Macromolecular Research, Vol. 21, No. 12, pp 1331-1337 (2013) www.springer.

com/13233
DOI 10.1007/s13233-013-1171-x pISSN 1598-5032 eISSN 2092-7673

Tumor Targeting and pH-Responsive Polyelectrolyte Complex Nanoparticles

Based on Hyaluronic Acid-Paclitaxel Conjugates and Chitosan for
Oral Delivery of Paclitaxel

Jiao Li1, Pingsheng Huang1, Longlong Chang1, Xingwen Long1, Anjie Dong1, Jinjian Liu2, Liping Chu2,
Fuqiang Hu3, Jianfeng Liu*,2, and Liandong Deng*,1
1
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China
2
Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine,
Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, P. R. China
3
College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, P. R. China

Received March 19, 2013; Revised May 8, 2013; Accepted May 17, 2013

Abstract: A new platform of paclitaxel (PTX) for application as an oral delivery system was developed, by com-
bining the pH sensitivity of polyelectrolyte complex nanoparticles (CNPs) and the active targeting of hyaluronic acid
(HA). Chitosan/hyaluronic acid-paclitaxel (CS/HA-PTX) CNPs were prepared by coating the CS onto the HA-PTX
nanoparticles (NPs), and characterized by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic reso-
nance (1H NMR), transmission electron microscopy (TEM) and high-performance liquid chromatography (HPLC).
HA-PTX conjugates could self-assemble into NPs in aqueous solution with an average size of 100±5 nm, and the
PTX content of HA-PTX conjugates was 10.6 wt%. The CS/HA-PTX CNPs had a smaller size and higher PTX content
when the ratio of positive charge to negative charge was 2:1. The in vitro release of PTX from CNPs was pH-responsive,
suggesting that the CS shell could prevent the breakage of the ester bond in HA-PTX NPs in acidic pH conditions.
HA-PTX NPs exhibited higher cellular uptake than free PTX against HepG2 cells via receptor-mediated endocyto-
sis. PTX could accumulate remarkably into tumor sites after oral administration of CNPs. These results indicate that
the CNP drug delivery system has great potential for applications in the oral administration of hydrophobic drugs.
Keywords: polyelectrolyte complex nanoparticles, pH-responsive, tumor targeting, oral administration, hyaluronic acid,
paclitaxel.

Introduction Various attempts have been made to protect hydrophobic

Oral administration is the most comfortable and conve-
nient method for drug delivery in the cases of chronic thera-
pies. It eliminates pain caused by injection and possible
infections associated with multiple daily injections.1-3 Nev-
ertheless, drugs, especially hydrophobic drugs must overcome
variously significant barriers before reach to the blood-
stream.4 Poor water solubility makes the hydrophobic drugs
hard to fully contact with the gastrointestinal (GI) mucous
membrane, which prevents the penetration and subsequent
absorption.5,6 First-pass meta bolism by Cytochrome P450
(CYP450)-dependent enzymes in the GI tract and liver
reduce the bioavailability of drugs.7 Moreover, P-glycopro-
tein (P-gp) are located in the apical membrane of epithelial
cells (e.g. in the intestinal wall), where they can actively extrude
a variety of structurally diverse drugs and drug metabolites.8
*Corresponding Authors. E-mails: dengliandong@yahoo.com.cn or
lewis78@163.com
drugs from the aggressive environments, such as developing
micellar formulations9-13 and polymer-drug approaches.14-20
Among these methods, polymer-drug approach has been
shown as a promising way for oral administration. Lee et al.17
studied the low molecular weight chitosan-paclitaxel (LMWC-
PTX) conjugates, which could greatly enhanced bioavail-
ability and prolonged retention time of the drug in the GI
tract due to the mucoadhesive property of LMWC. Milas et
al.18 found that the uptake, tumor retention, and antitumor
efficacy of poly(L-glutamic acid)-paclitaxel (PG-PTX) were
dramatically potentiated compared with free PTX. Conse-
quently, polymer-drug has been proved to be an efficient
way to improve bioavailability, increase half-life of hydro-
phobic drugs and enhance antitumor effects. Despite signif-
icant advances, lots of the existing polymer-drug for orally
administrated hydrophobic drugs has not reached the desired
performance, such as complex process of prepared,15,20 low-
biocompatibility of polymer19,21and the premature drug release
caused by the hydrolysis of ester bond or amide linkage in

