A new drug-loading technique with high efficaciency and sustained-releasing ability via the Pickering emulsion interfacial assembly of temperature/pH- sensitive nanogels

Article abstract of DOI:10.1016/j.reactfunctpolym.2013.08.004

pNIPAM nanogels, which exhibited rich sol-gel transition behavior, have been used extensively in the biomedical fields such as drug delivery, blood vessel embolization and tissue-engineering Owing to their limitation of 3D networks, pNIPAM nanogels have low drug-loading amount and poor sustained releasing properties. In the present research, Pickering emulsion combined with solvent evaporation (PESE) is developed firstly as a new drug-loading technique of pNIPAM nanogels in our knowledge. The entrapment efficiency (EE%) reached nearly 100%, and DOX loading amount (DL%) could reach 15%. Owing to ionic bonding interaction of DOX molecules and sulfonamide groups, DOX loaded PNS nanogels by PESE (PNS-D nanogels) show a slow releasing behavior, only 13.2% for 48 h in pure water, without any burst release. As the ionic strength of media increased, the DOX-releasing amount from PNS-D-10 nanogels increased to 29.5%, 41.6% and 48.0% respectively in 0.9 wt%, 3.0 wt% and 5.0 wt% of NaCl solutions for 48 h. The loading DOX has significant effect on the rheologic behavior of nanogel dispersions. PESE technique is hopeful to be developed as a universal hydrophobic drug loading method of nanogels, and will be extensively applied in drug delivery and tissue engineering.

Full text of DOI:10.1016/j.reactfunctpolym.2013.08.004

Reactive & Functional Polymers 73 (2013) 1537–1543
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Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
A new drug-loading technique with high efficaciency and
sustained-releasing ability via the Pickering emulsion interfacial
assembly of temperature/pH-sensitive nanogels
Guofeng Zhou a,c,1,3, Yanbing Zhao a,b, , Jindong Hu b,2, Liang Shen a,1, Wei Liu a,b,1,2, Xiangliang Yang a,b,1,2
⇑
a National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan City, PR China
b College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan City, PR China
c Interventional Radiology Department of Wuhan Union Hospital, Huazhong University of Science and Technology, Wuhan City 430022, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
pNIPAM nanogels, which exhibited rich sol–gel transition behavior, have been used extensively in the
biomedical fields such as drug delivery, blood vessel embolization and tissue-engineering Owing to their
limitation of 3D networks, pNIPAM nanogels have low drug-loading amount and poor sustained releasing
properties. In the present research, Pickering emulsion combined with solvent evaporation (PESE) is
developed firstly as a new drug-loading technique of pNIPAM nanogels in our knowledge. The entrap-
ment efficiency (EE%) reached nearly 100%, and DOX loading amount (DL%) could reach 15%. Owing to
ionic bonding interaction of DOX molecules and sulfonamide groups, DOX loaded PNS nanogels by PESE
(PNS-D nanogels) show a slow releasing behavior, only 13.2% for 48 h in pure water, without any burst
release. As the ionic strength of media increased, the DOX-releasing amount from PNS-D-10 nanogels
increased to 29.5%, 41.6% and 48.0% respectively in 0.9 wt%, 3.0 wt% and 5.0 wt% of NaCl solutions for
48 h. The loading DOX has significant effect on the rheologic behavior of nanogel dispersions. PESE tech-
nique is hopeful to be developed as a universal hydrophobic drug loading method of nanogels, and will be
extensively applied in drug delivery and tissue engineering.
Received 17 January 2013
Received in revised form 18 June 2013
Accepted 13 August 2013
Available online 22 August 2013
Keywords:
Pickering emulsion
Temperature/pH-sensitive
Nanogel
Drug loading
Sustained release
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
centrated pNIPAM nanogels has been developed as a blood–vessel-
embolic materials with good flowability and high mechanical
The temperature-responsive p(N-isopropylacrylamide) (pNI-
PAM) nanogels have been widely used as drug vehicles [1], biosen-
sing [2] and tissue-engineering scaffold materials [3]. Moreover,
multi-stimuli responsive nanogels could be developed by incorpo-
ration of functional monomers (e.g. acrylic acid), inorganic and me-
tal nanoparticles (such as Fe3O4, Au and Ag nanoparticles) [4–6].
