was gradually increasing for high molecular
weight alginate oxidized for 12 and 18 h. However, the results
showed that SR could slightly decrease when the oxidation
process was longer (i.e., 18 h). The longer the oxidation time,
the more aldehyde groups for cross-linking process. As a result,
the higher cross-link density confined the polymer relaxation
steps and reduced SR. On the other hand, longer oxidation
period caused polymer decomposition and reduced molecular
weight. The lower the molecular weight, the more the
compacted gel network is and consequently the lower SR.
The pH of the aqueous medium had a marginal effect on SR
and became effect-less after ∼1 h (Figure 6c). pH can partially
increase the protonation of amine groups available in the
solution, which may delay the cross-linking process and provide
a slightly longer relaxation period for polymer chains inside the
gel network. These results were ratified by PEI release test at
different pH within 5 days (Figure 6d), where the amount of
PEI release in the first day was negligible for all pH. However,
the carbonyl-amine bonds showed greater sensitivity to pH less
than 6 in the following days.
The degradation of hydrogel samples was investigated in
vitro and the results were presented in Figure 7. Among
Figure 7. Hydrogel degradation results. All data are expressed as the
average ± standard deviation of three independent experiments.
different hydrogels, bPEI (1%)+Alg-Ald (3%) was the most
stable sample over 60 days, while others lost their weight
considerably faster over the same period. As seen in Figure 7,
the initial concentration of bPEI solution strongly affects the
stability of the hydrogels and degradation rates, where bPEI
(10%) + Alg-Ald (3%) that did not form obvious gel
completely disappeared before day 30.
The dynamic mechanical behavior of bPEI (1%)/Alg-Ald
(3%) hydrogel was investigated by oscillatory rheology at 37
°C. First, the stress sweep at a constant frequency was
conducted to identify the linear viscoelastic region (LVR)
profiles of the hydrogel. The effect of the stress amplitude on
Figure 8. Rheological analysis of bPEI (1%)/Alg-Ald (3%) hydrogel. (a) Stress sweep and (b) frequency sweep.
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the cross-linked hydrogels is shown in Figure 8a. Over the
linear range, the applied stress results in strain. However,
beyond LVR, G′ (elastic modulus) sharply decreases due to the
structure breakdown upon increasing the deformation imposed.
Therefore, the pressure (∼200 Pa) at which the hydrogel
network begins its nonlinear viscoelastic behavior was
determined as critical shear stress. As the linear part happened
over the range of 0.5 to 200 Pa (Figure 8a), the frequency
sweep experiment was subsequently conducted at 10 Pa to
investigate the stability of three-dimensional cross-linked gel
networks over a frequency range from ∼0.6 to 600 rad s−1. As
shown in Figure 8b, there is a large viscoelastic plateau between
∼0.6 and 200 rad s−1, which is an indication of a stable hydrogel
structure. No cross-point between G′ and G″ was also
observed, hence representing the stability of the hydrogel
structure. In addition, the frequency independent behavior of
elastic modulus indicates the solid-like behavior of the hydrogel.
At low frequency, the polymer chains between the cross-link
points can rearrange themselves due to comparable relaxation
time scales, while at higher frequencies, they are impotent to
rearrange themselves within a short period of imposed motion
(chain stiffening) and show solid-like behavior.
3.7. Antibacterial Activity of Hydrogels. To evaluate the
antibacterial property of the hydrogels, the surface of the
hydrogels was challenged with model Gram-negative (E. coli)
and Gram-positive (B. pumilus) bacteria. Figure 9a and 9c
demonstrate the results of antibacterial tests in which the
surface of hydrogels prepared with varying amount of PEI was
incubated to 104 CFU ml−1 of bacteria. The empty tissue
culture wells and the same amount of PEI used for the
preparation of hydrogels were considered as negative and
positive controls, respectively. The antibacterial activity of
formulations as well as controls were evaluated by the colony
formation ability within 18 h postexperiment times. The results
showed that antibacterial activity of hydrogels increases with
the increase of PEI concentration in the formulations. On the
other hand, a bare 3 wt. % alginate solution show no
antibacterial activity against both E. coli and B. pumilus, similar
to empty wells (negative controls). For instance, at 1 wt. % PEI
the hydrogel showed ∼100 times greater antibacterial activity
compared with bare alginate. The inhibitory effect of hydrogels
was significantly higher at PEI concentration ≥2.5 wt. %, where
no colony was formed after 18 h incubation on agar plates.