The Polymer Society of Korea 1331

 J. Li et al.
Scheme I. Scheme representation for the preparation and appli-
cation of CS/HA-PTX CNPs.
acid environment.15,19
In this study, hyaluronic acid (HA) was used to conjugate
with PTX (a well-known mitotic inhibitor used in cancer
chemotherapy21,22) to synthesize HA-PTX conjugates via
esterification reaction. HA is generally regarded as non-toxic,
biodegradable and natural acidic polysaccharide, which is
one of the main components of the extracellular matrix.23 In
addition, the active targeting via HA and CD44 receptor
interaction has been found on the cell surface of several
malignant tumors, which bring about the broad applications
of HA-based polymers in active tumor targeting for antican-
cer drugs.24-28 Furthermore, hyaluronidase (HAase) is widely
distributed in the acidic tumor extracellular matrix, which
played a signicant role in the degradation of HA.29 Chitosan
(CS), a natural cationic polymer, with biocompatibility, bio-
degradability and low toxicity, was used to coat HA-PTX
nanoparticles (NPs) to prepare pH-responsive polyelectrolyte
camplex nanoparticles (CNPs).30 Several favorable advantages
were anticipated with this CNPs. First, preparation process of
CNPs was simple and feasible, and HA-PTX conjugates could
improve the water solubility of PTX, bypass CYP450-
dependent metabolism and overcome the P-gp-mediated
barrier to drug.17,19,31 Secondly, the pH-sensitive CNPs pro-
tect the PTX release from HA-PTX NPs in the GI tract.30,32,33
Finally, HA-PTX NPs have the active targeting tumor cell
via HA and CD44 receptor interaction. The illustration about
preparation and the pH-sensitive of CS/HA-PTX CNPs were
shown in Scheme I. The characteristi cs of the prepared CNPs
were well studied and the in vitro drug release was imple-
mented at different pH range. The cellular uptake and MTT
assay was performed on HepG2 and NIH-3T3 cell lines. Ex
vivo biodistribution of PTX via oral administration of CS/
HA-PTX CNPs were investigated.
Experimental
Materials. Chitosan (CS) was purchased from Aokang
1332
biological technology Co., LTD (Shandong, China) with the
molecular weight of 100 kD. Hyaluronic acid (HA) sodium
salt was purchased from FuRuiDa pharmaceutical Co., LTD
with the molecular weight of 29 kD (Shandong, China). Pacli-
taxel (PTX) was provided by TianFeng biological engineering
technology Co., LTD (Shenyang, China). Dicyclohexylcar-
bodiimide (DCC) and 4-dimethylaminopyridine (DMAP)
were purchased from J&K Chemical LTD. Dimethyl sul-
foxide (DMSO) was dried over calcium hydride (CaH2) for
48 h at room temperature and distilled under reduced pres-
sure just before use. Other reagents were commercially available
and used as received.
Preparation of Hyaluronic Acid-Paclitaxel (HA-PTX)
Conjugates and NPs. 1 g of HA was dissolved in 100 mL
of deionized water and dialyzed (MWCO: 8-14 kD) for
12 h in dilute acid followed by lyophilized. 50 mg of the
desalted HA powder was dissolved in 5 mL of anhydrous
DMSO, stirred for 2 h at 80 ºC. Then DCC and DMAP were
added to activate carboxylic groups in HA, stirred for 1 h at
room temperature. Subsequently, 25 mg of PTX was dis-
solved in 1 mL of anhydrous DMSO and added to the solu-
tions under dry N2 with stirring for another 24 h at 40 ºC.
The resultant solution was dialyzed against DMSO for 24 h
and deionized water for 48 h, and followed by lyophilized.
HA-PTX conjugates were dissolved in deionized water to
form the NPs under magnetic stirring.
Preparation of Polyelectrolyte CNPs (CS/HA-PTX CNPs).
