Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and efficient intracellular doxorubicin release

Article abstract of DOI:10.1016/j.jconrel.2014.11.012

Charge-conversional and reduction-sensitive polyvinyl alcohol (PVA) nanogels were developed for efficient cancer treatment by enhanced cell uptake and intracellular triggered doxorubicin (DOX) release. These PVA nanogels were prepared in a straightforward manner by inverse nanoprecipitation via click reaction with an average diameter of 118 nm. The introduction of COOH into the PVA nanogels efficiently improved the DOX encapsulation due to the electrostatic interaction. The in vitro release result showed that the decrease of electrostatic interaction between COOH and DOX under a mimicking endosomal pH, in combination with the cleavage of the intervening disulfide bonds in response to a high glutathione (GSH) concentration led to a fast and complete release of DOX. Furthermore, confocal laser scanning microscopy (CLSM) revealed that the ultra pH-sensitive terminal groups allowed nanogels to reverse their surface charge from negative to positive under a tumor extracellular pH (6.5-6.8) which facilitated cell internalization. MTT assays and real time cell analysis (RTCA) showed that these DOX-loaded charge-conversional and reducible PVA nanogels had much better cell toxicity than DOX-loaded non-charge-conversional or reduction-insensitive PVA nanogels following 48 h of incubation. These novel charge-conversional and stimuli-responsive PVA nanogels are highly promising for targeted intracellular anticancer drug release.

Full text of DOI:10.1016/j.jconrel.2014.11.012

COREL-07443; No of Pages 10
Journal of Controlled Release xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Charge-conversional and reduction-sensitive poly(vinyl alcohol)
nanogels for enhanced cell uptake and efficient intracellular
doxorubicin release
Wei Chen a, Katharina Achazi a, Boris Schade b, Rainer Haag a,
⁎
a
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 3, Berlin 14195, Germany
b
Research Center of Electron Microscopy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Fabeckstrasse 36a, Berlin 14195, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Charge-conversional and reduction-sensitive polyvinyl alcohol (PVA) nanogels were developed for efficient can-
cer treatment by enhanced cell uptake and intracellular triggered doxorubicin (DOX) release. These PVA
nanogels were prepared in a straightforward manner by inverse nanoprecipitation via “click” reaction with an
average diameter of 118 nm. The introduction of COOH into the PVA nanogels efficiently improved the DOX en-
capsulation due to the electrostatic interaction. The in vitro release result showed that the decrease of electrostat-
ic interaction between COOH and DOX under a mimicking endosomal pH, in combination with the cleavage of
the intervening disulfide bonds in response to a high glutathione (GSH) concentration led to a fast and complete
release of DOX. Furthermore, confocal laser scanning microscopy (CLSM) revealed that the ultra pH-sensitive
terminal groups allowed nanogels to reverse their surface charge from negative to positive under a tumor extra-
cellular pH (6.5–6.8) which facilitated cell internalization. MTT assays and real time cell analysis (RTCA) showed
that these DOX-loaded charge-conversional and reducible PVA nanogels had much better cell toxicity than DOX-
loaded non-charge-conversional or reduction-insensitive PVA nanogels following 48 h of incubation. These novel
charge-conversional and stimuli-responsive PVA nanogels are highly promising for targeted intracellular
anticancer drug release.
Received 2 October 2014
Accepted 16 November 2014
Available online xxxx
Keywords:
PVA nanogel
Inverse nanoprecipitation
Charge-conversion
Stimuli-responsive
Drug delivery
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
strategies have been developed to enhance tumor cell uptake of nano-
systems, including the decoration of active targeting ligands for
Drug delivery systems (DDSs) have appeared as a promising and
reliable approach for delivering potent drugs to the site of action in a
precise and timely fashion. Polymeric nanosystems which include
micelles, liposomes, nanoparticles, and nanogels have been the most
investigated DDSs, because of their prolonged circulation time, en-
hanced accumulation in the tumor sites via the enhanced permeability
and retention (EPR) effect, decreased adverse effects, and improved
drug tolerance [1–7]. Compared to other nanosystems, nanogels with
internally cross-linked 3D structures are highly interesting for control-
ling drug delivery at the target site in fast response to external stimuli
as well as for improving drug bioavailability [8–13]. It should be noted
that the traditional nanocarriers such as those with PEG-shielding can
only enhance the accumulation by EPR effect in tumor tissues, however,
the poor cellular internalization always leads to intracellular dosages of
anticancer drugs below the required therapeutic level, which limits the
efficacy of cancer chemotherapy [14–16]. This high stealth for long
circulation versus cellular internalization dilemma has been quite a
challenge for targeted tumor chemotherapy. In the past decade, several
receptor-mediated endocytosis and the usage of tumor extracellular mi-
croenvironment stimuli, i.e., tumoral acidity and temperature gradients
[17–19]. As compared to the strategy of active targeting ligand-
decoration, the nanosystems using the tumor pH for enhanced cellular
internalization is easy to prepare and can be exploited for the universal
tumor cells, since the mildly acidic extracellular pH (in the range of
6.5–6.8) exists in most tumor tissues, which is lower than that of normal
tissues and the blood stream (pH 7.2–7.4) [20,21]. Several ultra pH-
sensitive polymers including poly(β-amino ester), poly(methacryloyl
sulfadimethoxine), polyhistidine, and polymer with 2,3-dimethylmaleic
anhydride (DMMA) modified amine moieties, have been developed for
the construction of polymeric delivery systems in response to weakly
acidic tumor tissue microenvironments. For example, Kataoka et al.
first reported the concept of charge-conversional polymer based on
citraconic amides, which are stable under neutral conditions while
they can be cleaved only under slightly acidic pH conditions, thus lead-
ing to the transformation of negatively charged carboxylate functional-
ities into positively charged primary amines [22]. Recently, Wang et al.
further developed dual pH-sensitive prodrug nanoparticles using
two pH-sensitive linkages with different stabilities towards acidity,
DMMA-based amides, and hydrozone linkages, thereby achieving
⁎
Corresponding author. Tel.: +49 30 83852633; fax: +49 30 838452611.
