PH triggered doxorubicin delivery of PEGylated glycolipid conjugate micelles for tumor targeting therapy

Article abstract of DOI:10.1021/mp300002v

The main objective of this study was aimed at tumor microenvironment- responsive vesicle for targeting delivery of the anticancer drug, doxorubicin (DOX). A glucolipid-like conjugate (CS) was synthesized by the chemical reaction between chitosan and stearic acid, and polyethylene glycol (PEG) was then conjugated with CS via a pH-responsive cis-aconityl linkage to produce acid-sensitive PEGylated CS conjugates (PCCS). The conjugates with a critical micelle concentration (CMC) of 181.8 μg/mL could form micelles in aqueous phase, and presented excellent DOX loading capacity with a drug encapsulation efficiency up to 87.6%. Moreover, the PCCS micelles showed a weakly acid-triggered PEG cleavage manner. In vitro drug release from DOX-loaded PCCS micelles indicated a relatively faster DOX release in weakly acidic environments (pH 5.0 and 6.5). The CS micelles had excellent cellular uptake ability, which could be significantly reduced by the PEGylation. However, the cellular uptake ability of PCCS was enhanced comparing with insensitive PEGylated CS (PCS) micelles in weakly acidic condition imitating tumor tissue. Taking PCS micelles as a comparative group, the PCCS drug delivery system was demonstrated to show much more accumulation in tumor tissue, followed by a relatively better performance in antitumor activity together with a security benefit on xenograft tumor model.

Full text of DOI:10.1021/mp300002v

Article
pubs.acs.org/molecularpharmaceutics
pH Triggered Doxorubicin Delivery of PEGylated Glycolipid
Conjugate Micelles for Tumor Targeting Therapy
Fu-Qiang Hu, Yin-Ying Zhang, Jian You, Hong Yuan, and Yong-Zhong Du*
College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, P. R. China
S
* Supporting Information
ABSTRACT: The main objective of this study was aimed at tumor
microenvironment-responsive vesicle for targeting delivery of the
anticancer drug, doxorubicin (DOX). A glucolipid-like conjugate (CS)
was synthesized by the chemical reaction between chitosan and stearic
acid, and polyethylene glycol (PEG) was then conjugated with CS via a
pH-responsive cis-aconityl linkage to produce acid-sensitive PEGylated
CS conjugates (PCCS). The conjugates with a critical micelle
concentration (CMC) of 181.8 μg/mL could form micelles in aqueous
phase, and presented excellent DOX loading capacity with a drug
encapsulation efficiency up to 87.6%. Moreover, the PCCS micelles
showed a weakly acid-triggered PEG cleavage manner. In vitro drug
release from DOX-loaded PCCS micelles indicated a relatively faster
DOX release in weakly acidic environments (pH 5.0 and 6.5). The CS
micelles had excellent cellular uptake ability, which could be significantly reduced by the PEGylation. However, the cellular
uptake ability of PCCS was enhanced comparing with insensitive PEGylated CS (PCS) micelles in weakly acidic condition
imitating tumor tissue. Taking PCS micelles as a comparative group, the PCCS drug delivery system was demonstrated to show
much more accumulation in tumor tissue, followed by a relatively better performance in antitumor activity together with a
security benefit on xenograft tumor model.
KEYWORDS: polyethylene glycol, pH-sensitive, cis-aconityl linkage, polymeric micelles, chitosan, antitumor activity
micelles protonate or deprotonate in a crude way upon
variation of the pH. The change in ionization state thus leads to
a conformational alteration, followed by the unloading of the
drug conjugated to7−9 or encapsulated in10−12 the pH-
responsive polymeric micelles.
1. INTRODUCTION
Polymeric micelles have gained considerable attention as a
versatile nanomedicine platform for cancer therapeutic
applications since they were first proposed as drug carriers in
the 1980s.1,2 Consisting of hydrophilic and hydrophobic
segments, polymeric micelles are capable of forming a unique
core−shell architecture, which possesses a narrow size
distribution varying from 20 to 100 nm.3,4 Based on its size
and surface properties, the micelle can deliver the encapsulated
drug to tumor tissue in a remarkably efficient way due to the
well-known “enhanced permeability and retention (EPR)
effect”, where nanoparticles spontaneously accumulate in the
pathological area owing to the hydrophilic surface and long
blood duration. But even this is far away from people’s
expectation for an ideal drug delivery system for cancer
treatment.
The exploration of environment-sensitive nanoparticulate
carriers offers an interesting opportunity for both drug and
gene delivery where the delivery system becomes an active
participant, rather than passive vehicle, in the optimization of
therapy.5 Among all the intrinsic stimuli addressed through
smart drug carriers, pH is one of the most important triggers, as
a result of the relatively acidic environment at tumor which is
slightly distinguished from its surrounding normal tissue.6
Usually comprising amines, phosphoric acid or carboxylic acid
groups into the block copolymers, pH-sensitive polymeric
Biocompatible stearic acid grafted chitosan (CS) exhibits a
capability of rapid internalization and has been previously used
as a micelle-like drug carrier for oral drug delivery and
antitumor treatment, the latter of which include chemotherapy
and gene therapy.13−15 However, the toxicities are also
enhanced with the increased therapeutic effect of CS micelle
delivery system, because of its nonspecific cellular uptake.
Polyethylene glycol (PEG) is considered to be a hydrophilic
polymer approved by FDA for human use, which lacks
immunogenicity, antigenicity and toxicity.16 PEGylation of
the polymeric micelle presents advantages such as improved
water solubility, minimized recognization and phagocytization
by the reticuloendothelial system (RES), resulting in the
prolongation of circulation time. Unfortunately, there is a
potential obstacle for cellular uptake, a significant character of
PEGylation owing to the reduced cell surface lipid membrane
Received: January 2, 2012
Revised: July 13, 2012
Accepted: July 24, 2012
Published: July 24, 2012
© 2012 American Chemical Society
2469
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
interaction.17 Thus, it is suggested to be a good way to
introduce acid-cleavable linkage between PEG and micelles to
design acid-sensitive PEGylated polymeric micelles, which are
stable in vivo environments to reach prolonged circulation time
and reduced cellular uptake of the micelle delivery system by
normal cells. Furthermore, the PEG could cleave from the
micelles in tumor tissue (weakly acidic environment), and the
drug was delivered by the CS micelles into tumor cells. Among
all the acid-labile linkers, cis-anonityl acid and hydrazone
derivatives are the most prominent ones.18 Recent researches
have proposed successful conjugation of PEG to copolymer via
cis-aconitic anhydride19 and hydrazone.20
Herein, synthesized by coupling reaction between the amino
groups of chitosan and carboxyl group of stearic acid,21 CS was
used as backbone and further modified with PEG via pH-
sensitive cis-aconityl linkage to produce pH-sensitive
PEGylated CS conjugate (PCCS). In the meantime, the pH-
insensitive copolymer which conjugated PEG directly to CS
(PCS) was introduced for comparative purpose. Using DOX as
a model anticancer drug, the effects of the linkage style on drug-
encapsulating ability, in vitro drug release, cellular uptake and
cytotoxicity against immortalized rat liver BRL-3A cells and
human liver carcinoma BEL-7402 cells, as well as in vivo
distribution and anticancer activities were investigated in detail.