CS and HA-PTX conjugates were dissolved in acetic acid solu-
tion (pH 5.0) and deionized water, respectively. The concen-
trations of CS and HA-PTX conjugates were both 1.0 mg/mL.
Then, HA-PTX solution was added into the CS solution to
obtain CS/HA-PTX CNPs. The pH of the mixed solution was
adjusted with diluted NaOH or HCl to the designated value.
Characterization. Fourier-transform infrared spectroscopy
measurements (FTIR) were executed on a BIO-RAD FTS3000
system with KBr pellets. Nuclear magnetic resonance (1H
NMR) spectra were recorded on a Varian INOVA 500 MHz
nuclear magnetic resonance instrument using DMSO-d6 and
D2O as solvents, and tetramethylsilane (TMS) as internal stan-
dard. The size and zeta potentials of materials were mea-
sured on a BI 90 Plus/Zetaplus instrument of Brookhaven
Corporation at 25 ºC employing a laser source at an angle of
90º. Transmission electron microscope (TEM) was also
employed to visualize the possible morphologies of NPs.
TEM samples were prepared by adding two drops of NPs
(1.0 mg/mL) onto a copper grid with a carbon film and air-
dried at room temperature. High performance liquid chro-
matography (HPLC) was used to determine the amount of
paclitaxel conjugated to HA.
In vitro Release. The in vitro drug-release was evaluated
by the dialysis method. Dialysis bags containing 4.0 mL of
the NPs solution and 1.0 mL hyaluronidase-1 in release medium
were immersed in a thermostated gas bath containing 20 mL
release medium with different pH values (pH 7.4, 6.0, and
Macromol. Res., Vol. 21, No. 12, 2013
 Tumor Targeting and pH-Responsive Polyelectrolyte Complex Nanoparticles for Oral Administration
3.0) at 37 ºC. At predefined intervals, 10 mL of receiving
buffer were withdrawn and replaced with the same volume
of release medium.
In vitro Cellular Uptake. Rhodamine B (RB) had an
intrinsic fluorescence spectrum which often was used to moni-
tor localization of the drug.34 Rhodamine B (RB) conjugated
HA-PTX (RB-HA-PTX) was synthesized as the preparation
process of HA-PTX. The content of RB in the conjugate
was quantied by a UV-vis spectrophotometer. HepG2 cells
were seeded in a 24-well plate at a density of 1×104 cells/well.
When the cells reached about 80% conuence, the medium
was replaced by 100 mL of the RB-HA-PTX NPs or free
RB solution. After 4 h incubation, the above incubated cells
were rinsed three times with PBS to remove excess RB-
HA-PTX NPs or free RB. The cellular uptake was visual-
ized by an inverted fluorescence microscope. To prove the
receptor mediated endocytosis of HA-PTX NPs, the cellular
uptake extents of RB were monitored by flow cytometry.
HepG2 cells with a density of 1×104 cells/well were incu-
bated with free RB or RB-HA-PTX NPs for 4 h at 37 ºC.
For competitive inhibition study of RB-HA-PTX NPs, free
HA (29 kD) was additionally added to RB-HA-PTX NPs
without changing the concentration of RB.
In vitro Cytotoxicity. Cytotoxicity of HA-PTX NPs was
evaluated by MTT method using HepG2 cells and NIH-3T3
cells. They were cultured in the growth medium Dulbecco’s
modified Eagle’s medium (DMEM). Cells (1×104 cells/well)
were seeded in 96-well tissue culture plates and incubated
for 24 h with the free PTX and HA-PTX NPs. After incuba-
tion, 20 µL of MTT solution (5 mg/mL) was added to each
well of the plate. The incubation was continued for another
4 h. Then the MTT derivative was dissolved with DMSO
and the optical density of the solution was determined.
Ex vivo Distribution. For ex vivo distribution test, Balb/c