These results suggest that the protonated amines in PEI
molecules can directly influence its antibacterial behavior.
Figure 9b and 9d presented the colony formation ability of
bacteria as a function of increasing initial CFU for a hydrogel
prepared from 3 wt. % alginate+1 wt. % PEI. The data showed
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Figure 9. Antibacterial activity of hydrogels: Alg-Ald cross-linked with different concentration of PEI-25K were incubated with 104 CFU mL−1 (a) E.
coli or (c) B. pumilus for 18 h. Hydrogel surface (Alg-Ald: PEI 3:1 w/w) was challenged with serially diluted (b) E. coli or (d) B. pumilus in PBS for
18 h. (e) The time course of bacterial (E. coli) killing experiment. (f) Contact-dependent antibacterial effect. The control experiment (purple bars)
was performed by incubating bacteria (104 CFU mL−1) without hydrogels for the corresponding time points.

that the hydrogel is highly active at low bacteria concentrations.
In addition, a significant reduction in bacteria activity (∼1000
times) was observed at extremely high bacteria concentration
(108 CFU ml−1). This high concentration was ∼10 million
times greater than that is expected in a contaminated operating
theater.39,40
Figure 9e shows the time-dependent antibacterial test of the
hydrogel prepared from 1 wt. % PEI. The colony formation
ability of bacteria incubated with the hydrogel at various time-
points was evaluated and the data showed a time-dependent
activity where the number of colonies gradually decreased as
the incubation period extended.
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All data presented above suggests a contact-dependent
mechanism of action for antibacterial activity of the hydrogels.
To assess this hypothesis a set of quantitative and qualitative
experiments was performed in vitro. The hydrogel prepared
was loaded into a transwell tissue culture insert and the inset
was placed in the culture plate containing bacteria in 1 × PBS,
where the porous membrane of the inset separated the
hydrogel and bacteria. After 18 h, the effect of PEI molecules
leached from the hydrogel on the activity of bacteria was
determined. The results of bacteria-hydrogel contact test were
presented in Figure 9f. As can be seen, the number of colonies
formed after the experiment was very close to that of control
groups. The data suggest that most of PEI molecules reacted
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Figure 10. Viability of bacteria (E. coli) on the surface of hydrogel. E. coli viability was imaged using confocal microscopy after 18 h of incubation on
agar gel/hydrogel interface. (a) The bright field, (b) the fluorescence micrograph of bacteria stained with SYTO 9, and (c) the overlay image of
bacteria and gels. The white dash-line indicates the interface of agar gel/hydrogel. Scale bars: 100 μm.

Figure 11. Results of in vitro biocompatibility tests for cells cocultured with hydrogels. (a) Cell viability percentage after 24 h incubation with
hydrogels at various PEI concentrations. Note: Smooth muscle cells were cultivated in smooth muscle cell medium (SMCM) supplemented with 2%
of FBS, 1% of cell growth supplement, and 1% of penicillin-streptomycin antibiotic solution. The viability percentage of (b) HepG2 and (c) MDA-
MB-231 cells after 24 h treatment with hydrogels at various PEI concentrations. (d) Live/dead cell viability staining images of MDA-MB-231 cells
after three consecutive days cocultured with hydrogel (Alg-ald:PEI 3:1 w/w %). The green color demonstrates live cells and the red color
demonstrates dead cells. Dead cells were indicated by red dash-circles. All data represent as mean ± SD of n = 4 independent experiments. Scale
bars: 200 μm.

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with Alg-ald molecules and did not leach from the hydrogel as
previously indicated in Figure 6d (PEI concentration in
supernatant). This mechanism was qualitatively investigated
by bacteria-hydrogel contact experiment on agar plate. A
cylindrical portion of preformed agar gel was removed and the
hole was filled with the hydrogels. Next, the surfaces of
hydrogel and agar gel were exposed to bacteria solution at 104
CFU mL−1 for 18 h. Figure 10 shows both the bright field and
fluorescent image of bacteria stained with SYTO-9 fluorescent
dye. The lack of green fluorescence observed on the area
occupied by the hydrogel indicated that there were no viable
bacteria on the hydrogel surface. However, bacteria could
proliferate on the surrounding agar surface. On the basis of
these results, the hydrogel is a contacted-based bactericidal
surface, which kills adhering bacteria.
