Antifouling and biodegradable poly(N-hydroxyethyl acrylamide) (polyHEAA)-based nanogels

Article abstract of DOI:10.1039/c3ra42323a

We synthesize and characterize two types of poly(N-hydroxyethyl acrylamide) (polyHEAA)-based nanogels: antifouling poly(2-(methacryloyloxy) ethyl trimethyl ammonium-g-N-hydroxyethyl acrylamide) (polyTM-g-HEAA) by a new two-step polymerization method of inverse microemulsion ATRP and surface-initiated atom transfer radical polymerization (SI-ATRP), and pH-responsive biodegradable polyHEAA nanogels by the inverse microemulsion free radical polymerization method. PolyTM-g-HEAA nanogels with a core-shell structure by grafting antifouling polyHEAA onto the cationic polyTM core are tested for their antifouling property and stability in fetal bovine serum (FBS) and nanogels-induced cell toxicity. Results show that with the antifouling protection of polyHEAA, polyTM-g-HEAA nanogels significantly improve their long-term stability in FBS up to 7 days by preventing nonspecific protein adsorption, and they also improve cell viability to ～94% and exhibit almost neglectable cell toxicity. Further, polyHEAA nanogels cross-linked with acid-liable ethylidenebis(oxy-2,1-ethanediyl) ester (EOE) are synthesized, which exhibit both biodegradation and control-release of encapsulated rhodamine 6G (R6G) at acid conditions. Conjugation of transferrin ligands onto R6G-loaded polyHEAA nanogels further enhances cellular uptake efficiency and intracellular drug release for targeting drug delivery. This work demonstrates that polyHEAA-based nanogels with easy synthesis, excellent antifouling property and stability, biodegradability, low toxicity, and pH-responsive intracellular drug release are highly promising for targeted drug delivery systems for biomedical applications. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra42323a

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PAPER
Antifouling and biodegradable poly(N-hydroxyethyl
acrylamide) (polyHEAA)-based nanogels
Cite this: RSC Adv., 2013, 3, 19991
Chao Zhao,a Kunal Patel,b Lindsay Marie Aichinger,a Zhaoqian Liu,a Rundong Hu,a
a
a
a
b
c
a
*
Hong Chen, Xiaosi Li, Lingyan Li, Ge Zhang, Yung Chang and Jie Zheng
We synthesize and characterize two types of poly(N-hydroxyethyl acrylamide) (polyHEAA)-based nanogels:
antifouling poly(2-(methacryloyloxy) ethyl trimethyl ammonium-g-N-hydroxyethyl acrylamide) (polyTM-g-
HEAA) by a new two-step polymerization method of inverse microemulsion ATRP and surface-initiated
atom transfer radical polymerization (SI-ATRP), and pH-responsive biodegradable polyHEAA nanogels by
the inverse microemulsion free radical polymerization method. PolyTM-g-HEAA nanogels with a core–
shell structure by grafting antifouling polyHEAA onto the cationic polyTM core are tested for their
antifouling property and stability in fetal bovine serum (FBS) and nanogels-induced cell toxicity. Results
show that with the antifouling protection of polyHEAA, polyTM-g-HEAA nanogels significantly improve
their long-term stability in FBS up to 7 days by preventing nonspecific protein adsorption, and they also
improve cell viability to ꢀ94% and exhibit almost neglectable cell toxicity. Further, polyHEAA nanogels
cross-linked with acid-liable ethylidenebis(oxy-2,1-ethanediyl) ester (EOE) are synthesized, which exhibit
both biodegradation and control-release of encapsulated rhodamine 6G (R6G) at acid conditions.
Conjugation of transferrin ligands onto R6G-loaded polyHEAA nanogels further enhances cellular
uptake efficiency and intracellular drug release for targeting drug delivery. This work demonstrates that
polyHEAA-based nanogels with easy synthesis, excellent antifouling property and stability,
biodegradability, low toxicity, and pH-responsive intracellular drug release are highly promising for
targeted drug delivery systems for biomedical applications.
Received 10th May 2013
Accepted 12th August 2013
DOI: 10.1039/c3ra42323a
www.rsc.org/advances
short circulation time, low drug loading and uptake, poor
stability before targeting and biodegradability aer targeting, or
1. Introduction
Polymer-based nanodelivery systems offer many advantages for
targeted drug/gene delivery in a controlled-release manner,
which would have great potential to treat numerous diseases,
including cancers.1,2 Because of a wide variety of polymers with
different but tunable chemistries and structures, along with
many different synthesis methods, the nanodelivery systems can
be readily prepared with well-dened physical structures and
sizes, tunable chemical compositions, and desirable func-
tions.3–5 Additionally, these systems can also be conjugated with
bioactive ligands to realize specic recognization of cell recep-
tors for improving targeted delivery and reducing side effects.
Signicant progress has made in the development of polymer-
based nanodelivery systems including polymer micelles, lipo-
somes, hydrogels, nanoparticles, and nanogels.6,7 However, most
of nanodelivery systems are still far from optimal for (pre)clinical
applications. A number of limitations and challenges – including
any combination among them – have hindered the development
of effective and biological benign nanodelivery systems.