mice bearing Ehrlich ascites tumor (100 mm3) were fasted
for 12 h and then orally administrated with CS/RB-HA-PTX
CNPs. The mice were sacrificed at 24 h after oral adminis-
tration and the heart, liver, spleen, lung, kidney, stomach,
intestine and tumor were harvested, and examined using the
Kodak IS ex vivo FX imaging system.
All animals were housed individually in plastic cages in a
Scheme II. Synthesis process of HA-PTX conjugates.
Macromol. Res., Vol. 21, No. 12, 2013
controlled environment with free access to food and water.
All animal experiments were performed in accordance with
the People’s Republic of China national standard (GB/T
16886.6-1997).
Results and Discussion
Preparation and Characterization of HA-PTX Conju-
gates and NPs. Although PTX has shown remarkable potential
as an anticancer drug, its poor solubility in water led to poor
bioavailability. Polymer-drug conjugates have distinctive advan-
tages over conventional polymeric nanosized carriers due to
its good water solubility and increased drug half-life in the
body. In this paper, HA-PTX conjugates were synthesized via
esterification reaction in the presence of DCC and DMAP as
shown in Scheme II. HA was desalted to facilitate the conjugat-
ing of -COOH group with the -OH groups of PTX. According
to the previous reports, it appeared that the 2'-hydroxyl group
in paclitaxel was preferentially conjugated to HA rather than the
7'-hydroxyl group because it was less sterically hindered.6
HA-PTX conjugates were further confirmed by FTIR as
shown in Figure S1. As shown in Figure S1(A), hyaluronic
acid showed a peak at 1618 cm-1 associated with C-O stretching
of carboxylate anion, while it shifted to 1657 cm-1 once the
conjugate formed in Figure S1(B). A band around 1342 cm-1
also could be seen in in Figure S1(B), which was attributed
to C-O stretching frequency of ester linkage in the HA-PTX
conjugate.26
The synthesis of HA-PTX conjugates was verified by 1H
NMR spectra. As shown in Figure 1(A), peaks associated
with aromatic protons of PTX were observed at 7-8 ppm.
When D2O was used as a solvent, as shown in Figure 1(B),
characteristic peaks of PTX disappeared, while a strong
acetyl (-NHCOCH3) peak was identied at 1.8 ppm (a) along
with glucosidic H at 3.0-4.0 ppm (b) and anomeric H at 4.30
ppm (d) and 4.40 ppm (c). It was obvious that the disap-
peared of PTX special proton peaks in D2O was mainly due
to the embedded PTX in the inner core of HA-PTX NPs.13
As shown in Figure 1(C), in the case of DMSO-d6, the char-
acteristic peaks of PTX strongly appeared in the 1H NMR
spectra, which was attributed to the exposure of PTX in the
1333
 J. Li et al.
Figure 1. 1H NMR spectra of PTX in DMSO-d6 (A), HA-PTX in
D2O (B), and HA-PTX in DMSO-d6 (C).
organic phase. The shielding and deshielding effects of
paclitaxel and HA peaks made it difficulty to accurately
estimate the conjugation extent of paclitaxel to HA. HPLC
was used to measure the content of PTX in HA-PTX, which
was 10.6% by weight.
The size distribution of HA-PTX conjugates in solution
was measured by LPSA, and result indicated that HA-PTX
conjugates could self-assemble into micelle in aqueous medium
with an average diameter of 100±5 nm. TEM images showed
round shaped HA-PTX conjugate micelle with an average
diameter of around 50±2 nm as shown in Figure 2(A). The
sizes obtained from LPSA measurement was higher from
what is observed from TEM, which was expected in the
sense that in solution the micelles would be in an expanded
form due to the high hydrophilicity of the HA moieties. In