E-mail address: haag@chemie.fu-berlin.de (R. Haag).
http://dx.doi.org/10.1016/j.jconrel.2014.11.012
0168-3659/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012

2
W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
improved cellular internalization of nanoparticles under a tumor extra-
cellular pH and effective release of covalently conjugated DOX under
endosomal/lysosomal pH [23].
Poly(vinyl alcohol) (PVA) has an excellent history of biomedical
applications, specifically in the form of micro/hydrogel materials that
are used for enzyme immobilization, cell encapsulation, and clinical
applications as embolic bodies [37]. However, due to PVA's inhomoge-
neous interior, high porosity, and lack of polar PVA nanogels, it failed
to meet the demands of (nano)biotechnology, especially in the
nanomedicine area because of its inferior drug encapsulation capability
and uncontrollable release behavior. Therefore we developed a unique
charge-conversional and reducible PVA nanogel system for enhanced
cell uptake and efficient intracellular DOX release. These PVA nanogels
are prepared in a straightforward manner by inverse nanoprecipitation
via a “click” reaction with an ultra pH-sensitive terminator which
enables nanogels to reverse their surface charge from negative to
positive at tumor extracellular pH (6.5–6.8) for enhanced cell internali-
zation (Scheme 1). Subsequently, in vitro drug release and tumor cell
killing activity of DOX-loaded nanogels were investigated and the re-
sults compared with those obtained using DOX-loaded non-charge-
conversional or reduction-insensitive counterparts.
Another considerable challenge for cancer therapy is how to accom-
plish a rapid drug release after the nanosystems arrive at the patholog-
ical site for better therapeutic efficacy. In the past decade, a tremendous
effort has been directed to develop intelligent drug nanocarriers that re-
lease their payload in response to intrinsic intracellular signals, particu-
larly to endosomal/lysosomal pH and cytoplasmic glutathione (GSH)
[24–26]. Polymeric components with pH-sensitive groups (pH-induced
protonation/deprotonation or degradation) are used to produce drug-
formulation nanosystems that are relatively stable in the circulation
but also have the ability to rapidly release the entrapped drugs in the
tumor tissue (pH 6.8) as well as in the intracellular compartments,
such as endosomes (pH 5.5–6) and lysosomes (pH 4.5–5.0) of cells
[27–29]. Following escape from the endosome and by taking advantage
of the high redox potential in cytoplasms and nuclei of cancer cells,
which have much higher concentration of reducing glutathione (GSH)
tripeptide than body fluids and extracellular milieu (0.5–10 mM versus
2–20 μM GSH), reduction-sensitive nanosystems containing S–S bonds
have been designed and exploited for active cytoplasmic release of
various potent chemotherapeutics [30–32]. It should be noted that
polymeric nanosystems with dual and multi-stimuli responses have
shown unprecedented control over drug delivery and thus led to supe-
rior in vitro and/or in vivo anti-cancer efficacy, whereby these combined
responses take place either simultaneously or in a sequential manner at
the pathological site [33–35]. Very recently, we developed pH and redox
dual-responsive prodrug nanogels using an inverse nanoprecipitation
method without any surfactants. After cleavage of the hydrazone linkers
and degradation of S–S crosslinked networks under intracellular
conditions, conjugated DOX could be efficiently released from the
hyperbranched polyglycerol (hPG) nanogel matrix and internalized by
the tumor cells to induce cell death [36].
2. Experimental section
2.1. Materials
Cystamine bishydrochloride (Aldrich, 96%), succinic anhydride
(Aldrich, 99%), triethylamine (Acros, 99%), propargyl chloroformate
(Aldrich, 96%), propargylamine (Aldrich, 98%), 2,3-dimethylmaleic an-
hydride (DMMA, Acros, 97%), mesyl chloride (MsCl, Acros, 99%), sodium
azide (Acros, 99%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC, Acros, 97%), N-hydroxysuccinimide (NHS, Acros, 98%), doxorubi-
cin hydrochloride (DOX, Sigma, 98%) were used as received. Polyvinyl
alcohol (PVA, Mowiol 3-97, Mw = 16,000 g/mol) was provided by
Kuraray Europe GmbH (Germany). For cell culture experiments,
MCF-7 cells (DSMZ no.: 115) were cultured in RPMI supplemented
Scheme 1. Illustration of charge-conversional and reduction-sensitive PVA nanogels for enhanced cell uptake and efficient intracellular DOX release.
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012

W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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with 10% fetal calf serum, MEM nonessential amino acids, 1 mM sodium
2.6. Synthesis of azido-functionalized PVA (PVA-N3)
pyruvate, and 10 g/mL human insulin HeLa cells (DSMZ no.: ACC 57)
were cultured in RPMI supplemented with 10% fetal calf serum and
1 mM sodium pyruvate.
The synthesis of azido-functionalized PVA was carried out in a two-
step protocol [38]. In the first step, 10 mL pyridine and methylsulfonyl
chloride (0.84 mL, 10.90 mmol) were added to a solution of PVA
(2.00 g, 36.38 mmol of OH group) in DMSO (100 mL). The reaction
mixture was stirred overnight in a cold water bath; NaN3 (1.42 g,
21.8 mmol) was added into the solution. The reaction was allowed
to proceed with stirring overnight at 80 °C. After cooling to room
temperature, the polymer was isolated by precipitation in diethyl
ether/acetone (1/1), and the solid was further purified by dialysis in
water to give a light yellow solid after freeze-drying.