stirring at 60 °C. After stirring for 1 h, the organic phase was
slowly dropped into the aqueous phase. The reaction mixtures
were stirred at 80 °C for another 4 h and submitted to dialysis
against DI water using a dialysis membrane (MWCO: 7.0 kDa,
Spectrum Laboratories, Laguna Hills, CA) for 2 days. Then the
reaction solution was lyophilized and the lyophilized product
was purified with ethanol to remove the unreacted stearic acid.
Finally, the product CS was redispersed in DI water and
lyophilized.
The amino-substitution degree for CS was determined by
TNBS reaction.22 In brief, 2 mL of CS solution (containing 300
μg of CS) was added into 2 mL of NaHCO3 (4%, pH 8.5) and
2 mL of TNBS (0.1%) mixture solution. The reaction was
carried out by incubating the mixture at 37 °C for 2 h, followed
by the addition of 2 mL of HCl (2 N). The UV absorbance of
the final reaction mixture was measured at 344 nm by a UV
spectrophotometer (TU-1800PC, Beijing, Purkinje General
Instrument Co., Ltd., China). The degree of amino substitution
(SD, %) for CS was calculated using SD% process software.
To a round-bottom flask fitted with a pressure equalizing
dropping funnel were added mPEG-NH2 (500 mg) and
pyridine (5 mL). The stirred solution was cooled to 4 °C,
and CA (78 mg) in 10 mL of dioxane was added dropwise to
the PEG solution using the dropping funnel. Upon the
completion of the addition, the reaction mixture was stirred
another 6 h at 4 °C and was added into 80 mL of chilled diethyl
ether to precipitate the product. After the precipitate was
purified with chilled ether three times and isolated by
centrifugation at 4 °C (10000 rpm, 5 min), the reaction
intermediate so-called “mPEG-N-cis-aconityl” (mPEG-CA) was
obtained under drying in vacuum overnight.
mPEG-CA was then conjugated to CS copolymer in the
presence of water-soluble carbodiimide EDC. Briefly, 500 mg of
mPEG-CA, EDC and NHS were dissolved in 50 mL of DI
water. After the solution was stirred for 1 h to activate the
carboxylic acid of mPEG-CA, 200 mg of CS in 20 mL of DI
water was added, and the pH value of the reaction mixture was
adjusted to 7.0. The reaction was allowed to continue for 8 h at
room temperature. The resulting product was firstly purified by
pressure filtration in a stirred ultrafiltration cell (Amicon stirred
cell 8010, Millipore, USA) with an ultrafiltration membrane
(cutoff 5 kDa) from 50 to 5 mL. After repeating the dilution
and ultrafiltration procedure three times to remove unreacted
PEG and low molecular weight molecules, the reaction mixture
was lyophilized and the lyophilized product was washed with
ethanol. By redispersion in DI water and lyophilization, the
resulting product PCCS was received.
2. EXPERIMENTAL SECTION
2.1. Materials and Animals. Chitosan with average
molecular weight (Mw) of about 18 kDa was obtained by
enzymatic degradation21,22 of 95% deacetylated degree chitosan
(Mw = 450.0 kDa) which was supplied by Yuhuan Marine
Biochemistry Co., Ltd. (Zhejiang, China). Stearic acid (SA) was
purchased from Chemical Reagent Co., Ltd. (Shanghai, China).
Methoxy PEG with amine group (mPEG-NH2, Mw = 2000)
and methoxy PEG with aldehyde group (mPEG-ALD, Mw =
2000) were donated by Chinese Academy of Science. Cis-
aconitic anhydride (CA) was gifted by Alfa Aesar Co. (St.,
USA). N-Hydroxy-succinimide (NHS) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) were purchased
from Medpep Co., Ltd. (Shanghai China). 2,4,6-trinitrobenze-
nesulfonic acid (TNBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) and fluorescein isothiocya-
nate (FITC) were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Pyrene was purchased from Aldrich
Chemical Co. (USA). 1-Dodecylpyridinium chloride (DPC)
was purchased from Chemical Industries Co., Ltd. (Japan).
1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide
(DiR) was a product of Molecular Probes Inc. (Eugene, OR,
USA). Dulbecco’s modified Eagle’s medium (DMEM) and
trypsin-EDTA were purchased from Gibco-BRLC (MD, USA).
Fetal bovine serum (FBS) was purchased from Sijiqing Biologic
Co., Ltd. (Zhejiang, China). All other chemicals were of
analytical or chromatographic grade.
Male BALB/C nude mice (6−8 weeks old), ranging from 18
to 22 g, were provided by Shanghai SLAC Laboratory Animal
Co., Ltd. (Shanghai, China). All care and handling of animals
were performed in accordance with the guidelines of the animal
ethics committee of Zhejiang University (Hangzhou, China).
2.2. Synthesis and Characterization of PEGylated CS
Conjugate. The CS copolymer was synthesized via the
reaction of carboxyl groups of SA with amine groups of
chitosan in the presence of EDC.21 In brief, 0.5 g of chitosan
was dissolved in 33 mL of DI water. 0.825 g of SA and 5.5 g of
EDC were dissolved in 17 mL of ethanol and incubated under
PCS conjugates were prepared by coupling reaction of the
primary amine groups of CS conjugates and aldehyde side
group of mPEG-ALD using the method reported by Hu FQ et
al.23 Briefly, 500 mg of mPEG-ALD was added into a solution
of 200 mg of CS in DI water. The solution was stirred 6 h at
room temperature and then dialyzed against DI water using a
dialysis membrane (MWCO: 7.0 kDa) for 24 h. The final
product termed as PCS was harvested by lyophilization.
The structure of received CS, PCS and PCCS was confirmed
1
by H NMR spectra using an NMR spectrometer (AC-80,
Bruker Biospin, Germany). All samples were dissolved in D2O
with a concentration of 20 mg/mL for measurement.
PEGylation degree (PD, %) was calculated using formula 1,
by taking the characteristic peaks of PEG and CS into account.