Compared to linear pNIPAM polymers, pNIPAM nanogels with
nanometer-sized polymeric networks have unique microtopologi-
cal structure, and exhibit much richer sol–gel phase transition
behavior [7,8]. For example, concentrated pNIPAM nanogel disper-
sions showed three phase states (swollen gel, flowable sol and
shrunken gel) with the increasing temperature from 10 °C to
40 °C. Based on their temperature sensitive sol–gel transition, Con-
strength in transcatheter arterial embolization (TAE) therapy of li-
ver tumors in our recent research [9].
In order to improve therapeutic effect of tumors, many chemo-
therapeutic drugs (such as doxorubicin, paclitaxel, 5-fluorouracil)
was often used in combination with TAE, namely transcatheter
arterial chem-embolization (TACE) [10]. Unfortunately, the cura-
tive effect of TACE was limited by the low drug-loading amount
and poor sustained release of embolic materials. Recently many
nanomedicines (such as liposome, micelles, solid lipid nanoparti-
cles and polymeric nanoparticles), which have many advantages
including high loading capacity, good controlled release and bio-
compatibility etc., has been broadly applied in diagnosis and ther-
apy of tumors [11–14]. Most of polymeric nanoparticles, such as
polymeric micelle and dendrimer, could be co-dissolved first with
hydrophobic drugs in an organic solvent, and then the polymer and
drug were co-precipitated in an aqueous solution after removing
organic solvent, that is, so-called emulsion solvent evaporation
[15]. Being insoluble in any organic solvent, however, pNIPAM
nanogels could not load drug by emulsion solvent evaporation
method. In fact, the most common method is physic absorption
for the drug-loading of pNIPAM nanogels. For this reason, pNIPAM
⇑
Corresponding author at: National Engineering Research Center for Nanomed-
icine, Huazhong University of Science and Technology, Wuhan City, PR China. Tel./
fax: +86 27 68789303, tel./fax: +86 27 87792147.
E-mail addresses: zhaoyb@mail.hust.edu.cn, zhaoyb@hust.edu.cn (Y. Zhao).
Tel./fax: +86 27 68789303.
Tel./fax: +86 27 87792147.
Tel./fax: +86 27 87792234.
1
2
3
1381-5148/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.reactfunctpolym.2013.08.004

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G. Zhou et al. / Reactive & Functional Polymers 73 (2013) 1537–1543
B
A
C
D
Fig. 1. The size (solid sphere) and zeta potential (hollow square) curves of PNS nanogel as a function of temperature (Plot A) and pH (Plot B); TEM images of PNS nanogel dried
under 25 °C (Plot C) and 37 °C (Plot D).
Stirring and
ultrasound
Evaporation
of solvent
A
B
C
Fig. 2. The schematic diagram of the DOX-loading process of PNS nanogel by the Pickering emulsion evaporation technology. (A) the TEM picture of PNS nanogels before
Pickering emulsification; (B) the TEM picture of emulsion drops stabilized by PNS nanogels and (C) the TEM picture of PNS-D nanogels after solvent evaporation. The scale is
500 nm.
Table 1
The drug loading amount (DL%) and the entrapment efficiency (EE%) of PNS and PNS-D nanogels.
Z-average Size (nm, 25 °C)
PDI (25 °C)
The loading method
EE (%)
DL (wt%)
PNS nanogel
PNS nanogel
PNS-D-05
PNS-D-10
PNS-D-15
208
224.5
235
294.8
489
0.25
0.18
0.27
0.28
0.31
PAa
36.2 7.2
68.2 1.6
98.7 0.6
99.0 0.6
99.1 0.3
1.3 0.2
5.23 0.06
4.70 0.03
9.00 0.05
12.93 0.04
SDb
PESEc
PESE
PESE
a
b
c
Physical absorption: the mixing solution of DOX and nanogels were stirred overnight, and then dialyzed for removing the free DOX.
Solvent diffusion method: the DMSO solution of DOX and nanogels was added dropwise to water.