2.2. Characterization
1H NMR spectra were recorded on a Bruker ECX 400. The chemical
shifts were calibrated against residual solvent peaks as the internal stan-
dard. IR measurements were carried out on a Nicolet AVATAR 320 FT-IR
5 SXC that was equipped with a DTGS detector from 4000–600 cm−1
.
The size of nanogels was determined by dynamic light scattering
(DLS) at 25 °C using a Zetasizer Nano-ZS from Malvern Instruments
equipped with a 633 nm He–Ne laser. The morphology of nanogels
was observed using cryogenic transmission electron microscopy
(Cryo-TEM, Philips CM12). Microscopy was carried out at a 94 K sample
temperature using the low-dose protocol of the microscope at a primary
magnification of ×58,300 and an accelerating voltage of 100 kV
(LaB6-illumination).
2.7. Preparation of PVA nanogels by inverse nanoprecipitation via ‘click’
chemistry
PVA-COOH-alkynyl and PVA-N3 were dissolved separately in phos-
phate buffer (PB, pH 7.4, 10 mM) with a concentration of 10 mg/mL.
THPTA (110 mg/mL, 10 μL), CuSO4 (21 mg/mL, 10 μL), and NaAsc
(50 mg/mL, 10 μL) were sequentially added into 800 μL of PVA-
alkynyl solution. The solution was kept at 4 °C and mixed with 800 μL
of PVA-N3 solution. The mixed solution was quickly added into 20 mL
of acetone. After 24 h, the reaction was quenched by the addition of ex-
cess dimethylmaleic propargylamide (charge-conversional nanogel)
dissolved in acetone or propargylalcohol (non-charge-conversional
nanogel). After 10 min, 20 mL of PB was added and acetone was evapo-
rated to obtain PVA nanogels dispersed in water. The nanogels were
collected by centrifugation (5000 rpm) with a MWCO of 10,000, wash-
ing 5 times with Milli-Q-water, and freeze-drying. The nanogel samples
were redispersed in PB by sonication and characterized by DLS and
cryo-TEM. For the preparation of DOX-loaded nanogels, 160 or 320 μL
of DOX∙HCl solution in Milli-Q-water (5.0 mg/mL) was added into
800 μL of PVA-alkynyl solution, and the following steps were carried
out similar to the preparation of blank NGs except that the procedure
was performed in the dark.
2.3. Synthesis of propargyl-cystamine
Cystamine bishydrochloride (4.00 g, 17.6 mmol) and triethylamine
(7.40 mL, 3 equiv.) were dissolved in methanol (300 mL), followed by
drop wise addition of propargyl chloroformate (2.10 g, 17.6 mmol) dis-
solved in DCM at 0 °C (Scheme S1). After 4 h the solvent was evaporat-
ed, and NaH2PO4 aqueous solution was added (100 mL 1 M, pH 4.2). The
aqueous solution was extracted with diethyl ether three times to re-
move the byproduct, di-propargyl-cystamine. The aqueous solution
was basified to pH 9 by NaOH aqueous solution (1 M) and extracted
with ethyl acetate three times. The combined organic phases were
dried over MgSO4 and evaporated to yield the product as a viscous
yellow liquid. Yield: 1.32 g (32%). 1H NMR (400 MHz, CD3OD): δ 4.63
(2H, –CH2-C ≡ CH), 3.40 (2H, –NH-CH2-CH2–), 2.91 (2H, –CH2-NH2),
2.86 (1H, –C ≡ CH), 2.78 (4H, –CH2-SS-CH2–) (Fig. S1).
2.8. Reduction-sensitivity and surface charge-conversion of PVA nanogels
PVA nanogel suspension (1.0 mg/mL) was divided into two aliquots
of 1 mL, and 10 μL of GSH solution (1.0 M) was added to one of the two
aliquots with a final GSH concentration of 10 mM. The samples were
slowly stirred at 37 °C under a N2 atmosphere and the change in the
nanogel size was monitored over time by DLS. To monitor the surface
charge conversion, PVA nanogel suspension (1.0 mg/mL) was similarly
divided into two aliquots of 1 mL, whereby 25 μL of acetate buffer
(pH 6.8, 4 M) was added to one of the two aliquots with a final pH of
6.8. The surface charge of the nanogels was followed by zeta potential
measurements.
2.4. Synthesis of dimethylmaleic propargylamide
Propargylamine (0.50 g, 9.09 mol) and 2,3-dimethylmaleic anhy-
dride (1.14 g, 9.09 mmol) were sequentially dissolved in anhydrous
THF (25 mL), and the solution was stirred for 4 h at room temperature
(Scheme S2). The final product was recrystallized in THF. Yield: 1.50 g
(91%). 1H NMR (400 MHz, CD3OD): δ 3.70 (2H, –CH2-C ` CH), 2.01
(1H, –CH2-C ` CH), 1.89 (6H, –C(CH3) _ C(CH3)–) (Fig. S2).
2.5. Synthesis of carboxyl-alkynyl-functionalized PVA (PVA-COOH-alkynyl)
2.9. In vitro release of DOX from PVA nanogels
The synthesis of carboxyl-alkynyl functionalized PVA (PVA-COOH-
alkynyl) was carried out in two steps. Briefly, PVA (2.00 g, 36.38 mmol
of OH group) was dissolved in anhydrous DMSO (100 mL) at 50 °C
and the solution cooled down to room temperature. Succinic anhydride
(1.09 g, 10.90 mmol) and a catalytic amount of Et3N were sequentially
added to the reaction. After 24 h reaction, a sample was taken for deter-
mination of carboxyl functionality. The reaction solution was divided
into two aliquots. EDC (494.2 mg, 2.58 mmol) and NHS (296.7 mg,
2.58 mmol) were added to both aliquots (PVA-COOH, 5.17 mmol
of COOH group). After 30 min, propargyl-cystamine (604.0 mg,
2.58 mmol) and propargylamine (142.0 mg, 2.58 mmol) were separately
added into these two aliquots, and the reaction was allowed to proceed
with overnight stirring at room temperature. The polymers were isolated
by dialysis, first in ethanol for 8 h, then in water for another 48 h. After
that they were freeze-dried.