PD% = A3.2/A0.9 × SD%
(1)
2470
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
A3.2 and A0.9 represented the peak areas at 3.2 ppm and 0.9
ppm in 1H NMR spectra of the conjugates, which corresponded
to the protons of terminal −OCH3 group of PEG and −CH3 of
SA, respectively.
conjugate (10 mg) in 1 mL of PBS (pH 7.4) was sealed in a
dialysis bag (MWCO: 7.0 kDa), which was then placed in a
plastic tube containing 10 mL of PBS (pH 5.0, 6.5 and 7.4).
The tests were carried out in an incubator shaker (HZ-88125,
Scientific and Educational Equipment plant, Taicang, China),
which was shaken horizontally at 65 rpm. At predetermined
time intervals, a 1 mL sample was taken from the outer fluid
and replaced with 1 mL of fresh PBS. The amount of
degradative PEG was determined by measuring the absorbance
at 334 nm using an ultraviolet spectrophotometer in
comparison to the calibration curve.
2.6. In Vitro Release of DOX from Drug-Loaded
Micelles. In vitro DOX release from drug loaded micelles
was performed using the dialysis method. Briefly, 1 mL of drug
loaded micelle solution was added into the dialysis bag
(MWCO: 3.5 kDa) and immersed into a plastic tube containing
15 mL of PBS with different pH (pH 5.0, 6.5 and 7.4). The
tests were conducted in an incubator shaker, which was
maintained at 37 °C and shaken horizontally at 65 rpm. The
whole medium was removed and replaced with fresh medium at
predetermined time intervals. Drug concentrations were
determined by fluorescence spectrophotometer with the same
parameter settings as described in a previous section.
Additionally, the release assay for free DOX was conducted
in PBS (pH 7.4) with the same procedure to exclude the
potential interference which might be caused by the blocking
effect of the dialysis bag.
2.7. Cell Culture. Immortalized rat liver cells BRL-3A and
human liver tumor cells BEL-7402 were obtained from Institute
of Biochemistry and Cell Biology (Shanghai, China), and were
cultured in DMEM supplemented with 100 U/mL penicillin,
100 U/mL streptomycin, and 10% (v/v) FBS in a humidified
atmosphere containing 5% CO2 at 37 °C. Cells were
subcultured regularly using trypsin/EDTA.
2.8. In Vitro Cytotoxicity Assay. Cytotoxicities of DOX,
blank, and DOX-loaded micelles against BRL-3A and BEL-
7402 were evaluated using MTT assay. 104 cells/well were
seeded in a 96-well culture plate (Nalge Nunc International,
Naperville, IL, USA) and allowed to adhere for 24 h. Treated
with serial concentrations of DOX, blank, and DOX-loaded
micelles, cells were cultured for another 72 h. After that, cells
were incubated with 20 μL of MTT (5 mg/mL) each well for
further 4 h. The medium was then removed, and 200 μL of
DMSO was added into each well for dissolution of the
formazan crystals. After 10 min’s shaking, absorption was
measured at 570 nm in a micro plate reader (Bio-Rad, model
680, USA).
The critical micelle concentration (CMC) of obtained CS,
PCS and PCCS in PBS (pH 7.4) was determined by
fluorescence spectroscopy using pyrene as a probe.24
Fluorescence spectra were recorded by fluorometer (F-2500,
Hitachi Co., Japan), with the excitation wavelength at 337 nm
and the slit at 10 nm (excitation) and 2.5 nm (emission). The
intensities of the emission were monitored at a wavelength
range from 360 to 450 nm. The concentration of CS, PCS and
PCCS solutions was varied from 1.0 × 10−3 to 1.0 mg/mL
containing 5.93 × 10−7 M pyrene. Then the intensity ratio of
the first peak (I1, 374 nm) to the third peak (I3, 385 nm) in the
fluorescence spectra was plotted versus the sample concen-
tration for the determination of CMC.
2.3. Preparation of Blank and DOX-Loaded Micelles.
To prepare the blank CS, PCS and PCCS micelle solution, 10
mg of the conjugates were respectively dissolved in 10 mL of
PBS (pH 7.4) followed by treatment of probe-type ultra-
sonicator (400 W, 30 cycles with 2 s active−3 s duration, JY92-
II, Scientz Biotechnology Co., Ltd., China) at room temper-
ature.
Doxorubicin base (DOX) was obtained by reaction of
doxorubicin hydrochloride (DOX·HCl) with twice the number
of moles of triethylamine (TEA) in DMSO overnight.25 An
appropriate amount of DOX/DMSO stock solution (1 mg/
mL) was added into the three kinds of micelle solutions
respectively (DOX/conjugate = 5%, w/w), and the entire
mixture was subjected to dialysis against PBS (pH 7.4) for 24 h
(MWCO: 3.5 kDa). After that, the mixture solutions were
centrifuged at 4000 rpm for 10 min to remove precipitated free
drug and the final products were received, which were termed
as CS/DOX, PCS/DOX and PCCS/DOX, respectively.
2.4. Characterization of Blank and Drug-Loaded
Micelles. Micellar size and zeta potential of the blank and
drug loaded micelles in PBS (pH 7.4) were measured by
Zetasizer analyzer (3000 HS, Malvern Instruments Ltd., U.K.).
The morphological examinations of the blank and drug
loaded micelles were performed by transmission electron
microscopy (TEM) (JEOL JEM-1230, Japan). The samples
were placed on copper grids with films and then stained with
2% (w/v) phosphotungstic acid for viewing by TEM.
The DOX content encapsulated in drug loaded micelles was
measured by fluorescence spectrophotometer with the
excitation wavelength, emission wavelength and slit at 505,
565, and 5 nm, respectively. All the drug loaded micelle
solutions were diluted 100-fold by DMSO aqueous solution
(DMSO:H2O = 9:1, v:v) and subjected to ultrasound for 30
min to dissociate the micelles. The DOX content was measured
by the calibration curve method. The drug encapsulation
efficiency (EE, %) and drug loading content (DL, %) could be
calculated by formulas 2 and 3:
2.9. Cellular Uptake of Blank and Drug-Loaded
Micelles. FITC-labeled micelles were prepared by adding 2
mg/mL FITC ethanol solution into 1 mg/mL micellar aqueous
solution (conjugate:FITC = 1:1, mol:mol). The mixture
solution was stirred under aphotic condition for 6 h, and
then was submitted to dialysis (MWCO: 7.0 kDa) against pure
water overnight to remove ethanol and unreacted FITC.
Finally, FITC-labeled micelle solution was obtained for the
cellular uptake test.