3.8. Biocompatibility of Hydrogels. Since biocompati-
bility is a crucial factor for their successful applications of
synthetic biomaterials, the cytocompatibility of the hydrogels
was evaluated by MTS assay using six different cell lines. The
cell lines were selected from different tissues, where the drug
delivery system proposed in this article may be used. Among
four different hydrogels tested for cytocompatibility, the
hydrogel with the lowest PEI concentration (i.e., Alg-ald:PEI
3:1 wt. %) showed minimum toxicity and maximum cell
viability (Figure 11a). However, the number of live cells was
gradually decreased with increasing PEI concentration from 2.5
to 10 wt. %. Notably, cell proliferation assay revealed that the
hydrogel prepared with 1 wt. % of PEI should be more stable
than other hydrogels due to formation of a well-interconnected
network, which prevents the premature release of non-cross-
linked toxic PEI. A similar trend was found for the cell lines
incubated with medium molecular weight and high molecular
weight hydrogel at various PEI concentrations (Figure 11b and
c). Therefore, it can be concluded that molecular weight of the
oxidized alginate has no detrimental effect on cell growth and
proliferation. The extended cell viability test was performed for
3 days. Cells were stained using LIVE/DEAD assay and
observed using laser scanning confocal microscopy (Figure
11d). The results showed a few dead cells (red color), and cells
maintained normal cell morphology for 3 days. These results
demonstrated that the hydrogel (i.e., Alg-ald:PEI 3:1 w/w %) is
an appropriate candidate for long-term drug delivery
applications.
3.9. In Vitro Drug Release: Microparticles and Micro-
particles-Embedded Hydrogels. The molecular weight of
an anticancer agent and its affinity for the carrier’s molecules
are important parameters contributing to the design of a
successful controlled release system. Thus, the ability of
microparticles for the sustained release of verapamil (VRP),
paclitaxel (PTX), and cisplatin (Cis-DDP) as model drugs was
examined in vitro. Table S3 presents the results of drug loading
content (DLC) and encapsulation efficiency (EE) of the
particles. The release profiles of both formulations showed a
small initial burst of ∼11% VRP and ∼12% PTX for
formulation A1, and ∼13% VRP and ∼4% PTX for formulation
B1 (Figure S6). However, after an initial premature release, the
drugs were gradually released in a sustained manner over a
period of 60 days, indicating the progressive degradation of
polymeric microspheres for a sustained and long-term release.
In formulation A1, both drugs exhibited a near zero order
release rate, however, for VRP in formulation B1 (Figure S6b),
it showed a sharp increase in release profile after 30 days.
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In addition, for formulation A1 (Figure S6a), both PTX and
VRP release curves overlapped with each other in the first 10
days, after which VRP was released slightly faster, while the gap
between the two release curves was significantly larger in
formulation B1 (Figure S6b). Moreover, the slope of VRP
curve suddenly increased after 30 days, causing an even greater
deviation from PTX curve. Therefore, VRP was released
significantly faster and in a larger dosage, making it seemed to
be released first while PTX was released second. These results
indicated that highly versatile drug carriers could be designed
by simply swapping the drugs in a one-step CEHDA fabrication
technique to present a parallel or a sequential release of drugs.
This flexibility is an advantage of the CEHDA fabrication
technique compared to other techniques such as the emulsion/
solvent evaporation method.41,42 Emulsion/solvent evaporation
method involves a rapid stirring step that adversely promotes
the disintegration of the drugs and confines loading capacity of
hydrophilic drugs due to prolonged contact with aqueous
environment.
