Novel pH-sensitive polysialic acid based polymeric micelles for triggered intracellular release of hydrophobic drug

Article abstract of DOI:10.1016/j.carbpol.2015.12.041

Polysialic acid (PSA), a non-immunogenic and biodegradable natural polymer, is prone to hydrolysis under endo-lysosomal pH conditions. Here, we synthesized an intracellular pH-sensitive polysialic acid-ursolic acid conjugate by a condensation reaction. To further test the drug loading capability, we prepared paclitaxel-loaded polysialic acid-based amphiphilic copolymer micelle (PTX-loaded-PSAU) by a nanoprecipitation method. Results showed PTX-loaded-PSAU exhibited well-defined spherical shape and homogeneous distribution. The drug-loading was 4.5% with an entrapment efficiency of 67.5%. PTX released from PTX-loaded-PSAU was 15% and 42% in 72 h under simulated physiological condition (pH 7.4) and mild acidic conditions (pH 5.0), respectively. In addition, In vitro cytotoxicity assay showed that PTX-loaded-PSAU retained anti-tumor (SGC-7901) activity with a cell viability of 53.8% following 72 h incubation, indicating PTX-loaded-PSAU could efficiently release PTX into the tumor cells. These results indicated that the pH-responsive biodegradable PTX-loaded-PSAU possess superior extracellular stability and intracellular drug release ability.

Full text of DOI:10.1016/j.carbpol.2015.12.041

Carbohydrate Polymers 139 (2016) 75–81
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Novel pH-sensitive polysialic acid based polymeric micelles for
triggered intracellular release of hydrophobic drug
Wuxia Zhanga, Dongqi Donga, Peng Lib, Dongdong Wanga, Haibo Mua, Hong Niua,
Jinyou Duana,∗
a College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China
b College of Life Science, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Polysialic acid (PSA), a non-immunogenic and biodegradable natural polymer, is prone to hydroly-
sis under endo-lysosomal pH conditions. Here, we synthesized an intracellular pH-sensitive polysialic
acid-ursolic acid conjugate by a condensation reaction. To further test the drug loading capability, we
prepared paclitaxel-loaded polysialic acid-based amphiphilic copolymer micelle (PTX-loaded-PSAU) by
a nanoprecipitation method. Results showed PTX-loaded-PSAU exhibited well-defined spherical shape
and homogeneous distribution. The drug-loading was 4.5% with an entrapment efficiency of 67.5%. PTX
released from PTX-loaded-PSAU was 15% and 42% in 72 h under simulated physiological condition (pH
7.4) and mild acidic conditions (pH 5.0), respectively. In addition, In vitro cytotoxicity assay showed that
PTX-loaded-PSAU retained anti-tumor (SGC-7901) activity with a cell viability of 53.8% following 72 h
incubation, indicating PTX-loaded-PSAU could efficiently release PTX into the tumor cells. These results
indicated that the pH-responsive biodegradable PTX-loaded-PSAU possess superior extracellular stability
and intracellular drug release ability.
Received 6 October 2015
Received in revised form
15 December 2015
Accepted 16 December 2015
Available online 18 December 2015
Keywords:
Polysialic acid
Paclitaxel
pH-sensitive
Polymeric micelles
© 2015 Elsevier Ltd. All rights reserved.
1. Introduction
the physiological condition (pH 7.4), which provide an important
stimulus in vivo that could be used to trigger intracellular release
Tremendous efforts have been directed to the development
of targeted drug delivery systems because they promised to
resolve several key therapeutical issues associated with current
clinical practice including low treatment efficacy and significant
side effects (Langer, 1998; Soppimath, Aminabhavi, Kulkarni, &
Rudzinski, 2001). Until now, various drug delivery systems based
on liposomes, nanoparticles, polymeric conjugates and micelles
have been intensively explored (Yoo, Lee, & Park, 2002). Polymeric
micelles could alter the pharmacokinetic profile of drugs, reduce
off-target toxicity and side effects, prolong circulation in the blood
owing to its high water-solubility, and enhance the therapeu-
tic efficiency (Cayre, Chagneux, & Biggs, 2011; Kedar, Phutane,
Shidhaye, & Kadam, 2010). However, only few drugs could be
carried into the intracellular compartments of the cancer cell due
to the slow drug release from micelles (Shuai, Ai, Nasongkla, Kim,
& Gao, 2004; Sui, Liu, & Shen, 2011). The proton concentration of
late endosomes and lysosomes is 100-times lower (pH 5.0) than
of hydrophobic drugs (Duncan, 1992; Haag, 2004).