TEM, however, the entities were visualized in a dry state.
The Zeta potential distribution of HA-PTX in different pH
was shown in Figure 2(B), which could display that pKa of
the HA-PTX NPs was 2.1.
Preparation and Characterization of CS/HA-PTX CNPs.
CS has special features of adhering to the mucosal surface
and opening the tight junctions between epithelial cells, and
it was suggested that interactions of positively charged amino
groups of CS with negatively charged on cell surfaces induced
a redistribution of F-actin and the tight junction’s protein
ZO-1, which accompanied the increased paracellular per-
meability of NPs.3,35 Therefore, only NPs with a positive sur-
Figure 2. TEM photographs of HA-PTX NPs (A) and zeta potential
of HA-PTX aqueous solution in different pH environments (B).
1334
Table I. Size of CS/HA-PTX CNPs at Different Charge Ratios
CS:HA-PTX Charge CS:HA-PTX Weight Size (nm)
Ratios Ratios
1:2 1:5 -
1:1 2:5 -
3:2 3:5 350.7
2:1 4:5 208.2
3:1 6:5 181.3
4:1 8:5 163.4
face charge were selected for this study. CS/HA-PTX CNPs
was prepared by coating CS onto HA-PTX NPs via electro-
static interactions.
As shown in Table I, CS/HA-PTX CNPs were synthetized
at different charge ratios (the positively charged -NH3+ groups
on CS to the negatively charged-COO- groups on HA-PTX
NPs), which could form stable CNPs with the exception of
the charge ratios were approximately 1.0: 2.0 and 1.0: 1.0.
The diameters of the prepared CNPs were in the range of
160-350 nm. When the amount of the positively charged CS
significantly surpassed that of the negatively charged HA-
PTX, some of the excessive CS molecules were intertwined
around the surfaces of the formed CNPs. CS/HA-PTX CNPs
have an size of 208 nm at the charge ratio of 2:1 (4:5=CS:
HA-PTX) while it was 350 nm at 3:2 (3:5=CS: HA-PTX).
CNPs had a smaller size could be more favorable to across
the tight junctions between epithelial cells. Overall consid-
eration of the PTX content and the size of CNPs, we chose
the charge ratio of 2:1 as optimal synthesis condition.
Because of the difference of dissociation constant of CS
and HA-PTX, CS/HA-PTX CNPs were fabricated at differ-
ent pH values. In the presence of food, the stomach pH was
about 1.2-2.0, while the fasting pH of the stomach was 2.5-
3.7. The pH values in the duodenum, the jejunum and the
proximal ileum were 6.0-7.0, while the mean pH in the distal
ileum and in the body fluid at intercellular spaces between
enterocytes was about 7.4.36,37 Therefore, CNPs must be charac-
terized at different pH values, simulating the pH environ-
ments in the GI tract and at the intercellular spaces between
the enterocytes.
The sizes distribution and TEM images of CS/HA-PTX
CNPs at different pH conditions was shown in Table II and
Figure 3, respectively. As shown in Table II and Figure 3(A),
CNPs was disintegrated in pH 1.2 due to the poor electro-
static interactions between CS and HA-PTX NPs. In pH 3.0,
5.0, and 6.0, the ionized CS and HA-PTX were able to form
CNPs via electrostatic interactions, and the spherical shapes
of CNPs were shown in Figure 3(B), (C), and (D). As shown
in Table II, CS/HA-PTX CNPs showed the smallest sizes at pH
5.0, which illustrated that the combination of CS/HA-PTX
CNPs was the closest. As shown in Figure 3(E), CS could not
form -NH3+ via protonation owing to the dissociation con-
stant of CS, and resulted in the disintegration of CNPs.
Macromol. Res., Vol. 21, No. 12, 2013
 Tumor Targeting and pH-Responsive Polyelectrolyte Complex Nanoparticles for Oral Administration