Pickering emulsion solvent evaporation.
nanogels were limited to the loading of hydrophilic drugs. More-
over, an initial burst release was often found in pNIPAM nanogels
by physic absorption method.
Pickering emulsion, which was first reported by Pickering [16]
and Ramsden [17] about a century ago, was an emulsion that is sta-
bilized solely by solid, mostly inorganic nano/micro-particles in
the common sense. Its emulsified mechanism involved in the
three-phase contact angle h, which determined the particle posi-
tion in the oil–water interface. Recently, several groups found that
stimuli-responsive Pickering emulsion could be stabilized by some

G. Zhou et al. / Reactive & Functional Polymers 73 (2013) 1537–1543
1539
B
A
25oC
400
0.4
0.2
0.0
37oC
400
200
200
0
20
30
40
o
PNS-D-15
PNS-D-05
PNS-D-10
Temperature,
PNS nanogels
C
Fig. 3. (A) The size of PNS-D nanogels as a function of temperature in plot A. PNS nanogel (hollow square); PNS-D-05 nanogel (solid sphere); PNS-D-10 nanogel (hollow
triangle); PNS-D-15 (solid diamond) and (B) the influence of the DOX-loading amount of PNS nanogels on the size and size distribution (polydispersity index, PDI). The PDI
data was measured at 25 °C.
B
A
40
20
0
40
20
0
0
20
40
0
20
40
Time, h
Time, h
Fig. 4. (A) The influence of NaCl concentration on in vitro release of DOX from PNS-D-10 nanogel dispersions. 0 wt% (solid diamond), 0.9 wt% (solid triangle), 3 wt% (solid
sphere), 5 wt% (solid square) and (B) the influence of pH on in vitro release of DOX from PNS-D-10 nanogel dispersions. pH 5.0 (hollow square), pH 6.0 (hollow sphere), pH 7.4
(hollow triangle).
Fig. 5. The releasing mechanism of DOX from PNS-D nanogel.
40
40
A
B
Shrunken Gel
Shrunken Gel
30
20
10
30
20
10
0
SGT2
SGT2
SGT1
SGT1
Flowable Sol
Flowable Sol
Swollen Gel
Swollen Gel
4
8
12
4.0
8.0
12.0
Nanogel Conc. wt%
Nanogel Conc. wt%
Fig. 6. The temperature sensitive sol–gel phase transition diagrams of both concentrated dispersions of PNS nanogels (A) and PNS-D-10 nanogels (B). The nanogel
concentration was 10 wt%.

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G. Zhou et al. / Reactive & Functional Polymers 73 (2013) 1537–1543
1000
100
10
1000
1
2
3
1
2
A
C
B
G'
G'
G''
3
100
10
G''
1
1
0.1
0.1
0
10
20
30
40
50
0
10
20
30
40
50
o
o
Temperature, C
Temperature, C
1000
100
10
1000
100
10
D
G'
G''
G'
G''
1
1
3
3
1
2
1
2
0.1
0.1
0
10
20
30
40
50
0
10
20
30
40
50
o
o
Temperature, C
Temperature, C
Fig. 7. The dynamic viscoelastic behavior of PNS-D nanogel dispersions: PNS nanogel dispersion (A); PNS-D-5 nanogels dispersion (B); PNS-D-10 nanogels dispersion (C);
PNS-D-15 nanogels dispersion (D). The nanogel concentration was 10 wt%. Area 1, 2, 3 were represented swollen gel, flowable sol and shrunken gel respectively.
soft particles like ‘‘smart’’ nanogels/microgels, and evolved ‘‘on de-
mand’’ under external stimuli such as temperature, pH, and mag-
netic field [18–20]. Veronique Schmitt group’s research and our
previous work suggested that the deformability of soft nanogels
and the interfacial rheologic properties had more important influ-
ence on the emulsion stability, rather than the three-phase contact
angle h [21,22]. Until now, Pickering emulsion technique has been
mostly used to construct supracolloidal structures, such as perme-
able hollow capsules, colloidosomes, complex gels, and Janus par-
ticles [23,24]. Despite of many advantages such as surfactant-free,
more stable and higher internal phase ratio, it was surprising in our
knowledge that Pickering emulsion has been seldom reported in
the application in encapsulation and transport of drugs.