Polysialic acid (PSA), a homopolymer of sialic acid (SA) in either
␣-2,8 or ␣-2,9 linkages or a mixture, was first found in E. coli
(Escherichia coli) K-1 and K-235 by Barry (Barry & Goebel, 1957).
The 2,8 linked polysialic acid only induced a low level of anti-
body that could avoid the clearance of modified drugs/carriers
from the blood circulation and present long circulation time in vivo
(Fernandes & Gregoriadis, 1997, 2001). More importantly, it has
been proved that PSA is biodegradable and prone to hydrolysis
under endo-lysosomal pH conditions (Zhang et al., 2014). Also, the
degradation product of PSA, SA, could serve as a targeting ligand
for selectin, which is highly expressed in tumor vascular endothe-
lial cells (Greco et al., 2013; Jayant et al., 2007; Zhang et al., 2014). SA
could inhibit tumor metastasis by selectin targeting (Zeisig, Stahn,
Wenzel, Behrens, & Fichtner, 2004). Obviously, PSA had a great
potential to be an effective drug delivery material for targeting
cancer disease. Ursolic acid, a natural triterpene, structurally sim-
ilar to dexamethasone, exhibited antitumor effects in various cell
types (Kassi et al., 2007). Research has shown that ursolic acid was
relatively non-toxic, and have been used in cosmetics and health
products (Liu, 1995).
∗
Corresponding author.
E-mail address: jduan@nwsuaf.edu.cn (J. Duan).
http://dx.doi.org/10.1016/j.carbpol.2015.12.041
0144-8617/© 2015 Elsevier Ltd. All rights reserved.

76
W. Zhang et al. / Carbohydrate Polymers 139 (2016) 75–81
In the present work, we reported a novel pH-sensitive degrad-
resulting solution was cooled by ice bath. Acetic anhydride (600 ␮L,
6.3 mM) was added dropwise to the solution, and then the mixture
was warmed up to room temperature and stirred for another 8 h.
The solvent was removed in vacuo and the residual mixture was
dissolved with dichloromethane. The solution was washed sequen-
tially with 10% hydrochloric acid, water and brine. Pure U1 was
obtained by silica gel column chromatography.
Secondly, U1 (0.24 mM) dissolved in 5 mL anhydrous
dichloromethane (DCM) was cooled by ice bath. Oxalyl chlo-
ride (180 ␮L, 2.1 mM) was added dropwise. After 30 min, the
mixture was warmed up to room temperature and stirred for
another 24 h. The solvent was removed in vacuo and the solid was
dissolved with anhydrous DCM as a stock solution.
able micelle for targeted intracellular paclitaxel (PTX) release.
Firstly, the amphiphilic copolymer, polysialic acid-ursolic acid con-
jugate, designed as PSAU was synthesized and characterized. Then,
PTX was loaded in PSAU micelle by a nanoprecipitation method.
Finally, the particle size, drug entrapment efficiency, drug release
behavior in vitro and the anti-tumor activity of the PTX-loaded
PSAU micelle were investigated.
2. Materials and methods
2.1. Materials
Polysialic acid sodium salt (Mw = 1.6 × 104 Da), purchased from
Jiangsu Rui Guang Biotechnology Co., Ltd., was further dialyzed
against distilled water and lyophilized. Ursolic acid, oxalyl chlo-
ride, pyrene, paclitaxel and ethylenediamine were obtained from
Aladdin Reagent Int. Bovine serum albumin (BSA) and 3-(4,5-
dimethyltiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were
obtained from Sigma–Aldrich Co. All other chemicals and reagents
were of analytical grade. The SGC-7901 cell line was a kindly gift
from Professor Kan Ding in Shanghai Institute of Materia Medica,
Chinese Academy of Sciences.
Thirdly, to an ice-cold solution of anhydrous ethylenediamine
(12 mM) in 2 mL anhydrous DCM, the stock solution above was
added dropwise. After 30 min, the mixture was warmed up to
room temperature and stirred for another 12 h. The solution was
washed sequentially with 10% hydrochloric acid, water and brine.