The in vitro release of DOX from PVA nanogels was investigated at
37 °C under four different conditions: (i) PB (10 mM, pH 7.4), (ii) acetate
buffer (10 mM, pH 5.5), (iii) PB (10 mM, pH 7.4) containing 10 mM GSH,
and (iv) acetate buffer (100 mM, pH 5.5) containing 10 mM GSH.
DOX-loaded micelle suspension was divided into six aliquots and imme-
diately transferred to a dialysis tube with a MWCO of 12,000–14,000. The
dialysis tube was immersed into 20 mL of appropriate buffer and shaken
at 37 °C. At set time intervals, 5.0 mL of the release medium was taken
out and replenished with an equal volume of fresh medium. To avoid
oxidation of GSH, the release media were perfused with argon gas.
The concentration of DOX was determined by fluorescence measure-
ments (excitation at 480 nm). To determine the drug loading content,
DOX-loaded nanogel suspensions were freeze-dried, dissolved in
DMSO, and analyzed with UV spectroscopy. A calibration curve was ob-
tained using DOX/DMSO solutions with different DOX concentrations.
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
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4
W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
To determine the amount of released DOX, calibration curves were run
with DOX/phosphate buffer solutions with different DOX concentra-
tions at pH 7.4. The emission at 480 nm was recorded. Release experi-
ments were conducted in triplicate. The results are presented as the
average standard deviation.
(10 mM, pH 7.4) were added. The cells were incubated for another
48 h, and then 10 μL of MTT solution (5 mg/mL) was added. The cells
were incubated for 4 h, and the medium was replaced by 150 μL of
DMSO to dissolve the resulting purple crystals. The optical densities
were measured by a microplate reader at 570 nm. The experiments
were conducted in triplicate and the results were presented as the aver-
age standard deviation.
Drug loading content (DLC) and drug loading efficiency (DLE) were
calculated according to the following formula:
To evaluate whether the high drug efficacy was specially caused by
tumoral pH-activated endocytosis, MCF-7 cells seeded in a 96-well
plate at a density of 1 × 104 cells per well for 24 h were incubated at
pH 6.8 or 7.4 for 4 h with DOX-loaded nanogels or free DOX. After
that, the medium was replaced by 100 μL of fresh medium and the
cells were cultured for another 48 h. The same procedure was carried
out as mentioned above for the cytotoxicity measurement.
DLCðwt:%Þ ¼ ðweight of loaded drug=total weight of polymer and loaded drugÞ
ꢀ100%
DLEð%Þ ¼ ðweight of loaded drug=weight of drug in feedÞ ꢀ 100%:
2.10. Confocal laser scanning microscopy
MCF-7 cells were plated on microscope slides in a 24-well plate
(5 × 103 cells/well) using 1640 culture medium containing 10% FBS.
After 24 h incubation, the medium was replaced by 450 μL of fresh cul-
ture medium and 50 μL of prescribed amounts of DOX-loaded nanogels
or free DOX. After incubation for 4 h and 8 h, respectively, the culture
medium was removed and the cells were washed twice with PBS. The
cells were fixed with 4% paraformaldehyde and the cell nuclei were
stained with DAPI. Fluorescence images of cells were obtained with
Confocal Laser Scanning Microscope (Leica, Germany) and analyzed
by Leica 2.6.0 software.
To study the cellular uptake of charge-conversional nanogels at
different pHs, DOX was linked to PVA-COOH-(SS-alkynyl) using a
carbodiimide chemistry via the amino group of DOX and the carbox-
yl group of the polymer. DOX-conjugated nanogels were prepared as
mentioned above using dimethylmaleic propargylamide (charge-
conversional nanogel) or propargyl alcohol (non-charge-conversional
nanogel) as the terminators. Similarly, MCF-7 cells were plated on mi-
croscope slides in a 24-well plate (5 × 103 cells/well) using 1640 culture
medium containing 10% FBS. After 24 h incubation, the medium was re-
placed by 450 μL of fresh culture medium with two pHs (pH 6.8 or 7.4)
and 50 μL of prescribed amounts of DOX-loaded nanogels or free DOX.
After incubation for 1 h and 2 h, respectively, the culture medium was
removed and the cells were washed twice with PBS. The cells were
fixed with 4% paraformaldehyde for 20 min, incubated with early
endosome antibody-FITC (Invitrogen, Germany) at 37 °C for 1 h, and
the cell nuclei were stained with DAPI. Fluorescence images of cells
were obtained with CLSM.
3. Results and discussion
3.1. Synthesis of functional PVA derivatives
Carboxyl/alkynyl-functionalized PVA was prepared by two steps:
(i) carboxylation of PVA with succinic anhydride; (ii) conjugation of
propargyl-cystamine or propargylamine via carbodiimide chemistry
(Scheme 2). The 1H NMR displayed new signals at 2.42 that corresponded
to methylene protons in the vicinity of the carboxyl group, whereby the
carboxyl was calculated at 28.5% according to the integral ratio of δ 1.94
(methyl group on PVA side chain) and δ 2.42 (Fig. S3B). The conjugation
of propargyl-cystamine or proparylamine to carboxyl-functionalized
PVA efficiently proceeded with 9.5% functionality as expected (Fig. S3C).
PVA-N3 was accordingly synthesized with a functionality of 23.4% and
characterized by FT-IR (Fig. S4A) and elemental analysis [38].