EE% = Me/Ma × 100%
(2)
At the same time, BRL-3A and BEL-7402 cells were seeded
at a density of 105 cells/well in a 24-well plate (Nalge Nunc
International, Naperville, IL, USA), respectively. After 24 h, the
medium was replaced by 1 mL of fresh medium at pH 6.5 or
7.4, and the same amount of blank micelles (100 μg) as well as
a certain amount of DOX-loaded micelles (5 μg of DOX
equivalent) was added for further incubation. For quantitative
DL% = Me/(Me + Mc) × 100%
(3)
where Me was the mass of DOX encapsulated in micelles; Ma
was the mass of DOX added; Mc was the mass of conjugate.
2.5. Assays for the PEG Cleavage from PCCS. In vitro
degradation behavior of PEG from the TNBS derivative PCCS
was examined at 37 °C in PBS with different pH. PCCS
2471
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
study, the cells were washed with PBS three times and
harvested at a predetermined time point. After repeated
freezing and thawing, the cell lysate was centrifuged at 10,000
rpm for 3 min, and the protein content inside the cell was
measured using the Micro BCA protein assay kit. Meanwhile,
the content of blank micelles and DOX taken up by cells was
measured by fluorescence spectrometer. The cellular uptake
percentages of blank micelles and DOX were calculated from
formula 4:
control. The remaining two groups were injected with PCCS/
DOX micelles (2 and 4 mg of DOX-equiv/kg body weight).
The drug was administered intravenously via tail vein everyday
from day 0 to day 6.
Mice were then checked for survival every day, and tumor
size as well as body weight were monitored every three days
from day 0. After 27 days, all mice were sacrificed, followed by
the measurement of tumor weight.
Tumor volume and inhibition of tumor growth were
calculated using formulas 5 and 6:
P% = Ct/C0 × 100%
(4)
t
tumor volume = (a2 × b)/2
(5)
where Pt represents the cellular uptake percentage of the
micelles at time t; Ct and C0 are intracellular concentration of
blank micelles or DOX corrected by intracellular protein
concentration at time t and time 0, respectively.
2.10. In Vivo Biodistribution Studies in Tumor-Bearing
Mice. The near-infrared fluorescent probe of DiR was loaded
into micelles for the further in vivo fluorescent imaging
investigation. The micelles of the above three formulations
were dissolved in DI water followed by addition of DiR which
was dispersed in DMSO (DiR/conjugate = 1%, w/w). The
mixture was sonicated with a bath type sonicator and was
submitted to dialysis for 8 h following by centrifugation at 4000
rpm for 10 min to remove the free DiR.
The BEL-7402 cells bearing male BALB/C nude mice (6−8
weeks old), which were established by subcutaneous inocu-
lation of 5 × 106 BEL-7402 cells/mouse into the left hind flanks
of nude mice, were applied as armpit tumor model to
investigate the in vivo distribution of DiR-loaded micelles.
The test was carried out when the tumor volume reached 150
mm3.
inhibition of tumor growth (%) = (W − W)/W × 100%
c
t
c
(6)
where a and b mean the shorter and the longer diameter of the
tumor respectively, and Wt and Wc mean the average tumor
weight of treated and negative controlled group respectively.
2.12. Statistical Analysis. Data were expressed as means of
three or six separate experiments. Differences between groups
were assessed using unpaired two-tailed Student’s t test, and a
p-value <0.05 was considered statistically significant in all cases.
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characteristics of CS and
PEGylated CS Conjugates. In the present study, an acid-
sensitive cis-aconityl linkage was introduced to conjugate PEG
and CS to produce PCCS by a two-step esterification of amine
groups. The synthesis scheme is presented in Figure 1A. At the
For each tumor model, mice were injected with 0.2 mL of
physiological glucose, free DiR and DiR-loaded micelles with
the same DiR concentration via tail vein, respectively. Mice
were anesthetized by inhalation of diethyl ether. At preset time
points, the anesthetized mice were put into the chamber and
the fluorescent images were detected using the Maestro in vivo
imaging system (CRI Inc., Woburn, MA, USA). Fluorescence
images of treated mice were acquired with a near-infrared filter
set (excitation filter, 684−729 nm; emission filter, 745 nm long
pass) and an exposure time of 500 ms. The software acquired
multispectral image cubes in 10 nm steps in the spectral range
between 740 and 950 nm. For ex vivo analysis, the mice were
sacrificed by CO2 asphyxia 24 h postinjection and excised
organs together with tumors were collected. The ex vivo
fluorescent images of the organs were detected using the same
imaging procedure accorded with the in vivo measurements.
The real-time accumulation of DiR in the tumor site was
evaluated by signal-to-noise ratio: average fluorescence signal at
the region of interest (ROI) was drawn around the tumor to
average fluorescence signal at the opposite region (right hind
flank) with the same area indicating background tissue.
2.11. In Vivo Antitumor Activity. The in vivo antitumor
efficacy of the DOX-loaded micelles was evaluated using BEL-
7402 cells bearing armpit tumor model as demonstrated in
section 2.10. Treatments were started 7−10 days after
transplantation when the tumor volume reached approximately
100 mm3, and this day was designed as day 0. On day 0, mice
were divided randomly into seven groups of six mice. 0.2 mL of
glucose solution and 2 mg/kg body weight of DOX·HCl were
administered as the negative and positive control respectively.
CS/DOX and PCS/DOX with the same dosage of 2 mg of
DOX-equiv/kg body weight were administered as praeparatum
Figure 1. The synthesis route (A) and structure confirmation (1H
NMR spectra using D2O as solvent, B) of PCCS.
same time, PEG was grafted to CS directly by the Schiff
reaction to obtain acid-insensitive PCS, which was utilized for
comparative study. As the backbone of PCS and PCCS, CS was
synthesized by coupling reaction between the carboxyl group of
SA and the amino groups of chitosan as previously reported.26
The ratio of substituted amino groups to the general amino
groups of CS was termed as amino substituted degree (SD%),
which was determined to be 5.9% by using the TNBS method.
As shown in Figure 1B, successful PEGylation was
authenticated by the simultaneous appearance of the peaks at
2472
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
1
0.9 ppm, 3.2 ppm, 3.4−3.6 ppm and 6.5 ppm in the H NMR
spectra of PCCS, which were attributed to the −CH3 groups of
SA, the terminal −OCH3 groups and repeated −CH2CH2O−
units’ protons of PEG, and vinylic protons of CA, respectively.
The grafted ratio of PEG (PD%) was defined as the substituted
ratio molecule to the total primary amino groups of chitosan. It
was calculated using the proton integration method, by taking
the proton peak areas of the characteristic peaks of SA (−CH3
groups) and PEG (−OCH3 groups) into account. The PEG
graft ratios of PCS and PCCS are presented in Table 1. It could
result of the formulation of the core−shell morphology and
incorporation of pyrene into the micelles. The concentration at
break point corresponded to the CMC from Figure 2. It was
clear that the CMC increased after PEGylation, which may
attributed to the elongated hydrophilic chain.