Aminoethyl U1 (U2) was obtained by silica gel column chromatog-
raphy.
Finally, U2 (45 mg, 0.083 mM) was dissolved in DMF at 0 ◦C under
N2, and N,N-diisopropylethylamine (DIPEA, 45 ␮L, 0.26 mM) was
slowly added. The DMF solution was stirred further for 10 min.
Polysialic acid (40 mg) was dissolved in 12 mL formamide. DMAP
(5.2 mg, 0.043 mM) and EDC.HCl (48 mg, 0.25 mM) were added to
the formamide solution within 10 min. The DMF solution and form-
amide solution were mixed. The mixture was allowed to warm
up to room temperature and stirred overnight. The resulting solu-
tion was dialyzed against the excess amount of water/methanol (1:
3–1:1, v/v) for 1 day and distilled water for another 2 days, respec-
tively. After lyophilization, the polysialic acid-based amphiphilic
copolymers (PSAU) were obtained as a white powder.
2.2. Synthesis of the polysialic acid-based amphiphilic
copolymers (PSAU)
The polysialic acid-based amphiphilic copolymers were syn-
thesized in four reaction steps from PSA, ursolic acid and
ethylenediamine (Scheme 1). Specific steps were as follows:
Firstly, 200 mg ursolic acid (0.43 mM) was dissolved in 9 mL
anhydrous THF, and 3 mg DMAP (0.025 mM) was added. The
Scheme 1. Synthetic routes of polysialic acid-based amphiphilic copolymers (PSAU).

W. Zhang et al. / Carbohydrate Polymers 139 (2016) 75–81
77
2.3. Characterization of PSAU
peak area against PTX concentration was at least 0.998. The follow-
ing equations were applied to calculate the drug loading content
(Eq. (1)) and encapsulation efficiency (Eq. (2)).
The chemical structures of UA, U1, PSA and PSAU conjugate
were characterized by 1H NMR (Meng, Song, Yan, & Xia, 2010;
Vliegenthart, Dorland, van Halbeek, & Haverkamp, 1982). 1H NMR
spectra were recorded on a Bruker Avance III 500 MHz NMR spec-
trometer. Chemical shifts of UA and U1 in CDCl3 were referenced
to tetramethylsilane (TMS). While HDO (ı = 4.7 ppm) was used as a
reference for proton NMR chemical shifts of PSA in D2O and PSAU
in D2O/CD3OD.
wt of the PTX in micelles
wt of the micelles × 100%
Drug loading content (%) =
(1)
(2)
wt of the PTX in micelles
wt of the feeding PTX × 100%
Encapsulation efficiency (%) =
2.7. In vitro PTX release of the PSAU micelles
The degree of substitution (DS), defined as the number of U2 per
100 sugar residues of PSA polymer, was estimated by elemental
analyzer based on the different carbon content.
PTX release behavior was studied in vitro in phosphate buffered
saline (PBS, pH7.4, pH 5.0) solution. Briefly, the solutions of PTX-
loaded PSAU micelles were placed into dialysis membrane (MWCO
3500–5000 Da) and dialyzed against 10 mL PBS with 1% tween-80 at
37 ◦C in an air-bath shaker at 100 rpm. Then, 2 mL release media was
collected and replaced with an equal volume fresh PBS at defined
time intervals. 2.0 mL DCM was introduced to the release medium
to extract PTX. The PTX in DCM solution was separated from the
water and kept in a 5.0 mL flask until DCM was volatilized. Finally,
1.0 mL methanol was added to the flask to dissolve PTX. The con-
centration of PTX in methanol solution was determined by HPLC as
above.
The critical micelle concentration (CMC) was determined using
pyrene as a fluorescence probe. A solution of pyrene in acetone
was added to a vial and the solvent was allowed to evaporate
to form a thin film at the bottom of the vial. Polymeric micelle
solutions at different concentrations were added to the vials
and the final pyrene concentration was 5 × 10−7 M in water. The
concentrations of polymer micelles varied from 1 × 10−4 mg/mL
to 2.0 mg/mL. The solutions were kept on a shaker at room
temperature for 12 h to reach equilibrium prior to fluorescence
measurement. Fluorescence spectra were recorded on a RF-5301PC
fluorescence spectrophotometer at room temperature. The exci-
tation spectra were scanned from 300 to 360 nm at the emission
wavelength of 390 nm. Excitation and emission bandwidths were
5 nm and 10 nm, respectively. The fluorescence intensity ratio of
I338/I333 was analyzed as a function of micelle concentrations.