In the present work, the O/W-type Pickering emulsion was sta-
bilized only by dual temperature/pH-sensitive poly(N-isopropylac-
rylamide-co-methacrylate sulfadiazine) nanogel (PNS nanogel).
After removing oil phase, hydrophobic Doxorubicin (DOX, an anti-
tumor drug) dissolved in oil phase could be deposited into the net-
work of PNS nanogels. Similar to common emulsion–solvent-
evaporation method in the drug-loading of many nanomedicines,
this drug-loading method was designated as Pickering emulsion
solvent evaporation (PESE) method. We investigated the aggrega-
tion behavior of PNS nanogels in Pickering emulsion process by
TEM. The drug loading amount (DL%), the entrapment efficiency
(EE%) and in vitro releasing behavior of PNS nanogels by PESE
method were compared with that by other methods. In order to
the application on injectable hydrogel drug delivery or blood–ves-
sel-embolic materials, concentrated PNS nanogel dispersions have
been studied on their rheologic behavior.
chloride (purity P 97.0%, Sigma–Aldrich Co., Ltd., USA), sulfadia-
zine (purity P 99.0%, Sigma–Aldrich Co., Ltd., USA), Sodium dode-
cyl sulfate (SDS, purity P 86.0%, Kermel Chemical Industry Co.,
Ltd., China), potassium persulfate (KPS, purity P 99.5%, Kermel
Chemical Industry Co., Ltd., China) were used directly without fur-
ther purification. NaOH (purity P 96.0%, Sinopharm Chemical Re-
agent Co., Ltd., China), HCl (36.0–38.0%, Sinopharm Chemical
Reagent Co., Ltd., China), Chloroform (purity P 99.0%, Sinopharm
Chemical Reagent Co., Ltd., China), acetone (purity P 99.5%, Sinop-
harm Chemical Reagent Co., Ltd., China), Triethylolamine
(purity P 99.0%, Aladdin Reagent Co., Ltd., Shanghai city, China)
were commercially available. Milli-Q ultra-pure water was used
in all experiments. Doxorubicin hydrochloride (DOXꢀHCl, pur-
ity > 98.0%) was purchased from Zhejiang Hisun Pharmaceutical
Co., Ltd., China.
2.2. The synthesis of methacrylate sulfadiazine (MSD)
Methacrylate sulfadiazine (MSD) was synthesized according to
modified Bae’s method [25]. In brief, sulfadiazine (25 g, 100 mmol)
and NaOH (8 g, 200 mmol) were dissolved in 150 mL of acetone/
water (60/90 v/v) mixture solution. In the resulting kelly solution,
methacryloyl chloride (15 mL, 155 mmol) was added dropwise
with stirring in ice salt bath. After reacting for 3.0 h in dark, the
solution was adjusted to pH 5.0–6.0 by HCl solution (0.01 mol/L).
The precipitated product was collected by filtration and washed
three times with distilled water. The yellowish green solid (31 g,
97%) was obtained after drying in vacuum at 40 °C for 3 h. 1H
NMR (400 MHz, d6-DMSO): d = 1.95 (3H, ACH3), 5.5 (1H, @CH2),
5.8 (1H, @CH2), 7.8–8.0 (4H, AC6H4A), 8.0–8.5 (3H, AC4N2H3A),
10.1 (1H, ACONHA), 11.6 (1H, ASO2NHA).
2. Experimental
2.1. Materials
2.3. The preparation of PNS nanogel
N-isopropylacrylamide (NIPAM, purity > 98.0%, Tokyo Chemical
Industry Co., Ltd., Japan) and N,N0-methylenebisacrylamide (BIS,
purity P 98.0%, Kermel Chemical Industry Co., Ltd., China) were
recrystallized from n-hexane (purity P 97.0%, Sinopharm Chemi-
cal Reagent Co., Ltd., China) and methanol (purity P 99.5%, Sinop-
harm Chemical Reagent Co., Ltd., China), respectively. Methacryloyl
Poly(N-isopropylacrylamide-co-methacrylate
sulfadiazine)
(PNS) nanogel was prepared by precipitation polymerizaition [9].