3.2. Preparation and Characteritics of Blank and
Drug-Loaded Micelles. Table 1 presented the micellar
hydrodynamic diameter and polydispersity index of the three
micelles in phosphate buffered solution (PBS, pH 7.4). Based
on light scattering experiments, the CS micelles had a smaller
hydrodynamic diameter of 27.6 1.8 nm and higher positive
zeta potential of 39.7 1.6 mV in PBS. An increase in micellar
size could be observed after PEGylation, which could be
attributed to the stretch of PEG chains on the PEGylated
micelle surface toward water phase.28 Moreover, the zeta
potential of PCS and PCCS micelles was a little lower than that
of CS micelle, which might result from the reduced amino
groups after PEGylation as well as the barrier shield effect of
PEG chains on the micelles.
a
Table 1. Characteristics of CS, PCS and PCCS
zeta
potential
(mV)
SD or
material PD (%)
CMC
size (nm)
PI
(μg/mL)
CS
5.9
25
22.7
27.6 1.8
39.5 7.3
50.6 7.0
0.58 0.10
0.54 0.04
0.62 0.01
39.7 1.6
26.2 5.9
24.8 5.5
122.2
214.5
181.8
PCS
PCCS
a
PI presents the polydispersity index of the micelle size. Data
DOX was chosen as the model drug and loaded into the
polymeric micelles by the ultrasound and dialysis method. The
encapsulation efficiency (EE%) of all three DOX-loaded
micelles was above 85%, and the drug loading content (DL
%) could reach up to 4.0% when the DOX feeding amount was
5%. The physicochemical characteristics of DOX-loaded CS,
PCS and PCCS (CS/DOX, PCS/DOX and PCCS/DOX)
micelles were also summarized in Table 2. A decrease in size
was found after DOX loading, which might mainly be caused by
the enhancement of cohesive force of the hydrophobic
interaction.13 However, the micellar zeta potential after drug
loading showed no significant change compared to the
corresponding blank micelles. Transmission electron micros-
copy (TEM) has been widely applied as a powerful tool for
detecting the size and surface morphology of nanoparticles.
Figure 3 presented the TEM images and size distribution of
PCCS and PCCS/DOX micelles, indicating micelles of
spherical morphology, with average size similar to those
obtained by dynamic light scattering. In addition, no obvious
changes in physicochemical characteristics of DOX-loaded
micelles were observed as the pH of the medium turned into
weakly acidic condition (pH 6.5, see Supporting Information).
It may be due to the pKa of chitosan, which is about 6.5.
3.3. In Vitro Behavior of PEG Degradation and DOX
Release. The in vitro behavior of PEG degradation from PCCS
was conducted at 37 °C under different pH conditions of 5.0,
6.5, and 7.4. As shown in Figure 4A, the PEG cleavage profile of
PCCS was both time- and pH-dependent. Within 48 h, PCCS
micelles exhibited 72.6 2.6% PEG cleavage at pH 5.0 while a
relatively lower cleavage percentage of 52.6 4.4% at pH 7.4.
This trend remained the same as the time prolonged over the
course of 252 h. This result indicated that the cleavage of PEG
from PCCS micelles under acidic conditions was governed by
the acid-responsive characteristic of the cis-aconitic anhydride
linkage between the PEG molecules and the polymer backbone.
An acid-cleavable linkage of cis-aconitic anhydride can undergo
hydrolysis mediated by an assisted intramolecular degradation29
in an acidic environment, and thus, PEG can be released from
the PCCS micelles. These findings agreed with those reported
previously with the DOX-CSO-SA conjugate.22
represent the mean standard deviation (n = 3).
be figured out that PCS and PCCS had a similar PEG
substitution of 25% and 22.7%, which meant each of them had
approximately 37 PEG molecules grafted onto the CS
conjugates by controlling the feeding molar ratio of supplied
PEG and CS.
Due to the composition of hydrophilic sections (chitosan)
and hydrophobic sections (stearic acid), CS could easily self-
assemble to form micelles in aqueous medium. After
PEGylation, both the PCS and PCCS conjugates kept this
property. The critical micelle concentration (CMC) was an
important physical parameter used to character the self-
aggregation ability of amphiphilic conjugates. The CMC of
CS, PCS and PCCS were determined by fluorometry using
pyrene as a probe. Pyrene would preferentially partition into
hydrophobic cores of micelles resulting in a concurrent change
of its photophysical properties.27 Figure 2 showed the
Figure 2. Variation of intensity ratio (I1/I3) vs the logarithm of
□
○
concentration of CS ( ), PCS ( ) and PCCS (Δ). Unit of
concentration was μg/mL.
dependence of the intensity ratio of the first and third peak
in the emission spectrum (I1/I3) on logarithm concentration of
samples. It could be seen that the values of I1/I3 at lower
concentration remain constant at about 1.7 even when the
concentration increased to some extent. However, with further
increases in concentration, the ratios decrease abruptly as a
To study the correlation between the drug release rate and
effect of PEGylation as well as PEG cleavage of pH-sensitive
micelles, the DOX release from the DOX-loaded micelles was
investigated at three different pHs consistent with the PEG
2473
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
a
Table 2. Characteristics of DOX-Loaded Micelles
material
feed ratio (w/w,%)
size (nm)
PI
zeta potential (mV)
DL (%)
EE (%)
CS/DOX
5
5
5
21.8 1.3
28.5 5.0
26.5 1.3
0.57 0.14
0.61 0.02
0.63 0.03
32.8 3.5
27.7 5.0
28.2 1.0
4.35 0.14
4.14 0.19
4.19 0.13
91.05 3.13
86.46 4.05
87.59 2.76
PCS/DOX
PCCS/DOX
a
PI presents the polydispersity index of the micelle size. Data represent the mean standard deviation (n = 3).
degradation experiment. The results dictated in Figure 4B−D
revealed a significant impact of PEGylation on the drug release
rate. A small initial burst release of DOX from CS/DOX was
observed at all pHs and appeared to reach a 50% cumulative
release percentage only within 12 h. However, as to PCS/DOX,
the release of DOX was demonstrated to be much slower,
which took nearly 72 h to contribute half drug release. In
particular, PCCS/DOX exhibited a moderate drug release rate
as compared to CS/DOX and PCS/DOX. In addition, it was
interesting to note that the drug release rate of PCCS/DOX
was slightly accelerated by decreasing pH. According to the
work done by P. R. Cullis and colleagues, it might be the
favorable partition of DOX that caused the faster drug release
in an acidic environment.30,31 However, it was observed that
the drug release rate of PCCS/DOX was influenced by pH
while the other two groups (CS/DOX and PCS/DOX)
represented no significant change against pH variation. The
phenomenon might be explained as follows: first, the PEG
bonding on the surface of the micelles created a barrier layer,
which hampered DOX release from the hydrophobic core.32 As
accelerated PEG cleavage occurred in PCCS micelles in acid
medium solution, the shielding effect of PEG chains reduced
and led to a faster DOX release. Second, the appreciably smaller
particle size of CS/DOX resulted in greater surface area than
PCS/DOX, promoting DOX diffusion and dissolution from
polymer.