2.8. Cell viability assays
The cytotoxicity of drug-free polymeric micelles, PTX-loaded
PSAU micelles and free PTX were evaluated by MTT assay (Le Garrec
et al., 2004). SGC-7901 cells (5 × 104 cells/well) were plated in a
96-well plate in RPMI-1640 (containing 10% FBS, 100 IU/mL peni-
cillin and 100 ␮g/mL streptomycin) and incubated at 37 ◦C for 4 h.
Then polymeric micelles (100 ␮g/mL), PTX-loaded PSAU micelles
(100 ␮g/mL, containing drug dosage: 4.5 ␮g/mL) or free PTX (drug
dosage: 4.5 ␮g/mL) were added and incubated for 24 h or 72 h.
DMSO was used to solubilize PTX. Then, 20 ␮L MTT (5.0 mg/mL) was
added and incubated for another 4 h at 37 ◦C. The 96-well plate was
centrifuged for 5 min (3500 rpm) and then the medium was aspi-
rated. The MTT-formazan generated by live cells was dissolved in
150 ␮L DMSO, and the absorbance was measured at 570 nm using a
microplatereader (Perlong DNM-9062, China). The cell viability (%)
was determined by comparing the absorbance at 570 nm with con-
trol wells containing cell culture medium alone. Data are presented
as mean SD (n = 4).
2.4. Preparation of PTX- loaded PSAU micelles
PTX loaded micelles were prepared by a nanoprecipitation
method with minor modification (Li et al., 2008, 2010). Briefly,
12 mg of PSAU and 0.8 mg PTX were dissolved in an aliquot of
methanol, which was added dropwise into 8 volumes of dis-
tilled water at room temperature. The solution was dialyzed in
a dialysis membrane (MWCO 3500–5000 Da, Sigma–Aldrich Co.,
USA) to remove methanol thoroughly. Solutions of drug-loaded
micelles and empty micelles were then lyophilized for further
utilization.
2.5. Characterization of PTX-loaded PSAU and drug-free
polymeric micelles
3. Results and discussion
The shape and particle size of the micelles were investigated
by field emission scanning electron microscopy (FESEM). Micelles
solution was placed on a clean silicon chip surface and then air-
dried overnight. The image was obtained with a Hitachi S-4800
FESEM system.
3.1. Synthesis and characterization of PSAU
The synthetic procedure of PSAU was shown in Scheme 1. To
obtain compound U1, ursolic acid was esterified with acetic anhy-
dride. In order to conjugate U1 to PSA, the carboxyl group of U1
was first converted to U2 which possessed a free amine group in
the presence of ethylenediamine. PSAU was synthesized through
covalently linking amino group with the carboxyl groups on the
backbone of PSA. The PSAU structure was identified by proton
nuclear magnetic resonance (1H NMR) spectroscopy. As showed
in Fig. 1, the peaks at 5.30 ppm and 3.25–3.28 ppm were attributed
2.6. Drug loading content and encapsulation efficiency
Drug loading capability of the PTX-loaded PSAU was determined
using high-performance liquid chromatography (HPLC) (Tao, Xu,
Chen, Bai, & Liu, 2012). Briefly, 40 ␮L PTX-loaded micelles solution
was diluted with methanol to10 mL. The solution was centrifuged
at 10,000 rpm for 8 min, and then 20 ␮L of supernatant was injected
into the chromatographic system. The HPLC system (515 HPLC
Pump, waters, USA) was equipped with a Lichrospher C18 col-
umn (4.6 mm × 150 mm, 5 m) with a mobile phase of methanol and
water (70:30). The flow rate and column temperature were set at
1 mL/min and 30 ◦C, respectively. The signals were recorded by UV
detector at 227 nm. A calibration line was conducted to determine
PTX concentrations in the range of 0.5–50 mg/L, and the R2-value of
to the protons of the olefinic double bonds and
O CH of UA
in CDCl3, respectively. Meanwhile, the peaks at 0.82–1.13 ppm
and 1.28–2.25 ppm belonged to the protons of the methyl group
and CH or CH2 of UA in CDCl3, respectively. The peaks at
0.82–2.25 ppm were the characteristic resonances of pentacyclic
triterpene (Fig. 1a). In comparison with 1H NMR spectrum in Fig. 1a,
a new peak at 2.08 ppm (Fig. 1b) appeared, suggesting the success-
ful O-acetylation.