In brief, NIPAM (10.0 g, 88.4 mmol), MBA (0.1 g, 0.6 mmol), MSD
(2.4 g, 7.5 mmol) and SDS (0.27 g, 0.92 mmol) were dissolved in
NaOH solution (1.5 mg/mL, 800 mL). The resulting solutions were
degassed for at least 60 min by bubbling N2 and heating to 70 °C

G. Zhou et al. / Reactive & Functional Polymers 73 (2013) 1537–1543
1541
under stirring. KPS (0.4 g, 1.5 mmol) was then added as polymeri-
zation initiator, and the reaction was maintained under an N2
atmosphere at 70 °C for 5.0 h. The resulting PNS nanogels were dia-
lyzed (the cutoff molecular weight is 14,000 Da) against water for
2 weeks to remove unreacted monomers and other small mole-
cules. PNS nanogels were lyophilized and stored in a desiccator
at room temperature.
persions (0.25 wt%, 1.5 mL) were added into a ultrafiltration tube
(Minipore, MwCO 10 K, 4 mL), and then centrifuged (3000 rpm,
1.0 h). The resulting supernatant was diluted 200-fold, and free
DOX was measured by fluorospectrophotometry (F4500, Hitachi
Co., Japan). The measured condition was: kex = 500 nm, kem = 556 -
nm, PMT Voltage = 700 V. The drug loading amount (DL%) and the
entrapment efficiency (EE%) were calculated respectively as
following:
2.4. The preparation of Pickering emulsion stabilized by PNS nanogels
W0 ꢂ Cf ꢀ Vf
DL% ¼
ð1Þ
Cn ꢀ Vn
Chloroform (1 mL) and Triethylolamine (TEA, 5.0 lL) were
added into PNS nanogel dispersion (1.0 wt%, 3 mL). The prelimin-
ary emulsion was obtained after shearing the mixture at
13,000 rpm in ice-water bath for 10 min (FA25 homogenizer, FLU-
KO Equipment Shanghai Co., Ltd., China). The stable Pickering
nano-emulsion was prepared by ultrasonicating the preliminary
emulsion using an ultrasonic cell disruptor (JY92-IIN, 800 W, 20–
25 kHz, Ningbo Scientz Biotechnology Co., Ltd., China). The diame-
ter of the ultrasonic amplitude transformer is 2 mm. In order to
avoid too high temperature caused by ultrasonicating for long
time, the emulsion was in ice bath, and the interval time was set
as 5 s after 5 s of working time. The total working time was 75 s.
W0 ꢂ Cf Vf
EE% ¼
ð2Þ
W0
Here, W0 is the feeding amount of DOX, Cf and Vf is the concentra-
tion and volume of free DOX, respectively. Cn and Vn is the concen-
tration and volume of nanogel dispersion.
3. Results and discussion
3.1. The preparation of poly(N-isopropylacrylamide-co-methacrylate
sulfadiazine) (PNS) nanogel
2.5. The preparation of Doxorubicin-loaded PNS nanogels (PNS-D
nanogels)
Sulfonamide, a well known antibacterial agent, has been used
recently as a new pH-sensitive group due to its weak acidic nature
(pKa = 6.5–7.0) [25–27]. Some antitumor drug carriers, which
based on the pH-sensitivity of sulfonamide, have been constructed
since most solid tumors have lower extracellular pH (<6.8) than
their surrounding tissues and blood (pH 7.4) [28–30]. In the pres-
ent work, PNS nanogels were prepared by precipitation copolymer-
ization of NIPAM and resultant MSD monomer. From Fig. 1 A, the
size of PNS nanogel decreased from 158 nm at 25 °C to 50 nm at
50 °C, and the zeta potential increased from ꢂ9 mV at 25 °C to
ꢂ32.6 mV at 50 °C. Just like pNIPAM nanogels (classic temperature
sensitive nanogels [9]), PNS nanogels exhibit a sudden change in
size and zeta potential as temperature increased, that is, so called
volume-phase transition. Its volume-phase transition temperature
(VPTT) was about 32–35 °C. Fig. 1B exhibited the pH-sensitivity of
PNS nanogel. With the increasing pH, the size of PNS nanogel de-
creased from 255 nm at pH 2.2 to 90 nm at pH 8.0, and the zeta po-
tential increased from ꢂ9 mV at pH 2.2 to ꢂ22.2 mV at pH 8.0. The
TEM pictures (Fig. 1C and D) showed that PNS nanogels were
spherical and hairy morphology. All of these data indicated that
PNS nanogels were highly deformable under the external stimuli
such as temperature and/or pH.