Figure 3. Size distribution obtained by DLS and TEM images of
PCCS and PCCS/DOX micelles: size distribution (A) and TEM
image (C) of PCCS micelles; size distribution (B) and TEM image
(D) of PCCS/DOX micelles.
Figure 4. In vitro behavior of PEG degradation and DOX release in PBS with different pHs: PEG degradation profile of PCCS (A); release profiles of
DOX from CS/DOX (B), PCS/DOX (C) and PCCS/DOX (D). Data represent the mean standard deviation (n = 3). * and ** represent P <
0.05 and P < 0.01, respectively.
2474
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
3.4. Cellular Uptake of Blank and Drug-Loaded
Micelles. To deeply investigate the relative amount of micelle
and drug endocytosis happening in the two cell lines, the
quantitative cellular uptake study was conducted. In this way,
the potent interference caused by different FITC (fluorescein
isothiocyanate)-labeling efficiency for different conjugates could
be excluded. As shown in Figure 5A,B, for normal liver cells
combined with a slight increase in uptake percentage of PCCS
under weakly acidic environment compared to normal
physiological conditions, which might be partly attributed to
the higher proportion of PEG cleavage under low pH
conditions. It was a promising result indicating a specific
cellular uptake capacity of PCCS micelles, which might result in
improved therapeutic efficiency as well as reduced side effects.
Furthermore, the intracellular uptake of free DOX and DOX-
loaded micelles was also conducted in a quantitative way. It
could be observed from Figure 5C that the cellular uptake of
DOX showed an overall trend of time dependent irrespective of
free or encapsulated drug. Compared to CS/DOX, a significant
decrease in cellular uptake was observed when cells were
incubated with PCS/DOX (P < 0.05), likely due to the poor
internalization of PCS that was demonstrated previously.
Besides, taking the quantitative data of CS/DOX and PCS/
DOX into account, there presented a medium cellular DOX
uptake percentage of about 20% for PCCS/DOX after 24 h
incubation, being parallel with the result of internalization of
blank micelles.
3.5. In Vitro Cytotoxicity Assay. Figure 6 shows the
cellular growth inhibition of BRL-3A and BEL-7402 cells after
incubation with CS, PCS and PCCS conjugates for 72 h.
Obviously, modification of PEG reduced the cytotoxicity,
representing an increased IC50 value from 250 to 300 μg/mL to
approximately 500 μg/mL. Moreover, the cellular growth
inhibition percentage was even less than 50% when incubated
with PCCS conjugates at the concentration of 1000 μg/mL.
These results indicate that the present micelles show a high
biocompatibility and a low cytotoxicity against the cultured cells
as drug carriers.
Cytotoxicities of free DOX and DOX-loaded micelles against
BEL-7402 were also conducted by MTT assays with cell
incubation time of 72 h, and the IC50 values are summarized in
Figure 5D. Compared with CS/DOX micelles, the cytotoxicity
of DOX-loaded micelles against BEL-7402 cells decreased after
the modification of PEG. However, with the introduction of
acid-sensitive linkage, the cytotoxicity of DOX-loaded PCCS
micelles increased by 2−3-fold as compared to PCS/DOX
micelles (P < 0.05), equivalent to the free DOX. It was an
observation consistent with the medium rate of DOX release
and endocytic uptake for PCCS/DOX.
Figure 5. Comparison of cellular uptake percentage and IC50 value of
blank or drug-loaded micelles: Cellular uptake of micelles on BRL-3A
cells (A) and BEL-7402 cells (B); cellular uptake of DOX on BEL-
7402 cells (C); IC50 values of free DOX and DOX loaded micelles
against BEL-7402 cells. Data represent the mean standard deviation
(n = 3).
BRL-3A under physiological pH conditions, the quantitative
cellular uptake percentage of CS was above 18% after 24 h but
the value significantly reduced to less than 3% (P < 0.05) after
pH-insensitive PEG modification. As to liver tumor cells BEL-
7402, the same trend of decreasing cellular uptake after
PEGylation was observed. However, there was a relatively
higher cellular internalization percentage for PCCS than PCS
3.6. In Vivo Biodistribution Studies in Tumor-Bearing
Mice. DiR-loaded micelles were given intravenously into BEL-
Figure 6. Cytotoxicity of CS, PCS and PCCS conjugate against BRL-3A (A) and BEL-7402 (B) cells for 72 h. Data represent the mean standard
deviation (n = 3).
2475
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
7402 bearing tumor model through the tail vein in order to
trace in vivo real-time biodistribution of PCCS micelles. Whole-
body fluorescence at 1 h, 6 h, and 24 h postinjection of DiR or
DiR-loaded micelles is presented in Figure 7A,C. Visible
in terms of the PCCS group, a similar trend of increasing
fluorescence was observed in just the same way as for the PCS
group. Particularly, the fluorescent image confirmed a higher
fluorescence accumulation in the tumors of the PCCS group
when compared to the other two groups. Meanwhile, as for the
mice treated with free DiR, no obvious fluorescent signals were
observed in the site of tumor from 6 to 24 h postinjection. This
is similar to the work done by Qiang Zhang and colleagues, in
which the researchers commented that free DiR was eliminated
from the body so quickly that it was not detectable and only a
thimbleful remained in the liver.33
Fluorescence images of excised tissues (tumor, heart, spleen,
lung, kidney and liver) (Figure 7B) were obtained 24 h after
injection. The results further confirmed the highest fluores-
cence accumulation in the tumor of PCCS micelles group,
combined with a weaker signal of DiR in liver and spleen which
had high macrophage uptake nature. Similar to the results
obtained from the whole-body imaging, the mice given free DiR
showed barely any fluorescence signal in tumor 24 h
postinjection, suggesting that the probable interference caused
by free DiR could be excluded and consequently the differences
of the fluorescence signal between the groups were mainly
attributed to the drug carrier itself.
These current results suggested that, first, the PEGylation of
micelles could prolong the retention of DiR in the body, and
second, the drug delivery system of pH-sensitive PCCS carrying
DiR exhibited higher accumulation in solid tumor which might
lead to better targeting efficiency as compared with the PCS
group.