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W. Zhang et al. / Carbohydrate Polymers 139 (2016) 75–81
Fig. 1. 1H NMR spectrum of UA in CDCl3(a), U1 in CDCl3 (b), PSA in D2O (c) and PSAU conjugates in D2O and CD3OD (2:1,v/v) (d).
The peaks at 2.01 ppm were attributed to the protons of the
been widely used as a hydrophobic fluorescence probe for micelle
methyl group of PSA. The peaks at 1.68 ppm and 2.60–2.62 ppm
belonged to the protons of CH2 of saccharide ring of PSA. The
peaks at 3.52–4.12 ppm present the other protons of PSA in D2O
(Fig. 1c). 1H NMR spectrum of PSAU in D2O and CD3OD (2:1,
v/v) showed peaks appearing at 0.7–1.5 ppm due to U1 moieties
(Fig. 1d). These results indicated that ursolic acid was successfully
connected to the PSA.
formation, in which, the ratio I338/I333 from excitation spectra,
could be used as an index of micelle hydrophobicity (Urbano, Silva,
Olea, Fuentes, & Martinez, 2008). As shown in Fig. 2, the intersec-
tion of the extrapolated straight line segments yielded the CMC
value of 0.11 mg/mL for PSAU copolymers in water at room tem-
perature. The low CMC value suggested that micelle formed from
PSAU copolymers would have a good stability in solution.
The degree of substitution (DS, defined as the number of U2
per 100 sugar residues of PSA polymer) was calculated by mea-
suring the content of carbon in U2, PSA and PSA-U copolymer using
elemental analysis. The content of carbon element was shown in
Table 1. According to the following equation, the DS of U2 in PSA-U
conjugate was 3.3.
(75.44% × W1 × n + W2 × 36.75% × 100)
= 39.02%
(W1 × n + W2 × 100 − 40 × n)
Among them: W1 is the molar mass of U2, (540.8 g/mol); W2
presents the molar mass of PSA unit, (313 g/mol); n is the substi-
tution degree of U2 in PSA; 40 is the molar mass (NaOH) would be
lost when PSA was connected with U2.
The CMC of amphiphilic PSAU copolymers was determined by
a fluorescence spectrometry using pyrene as a probe. Pyrene has
Table 1
The content of carbon element.
Compound
U2
PSA
PSA-U
39.02
Fig. 2. I338/I333 variation of pyrene excitation spectra versus PSA-U1 concentration
in water. The intersection of the extrapolated straight line segments yields the CMC
value (0.11 mg/mL).
Carbon content (%)
75.44
36.75

W. Zhang et al. / Carbohydrate Polymers 139 (2016) 75–81
79
Scheme 2. Schematic illustration of PTX loading and triggered release from PTX-loaded PSAU micelles in intracellular microenvironment.
3.2. Preparation and characterization of PSAU and PTX-loaded
PSAU micelles
were considered suitable for passive delivery of PTX to targeted
tumors through the EPR effect (Maeda, Wu, Sawa, Matsumura, &
Hori, 2000). By calculating the feeding ratio of copolymer and PTX,
the drug loading content of PTX into PSAU micelles was detected
as 4.5% and the encapsulation efficiency was 67.5%.
The anticancer drug, paclitaxel (PTX), is known to have the
highly hydrophobic property (Wang et al., 2008). Therefore, we
designed PSAU micelles as a carrier for PTX to increase its aque-
ous solubility. PTX was physically incorporated into PSAU micelles
using a nano-precipitation method at room temperature. The
schematic illustration of PTX loading was showed in Scheme 2.
The size and morphology of PSAU and PTX-loaded PSAU micelles
were analyzed with Field emission scanning electron microscopy
(FESEM). As shown in Fig. 3, the sizes of PSAU micelles ranged from
120 to 150 nm. The particle size increased and ranged from 120
to 180 nm when PTX was incorporated. PSAU micelles showed the
nearly spherical shape with increased particle size after drug load-
ing (Fig. 3b). Such small sizes of these PTX-loaded PSAU micelles
3.3. In vitro pH-responsive drug release of PTX-loaded PSAU
micelles
In order to investigate the effect of pH on the drug release of PTX-
loaded PSAU micelles, we have measured the cumulative release
under physiological condition (PBS, pH 7.4) and at pH 5.0 over a
period of time.