To remedy the limitations for current nanodelivery systems,
numerous strategies and techniques have been developed to
improve the targeted delivery efficacy. First, many antifouling
materials, which possess strong resistance to nonspecic
protein adsorption known as the stealth effect,8–10 have been
exclusively used as delivery carriers to increase blood circulation
time, structural stability, and the solubility of many low soluble
drugs.11–13 However, even the most widely used poly(ethylene
glycol) (PEG)-based drug delivery systems14–18 have oen demo-
nstrated in vitro instability, due to the oxidation and decompo-
sition of ethylene units in most common biochemical solution
containing transition-metal ions or in the presence of oxygen.19,20
Recently, zwitterionic-based water-soluble polymers such as
sulfobetaine and carboxybetaine have demonstrated their long-
term stability in vitro due to the super-low fouling properties.21–24
Second, prodrug approaches using polymeric drug conjugates
are oen used to increase the higher drug loading capacity and
to improve the targeted delivery of drugs to a specic location.25
Furthermore, upon completion of drug release, the degradability
of the nanodelivery systems are highly preferred to eliminate
aDepartment of Chemical and Biomolecular Engineering, The University of Akron,
Akron, Ohio, USA, 44325. E-mail: zhengj@uakron.edu
bDepartment of Biomedical Engineering, The University of Akron, Akron, Ohio, USA,
44325
cR&D Center for Membrane Technology and Department of Chemical Engineering
Chung Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan
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the need of surgery for removing residuals from human body, EOE cross-linker provides degradable sites at acid conditions, and
which will avoid any possible immune response and cytotoxic transferrin is used for improving cellular uptake. The polyHEAA–
side effect. Biodegradable cross-linkers in polymer-based EOE–transferrin nanogels combined these characteristics enable
delivery systems in response to external stimuli such as to retain large drug storage and high stability at neutral condi-
temperature, pH, and light are oen used for the sustained tions, while degrading intosmall fragmentstotrigger intracellular
release of drugs over a prolonged period.26,27 All of these strate- drug release at acid conditions. Completion of this work, along
gies have recently attracted signicant attention for the devel- with our previous works, will provide a complete framework for
opment of effective polymer-based delivery systems for current polyHEAA-based antifouling materials with different polymer
drugs, which are equally important for the development of new architectures from polymer brushes, hydrogels, nanoparticles, to
drugs, which is a very time-consuming and costly procedure.27–29 nanogels targeting for a wide range of applications of antifouling
We have recently synthesized a series of poly(N-hydroxyethyl coating, wound dressing, and drug delivery.
acrylamide) (polyHEAA)-based materials for different bio-
applications. PolyHEAA brushes exhibited strong and long-term
2. Materials and methods
2.1. Materials
surface resistance to protein adsorption (<0.3 ng cmꢁ2) from
undiluted blood plasma and serum, as well as cell adhesion and
bacterial colonization for up to 3 days.30 PolyHEAA-coated gold
nanoparticles (HEAA–AuNPs) also exhibited long-term super-
low fouling ability and structural stability in undiluted human
blood serum and plasma, without inducing any particle aggre-
gation and protein adsorption up to 7 days.30 Hybrid polyHEAA/
salicylate hydrogels achieved both antifouling and antimicro-
bial activities.7 pH-sensitive poly(HEAA/acrylic acid) nanogels
exhibited superlow fouling ability, ultrastability, low toxicity,
and high cellular uptake for controlled release of drug in vitro.31
Similar to PEG-based materials, polyHEAA achieves its super-
low fouling ability by forming a strong hydration layer around
amide and hydroxyl groups, which create a high energy and
physical barrier to prevent protein adsorption.
Encouraged by the excellent antifouling performance of
polyHEAA materials, here we extended this successful super-low
fouling polyHEAA to two polyHEAA-based nanogels – antifouling
poly(2-(methacryloyloxy) ethyl trimethyl ammonium-g-N-hydroxy-
ethyl acrylamide) (polyTM-g-HEAA) nanogels with a core–shell
structure and pH-responsive biodegradable polyHEAA nanogels
– to demonstrate that the introduction of HEAA as a protective
antifouling sheath enables to greatly improve biocompatibility
and stability, to reduce cell toxicity, and to realize cellular uptake
efficacy and subsequent intracellular drug delivery. First,
cationic polymers have been widely used as gene/drug delivery
carriers,32,33 simply because the cationic polymers enable to
interact strongly with most of anionic cell membrane to facilitate
the transport of polymer carriers and drugs into intracellular
targets. On the other hand, the cationic nature of the polymers
also induce signicant non-specic protein adsorption on the
surface of cationic carriers, which will decrease the circulation
time of carriers in vivo and block drug diffusion from carriers,
and also induce high toxicity to normal human tissue cells.34 To
overcome such limitations, we develop a new two-step ATRP
method to prepare polyTM-g-HEAA nanogels with a core–shell
structure by graing antifouling polyHEAA onto the surface of
polyTM nanogels via SI-ATRP. The stability and toxicity of poly-
TM-g-HEAA nanogels were tested by dynamic light scattering
(DLS) and live/dead SH-SY5Y cell assay, respectively. Further, we
synthesize biodegradable and pH-responsive polyHEAA nano-
gels cross-linked with ethylidenebis(oxy-2,1-ethanediyl) ester
N-Hydroxyethyl acrylamide (HEAA, 97%), 1,1-dimethoxyethane
(97%), 2-hydroxyethylacrylate (96%), p-toluenesulfonic acid
monohydrate (p-TSA, 98.5%), acryloyl chloride (97%), 2,20-bipyr-
idyl (BPY, 99%), copper(I) bromide (99.999%), copper(I) chlorine
(CuCl, 99.999%), N,N0-methylene-bis-acrylamide (MBAA),
ethylene bis(2-bromoisobutyrate) (97%), 2-(methacryloyloxy)
ethyl trimethyl ammonium (TM, 80 wt% in H2O), span 80, tween
80, rhodamine 6G (R6G, 99%), transferrin human (98%), N,N0-
disuccinimidyl carbonate (DSC, 95%), 4-(dimethylamino)pyri-
dine (DMAP, 99%), anhydrous N,N-dimethylformamide (DMF,
99.8%), benzene (anhydrous, 99.8%), hexane (95%), phosphate
buffer saline (PBS, pH 7.4, 0.15 M, 138 mM NaCl, 2.7 mM KCl),
ethanol (absolute 200 proof), tetrahydrofuran (THF HPLC),
dichloromethane (99.9%), sodium hydroxide (NaOH, 99.99%),
ethyl acetate (99.8%), and hydrochloric acid (HCl, 37%) were
purchased from Sigma-Aldrich (Milwaukee, WI). Tris[2-(dime-
thylamino)ethyl]amine (Me6TREN, 99%) was purchased from
Alfa Aesar (Ward Hill, MA). Fetal bovine serum (FBS) was
purchased from BioChemed Services (Winchester, VA). 2,20-Azo-
bis(4-methoxy-2.4-dimethyl valeronitrile) (V-70) was purchased
from Wako Pure Chemical Industries (Osaka, Japan). Water used
in these experiments was puried by a Millipore water purica-
tion system with a minimum resistivity of 18.0 MU cm.