3.7. In Vivo Antitumor Activity. The in vivo antitumor
efficacy of three different kinds of DOX-loaded micelles was
evaluated in BEL-7402 bearing mouse model, in comparison
with those of physiological glucose and DOX·HCl. The time-
dependent changes in the tumor volume are plotted and
presented in Figure 8A. It can be seen that, at the end of the
test (27 days), the tumor growth rates of mice treated with
DOX·HCl and DOX-loaded micelles were suppressed
effectively. Specifically, after iv injection with the same dosage
of 2 mg/kg DOX-equiv for 7 days, DOX·HCl, CS/DOX and
PCS/DOX groups displayed tumor inhibition rates of 80.98%,
84.21% and 50.60%, respectively. Although a more optimal
therapeutic effect was expected to be seen when employing
PEGylated nanoparticles since accumulation of PEGylated drug
carriers in tumors was favored by their long circulation time
and EPR effect,34 PCS/DOX conducted a poor performance
throughout the present in vivo antitumor study. This result
might be attributed to the combined influence of poor
internalization and slow drug release for the PCS drug delivery
system. As to PCCS/DOX, the tumor inhibition rate for dosage
of 2 mg/kg DOX-equiv was 68.8%, preceding that of the PCS/
DOX group, and the value reached up to 75.7% when doubling
the dosage to 4 mg/kg. That is to say, there was a relatively
better performance for PCCS/DOX compared to pH
insensitive PCS/DOX, likely attributed to the enhancement
of cellular uptake caused by PEG cleavage in vivo (especially in
tumor microenvironment). However, it seemed to be late for
the coming of significant antitumor activity of PCCS/DOX. As
it is known to all, the distribution for drug carriers into the
tumor was only a premise for chemotherapeutics to work. The
efficacy occurred only when pulse drug concentration was
achieved or in other words enough drug molecules accumulated
in tumor cells. Additionally, as shown in Figure 8B, the relative
body weight of mice in both DOX·HCl and CS/DOX group
Figure 7. In vivo fluorescent images of BEL-7402 tumor bearing mice
of armpit model given free DiR and DiR-loaded micelles via tail vein, 1
h, 6 h and 24 h after administration, respectively (A), and fluorescent
image of tissues ex vivo (B) 24 h postinjection. Relative fluorscence
signal of DiR distributed in tumors in comparison with background
over time in living mice model (C).
fluorescence could be detected in tumors of the CS group as
early as 1 h after injection, and exhibited increasing signal
intensity at 6 h while decreased at 24 h postinjection. As to the
PCS group, the fluorescence signal presented a delayed
appearance and conducted a continuous accumulation in
intensity through all experimental time points. Additionally,
2476
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
Figure 8. In vivo antitumor activity of DOX·HCl and DOX-loaded micelles after iv injection via tail vein in tumor bearing mice: mouse tumor
volume (A) and mouse relative body weight (B) changes within 27 days. Data represent the mean standard deviation (n = 6).
(3) Kataoka, K.; Harada, A.; Nagasaki, Y. Block copolymer micelles
for drug delivery: design, characterization and biological significance.
Adv. Drug Delivery Rev. 2001, 47, 113−131.
(4) Gaucher, G.; Dufresne, M. H.; Sant, V. P.; Kang, N.; Maysinger,
D.; Leroux, J. C. Block copolymer micelles: preparation, character-
ization and application in drug delivery. J. Controlled Release 2005, 109,
169−188.
(5) Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M. A review of
stimuli-responsive nanocarriers for drug and gene delivery. J. Controlled
Release 2008, 126, 187−204.
(6) Engin, K.; Leeper, D. B.; Cater, J. R.; Thistlethwaite, A. J.;
Tupchong, L.; McFarlane, J. D. Extracellular pH distribution in human
tumours. Int. J. Hyperthermia 1995, 11, 211−216.
(7) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S.
Amphiphilic multi-arm-block copolymer conjugated with doxorubicin
via pH-sensitive hydrazone bond for tumor-targeted drug delivery.
Biomaterials 2009, 30, 5757−5766.
(8) Bae, Y.; Nishiyama, N.; Fukushima, S.; Koyama, H.; Yasuhiro, M.;
Kataoka, K. Preparation and biological characterization of polymeric
micelle drug carriers with intracellular pH-triggered drug release
property: tumor permeability, controlled subcellular drug distribution,
and enhanced in vivo antitumor efficacy. Bioconjugate Chem. 2005, 16,
122−130.
(9) Park, J. H.; Cho, Y. W.; Son, Y. J.; Kim, K.; Chung, H.; Jeong, S.
Y.; Choi, K.; Park, C. R.; Park, R. W.; Kim, I. S. Preparation and
characterization of self-assembled nanoparticles based on glycol
chitosan bearing adriamycin. Colloid Polym. Sci. 2006, 284, 763−770.
(10) Gao, Z. G.; Lee, D. H.; Kim, D. I.; Bae, Y. H. Doxorubicin
loaded pH-sensitive micelle targeting acidic extracellular pH of human
ovarian A2780 tumor in mice. J. Drug Targeting 2005, 13, 391−397.
(11) Lee, E. S.; Na, K.; Bae, Y. H. Doxorubicin loaded pH-sensitive
polymeric micelles for reversal of resistant MCF-7 tumor. J. Controlled
Release 2005, 103, 405−418.
(12) Lee, E. S.; Na, K.; Bae, Y. H. Super pH-sensitive multifunctional
polymeric micelle. Nano Lett. 2005, 5, 325−329.
(13) Hu, F. Q.; Zhao, M. D.; Yuan, H.; You, J.; Du, Y. Z.; Zeng, S. A
novel chitosan oligosaccharide-stearic acid micelles for gene delivery:
properties and in vitro transfection studies. Int. J. Pharm. 2006, 315,
158−166.
(14) Hu, F. Q.; Wu, X.; Du, Y. Z.; You, J.; Yuan, H. Cellular uptake
and cytotoxicity of shell crosslinked stearic acid-grafted chitosan
oligosaccharide micelles encapsulating doxorubicin. Eur. J. Pharm.
Biopharm. 2008, 69, 117−125.
(15) Yuan, H.; Lu, L.; Du, Y. Z.; Hu, F. Q. Stearic acid-g-chitosan
polymeric micelle for oral drug delivery: in vitro transport and in vivo
absorption. Mol. Pharmaceutics 2011, 8, 225−238.
(16) Pasut, G.; Veronese, F. M. Polymer-drug conjugation, recent
achievements and general strategies. Prog. Polym. Sci. 2007, 32, 933−
961.
exhibited a continual decrease during the administration, while
the experimental group even treated with a high dosage of
PCCS/DOX (4 mg/kg) remained essentially the same
throughout the whole study just as the negative group. These
results indicated that PCCS/DOX micelles could reduce
toxicity to normal tissue while maintaining therapeutic effects.