Pickering emulsion solvent evaporation (PESE) method was
developed for the preparation of Doxorubicin-loaded PNS nanogels
(PNS-D nanogels). According to the feeding ratio of Doxorubicin
and nanogel, PNS-D nanogels were designated as PNS-D-15, PNS-
D-10, PNS-D-05, respectively. For example, PNS-D-15 was pre-
pared as follows: DOXꢀHCl (4.5 mg, 7.8 ꢁ 10ꢂ3 mmol) was dis-
solved in Chloroform (1 mL, containing 5.0 lL TEA). The solution
was added into PNS nanogel dispersion (1.0 wt%, 3 mL), and then
was emulsified to a preliminary emulsion in ice-water bath by
shearing at 13,000 rpm for 10 min. The stable Pickering nano-
emulsion was prepared by also ultrasonicating the preliminary
emulsion according to above ultrasonication condition. After
removing chloroform by a rotatory evaporator, the resulting PNS-
D nanogel dispersion was lyophilized and stored in dark.
2.6. Characterization
The hydrodynamic diameters and the zeta potentials of PNS
nanogels and the emulsion droplet were determined by dynamic
light scattering (DLS, Zetasizer Nano-ZS 90, Malvern Instrument
Limited, UK) using a He–Ne laser source (k = 633 nm) with the
scattering angle of 90°. All samples were diluted with ultrapure
water to 0.5 mg mLꢂ1. The micromorphology of nanogels and
emulsion droplets were characterized by Transmission Electron
Microscope (TEM, Tecnai G2 20, FEI Corp., Netherlands) at
180 kV. A drop of nanogel dispersion or Pickering emulsion was
added onto a carbon-coated copper grid, and then dried in air.
The samples were negatively stained with 1 wt% phosphotungstic
acid solution. The sol–gel phase transitions were measured by
the inverting-vial method from 5 °C to 50 °C in water bath. The dy-
namic viscoelastic properties of nanogel dispersions were obtained
using a strain-controlled rheometer (ARES-G2, TA Instruments-
3.2. The drug loading of PNS nanogels using Pickering emulsion solvent
evaporation (PESE)
Due to their stimuli-responsive deformability, soft pNIPAM
nanogels have been extensively applied as novel emulsifier, and
the resultant Pickering emulsion can evolve ‘‘on demand’’ under
some external stimuli, such as temperature, pH, and magnetic field
[21]. Based on the emulsifying ability of pNIPAM nanogels, Picker-
ing emulsion solvent evaporation (PESE) technology was devel-
oped as a new drug loading method as shown in Fig. 2. In our
previous works, we found that an unstable emulsion (preliminary
Waters LLC, USA) with a parallel plate (
U
= 40 mm, gap set at
emulsion) which size was in 1.0–5.0 lm was formed by shearing
0.9 mm) in the range of 20–40 °C with following parameters: strain
the mixture of oil phase and aqueous nanogel dispersion phase
at 13,000 rpm. The nanogels accumulated on O/W interfaces to
form interfacial hydrogel layers. The interfacial hydrogel layers
surrounding oil droplets, which form capsule-like microscopic
suprastructures, provided strong interfacial hindrance to resist
droplet–droplet coalescence. After further dispersed by ultrasound
(strong energy input), the microscopic suprastructures were disin-
was 0.2%, heating rate was 1 °C minꢂ1, and frequency was 1.0 Hz.
2.7. Drug loading and in vitro releasing behavior
In order to determine the drug loading amount and the entrap-
ment efficiency of PNS-D nanogel dispersions, PNS-D nanogel dis-

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G. Zhou et al. / Reactive & Functional Polymers 73 (2013) 1537–1543
tegrated into the same capsule-like nanoscale suprastructures with
near monodispersity (Fig. 2B). It is noteworthy that the nanoscale
suprastructures has the same size as PNS nanogels (about
200 nm). As suggested in our previous works [22], input of ultra-
sound energy might trigger the dehydration of nanogels and make
PNS nanogels shrunken. The shrunken nanogels absorbed on the
oil/water interface, and decreased interfacial tension by covering
on larger area of oil/water interface owing to its deformability.