Figure 3. TEM photographs of the synthesis of CS/HA-PTX CNPs

in different pH values.

Table II. Size and Zeta Potential of CS/HA-PTX CNPs at

Different pH Values
pH Values Size (nm) Zeta (mV)
1.2 - 20.8
3.0 234.5 28.5
5.0 208.3 26.4
6.0 335.2 23.5
7.4 - -11.6 Figure 5. Release profiles of PTX from HA-PTX NPs (a) and
from CS/HA-PTX CNPs (b) in pH 3.0 (A); release profiles of
PTX from the CS/HA-PTX CNPs simulating the pH environ-
The formation of CS/HA-PTX CNPs prepared at pH 5.0 ments in the fasting stomach, intestine and the body uid at inter-
with the charge ratio of 2:1 was further confirmed through cellular spaces, respectively (B).
FTIR spectra as is shown in Figure 4. A band appeared at
2106 cm-1 in Figure 4(A) was due to the excessive -NH3+ groups stomach (pH 3.0) were exhibited. The cumulative amount
of CS/HA-PTX CNPs.3 Furthermore, a peak, at 1412 cm-1, of PTX released from HA-PTX NPs was 19% at 2 h, while
arose in the CS/HA-PTX CNPs but was not observed in covered with CS, the cumulative released amount decreased
Figure 4(B) or (C). This phenomenon was attributed to the to 7%. It was indicated that the CS shell of CS/HA-PTX
interaction of the -NH3+ groups of CS with the -COO- groups CNPs could effectively avoid the cleavage of the ester link-
of HA-PTX NPs.38 All these data illustrated that the CS age in HA-PTX NPs, inhibit the quick release of PTX under
combined to HA-PTX NPs with an electrostatic interaction. acidic environment and enhance the bioavailability of PTX.
In vitro Release. To confirm the pH-sensitive of CS/HA- From the photographs of CNPs in different pH values, we
PTX CNPs, in vitro PTX release was examined. As shown could know that CNPs prepared in our study may be orally
in Figure 5(A), the release profiles of PTX from HA-PTX administered only before meal. Figure 5(B) simulated the
NPs (a) and CS/HA-PTX CNPs (b) at the fasting pH of the release of PTX from CS/HA-PTX CNPs via oral adminis-
tration. The cumulative amount of PTX released from CNPs
was about 11% at the fasting pH of the stomach, while it
was approximately 36% at pH 6.0. At pH 7.4, CNPs became
unstable and disintegrated, resulting in a fairly fast release
of HA-PTX NPs, which could be easily cleave at weakly acidic
tumor site. These results illustrated that the CS/HA-PTX CNPs
could be orally administered in the fasting conditions.
In vitro Cellular Uptake. Due to PTX released from CS/
HA-PTX CNPs in the form of HA-PTX NPs at pH 7.4, in
vitro cellular uptakes of PTX and HA-PTX NPs against HepG2
cells were monitored by an inverted fluorescence micro-
scope. RB-labeled HA-PTX conjugate (RB-HA-PTX) was
synthesized to simulate the endocytosis of HA-PTX NPs.
As shown in Figure 6, free RB was predominantly localized
in the nuclei, suggesting that it was passive diffusion into
Figure 4. FTIR spectra of CS/HA-PTX (A), CS (B), and HA-PTX (C). the cells. On the other hand, RB-HA-PTX NPs exhibited the