3.2. Formation of reduction-sensitive crosslinked nanogels
Copper-catalyzed Huisgen [2 + 3] cycloaddition was selected as a
crosslinking reaction due to its high conversion, fast reaction kinetics,
and good bioorthogonality [39]. The aqueous solutions of PVA-COOH-
(SS-alkynyl) and PVA-N3 were separately prepared with a concentra-
tion of 10 mg/mL. After cooling to 4 °C, a catalytic amount of THPA/
CuSO4/NaAsc was added, and PVA nanoaggregates were formed by pre-
cipitation into acetone. The polymer concentration drastically increased
during this process, which induced gelation reactions and PVA network
formation. After gelation, excessive propargyl-cystamine was added
to quench the remaining reactive azido groups. Finally, the charge-
conversional reducible PVA nanogels (CC-SS-NGs) were collected by
evaporating acetone, washing with PB, and freeze-drying. Interestingly,
CC-SS-NGs with an average size of 118 nm were determined by dynam-
ic laser scattering (DLS) (Fig. 1A), which is larger when dispersed in ac-
etone (98 nm) due to their swelling property in water. The cryo-TEM
micrograph showed that these nanogels had a spherical morphology
with particle sizes in accord with those determined by DLS (Fig. 1B).
Meanwhile, the charge-conversional nanogels without S-S bonds
(CC-NSS-NGs) were prepared with an average size of 88 nm as a
reduction-insensitive control (Fig. 1A). To confirm whether the
“click” reaction went well during the nanogel formation process,
nanogels diluted with DMSO were measured by DLS. The result showed
that both of the chemical crosslinked CC-SS-NGs and CC-NSS-NGs were
only swollen in DMSO, in contrast to the hydrogen-bonding induced
physically crosslinked PVA nanogels, which dissolved into unimer in
DMSO (Fig. 1A). FT-IR also showed that during the nanogels' formation
the peak at 2000 cm−1 attributed to azido group decreased after
crosslinking and disappeared with the addition of a terminator (Fig. S4).
It is confirmed that combining this inverse nanoprecipitation technique
with a click reaction is a very facile and efficient method for the prepara-
tion of hydrophilic nanogel particles.
2.11. Real time cell analysis (RTCA) of DOX-loaded PVA nanogels
RTCA was used for dynamically monitoring the cell proliferation
and viability in real time, based on the Real-Time Cell Electronic Sensor
(RT-CES) system. 50 μL of culture medium was added to each well of the
E-plate 96 (Roche, Mannheim, Germany) for the background measure-
ment, followed by adding 40 μL of MCF-7 cell suspension into the sensor
wells (1 × 104 cells/well). After 24 h incubation, 10 μL of nanogel sam-
ples at different concentrations in PB were added into each well. Four
replicates for each sample were measured and the results were report-
ed as the mean value. The E-plate was incubated with 5% CO2 at 37 °C
and monitored on the RTCA SP system (Roche, Mannheim, Germany)
with time intervals of at least 15 min for 48 h after treatment. Analysis
was performed using the RTCA software version 2.0.
2.12. Cytotoxicity of DOX-loaded PVA nanogels
The cytotoxicity of blank and DOX-loaded PVA nanogels was
studied by MTT assay using MCF-7 and HeLa cells. Cells were seeded
into a 96-well plate at a density of 1 × 104 cells per well in 90 μL of 1640
culture medium containing 10% FBS and incubated at 37 °C with 5% CO2.
After 24 h, 10 μL of nanogel samples at different concentrations in PB
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
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W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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Scheme 2. Synthesis of carboxyl-alkynyl functionalized PVA (PVA-COOH-alkynyl). Conditions: (i) succinic anhydride, Et3N, DMSO, room temperature, overnight; (ii) propargyl amine or
propargyl-cystamine, EDC, NHS, DMSO, room temperature, overnight.
3.3. Surface charge-conversion and reduction-sensitivity
(Fig. S5), which indicates that the nanogels kept their spherical struc-
ture before cellular uptake and subsequent restrictions from drug
leaching.
It has been demonstrated that the amide bond neighboring to the
dimethylmaleic group was pH-labile even under such slightly acidic
conditions as pH 6.8 [40,41]. The CC-SS-NGs were therefore expected
to show a similar charge-conversional behavior in the tumoral extracel-
lular pH environment, i.e., at pH 6.8. Initially, the CC-SS-NGs were neg-
atively charged at pH 7.4. When the pH value of the medium was
adjusted to pH 6.8 and kept in equilibrium for 2 min, however, the
zeta potential of these nanogels significantly increased and became
positive within 10 min because the cleavage of the amide bond in the
ultra pH-sensitive terminator transformed the carboxyl groups to
amino groups (Fig. 2A). Despite the slow increase of the nanogels' zeta
potential upon incubation at pH 7.4, the charge remained negative for
3 h. Interestingly, we observed that the zeta potential of non-charge-
conversional reduction-sensitive nanogels (NCC-SS-NGs, using propar-
gyl alcohol as the pH-insensitive terminal group) did not significantly
change at both pH 6.8 and 7.4 and stayed negative for the whole moni-
toring process (Fig. 2A). Since cell membranes are generally negatively
charged, the charge-conversion behavior of CC-SS-NGs in the tumoral
extracellular pH environment improved their internalization by tumor
cells. It should be noted that, although the zeta potential of CC-SS-NGs
quickly increased at pH 6.8, the NG size did not change after 18 h
The reduction-sensitivity of PVA nanogels was studied by monitor-
ing the nanogels' size over time in response to 10 mM GSH using DLS.