In the case of Cis-DDP and PTX loaded microparticles
(Table S4), the release behavior was considerably different
from what was previously mentioned above. For the case of
Cis-DDP dissolved in water, the release of Cis-DDP from both
formulation B2 and C2 (Figure 12) displayed an initial burst of
approximately 40%, followed by a sustained release throughout
a period of 45 days in a near zero-order rate. This initial burst
release can be attributed to the premature release of drug
molecules from the surface of particles or the diffusion of drugs
placed close to the surface. Furthermore, the hydrophilic nature
of Cis-DDP may contribute to higher premature release, as it is
more readily available for release into surrounding medium. In
contrast, the release of PTX from both formulation A2 and C2
did not show significant initial burst, but just a progressive and
sustained release throughout the study. The steady release of
PTX could be explained by the drug diffusion within the
microspheres, hence giving a predictable trend. As time
proceeded, the continuous penetration of aqueous medium
caused polymer degradation, pore formation, thinning of the
shell layer,43 and consequently faster release of drug into
surrounding medium.
As the limited dissolution of Cis-DDP in water (<2.5 mg
mL−1) considerably reduced drug loading efficiency, we used
dimethyl sulfoxide (DMSO) known as a suitable solvent for
Cis-DDP (>25 mg mL−1). In this case, the drug loading
content was significantly improved (Table S4). However, a
huge initial burst release of Cis-DDP (∼82% for formulation B3
and ∼78% for formulation C3) was observed within the first
day of experiments (Figure 12d). It was due to the miscibility of
DMSO and DCM that led to the undesirable diffusion of core
solution into shell counterpart during particle fabrication, where
the effective thickness of the shell compartment markedly
decreased. Therefore, Cis-DDP could readily pass the shell
barrier and dissolve into the surrounding medium.
To control the initial burst of Cis-DDP, the previously
introduced hydrogel was employed to entrap the drug-loaded
microspheres. Thus, the initial burst was significantly reduced
to ∼40% (Figure 12d) becaue of a more complex diffusion
pathway to be passed. The hydrogel matrix functioned as an
additional barrier to restrict direct exposure of microparticles to
PBS medium, thereby, reducing its high initial burst as well as
making its release more sustainable over a longer period.
As shown in Figure 12e, after preparation of composite
formulation, 75 w/w % microspheres-loaded hydrogel exhibited
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Figure 12. Release profiles of Cis-DDP and PTX from core−shell
microparticles: (a) the scheme of microparticles loaded with Cis-DDP
and PTX (formulation A2, PTX (shell), formulation B2, Cis-DDP
(core), formulation C2, PTX (shell) and Cis-DDP (core)); (b) the
release profiles of formulation A2 and B2 (Cis-DDP was dissolved in
water); (c) the release profile of formulation C2 (Cis-DDP was
dissolved in water); (d) the initial step of Cis-DDP release profile from
formulation B3 and C3 (Cis-DDP was dissolved in DMSO) with and
without hydrogel; (e) the release profile of formulation B3 (Cis-DDP
was dissolved in DMSO) and hydrogel at 25, 50, and 75 w/w %
loading ratios; (f) the release profile of formulation C3 (Cis-DDP was
dissolved in DMSO) and hydrogel at 50 w/w % loading ratio. pH of
PEI-25k and Alg-Ald solutions were adjusted at physiological pH (pH
∼7.0) using hydrochloric acid (32.5%) prior to hydrogel formation.
Data represents average ± standard deviation of three independent
measurements, n = 3.
Figure 13. (a) Bright-field images of tumor spheroids treated with combination of Cis-DDP and PTX: free drug (second column) and composite
formulation (third column) (scale bar = 200 μm). (b) Growth of tumor cell spheroids upon exposure to microparticle formulations over 10 days. (c)
Cell viability of tumor spheroids upon exposure to microparticle formulations at day 10. Data represents average ± standard deviation of three (n =
3) independent measurements.
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a slightly lower initial burst and cumulative release as compared
to 25 w/w % and 50 w/w % composite formulations, though all
three release curves had very similar shape and gradient. The
composite formulation C3 showed similar trend for Cis-DDP,
while release rate of PTX was undoubtedly reduced (Figure
12f). Regardless of the initial burst release of Cis-DDP, the new
system showed a simultaneous release of both agents in a
controlled and sustained manner, here is highly promising for
the synergistic treatment of tumors such as triple-negative
breast cancer (TNBC).