2.2. Synthesis of ethylidenebis(oxy-2,1-ethanediyl) ester
(EOE) crosslinker
EOE was synthesized through transacetalization of 1,1-dime-
thoxyethane (2.4 g, 26 mmol) with 2-hydroxyethylacrylate (6.0 g,
52 mmol) in the presence of p-TSA catalyst (110 mg, 0.64 mmol)
in dry benzene (200 mL) at room temperature for 2 h, then the
methanol was continuously removed by distillation of the
benzene-methanol azeotrope (58 ꢂC) for 1 h. Aer the reaction,
the solvent was removed by a rotary evaporation process, and
the crude product was puried by ash column chromatog-
raphy on silica gel with 3 : 1 hexane/ethyl acetate and nally
with 1 : 1 hexane/ethyl acetate. The EOE crosslinker was
ꢂ
obtained as a transparent liquid and was stored at ꢁ20 C.
2.3. Preparation of nanogels
(EOE) and conjugated with transferrin ligands for cell targeting, As shown in Scheme 1, polyTM-g-HEAA nanogels with core–
in which HEAA serves as an excellent non-fouling background, shell structure were prepared by two-step polymerization
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for 4 hours. The same experimental conditions were used to
synthesize polyHEAA nanogels with loaded R6G, by adding an
additional R6G (4.79 mg, 0.01 mmol) to the aqueous stock
solution.
Aer the polymerization, all nanogels were puried using
the same method as follows: 30 mL of THF were added into the
reaction solution and stirred for 1 h to remove surfactants.
The mixture was centrifuged for 5 minutes at 8000 rpm. The
supernatant was discarded, and the precipitate was washed
with 30 mL of DI water. The nal precipitate was stored at 4 ꢂC
for further characterization.
2.4. Characterization of nanogels
The hydrodynamic diameter and zeta potential of nanogels
were evaluated by a Zetasizer Nano ZS dynamic light scattering
(DLS) instrument (Malvern, U.K.). The wavelength of 633 nm
and the scattering angle of 173ꢂ were xed at room temperature.
The dispersant refractive index and the viscosity of water were
set to be 1.330 and 0.8872 cP, respectively. 1H nuclear magnetic
resonance (NMR) spectra were recorded using a Varian Mercury
300 MHz instruments, employing DMSO-d6 (dimethylsulfoxide)
as solvents.
Scheme 1 Synthesis of polyTM-g-HEAA nanogel with a core–shell structure.
method. First, polyTM nanogels were prepared using an inverse
microemulsion atom transfer radical polymerization (ATRP)
method. A continuous-phase solution contained 40 mL of
hexane, 1.4 g of tween 80, 1.6 g of span 80, and ethylene bis(2-
bromoisobutyrate) (8 mg, 0.022 mmol). Aqueous monomer
stock solution was prepared by dissolving TM (415 mL, 1.8
mmol) and MBAA (18.4 mg, 0.12 mmol) in 2 mL of DI water.
CuBr (14.3 mg, 0.1 mmol) and BPY (31.2 mg, 0.2 mmol) were
placed in a reaction ask, followed by evacuation and nitrogen
injection by three times. Degassed aqueous monomer stock
solution was rst added into the ask to dissolve the catalyst,
and then the degassed continuous-phase solution was added
into the ask, followed by vigorous shaking and sonication for
2 minutes. The reaction was maintained at room temperature
with stirring and was protected under nitrogen for 4 hours.
Second, SI-ATRP method was used to gra polyHEAA onto the
surface of polyTM nanogels. CuCl (16.8 mg, 0.170 mmol) and
25 mg polyTM nanogel were placed in a reaction tube sealed
with rubber septum stoppers and under nitrogen protection.
Degassed ethanol (2 mL) was added to the tube. HEAA (1 g, 8.69
mmol) and Me6TREN (40 mg, 0.174 mmol) were dissolved in
Milli-Q water (5 mL) and ethanol (3 mL), and deoxygenated by
passing a continuous stream of dry nitrogen through the solu-
tion while stirring. The solution with monomers was trans-
ferred to the tube using a syringe under nitrogen protection.
The mixture was kept at room temperature with stirring and
protected by dry nitrogen for 20 hours.