The results showed that CC-SS-NGs quickly dissociated in the presence
of 10 mM GSH, whereby a large population was observed that had re-
duced sizes of about 60 nm and 30 nm at 1 h and 6 h, respectively
(Fig. 2B). Unexpectedly, the nanogels did not degrade into soluble
unimers. This was probably due to the presence of the hydrogen bond-
ing and propargyl-cystamine hydrophobic modification, which resulted
in small PVA aggregations in the aqueous solution. In contrast, little size
change was detected for CC-NSS-NGs within 24 h under the same re-
ducing conditions (Fig. 2B), as well as for the CC-SS-NGs within 24 h
in the absence of GSH. It is therefore evident that CC-SS-NGs can be
rapidly disrupted under intracellular-mimicking reducing conditions.
3.4. Loading and triggered release of DOX
DOX can be readily encapsulated into nanogels by inverse nano-
coprecipitation in acetone, followed by a “click” reaction to form
the DOX-loaded nanogels. These nanogels can be quenched by
dimethylmaleic propargylamide (charge-conversional nanogels) or
CC-SS-NG
CC-SS-NG in DMSO
CC-NSS-NG
B
A
36
32
CC-NSS-NG in DMSO
PVA physical NG
28
PVA physical NG in DMSO
24
20
16
12
8
4
0
1
10
Size (nm)
100
1000
Fig. 1. Size distribution of PVA nanogels determined by DLS (A), and cryo-TEM image of charge-conversional reduction-sensitive PVA nanogels (CC-SS-NGs) (B).
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012

6
W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
CC-SS-NG 0 h
CC-SS-NG, pH 6.8
A
B
24
20
16
12
8
10
8
CC-SS-NG 1 h, 10 mM GSH
CC-SS-NG 6 h, 10 mM GSH
CC-SS-NG 24 h
CC-NSS-NG 0 h
CC-NSS-NG 24 h, 10 mM GSH
CC-SS-NG, pH 7.4
NCC-SS-NG, pH 6.8
NCC-SS-NG, pH 7.4
6
4
2
0
-2
-4
-6
-8
-10
4
0
0
40
80
120
160
200
10
100
1000
Time (min)
Size (nm)
Fig. 2. Changes in the zeta potential of PVA nanogels at pH 6.8 and 7.4 (A), and in the nanogel sizes in response to 10 mM GSH (B).
propargyl alcohol (non-charge-conversional nanogels), and, the
unloaded DOX can be removed by centrifugation. The results showed
that both CC-SS-NGs and CC-NSS-NGs had a higher loading capability
for DOX than the physically crosslinked PVA nanogels (Fig. S6). This
can be attributed to the following factors: (1) the presence of carbox-
yl groups in the nanogels that significantly improved the DOX-
loading content due to electrostatic interactions [42] and (2) the
propargyl-cystamine hydrophobic modification and the chemically
crosslinked networks in the nanogels, which provided the hydro-
phobic interaction with DOX. Due to the similar degrees of function-
ality (DF) of the same PVA, CC-SS-NGs and CC-NSS-NGs exhibited
similar DOX loading levels with the drug loading efficiency (DLE)
ranging from 67.4 to 79.2% at theoretical drug loading contents
(DLC) of 5 and 10 wt.% (Table 1). The DLE decreased as the theoreti-
cal DLC increased. The average size of CC-SS-NGs expanded from 118
to 154 nm after loading 4.0 wt.% DOX and then rose to 177 nm upon a
further increase in the DLC to 7.1 wt.%. All the DOX-loaded nanogels
were shown to be sufficiently stable with no size change after 24 h in
the presence of 10% FBS.
The in vitro release of DOX from nanogels was investigated at 37 °C
in the following four different media: (i) pH 7.4, (ii) pH 5.5, (iii) pH 7.4
and 10 mM GSH, and (iv) pH 5.5 and 10 mM GSH. The DOX release at
physiological pH (pH 7.4) was highly restricted with a released amount
of ca. 23% after 48 h (Fig. 3A). The release of DOX was significantly accel-
erated at pH 5.5 with 63% of DOX released after 48 h under otherwise
the same conditions and was likely due to the decrease of the electro-
static interaction between nanogels and DOX. A similar pH-dependent
DOX release behavior could be also observed from the poly(acrylic
acid)-DOX complex [43]. The DOX release rate accelerated under the re-
ducing environment containing 10 mM GSH at pH 7.4, in which 58% of
drug was released in 48 h. Notably, the fastest and almost complete
drug release was observed at pH 5.5 in the presence of 10 mM GSH,
wherein 95% of DOX was released in 10 h (Fig. 3A). CC-SS-NGs were
rapidly destabilized and the electrostatic interaction with DOX became
weaker under the dual stimuli. In contrast, DOX release from CC-NSS-
NGs was not influenced so much by the presence of GSH, both at
pH 5.0 and pH 7.4 (Fig. 3B), in which only 8% difference in cumulative
DOX release was observed most likely due to the diffusion of GSH
into the nanogels. Li et al. also reported that the anticancer drug pac-
litaxel (PTX) release from the (ortho ester)-containing and disulfide-
crosslinked nanogels was greatly accelerated by a cooperative effect
of both acid-triggered hydrolysis and DTT-induced degradation [44].
These results clearly indicate that the DOX released from CC-SS-NGs
proceeds in a controlled manner and can be activated by a synergistic
trigger of low pH and reductive environments.
3.5. Cellular uptake and intracellular release of DOX
To further demonstrate that nanogels can be more efficiently internal-
ized by cancer cells at extracellular pH, we investigated the cellular uptake
behaviors of DOX-conjugated nanogels at pH 7.4 and 6.8. MCF-7 cells
were incubated with DOX-conjugated CC-SS-NGs and NCC-SS-NGs at
each pH for 1 h and 2 h, and their cellular distribution was observed
with confocal laser scanning microscopy (CLSM). It was demonstrated
that charge-conversional CC-SS-NGs were internalized at pH 6.8 after
1 h incubation and intensively distributed in the cytoplasm after 2 h over-
laying with the endosomes (Fig. 4A and B), while it was rarely observed in
cells incubated with CC-SS-NGs at pH 7.4 after 2 h (Fig. 4C). The CLSM im-
ages also showed that the DOX-conjugated, non-charge-conversional
NCC-SS-NGs, as a negative control, had a very low cellular uptake at
both pH 6.8 and 7.4 for 2 h, similar to that of CC-SS-NGs at pH 7.4
(Fig. S7). It was concluded that the charge-conversional CC-SS-NGs
indeed exhibited significantly enhanced cellular internalization at the
tumoral extracellular pH.