Consequently the nano-emulsion stabilized by PNS nanogels was
very stable, and sustained unchanged in size and morphology for
several days (Fig. 2B).
At last, the hollow oil-in-water droplets (Fig. 2B) were changed
to solid nanoparticles (Fig. 2C) after removing chloroform by rotary
evaporation. It was obvious that PNS nanogels were re-dispersed in
water due to the remove of chloroform, and hydrophobic DOX was
transferred into the 3D networks of PNS nanogels. In this process,
the properties of droplets have important effect on the drug-load-
ing of PNS nanogels. The inhomogeneous droplets of preliminary
emulsion became unstable, and resulted in the precipitation of
DOX and nanogels in solvent evaporation process.
ing amount increased from PNS-D-10 nanogels. At 48 h, their re-
leases reached 29.5%, 41.6% and 48.0% respectively in 0.9 wt%,
3.0 wt% and 5.0 wt% of NaCl solutions (Fig. 4A). It indicated that
PNS-D-10 nanogels showed a controlled slow but constant release
in salt solution.
PNS-D-10 nanogels showed a pH dependence of in vitro DOX re-
lease as shown in Fig. 4B. Similarly with its release in pure water,
the cumulative release of DOX was very low (only 11.0% for 48 h)
for PNS-D-10 nanogels at pH 7.4 (the pH value of normal tissues
and blood). It indicated that PNS-D-10 nanogels induced little sys-
temic toxicity due to their small release in blood. As the pH of med-
ia decreased, the release of DOX from PNS-D-10 nanogels
increased, for example, its release reached up to 40% for 48 h at
pH 5.0. It was noteworthy that PNS-D-10 nanogels showed a slow
and constant release at pH 6.0 (the pH value of tumor tissues).
Their DOX-releasing amount reached 21.1% for 48 h, and still per-
sistently increased with the increase of releasing time. The results
suggested that PNS-D-10 nanogels could maintain a sustained drug
release in tumor tissue, which resulted in the improvement of DOX
concentration in tumor tissue and the enhancement of therapeutic
efficacy for tumors.
As shown in Table 1, three stable PNS-D nanogel dispersions
(PNS-D-05, PNS-D-10 and PNS-D-15 were prepared using PESE
method. Fig. 3A showed that the sizes of three PNS-D nanogels
have the same temperature dependence as PNS nanogels. That is,
all PNS-D nanogels exhibited a significant drop in size (volume
phase transition) with the increasing temperature, and their VPTTs
were about 32–35 °C. It indicated that the loading DOX had nearly
no influence on their VPTTs. Fig. 3B showed the influence of DOX-
loading amount on the size and polydispersity index (PDI) of PNS-
D nanogels. Their sizes increased as the DOX-loading amount in-
creased at either 25 °C or 37 °C. With the increase of DOX-loading
amount, the PDIs of PNS-D nanogels also increased from 0.12 (PNS
nanogels) to 0.44 (PNS-D-15 nanogels). It was likely to be because
hydrophobic DOX molecules resulted in the aggregation of PNS-D
nanogels, which decreased the stability of nanogel dispersions.
When the feeding ratio of DOX and nanogels was higher than
0.15, in fact, stable PNS-D nanogel dispersions could be not pre-
pared using PESE method owing to the precipitation of DOX and
nanogels.
The drug loading amount (DL) and the entrapment efficiency
(EE) are two important parameters in the evaluation of drug-load-
ing capability. As shown in Table 1, PNS nanogels could reach high-
er DL value by solvent diffusion method. However, low EE value
indicated that a large amount of DOX was not encapsulated in
PNS nanogels. It could be attributed to the fact that a large amount
of DOX molecules migrated into water in the diffusion of DMSO to
water. Compared with the common drug-loading technology of
nanogels, such as the solvent diffusion and the physical absorption,
PNS nanogels had higher DL and EE values using PESE technology.