2.5. Release of encapsulated drug from polyHEAA nanogels
40 milligrams of the puried nanogels with R6G drugs was re-
suspended in 20 mL of buffer solution (stock solution) and le
ꢂ
at 37 C. Then, 1 mL of solution was taken from the nanogel
solution. Considering that the R6G drugs that are loaded inside
the nanogels and released in the solution can generate UV
absorption signal. At t ¼ 0, almost all of R6G were entrapped
inside the nanogels aer numerous washing with water. The
initial UV density of the stock solution actually represents a
total amount of R6G inside the nanogels. Aer that, the R6G
drugs will be released from the nanogels into the solution. At
different time points, 1 mL of solution was taken from the stock
solution, placed into a 3 kDa molecular-weight-cutoff Amicon
Ultra centrifugal lter device (Millipore, Burlington, MA), and
centrifuged at 14 000 rpm for 30 minutes to collect ow-through
solution for drug detection. The UV-vis density of R6G in the
ltrate solution was measured by a Beckman model DU 530
spectrometer with cuvettes of 1 or 0.5 cm path length at room
temperature. The percentage of released drugs was dened as
the ratio of the UV-vis density of ow-through solution at
different time points to the initial UV-vis density of stock solu-
UV densityjflow through
PolyHEAA nanogels were prepared by an inverse micro-
emulsion free radical polymerization method as reported in our
previous work.31 To synthesize polyHEAA nanogel, the contin-
uous-phase solution contained 40 mL of hexane, 1.4 g of tween
80, 1.6 g of span 80, and V-70 (8 mg, 0.026 mmol). Aqueous
monomer stock solution was prepared by dissolving HEAA
(415 mL, 4 mmol) of and EOE crosslinker (5 mL, 0.02 mmol) into
2 mL of DI water. Then the aqueous stock solution was added to
a 100 mL ask containing 40 mL of continuous-phase solution,
solution
-
tion Drug release ð%Þ ¼
ꢃ 100%.
UV densityjstock
solution
Different amounts of HCl were added into PBS buffer (pH ¼ 7.4)
to prepare the buffers with pH 1.6 and 3.6.
2.6. Conjugation of transferrin to the surface of polyHEAA
nanogels
followed by vigorous shaking and sonication for 2 minutes. The To activate the terminated hydroxyl groups of polyHEAA nano-
asks were purged with nitrogen at 0 ꢂC for 30 minutes to gels, 20 mg polyHEAA nanogels were dispersed in dry DMF
remove dissolved oxygen. During polymerization, the reaction solution containing 0.1 M DSC and 0.1 M DMAP for 24 h at
was kept at 40 ꢂC with stirring and was protected under nitrogen room temperature under an argon atmosphere. Aer the
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reaction, the reaction solution was centrifuged for 30 minutes at
14 000 rpm, and 10 mg precipitate was mixed with 500 mL
aqueous solution of transferrin (10 mg mLꢁ1). The reaction was
kept at room temperature with stirring for 24 h. Aer the
reaction, the reaction solution was centrifuged for 30 minutes at
14 000 rpm, and the precipitates were collected for cell uptake
assay.
2.7. Cell culture
All chemicals were purchased from Life Technologies unless
otherwise stated. Human neuroblastoma (SH-SY5Y) cells
(ATCC) were used as model neurons, and cultured in 75 cm2
T-asks (Corning) in sterile-ltered Eagle's Minimum Essential
Medium and Ham's F-12 medium mixed at a 1 : 1 ratio con-
taining 10% fetal bovine serum, and 1% penicillin/strepto-
ꢂ
mycin. Flasks were incubated in a humidied 37 C incubator
with 5% CO2. Cells were cultured to conuence, and harvested
using 0.25 mg mLꢁ1 Trypsin/EDTA solution (Lonza). Cells were
re-suspended in Opti-MEM reduced serum medium and coun-
ted using a hemacytometer. Cells were then placed in a 24-well
tissue culture plate with approximately 100 000 cells per well in
500 mL of medium, which allow cells to attach for 24 hours
inside the incubator.
Fig. 1 1H NMR spectrum of polyTM-g-HEAA nanogels.
3. Results and discussion
3.1. Synthesis and characterization of antifouling polyTM-g-
HEAA core–shell nanogels
PolyTM-g-HEAA core–shell nanogels were prepared via a new
two-step ATRP method: cationic polyTM cores were rst
prepared via the inverse microemulsion ATRP, followed by the
graing of polyHEAA onto the surface of polyTM nanogels via
the surface-initiated-ATRP method (Scheme 1). 1H NMR was
used to conrm the chemical structure of polyTM-g-HEAA
nanogels. The chemical shis of all the protons in the copoly-
mers can be clearly identied in Fig. 1. Specically, the chem-
ical shis in the region of 3.3–3.5 ppm (peak g) were assigned to
the protons of –N+(CH3)3 group of TM segment. The apparent
signals at ꢀ7.6–7.8 ppm (peak a) and 5.0–5.1 ppm (peak b) were
characteristic peaks of the protons of –CO–NH– group and –OH
group of HEAA segment, respectively. Copolymer compositions
were also determined from the 1H NMR by comparising the
peak areas corresponding to the protons of –N+(CH3)3 group at
3.3–3.5 ppm and the protons of –CO–NH– group at 7.6–7.8 ppm.
The mole ratio of TM vs. HEAA in polyTM-g-HEAA nanogels was
estimated as ꢀ1 : 2.5, while the molar ratio of MBAA to HEAA in
the polyTM-g-HEAA nanogel was estimated as ꢀ2.7%. We
should note that in the second SI-ATRP step, a large amount of
HEAA was used to ensure the saturated graing polymerization
occurred on the surface of polyTM cores, but only a small
percentage of HEAA monomers can be graed on the polyTM
surfaces. This is the interpretation for the low conversion of
HEAA monomers between the large excess use of HEAA to TM
2.8. Live/dead cell assay for polyTM-g-HEAA nanogels
A live/dead assay was used to obtain cell viability/cytotoxicity
data and to observe live and dead cells. 0.25 mg of polyTM and
polyTM-g-HEAA nanogels were added to each well and pipetted
up and down a few times. The cells were then le for 24 hours in
ꢂ
a humidied incubator with 5% CO2 at 37 C, followed by the
assessment of cell toxicity. Calcein AM was added to each well to
a nal concentration of 2 mM to distinguish the presence of live
cells with a uorescence excitation/emission of 494/517 nm,
while ethidium homodimer-1 was added to each well to a nal
concentration of 5 mM to distinguish the presence of dead cells
with a uorescence excitation/emission of 528/617 nm. Aer
being incubated for 15 min to activate the uorescent dyes at
room temperature, the uorescence images of the live and
dead cells was collected using a Zeiss Axiovert 40 CFL
inverted microscope tted with lters at 510 nm and 600 nm.