3.10. Drug Release Effect on 3D MDA-MB-231
Spheroids. Although the traditional 2D cell-culture is suitable
for mechanism study and rigorously control the experimental
and treatment conditions, it usually provides overestimated or
underestimated results that may not be reproduced in vivo.
Therefore, a 3D tumor spheroid model is employed in the
current study as it demonstrates dose−response relationships
that may better predict tumor responses in vivo in future
studies. MDA-MB-231 spheroids in a prolonged study of 10
days were used to evaluate the efficacy of combined
formulation released from microparticles/hydrogel composite.
As shown in Figure 13, the composite formulation exerted a
greater inhibitory effect on the growth of tumor spheroids, as
compared with control and free drug groups. The free drug
treatment temporarily inhibited the growth of spheroids and
the size of the spheroids remained constant or increased within
10 days. In contrast, the spheroids slowly responded to the
treatment of composite formulation at the early stage, while the
responses were pronounced over 10 days and significantly
inhibited the growth of spheroids (Figure 13b and 13c). The
fast but short period response of spheroids to free drug
treatment was due to exposure with high concentration of
drugs. However, to mimic the in vivo conditions, concentration
of drugs was gradually reduced over 48 h. Therefore, the
spheroid could restore their proliferation conditions after ∼8
days. Despite the free drug results, higher drug efficacy
observed for spheroids incubated with the composite
formulation could be attributed to the controlled and sustained
release of Cis-DDP and PTX from the new delivery systems.
Although the initial drug concentrations were not sufficient to
cause significant cell death, the gradual accumulation of drug
DOI: 10.1021/acsami.6b03041
ACS Appl. Mater. Interfaces 2016, 8, 22785−22800
ACS Applied Materials & Interfaces
within the spheroid killed cells in the outer rim of the spheroid,
where it promoted penetration of drugs into the primed tumor
and caused a substantial cell death.
4. CONCLUSIONS
Cis-DDP and PTX loaded core−shell microparticles were
fabricated using the CEHDA techniques. A CFD simulation
model was developed and utilized for the optimization of
CEHDA operating parameters prior to fabrication of micro-
particles. The Alg-ald:PEI hydrogel was successfully synthesized
and evaluated for entrapment of microparticles inside a porous
matrix, modulating release profiles, and antibacterial activity
against Gram-positive and Gram-negative bacterial via a
contact-dependent mechanism. Moreover, three-dimensional
MDA-MB-231 spheroid studies demonstrated that the
combination of Cis-DDP and PTX released from the proposed
microparticles/hydrogel formulations could provide greater
efficacy and gradually reduce the size of spheroids. In contrast,
the free drug treatment was impotent to effectively reduce cell
viability in tumor spheroid in vitro. The new composite
formulation may hold great promise for practical applications in
cancer chemotherapy through localized delivery of multiple
agents in a controlled and sustained manner.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.6b03041.
Tables representing the CFD model description and
governing equations, drug loading content and encapsu-
lation efficiency of VRP and PTX loaded microparticles,
and drug loading content and encapsulation efficiency of
Cis-DDP and PTX loaded microparticles and figures
showign the morphology of injectable hydrogel com-
posed of Alg-Ald and PEI-25k at various concentrations,
solid-state 13C NMR spectrum of bare sodium alginate,
TGA and DTG curves of the hydrogel, the deswelling
kinetics of the hydrogel; SEM micrographs of hydrogel
morphologies before and after loading with core−shell
microparticles, TGA and DTG curves of the hydrogel at
different heating rates, weight changes of the hydrogel
over time, and in vitro release profiles of PTX and VRP
from double-walled microparticles (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel.: +65-65165079. Fax: +65-67791936. E-mail: chewch@
nus.edu.sg.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work is financially supported by A*STAR and National
University of Singapore under the project/grant numbers
APG2013/40A (A*STAR BMRC Strategic Positioning Fund,
A*STAR-P&G Collaboration, R279-000-487-305) and R261-
509-001-646 (3D Printing Initiatives), respectively. Pooya
Davoodi greatly appreciates the National University of
Singapore for PhD graduate research scholarship. We thank
Dr. Marc V Garland and Mrs. Li Li Ong for their help with
solid-state 13C NMR experiments.
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Research Article
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