Near 100% of EE value showed that all DOX molecules were mi-
grated into the 3D networks of PNS nanogels, and the highest DL
value arrived at about 15 wt%.
As shown in Fig. 5, the ionic bonding interaction between DOX
molecules and sulfonamide groups of PNS nanogel networks could
be responsible for the pH- and/or ion-dependent release. The tight
complex by ionic bonding interaction between DOX molecules and
sulfonamide groups resulted in the slow release of PNS-D-10 nano-
gels at pH 7.4. As pH decreased or ionic strength increased, the io-
nic bonding interaction was broken due to the ionizing of DOX
molecules. Therefore, free DOX molecules were released from the
networks of PNS-D-10 nanogels.
3.4. The sol–gel phase transition behavior of PNS nanogel
The sol–gel phase transition behavior of PNS and PNS-D-10
nanogel dispersions was shown in Fig. 6. With the increasing tem-
perature and nanogel concentration, both nanogel dispersions suc-
cessively exhibited three phase states: swollen gel, flowable sol
and shrunken gel, in according with our previous works [6]. There-
fore, there were two sol–gel phase transition temperatures (SGT1
and SGT2), which were corresponding respectively to the transi-
tion from swollen gel to flowable sol and the transition from flow-
able sol to shrunken gel. The SGT1 increased as the nanogel
concentration increased. However, the nanogel concentration has
nearly no influence on the SGT2. It could be attributed to the differ-
ent gelating mechanism. The swollen gel was formed by the vol-
ume blocking mechanism according to the hardsphere theory
[36,37], while the shrunken gel was formed due to the enhanced
interaction (hydrophobic force and electrostatic force) between
nanogel particles, in accordance with the softsphere theory [38].
The DOX loading amount has different influence on the two sol–
gel phase transition. The SGT1 value obviously increased after DOX
loading by the PESE method. For example, the SGT1 value in-
creased from 11.9 °C of PNS nanogels to 18.2 °C of PNS-D-10 nano-
gels at 8.0 wt%. This could be attributed to the fact that the loading
DOX made nanogel size increased as shown in Fig. 3. Therefore, the
swollen gel could occur at higher temperature. On the other hand,
the SGT2 value has slightly decreased (e.g. from 31.5 °C of PNS
nanogels to 26.9 °C of PNS-D-10 nanogels at 8.0 wt%). It was be-
cause that the loading DOX by PESE method enhanced hydropho-
bic interaction of nanogel particles.
3.3. In vitro sustained releasing behavior of PNS-D nanogels
Recent studies suggested that sustained drug release could im-
prove local DOX-concentration in tumor tissue and reduce its sys-
temic concentration in TACE therapy. For example, drug-eluting
beads (DEBs) such as DC Bead™ and QuadraSphere™ remained
much higher DOX concentration in tumor tissue up to 14 days,
whereas systemic drug concentration is kept at minimal level
[31–35]. Similarly with these drug-eluting beads, PNS-D-10 nano-
gels exhibited a slow release without any burst release. Their
cumulative release only reached 13.2% for 48 h (Fig. 4A), and about
76.8% of DOX-loading amount could be not released in pure water.
As the ionic strength of media increased, however, the DOX-releas-
The influence of DOX on the viscoelastic behavior of concen-
trated PNS-D-10 dispersions was characterized by a strain-con-
trolled rheometer in Fig. 7. Four nanogel dispersions had the
similar viscoelastic behavior. That is, the G0 and G00 values were
low below 25 °C, increased abruptly at the range of 28–30 °C,
and slightly decreased above 30 °C. Area 1, 2 and 3 represented

G. Zhou et al. / Reactive & Functional Polymers 73 (2013) 1537–1543
1543
swollen gel, flowable sol and shrunken gel respectively. In swollen
References
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This work was financially supported by the grant from National
Basic Research Program of China (973 Program, 2012CB932500),
National High Technology Research and Development Program of
China (SS2012AA023804), and the National Science Foundation
of China (NSFC, 31170960/C1007). We also thank the Analysis
and Test Center of Huazhong University of Science and Technology
for the related analysis.