Cell viability percentages were calculated using a Synergy H1
microplate reader (BioTek) with background subtraction. For
cytotoxicity assay, six independent values were collected and the
error bars in each gure represent the standard error of these
six independent experiments. A result was considered statisti-
cally signicantly different when p # 0.05.
2.9. Cell uptake assay for polyHEAA nanogels
R6G-loaded polyHEAA nanogels (0.05 mg and 0.1 mg) with or for preparing the polyTM-g-HEAA nanogels and the small ratio
without transferrin conjugation were added to each well, of HEAA to TM (2.5 : 1) in the resulting polyTM-g-HEAA.
respectively, and pipetted up and down a few times. Cells were
To verify whether the graed polyHEAA shell provides anti-
incubated for 24 h. Aer washing the coverlips 3 times with fouling protection to the polyTM core, the average hydrody-
10 mM PBS, the cells were xed with 3–4% paraformaldehyde, namic diameters of nanogels in the absence and presence of
followed by 3 washes with PBS. The cells were examined under polyHEAA shell in fetal bovine serum (FBS) was determined
20ꢃ magnications through differential interference contrast and compared by DLS. Fig. 2 clearly showed that polyTM-g-
and epiuorescence.
HEAA nanogels were able to retain their initial hydrodynamic
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self-aggregation of nanogels. The latter event should occur aer
the nonspecic adsorption of proteins. PolyTM nanogels
themselves actually can remain their sizes unchanged in PBS for
very long time, but upon the addition of plasma or serum,
nonspecic protein adsorption on the surface of polyTM
nanogel will further trigger the self-aggregation of the nanogels
via nonspecic protein interactions. Meanwhile, the increase of
initial hydrodynamic diameter from 58 nm for polyTM nanogels
to 140 nm for polyTM-g-HEAA nanogel in FBS also indicates the
successful gra of polyHEAA onto the polyTM nanogel. Clearly,
the incorporation of polyHEAA onto the polyTM core conrms
antifouling protection effect.
Since most of synthetical nanogels are oen cytotoxic, we
further evaluated the cytotoxicity of polyTM-g-HEAA nanogels
using the live/dead SH-SY5Y cell assay as compared to pure
polyTM nanogels without polyHEAA protection. Aer 24-hour
incubation of SH-SY5Y cells with different nanogels, live and
dead cells were quantitatively measured using a Synergy H1
microplate reader (Fig. 3) and qualitatively imaged using uo-
rescence microscopy (Fig. 4). With nanogel concentration of
0.5 mg mLꢁ1, cationic polyTM nanogels were highly toxic to
cells and induced ꢀ60% cell death, as compared to 98% cell
viability in the absence of nanogels as a control. Conversely,
polyTM-g-HEAA nanogels retained ꢀ94% relative cell viability
and exhibited almost neglectable cell toxicity. Meanwhile,
visual inspection of live and dead cells using uorescence
microscopy also conrmed few cell death induced by polyTM-g-
HEAA nanogels (Fig. 4b), as compared to massive cell death
induced by polyTM nanogels (Fig. 4c). High instability and
toxicity of polyTM nanogels are very likely attributed to their
cationic surfaces. Strong electrostatic interactions between
positively charged nanogels and negatively charged proteins in
FBS and cell membranes would cause strong protein adsorption
on the gel surfaces and strong membrane-binding and
membrane disruption, respectively. As expected, Fig. 5 showed
that x potentials of the polyTM nanogels in PBS were +30 mV,
indicating that the polyTM nanogels carrying strong positive
charges. While aer graing polyHEAA onto polyTM nanogels,
the x potentials of polyTM-g-HEAA nanogels decreased to
+3 mV, indicating the very weak surface charges. Taken
together, incorporation of antifouling HEAA groups into the
cationic core of polyTM nanogels signicantly improves the
biocompatibility, stability, and cell viability of nanogels.
Fig. 2 Time-dependent hydrodynamic diameter of polyTM nanogels and poly-
TM-g-HEAA nanogels in FBS. Error bars represent standard deviation from 3
independent measurements.
Fig. 3 Viability of SH-SY5Y cells following 24 h incubation with polyTM-g-HEAA
and polyTM nanogels (0.5 mg mLꢁ1), as normalized by cell viability in medium
only. Error bars represent standard deviation from 3 independent measurements.
diameters at 140 nm. The barely changed sizes of polyHEAA
nanogels indicate that HEAA groups can effectively prevent both
nonspecic protein adsorption on the nanogels and the self-
aggregation of nanogels, resulting in more homogeneously
dispersed and stable nanogels without aggregation in FBS over
a long period time (up to 48 h). Conversely, cationic polyTM
nanogels quickly increased their initial size from 58 nm to 330
nm within the rst 5 min and then continuously increased to
607 nm aer 48 h incubation in blood plasma. Such dramatic
size increase is likely attributed to (i) the nonspecic adsorption
of proteins from blood plasma onto the nanogels and (ii) the
Fig. 4 Representative fluorescence microscopy images of SH-SY5Y cells after 24 h incubation with (a) medium only, (b) polyTM-g-HEAA nanogels (0.5 mg mLꢁ1), and
(c) polyTM nanogels (0.5 mg mLꢁ1). Live cells are stained green, while dead cells are stained red.
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polyHEAA nanogels decreased to 10 nm, 18 nm, and 110 nm,
respectively (Fig. 6b–d), indicating that nanogels indeed
degrade. Further, a drastic decrease in gel sizes particularly at
acid conditions suggests an accelerated hydrolysis leading to a
faster degradation, while a slow degradation at neutral pH by
only 15% actually indicates relative stability of polyHEAA
nanogels against hydrolysis.