It could be seen that as the drug-loaded PSAU was immersed in
pH 5.0 buffer at 37 ◦C, paclitaxel was gradually released from PTX-
loaded PSAU micelles and the percentage of cumulative release
Fig. 3. FESEM photographs of blank PSAU micelles (a) and PTX-loaded PSAU micelles (b).

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W. Zhang et al. / Carbohydrate Polymers 139 (2016) 75–81
from PTX-loaded PSAU micelles intracellular microenvironment
was showed in Scheme 2.
Controlled delivery devices that utilize biodegradable polymers
have a significant advantage over competing delivery systems in
that there is no need for surgical removal of the device (Sahoo,
Parida, Rout, & Bindhani, 2012). PSA is a relatively unexplored
biodegradable material for the prevention of premature clearance
(Bader, Silvers, & Zhang, 2011). The pH-responsive biodegradable
PSAU micelles offers a novel and elegant approach to resolve the
extracellular stability versus intracellular drug release dilemma of
micellar drugs and provide a novel platform for tumor-targeting
delivery of anti-cancer drugs.
4. Conclusion
Fig. 4. In vitro cumulative PTX release profiles from PTX-loaded micelles in PBS
solution with different pH value.
In this study, the amphiphilic polymer, PSAU was synthesized
and characterized by 1H NMR and elemental analysis. The parti-
cle size of PTX-loaded PSAU micelles ranged from 120 to 180 nm.
The PTX-loaded PSAU micelles which possessed superior stability
at physiological pH were able to release loaded PTX under a mild
acidic condition mimicking that of the endo/lysosomal compart-
ments. In vitro cytotoxicity studies revealed that PSAU micelles
showed nearly noncytotoxicity against SGC-7901 cells while the
PTX-loaded micelles (PTX dosage: 4.5 ␮g/mL) showed high cyto-
toxicity against SGC-7901 cells after incubation for 72 h. Taken
together, PSAU micelles seemed to be a potential drug delivery
system of PTX for cancer chemotherapy.
of drug reached to 42% within 72 h (Fig. 4). In the medium of pH
7.4, only 15% PTX was released during 72 h. Hence the drug release
depended upon the pH of the media. The initial release might be
ascribed to the dissolution and diffusion of drug that was not com-
pactly loaded in the cores of the micelles. It appeared, therefore,
that pH sensitive degradable micelles on one hand possessed good
stability at physiological pH and on the other hand could release
drug at mild acidic pH, mimicking that of endosomal and lysosomal
compartments owing to PSA acidic hydrolysis.
3.4. In vitro cytotoxicity assay
Conflict of interest
The cytotoxicity is an important issue for both the drug and the
carrier, thus the in vitro toxicity of blank micelles, free PTX, PTX-
loaded PSAU micelles were evaluated in SGC-7901 cell line for 24 h
and 72 h by MTT assay.
We declare that we have no conflict of interest.
Acknowledgments
The results revealed that empty PSAU micelles had low cyto-
toxicities (cell viability >80%) at 100 ␮g/mL during the incubation
time (Fig. 5). However, both the free-PTX and the PTX-loaded PSAU
micelles showed time-dependent cytotoxicity. After incubation for
72 h, the free-PTX and the PTX-loaded PSAU micelles (PTX dosage:
4.5 ␮g/mL) showed higher cytotoxicity against SGC-7901 cells (cell
viabilities 35.3% versus 53.8%). Compared with the cytotoxicity of
free PTX, PTX-loaded PSAU micelles showed a slightly lower cyto-
toxicity at the concentration tested for 72 h incubation. This may
be attributed to the sustained release property of PTX from PSAU
micelles and only part of the total encapsulated PTX released from
the PSAU micelles during incubation. The result suggested that PTX-
loaded PSAU micelles were automatically dissociated inside the
tumor cells. The schematic illustration of triggered release of PTX
This work was supported by Program for New Century Excel-
lent Talents in University (NCET-13-0480) and the National Natural
Science Foundation of China (NSFC) (Grant 31270860).
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