Macromol. Res., Vol. 21, No. 12, 2013 1335

 J. Li et al.
Figure 6. Fluorescence pictures of HepG2 cells incubated with
RB and RB-HA-PTX NPs at an equivalent RB concentration of
10 µg/mL for 4 h at 37 ºC.
same brightness throughout the cytoplasm as well as in the nuclei.
The remarkable difference in the distribution of RB suggested
that NPs were taken up by tumor cells mainly via an endocytic
pathway. The enhanced cellular uptake of RB-HA-PTX NPs
would result in a higher concentration of PTX in the tumor.
An experiment was performed to further confirm whether
the HA-PTX NPs were taken up by HepG2 cells via HA-
specic receptor mediated endocytosis. Added free HA in the
RB-HA-PTX NPs without changing the concentration of
RB was achieved. The fluorescence intensity of RB-HA-
PTX NPs was extraordinarily higher than that of free RB as
was shown in Figure S2, while it was substantially reduced
with the adding of free HA in the NPs. These results clearly
illustrated that HA receptor-mediated endocytosis was dis-
played dominating role in efficient intracellular delivery of
HA-PTX NPs. That is to say, the receptor-mediated uptake of
HA-PTX NPs could protect PTX from pumping out by the
P-glycoprotein (P-gp) pump on the cellular membrane.
Therefore, the anti-tumor effect of HA-PTX NPs could sub-
stantially increase along with the enhanced cellular uptake.
In vitro Cytotoxicity. The cytotoxicity of HA, free PTX
and HA-PTX NPs against HepG2 cells were evaluated. As
shown in Figure 7(A), a slight toxicity (around 21%) was
detectable for NIH-3T3 cells treated with HA at the concen-
Figure 7. In vitro cytotoxicity of HA against HepG2 cells and NIH-3T3 cells at 24 h (A), in vitro cytotoxicity of PTX and HA-PTX NPs
against HepG2 cells (B).
1336
Figure 8. Ex vivo fluorescence intensity images of the tumors and
major organs after oral administration of CS/RB-HA-PTX CNPs and RB.
tration of 100 µg/mL, whereas for HepG2 cells no obvious
toxicity was observed regardless of the concentration of the
added HA. Figure 7(B) depicted that the cytotoxicity of PTX
and HA-PTX NPs was dose-dependent. HA-PTX NPs were
less active than free PTX against HepG2 cells, which might
be due to the slow release rate of PTX from the NPs.39,40 Prelimi-
nary results revealed that the PTX was efficiently released
from the NPs in the case of HAase. Thus, the HA-PTX NPs
in tumor tissue could display effective antitumor activity.
Ex vivo Biodistribution. In order to ascertain whether the
PTX after oral administration of CS/HA-PTX CNPs could
efficiently accumulate at tumor sites, the Balb/c mice were
used to examine the PTX biodistribution with the treatment
of CS/RB-HA-PTX CNPs in Ehrlich ascites tumor model.
The ex vivo fluorescent images of the excised organs further
confirmed that RB from CS/RB-HA-PTX CNPs could out-
standing accumulate in the tumor as was shown in Figure 8.
However, most of RB distributed in the liver and kidney
after free PTX oral, only a little distributed in the tumor. These
results indicate that CS/RB-HA-PTX CNPs could target and
successfully accumulate in the tumor tissues, due to tumor
targeting and prominent EPR effect.
Conclusions
In this study, CNPs with pH-sensitive and tumor targeting
characteristic were prepared through electrostatic interac-
Macromol. Res., Vol. 21, No. 12, 2013
 Tumor Targeting and pH-Responsive Polyelectrolyte Complex Nanoparticles for Oral Administration
tion for oral delivery of PTX. The TEM photographs and in
vitro release study demonstrated that the release of PTX
from CNPs was pH-dependent. HA-PTX NPs with the showed
increased extent of cellular uptake for HepG2 cells than free
PTX confirming that the NPs taken up by cells via endocytosis.
Flow cytometry data clearly supported the important role of
HA receptor-mediated endocytosis in efficient intracellular
delivery of HA-PTX NPs. In vitro cytotoxicity experiment
demonstrated that HA was biocompatible to HepG2 cells
and NIH-3T3 cells, and PTX could be released from and
HA-PTX NPs without losing cytotoxicity. The ex vivo bio-
distribution experiments also confirmed that PTX could
effectively accumulate in the tumor sites after oral adminis-
tration of CS/HA-PTX CNPs. These results suggested that
the CNPs developed in this work could be employed as a
potential carrier for the oral delivery of hydrophobic drug.
Acknowledgments. This work had been supported by a
grant from 973 Program (2009CB930300), the National Natural
Science Foundation of China (Number 31271073) and the National
Natural Science Foundation of China (Number 81171371).
Supporting Information: FTIR spectra of HA-PTX (A)
and HA (B). FACS analysis of HA-PTX NPs taken up by
HepG2 cells via HA-specific receptor mediated endocyto-
sis. Control (A); RB-HA-PTX NPs with free HA (B); free RB
(C); RB-HA-PTX NPs (D). The materials are available via
the Internet at http://www.springer.com/13233.
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