The degradation behavior of polyHEAA nanogels is not only
tunable by varying the pH values, but also used to regulate drug
release from the polyHEAA nanogels aer uptake by cells. The
acid-triggered release of drugs from polyHEAA nanogels cross-
linked with 0.5% EOE was investigated. Positively charged R6G
was used as model drug. To qualify the amount of R6G released
from nanogels to aqueous solution, the concentration of
Fig. 5 Zeta potential of polyTM and polyTM-g-HEAA nanogels in PBS buffer
(pH
¼
7.4). Error bars represent standard deviation from 3 independent
measurements.
ꢂ
released R6G was measured by UV-vis density at 25 C. Fig. 7
shows the acidic-responsive release kinetics of R6G from poly-
HEAA nanogels cross-linked with EOE in PBS solutions with
different pH. It can be seen clearly that at a strong acid condi-
tion of pH ¼ 1.6, a relative fast drug-release from nanogels
within 25 h was observed, in which ca. 2.0%, 16.2%, 34.2%
drugs were released in 5, 10, 25 h, respectively. Similar drug-
release trend was also observed but to a smaller extent at pH of
3.6. In contrast, minimal drug release (ca. 7.5%) was observed
within 120 h at pH 7.4 under otherwise the same conditions.
Overall, the cumulative percentages of released drugs aer
120 h were 70% at pH 1.6, 45% at pH 3.6, and 7.5% at pH 7.4,
respectively, further conrming that this acid-triggered drug
release behavior is likely due to the degradability of a large
fraction of nanogels at acid conditions, while still retaining a
relative large drug storage and high stability of polyHEAA
nanogels at physiological condition (Scheme 2B). Overall, the
more acid condition, the higher degree of nanogel degradation,
the more drug release percentage, explaining the fact that
saturated drug release is in the order of pH values: 70% at pH ¼
1.6, 40% at pH ¼ 3.6, and 7.5% at pH ¼ 7.4. The biodegradable
polyHEAA nanogels with easy synthesis, enhanced antifouling
3.2. Synthesis and characterization of biodegradable
polyHEAA nanogels
Besides the biocompatibility of nanogels, biodegradability is
another important factor for their biomedical uses. Here, we
prepared biodegradable polyHEAA nanogels cross-linked with
0.5% EOE via an inverse microemulsion free radical polymeri-
zation method. EOE as a degradable crosslinker is stable under
neutral and alkaline conditions, however it readily undergoes
hydrolysis in aqueous acidic solutions35 (Scheme 2A). It is
widely accepted that the intracellular pH of tumors is more
acidic (pH 4.0–5.5) than that of blood and normal tissues.
Therefore, the pH-responsive materials, such as EOE used in
this work, offer a general advantage for drug release and cancer
therapies, as compared to many other stimuli-responsive
materials. The degradability of polyHEAA nanogels was deter-
mined by monitoring the hydrodynamic diameter of nanogels
in PBS solutions of different pH values. As shown in Fig. 6,
polyHEAA nanogels had initial average hydrodynamic diameter
of ꢀ130 nm (Fig. 6a). Aer incubating nanogels in PBS solutions
with pH 1.6, 3.6, and 7.4 aer 120 h, average diameters of
Scheme 2 (A) Hydrolysis of an EOE crosslinker and (B) acid-triggered degradation of polyHEAA nanogel and subsequent R6G release from degraded nanogels.
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Fig. 6 Hydrodynamic diameter distributions of polyHEAA nanogels conjugated with EOE crosslinkers (a) before hydrolysis (initial diameters) and after hydrolysis when
incubating nanogels in PBS solutions at (b) pH ¼ 1.6, (c) pH ¼ 3.6, and (d) pH ¼ 7.4 for 120 h at 37 ꢂC.
Our previous live/dead assays have revealed that polyHEAA
nanogels were nontoxic to SH-SY5Y cells with cell viabilities
93.4% up to a tested concentration of 0.5 mg mLꢁ1. Here, we
extended the study to examine the SH-SY5Y cellular uptake and
subsequent intracellular drug release behaviors of R6G-loaded
polyHEAA nanogels with biodegradable cross-linkers. To realize
efficient and specic intracellular drug delivery to tumor cells,
we conjugated transferrin ligands onto the surface of polyHEAA
nanogels using an activation-coupling approach, i.e. the
hydroxyl groups of HEAA monomers are rst activated with
N,N0-disuccinimidyl carbonate, followed by subsequent
coupling to a primary amine of transferrin. Transferrin, an iron-
Fig. 7 Time-dependent release profiles of R6G from degradable polyHEAA
binding glycoprotein, is a well-studied ligand that facilitates
efficient and specic tumor cell uptake of nanodelivery systems
via receptor-mediated endocytosis. Fig. 8 compares differential
interference contrast (le), the corresponding epiuorescent
nanogels in PBS solution of different pH values at 37 ꢂC. Error bars represent
standard deviation from 3 independent measurements.
ability and stability, and pH-triggered drug release behaviors images (middle), and the merged images (right) of SH-SY5Y
are highly interesting as “smart” vehicles for active intracellular cells aer 24 h incubation with R6G-loaded polyHEAA nanogels
drug release.
with and without conjugated transferrin. In all concentrations
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Fig. 8 Differential interference contrast images (left panel), epifluorescent images of rhodamine 6G (color in red) (middle panel), and merged images (right panel) of
SH-SY5Y cells after 24 h incubation with (a) 0.1 mg mLꢁ1 and (b) 0.2 mg mLꢁ1 of R6G-loaded polyHEAA nanogels with transferrin, (c) 0.1 mg mLꢁ1 and (d) 0.2 mg mLꢁ1
of R6G-loaded polyHEAA nanogels without transferrin. The scale bars correspond to 50 mm in all the images.