To demonstrate that DOX could be efficiently released from the
nanogels following cellular uptake and be further internalized with
the cell nucleus, we investigated the intracellular DOX release from
the DOX-loaded nanogels in MCF-7 cells using CLSM. Interestingly, sig-
nificant DOX fluorescence was observed in the cytoplasm and nuclei of
MCF-7 cells following 4 h incubation with CC-SS-NGs (Fig. 5A). The DOX
fluorescence became even stronger in the cell nuclei at a longer incuba-
tion time of 8 h (Fig. 5B), which was notably similar for MCF-7 cells fol-
lowing 4 h incubation with free DOX (positive control, Fig. 5E). In
contrast, very weak DOX fluorescence was observed in the peri-nuclei
region of cells incubated for 4 h with CC-NSS-NGs (reduction-insensitive
control) under otherwise the same conditions (Fig. 5C), and this re-
leased DOX was found in the cell nuclei after 8 h incubation (Fig. 5D).
The different intracellular drug release behaviors of CC-SS-NGs and
CC-NSS-NGs were most likely due to the fast and complete DOX release
from CC-SS-NGs following their escape from the endosomes as a result
Table 1
Characterization of DOX-loaded PVA nanogels.
Nanogels
DLC (wt.%)
Theory
DLE (%)
Sizeb (nm)
PDIb
Determineda
CC-SS-NG
5
10
5
4.0
7.1
3.8
6.7
79.2
70.5
77.0
67.4
154.1
177.3
91.3
0.18
0.23
0.12
0.11
CC-NSS-NG
10
105.7
a
Determined by UV measurements.
Determined by DLS measurements.
b
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012

W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
7
SS NG, pH 7.4
SS NG, pH 7.4, 10 mM GSH
SS NG, pH 5.5
SS NG, pH 5.5, 10 mM GSH
NSS NG, pH 7.4
NSS NG, pH 7.4, 10 mM GSH
NSS NG, pH 5.5
A
B
100
80
60
40
20
0
100
80
60
40
20
0
NSS NG, PH 5.5, 10 mM GSH
0
10
20
30
40
50
0
10
20
30
40
50
Time (h)
Time (h)
Fig. 3. pH and/or redox-triggered release of DOX from CC-SS-NGs (A) and CC-NSS-NGs (B) at 37 °C at pH 7.4 or 5.5 in the presence or absence of 10 mM GSH.
of reduction-triggered nanogel dissociation in the cytosol, which is in
line with the in vitro release under dual stimuli. Although the DOX
release from CC-NSS-NGs could be accelerated under endosomal pH, it
would have been significantly restricted in the cytosol (reducing envi-
ronment and neutral pH), as indicated in Fig 3B. These results demon-
strate that pH and reduction dual-responsive CC-SS-NGs mediate a
more efficient intracellular anticancer drug release than reduction-
insensitive nanogels.
3.6. Tumor cytotoxicity of DOX-loaded nanogels
MTT assays in MCF-7 and HeLa cells revealed that the blank PVA
nanogels were practically non-toxic (cell viabilities ≥ 80%) up to a test-
ed concentration of 1.0 mg/mL (Fig. 6A and S8A), which confirms that
these degradable nanogels have good biocompatibility. DOX-loaded
nanogels, however, displayed significant cell killing activity towards
MCF-7 and HeLa cells following 48 h incubation (Fig. 6B and S8B). It
Fig. 4. CLSM images of MCF-7 cells following 1 or 2 h incubation with DOX-conjugated charge-conversional CC-SS-NGs and free DOX (5 μg/mL). The images show for each panel from left to
right cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red), early endosome labeled by antibody-FITC (green), and overlays of three images. The scale bars correspond to 25 μm
in all the images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012

8
W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
Fig. 5. CLSM images of MCF-7 cells after 4 or 8 h incubation with DOX-loaded PVA nanogels and free DOX (5 μg/mL). The images in each panel depict from left to right cell nuclei stained by
DAPI (blue), DOX fluorescence in cells (red), and overlays of both images. The scale bars correspond to 25 μm in all the images. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
should be noted that DOX-loaded CC-SS-NGs had low half inhibitory
concentration (IC50) values of 0.32 and 0.45 μg DOX equiv./mL for
MCF-7 and HeLa cells, respectively, which was lower than those obtain-
ed with the reduction-insensitive CC-NSS-NGs under otherwise the
same conditions (IC50 = 2.04 and 1.42 μg DOX equiv./mL for MCF-7
and HeLa cells, respectively). This higher cell killing activity of CC-SS-
NGs is in accordance with the CLSM observations that CC-SS-NGs medi-
ated a faster and better intracellular drug release than the correspond-
ing reduction-insensitive CC-NSS-NGs. Interestingly, it was found that
both DOX-loaded charge-conversional CC-SS-NGs and CC-NSS-NGs pre-
sented superior cell killing activity towards the cancer cells compared to
the corresponding non-charge converted NCC-SS-NGs and NCC-NSS-
NGs, which was attributed to enhanced cellular uptake via the cleavage
of pH-labile amide bonds.