4. CONCLUSIONS
A pH-sensitive PCCS conjugate was synthesized successfully in
this work. PEGylation significantly reduced cytotoxicity of the
conjugate, and acid-triggered PEG degradation combined with
DOX release was demonstrated to be feasible in efficient
internalization to tumor cells, not only for vehicles but also for
encapsulated DOX. Furthermore, in vivo studies indicated that
PCCS/DOX conducted well in inhibiting the growth of the
solid tumor while reducing the toxicity against animal body.
Overall, the results suggested that the PCCS micelle was a
promising candidate for smart drug delivery.
ASSOCIATED CONTENT
* Supporting Information
■
S
The physicochemical characteristics of DOX-loaded micelles at
pH 6.5. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*College of Pharmaceutical Sciences, Zhejiang University, 388
Yuhangtang Road, Hangzhou 310058, P. R. China. Tel
(fax):86-571-88208439. E-mail: duyongzhong@zju.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for financial support from the National Basic
Research Program of China (973 Program) under Contract
2009CB930300, the National HighTech Research and
Development Program (863) of China (2007AA03Z318),
and the National Nature Science Foundation of China under
Contract 81072583.
REFERENCES
■
(1) Sutton, D.; Nasongkla, N.; Blanco, E.; Gao, J. Functionalized
micellar systems for cancer targeted drug delivery. Pharm. Res. 2007,
24, 1029−1046.
(2) Jones, M. C.; Leroux, J. C. Polymeric micelles-a new generation
of colloidal drug carriers. Eur. J. Pharm. Biopharm. 1999, 48, 101−111.
(17) Nie, Y.; Gunther, M.; Gu, Z.; Wagner, E. Pyridylhydrazone-
̈
based PEGylation for pH-reversible lipopolyplex shielding. Biomate-
rials 2010, 32, 858−869.
2477
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478

Molecular Pharmaceutics
Article
(18) Schmaljohann, D. Thermo-and pH-responsive polymers in drug
delivery. Adv. Drug Delivery Rev. 2006, 58, 1655−1670.
(19) DuBois Clochard, M. C.; Rankin, S.; Brocchini, S. Synthesis of
soluble polymers for medicine that degrade by intramolecular acid
catalysis. Macromol. Rapid Commun. 2000, 21, 853−859.
(20) Zhou, L.; Yu, L.; Ding, M.; Li, J.; Tan, H.; Wang, Z.; Fu, Q.
Synthesis and Characterization of pH-Sensitive Biodegradable Polyur-
ethane for Potential Drug Delivery Applications. Macromolecules 2011,
44, 857−864.
(21) Hu, F. Q.; Ren, G. F.; Yuan, H.; Du, Y. Z.; Zeng, S. Shell cross-
linked stearic acid grafted chitosan oligosaccharide self-aggregated
micelles for controlled release of paclitaxel. Colloids Surf., B 2006, 50,
97−103.
(22) Hu, F. Q.; Liu, L. N.; Du, Y. Z.; Yuan, H. Synthesis and
antitumor activity of doxorubicin conjugated stearic acid-g-chitosan
oligosaccharide polymeric micelles. Biomaterials 2009, 30, 6955−6963.
(23) Hu, F. Q.; Meng, P.; Dai, Y. Q.; Du, Y. Z.; You, J.; Wei, X. H.;
Yuan, H. PEGylated chitosan-based polymer micelle as an intracellular
delivery carrier for anti-tumor targeting therapy. Eur. J. Pharm.
Biopharm. 2008, 70, 749−757.
(24) Slaughter, J. N.; Schmidt, K. M.; Byram, J. L.; Mecozzi, S.
Synthesis and self-assembly properties of a novel [poly (ethylene
glycol)]-fluorocarbon-phospholipid triblock copolymer. Tetrahedron
Lett. 2007, 48, 3879−3882.
(25) Kohori, F.; Yokoyama, M.; Sakai, K.; Okano, T. Process design
for efficient and controlled drug incorporation into polymeric micelle
carrier systems. J. Controlled Release 2002, 78, 155−163.
(26) Hu, F. Q.; Li, Y. H.; Yuan, H.; Zeng, S. Novel self-aggregates of
chitosan oligosaccharide grafted stearic acid: preparation, character-
ization and protein association. Pharmazie 2006, 61, 194−198.
(27) Gao, J.; Ming, J.; He, B.; Fan, Y.; Gu, Z.; Zhang, X. Preparation
and characterization of novel polymeric micelles for 9-nitro-20 (S)-
camptothecin delivery. Eur. J. Pharm. Sci. 2008, 34, 85−93.
(28) Eliyahu, H.; Joseph, A.; Schillemans, J. P.; Azzam, T.; Domb, A.
J.; Barenholz, Y. Characterization and in vivo performance of dextran-
spermine polyplexes and DOTAP/cholesterol lipoplexes administered
locally and systemically. Biomaterials 2007, 28, 2339−2349.
(29) DuBois Clochard, M. C.; Rankin, S.; Brocchini, S. Synthesis of
soluble polymers for medicine that degrade by intramolecular acid
catalysis. Macromol. Rapid Commun. 2000, 21, 853−859.
(30) Mayer, L. D.; Tai, L. C. L.; Bally, M. B.; Mitilenes, G. N.;
Ginsberg, R. S.; Cullis, P. R. Characterization of liposomal systems
containing doxorubicin entrapped in response to pH gradients.
Biochim. Biophys. Acta, Biomembr. 1990, 1025, 143−151.
(31) Harrigan, P. R.; Wong, K. F.; Redelmeier, T. E.; Wheeler, J. J.;
Cullis, P. R. Accumulation of doxorubicin and other lipophilic amines
into large unilamellar vesicles in response to transmembrane pH
gradients. Biochim. Biophys. Acta, Biomembr. 1993, 1149, 329−338.
(32) Bhadra, D.; Bhadra, S.; Jain, S.; Jain, N. K. A PEGylated
dendritic nanoparticulate carrier of fluorouracil. Int. J. Pharm. 2003,
257, 111−124.
(33) Xiang, Y.; Liang, L.; Wang, X.; Wang, J.; Zhang, X.; Zhang, Q.
Chloride channel-mediated brain glioma targeting of chlorotoxin-
modified doxorubicine-loaded liposomes. J. Controlled Release 2011,
152, 402−410.
(34) Xin, H.; Chen, L.; Gu, J.; Ren, X.; et al. Enhanced anti-
glioblastoma efficacy by PTX-loaded PEGylated poly(ε-caprolactone)
nanoparticles: In vitro and in vivo evaluation. Int. J. Pharm. 2010, 402,
238−247.
2478
dx.doi.org/10.1021/mp300002v | Mol. Pharmaceutics 2012, 9, 2469−2478