tested, cells treated with R6G-loaded polyHEAA nanogels core, which switches positively charged gel surface to a neutral
without conjugated transferrins displayed weak uorescence and hydrophilic surface, polyTM-g-HEAA nanogels dramatically
(Fig. 8c–d). In contrast, upon conjugation with transferrin onto improve their long-term stability in FBS and reduce toxicity to
R6G-loaded polyHEAA nanogels, very bright uorescence SH-SY5Y cells. Further, biodegradable polyHEAA nanogels
arising from R6G was observed in SH-SY5Y cells, indicating the cross-linked with acid-responsive EOE also exhibit fast degra-
internalization of nanogels and the release of drugs inside cells. dation upon hydrolysis and drug release at acid conditions,
Aer nanogels cellular uptake and drug releases, we did not while retaining relative large drug storage and high stability at
observe apparent cell death, further conrming that these physiological condition. Conjugation of transferrin onto poly-
biodegradable nanogels have excellent biocompatibility. Over- HEAA nanogels further improves the efficiency of cellular
all, polyHEAA nanogels with excellent antifouling property, uptake and subsequent intracellular drug release for targeting
biodegradability, low toxicity, and pH-responsive intracellular drug delivery. Overall, these HEAA-based nanogels exhibit
drug release are highly promising for targeted cancer therapy.
excellent biocompatibility, enhanced stability, low cell toxicity,
and pH-triggered degradability and intracellular drug release,
which make them as great potential nanodelivery systems to be
widely used in biomedical applications.
4. Conclusions
This study has made a proof of concept that HEAA-functional-
ized polymers can be prepared for the construction of anti-
fouling gra copolymer nanogels and pH-responsive
Acknowledgements
biodegradable nanogels for different biomedical applications. J.Z. thanks the support from NSF grants (CAREER Award CBET-
Upon graing of an antifouling polyHEAA shell to the polyTM 0952624 and CBET-1158447).
19998 | RSC Adv., 2013, 3, 19991–20000
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15 H. Otsuka, T. Satomi, K. Ueno, Y. Mitamura and T. Tateishi,
Synthesis of polypyridine-gra-PEG copolymer for long-term
stability of nonfouling character, in Transactions of the
Materials Research Society of Japan, ed. S. Somiya and M.
Doyama, Elsevier Science Bv, Amsterdam, 2006, vol. 31, no.
3, pp. 627–631.
References
1 D. Putnam, C. A. Gentry, D. W. Pack and R. Langer, Polymer-
based gene delivery with low cytotoxicity by a unique balance
of side-chain termini, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
1200–1205.
2 D. M. Lynn, M. M. Amiji and R. Langer, pH-responsive
polymer microspheres: Rapid release of encapsulated
material within the range of intracellular pH, Angew.
Chem., Int. Ed., 2001, 40, 1707–1710.
16 P. Kingshott, J. Wei, D. Bagge-Ravn, N. Gadegaard and
L. Gram, Covalent attachment of poly(ethylene glycol) to
surfaces, critical for reducing bacterial adhesion, Langmuir,
2003, 19, 6912–6921.
3 S. P. Hudson, R. Langer, G. R. Fink and D. S. Kohane,
Injectable in situ cross-linking hydrogels for local
antifungal therapy, Biomaterials, 2010, 31, 1444–1452.
4 D. N. Nguyen, J. J. Green, J. M. Chan, R. Longer and
D. G. Anderson, Polymeric Materials for Gene Delivery and
DNA Vaccination, Adv. Mater., 2009, 21, 847–867.
5 K. E. Uhrich, S. E. M. Ibim, D. R. Larrier, R. Langer and
C. T. Laurencin, Chemical changes during in vivo
degradation of poly(anhydride-imide) matrices, Biomaterials,
1998, 19, 2045–2050.
17 G. M. Harbers, K. Emoto, C. Greef, S. W. Metzger,
H. N. Woodward, J. J. Mascali, et al. Functionalized
poly(ethylene glycol)-based bioassay surface chemistry that
facilitates bio-immobilization and inhibits nonspecic
protein, bacterial, and mammalian cell adhesion, Chem.
Mater., 2007, 19, 4405–4414.
18 J.-C. Yang, C. Zhao, I. F. Hsieh, S. Subramanian, L. Liu,
G. Cheng, et al. Strong resistance of poly(ethylene glycol)
based L-tyrosine polyurethanes to protein adsorption and
cell adhesion, Polym. Int., 2012, 61, 616–621.
6 X. Wang, X. Sun, G. Jiang, R. Wang, R. Hu, X. Xi, et al.
Synthesis of biomimetic hyperbranched zwitterionic
polymers as targeting drug delivery carriers, J. Appl. Polym.
Sci., 2013, 128, 3289–3294.
7 C. Zhao, X. Li, L. Li, G. Cheng, X. Gong and J. Zheng, Dual
functionality of antimicrobial and antifouling of poly(N-
hydroxyethylacrylamide)/salicylate hydrogels, Langmuir,
2013, 29, 1517–1524.
8 K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert,
Poly(ethylene glycol) in Drug Delivery: Pros and Cons as
Well as Potential Alternatives, Angew. Chem., Int. Ed., 2010,
49, 6288–6308.
19 D. W. Branch, B. C. Wheeler, G. J. Brewer and
D. E. Leckband, Long-term stability of graed polyethylene
glycol surfaces for use with microstamped substrates in
neuronal cell culture, Biomaterials, 2001, 22, 1035–1047.
20 A. Roosjen, J. de Vries, H. C. van der Mei, W. Norde and
H. J. Busscher, Stability and effectiveness against bacterial
adhesion of poly(ethylene oxide) coatings in biological
uids, J. Biomed. Mater. Res., Part B, 2005, 73, 347–354.