24 h undisturbed cell growth, the substances were supplied with
nanogel samples at a DOX concentration of 5 μg/mL. Cytotoxicity effects
of DOX-loaded nanogels were recorded in a time-dependent decrease of
the CI value. As predicted, DOX-loaded CC-SS-NGs efficiently inhibited
cell growth, and the CI value started to decrease after 14 h treatment
(Fig. 7A), which seemed longer than the detected time for intracellular
release from CLSM, which might be due to the fact the released DOX
needed more time to intercalate with the DNA in cell nuclei in order
to induce cell death after cellular uptake [45]. It should be noted that
the inhibition of cell growth treated with both DOX-loaded CC-NSS-
NGs and NCC-NSS-NGs was much lower than with DOX-loaded CC-SS-
NGs, which was mainly due to insufficient intracellular drug release
from the reduction-insensitive nanogel carriers. Notably, as the same
reduction-sensitive carriers, DOX-loaded non-charge-conversional
NCC-SS-NGs showed a characteristic delay in the onset of cytotoxicity
(approximately 26 h) as compared to the charge-conversional CC-SS-
NGs. To further study the pH-induced cellular uptake and improved
cell killing activity of the DOX-loaded nanogels, MCF-7 cells were
incubated at pH 6.8 or 7.4 for only 4 h with DOX-loaded nanogels. The
The cytotoxicity of MCF-7 cells incubated with DOX-loaded nanogels
was also monitored by a RTCA assay that could reflect the real-time cell
number, cell morphology, and degree of cell adhesion. Adherent cells
attached to the bottom of the well were able to cause an increase in
impedance and displayed an increase in the cell index (CI) value. After
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
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W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
9
CC-SS-NG
NCC-SS-NG
CC-NSS-NG
NCC-NSS-NG
B
A
100
80
60
40
20
0
120
100
80
60
40
20
0
PVA nanogels
IC50 (ug/mL)
CC-SS-NG
NCC-SS-NG
CC-NSS-NG
NCC-NSS-NG
Free DOX
0.32
0.65
2.04
2.97
0.18
0.05
0.1
0.2
0.5
1
1E-3
0.01
0.1
1
10
Nanogel concentration (mg/mL)
DOX concentration (ug/mL)
Fig. 6. Cytotoxicity of blank (A) and DOX-loaded (B) PVA nanogels using MCF-7 cells. The cells were incubated with nanogels for 48 h. The data are presented as the average standard
deviation (n = 4).
culture medium was removed and replenished with fresh culture
medium and the cells were cultured for another 48 h. Interestingly,
MCF-7 cells treated with DOX-loaded CC-SS-NGs at pH 6.8 displayed a
lower cell viability of 43.6% than those incubated with DOX-loaded
CC-SS-NGs at pH 7.4 as well as with DOX-loaded non-charge converted
NCC-SS-NGs at pH 6.8 (cell viability: 71.0%) under otherwise the same
conditions (Fig. 7B). It should be noted that, although CC-SS-NGs and
CC-NSS-NGs are both charge-conversional nanogels, DOX-loaded
reduction-sensitive CC-SS-NGs displayed better cell killing activity than
the reduction-insensitive CC-NSS-NGs (cell viability: 58.9%), with a cell
killing activity very close to that of free DOX (cell viability: 47.5%). These
results demonstrate that CC-SS-NGs can facilitate efficient cellular uptake
and drug delivery to achieve remarkable triggered cell killing activity.
in significant tumor cell killing activity. This is a new design and study of
degradable PVA nanogel systems that respond to tumoral extracellular
pH, intracellular endosomal pH, as well as cytoplasmic glutathione for
enhanced cellular uptake and a more efficient intracellular release of an-
ticancer drugs. These intelligent blank nanogels have little cytotoxicity
and promote a fast and maximum drug release inside the cancer cells
in which the drug release is being activated during the whole extracel-
lular/intracellular trafficking process. These charge-conversional and
dual-responsive degradable nanogel systems provide a promising
platform for a targeted and controlled intracellular release of potent
chemotherapeutics.
Acknowledgments
4. Conclusions
We thank Dr. Markus Meise (Head of Research & Technical Services
in Kuraray Europe GmbH) for providing the PVA samples. We also thank
Dr. Pamela Winchester for carefully proofreading this manuscript. This
work was financed by the German Research Foundation in the context
We have demonstrated that charge-conversional and reducible PVA
nanogels efficiently deliver and release DOX into cancer cells and result
CC-SS-NG
NCC-SS-NG
CC-NSS-NG
NCC-NSS-NG
Free DOX
B
A
***
100
80
60
40
20
0
5
CC-SS-NG
NCC-SS-NG
CC-NSS-NG
NCC-NSS-NG
Free DOX
**
***
4
3
Blank control
2
Samples adding
1
0
pH 6.8
pH 7.4
0
10
20
30
40
50
60
70
80
Time (h)
Fig. 7. (A) Cytotoxicity profiles determined online with an xCelligence RTCA device. Cell proliferation is given as cell index recorded over time. The nanogel samples or free DOX were added
into the MCF-7 cells after 24 h cell seeding. DOX dosage was 5 μg/mL. (B) Tumor cell killing activity of DOX-loaded nanogels in MCF-7 cells using MTT assays. The cells were cultured with
DOX-loaded nanogels at pH 6.8 or 7.4. DOX dosage was 10 μg/mL. After 4 h incubation, the medium was replaced by 100 μL of fresh culture medium, and the cells were cultured for another
48 h. Cells without treatment were used as a blank control. Data are presented as the average standard deviation (n = 4, Student's t-test, **p b 0.01, ***p b 0.001).
Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012

10
W. Chen et al. / Journal of Controlled Release xxx (2014) xxx–xxx
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Please cite this article as: W. Chen, et al., Charge-conversional and reduction-sensitive poly(vinyl alcohol) nanogels for enhanced cell uptake and
efficient intracellular doxo..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.11.012