21 G. Cheng, H. Xue, G. Li and S. Jiang, Integrated
Antimicrobial and Nonfouling Hydrogels to Inhibit the
Growth of Planktonic Bacterial Cells and Keep the Surface
Clean, Langmuir, 2010, 26, 10425–10428.
9 S. Chen, L. Li, C. Zhao and J. Zheng, Surface hydration:
principles and applications toward low-fouling/nonfouling
biomaterials, Polymer, 2010, 51, 5283–5293.
10 Y. Chang, W. Yandi, W.-Y. Chen, Y.-J. Shih, C.-C. Yang,
Y. Chang, et al. Tunable bioadhesive copolymer hydrogels
of thermoresponsive poly(N-isopropyl acrylamide) containing
zwitterionic polysulfobetaine, Biomacromolecules, 2010, 11,
1101–1110.
22 W. Yang, H. Xue, L. R. Carr, J. Wang and S. Jiang,
Zwitterionic poly(carboxybetaine) hydrogels for glucose
biosensors in complex media, Biosens. Bioelectron., 2011,
26, 2454–2459.
23 S. Jiang and Z. Cao, Ultralow-Fouling, Functionalizable, and
Hydrolyzable. Zwitterionic Materials and Their Derivatives
for Biological Applications, Adv. Mater., 2010, 22, 920–
932.
11 C. Zhao, L. Li and J. Zheng, Achieving highly effective
nonfouling performance for surface-graed poly(HPMA)
via atom-transfer radical polymerization, Langmuir, 2010,
26, 17375–17382.
12 C. Zhao, L.-Y. Li, M.-M. Guo and J. Zheng, Functional
polymer thin lms designed for antifouling materials and
biosensors, Chem. Pap., 2012, 66, 323–339.
13 C. Zhao, L. Li, Q. Wang, Q. Yu and J. Zheng, Effect of lm
thickness on the antifouling performance of poly(hydroxy-
functional methacrylates) graed surfaces, Langmuir, 2011,
27, 4906–4913.
24 Y. Chang, S.-H. Shu, Y.-J. Shih, C.-W. Chu, R.-C. Ruaan and
W.-Y. Chen, Hemocompatible mixed-charge copolymer
brushes of pseudozwitterionic surfaces resistant to
nonspecic plasma protein fouling, Langmuir, 2009, 26,
3522–3530.
25 W. Gao, B. Xiang, T.-T. Meng, F. Liu and X.-R. Qi,
Chemotherapeutic drug delivery to cancer cells using a
combination of folate targeting and tumor microenviron-
ment-sensitive polypeptides, Biomaterials, 2013, 34, 4137–
4149.
26 X. Sun, S. Chen, J. Han and Z. Zhang, Mannosylated
biodegradable polyethyleneimine for targeted DNA delivery
to dendritic cells, Int. J. Nanomed., 2012, 7, 2929–2942.
27 Y. Wang, G. Wu, X. Li, J. Chen, Y. Wang and J. Ma,
¨
14 V. Zoulalian, S. Zurcher, S. Tosatti, M. Textor, S. Monge and
J.-J. Robin, Self-Assembly of Poly(ethylene glycol)–Poly(alkyl
phosphonate) Terpolymers on Titanium Oxide Surfaces:
Synthesis, Interface Characterization, Investigation of
Nonfouling Properties, and Long-Term Stability, Langmuir,
2009, 26, 74–82.
Temperature-triggered
redox-degradable
poly(ether
urethane) nanoparticles for controlled drug delivery,
J. Mater. Chem., 2012, 22, 25217–25226.
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 19991–20000 | 19999

View Article Online
RSC Advances
Paper
28 R. Bird, T. Freemont and B. R. Saunders, Tuning the 32 B. Yang, S. Y. Geng, X. M. Liu, J. T. Wang, Y. K. Chen,
properties of pH-responsive and redox sensitive hollow
particles and gels using copolymer composition, So
Matter, 2012, 8, 1047–1057.
Y. L. Wang, et al. Positively charged cholesterol derivative
combined with liposomes as an efficient drug delivery
system, in vitro and in vivo study, So Matter, 2012, 8, 518–
525.
29 S. Carregal-Romero, M. Ochs, P. Rivera-Gil, C. Ganas,
A. M. Pavlov, G. B. Sukhorukov, et al. NIR-light triggered 33 F. Fayazpour, B. Lucas, C. Alvarez-Lorenzo, N. N. Sanders,
delivery of macromolecules into the cytosol, J. Controlled
Release, 2012, 159, 120–127.
30 C. Zhao and J. Zheng, Synthesis and characterization of
J. Demeester and S. C. De Smedt, Physicochemical and
transfection properties of cationic hydroxyethylcellulose/
DNA nanoparticles, Biomacromolecules, 2006, 7, 2856–2862.
poly(N-hydroxyethylacrylamide) for long-term antifouling 34 C. F. Jones, R. A. Campbell, Z. Franks, C. C. Gibson,
ability, Biomacromolecules, 2011, 12, 4071–4079.
31 C. Zhao, Q. Chen, K. Patel, L. Li, X. Li, Q. Wang, et al.
Synthesis and characterization of pH-sensitive poly(N-2-
G. Thiagarajan, A. Vieira-de-Abreu, et al. Cationic PAMAM
Dendrimers Disrupt Key Platelet Functions, Mol.
Pharmaceutics, 2012, 9, 1599–1611.
´
hydroxyethyl acrylamide)-acrylic acid (poly(HEAA/AA)) 35 E. R. Gillies, A. P. Goodwin and J. M. J. Frechet, Acetals as
nanogels with antifouling protection for controlled release,
So Matter, 2012, 8, 7848–7857.
pH-Sensitive Linkages for Drug Delivery, Bioconjugate
Chem., 2004, 15, 1254–1263.
20000 | RSC Adv., 2013, 3, 19991–20000
This journal is ª The Royal Society of Chemistry 2013