International Journal of Pharmaceutics 573 (2020) 118840

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Evaluation of intestinal permeation enhancement with carboxymethyl T

chitosan-rhein polymeric micelles for oral delivery of paclitaxel
Xiaoying Wanga, Liangzhen Qiua, Xiaying Wanga, Huizhi Ouyanga, Tonglei Lib, Lifeng Hana,
⁎ ⁎
Xue Zhanga, Wei Xua, , Kedan Chua,
a
Pharmacy College, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
b
Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907, United States

A R T I C LE I N FO A B S T R A C T

Keywords: Polymeric micelles (PMs) are currently under investigation as potential nanocarriers for oral administration of
Polymeric micelles paclitaxel (PTX). Previously, we developed amphiphilic carboxymethyl chitosan-rhein (CR) conjugate for oral
Intestinal permeation enhancement delivery of PTX. PTX-loaded CR PMs exhibited a homogeneous and small size (< 200 nm) with a drug loading
Carboxymethyl chitosan-rhein conjugate capacity (DL) of 35.46 ± 1.07%. However, The absorption parameters of PTX using CR PMs have not been
Paclitaxel
studied before. Here, we evaluated the intestinal permeation of CR PMs by in situ intestinal absorption experi-
ments. PTX-loaded CR PMs enhanced the absorption of PTX in the intestine without causing significant intestinal
villi injury. Compared to the P-glycoprotein (P-gp) inhibition of verapamil, the transport mechanism of CR PMs
across intestinal epithelial cells may bypass P-gp efflux. Caco-2 cell uptake assays also confirmed that CR PMs
can be taken up into the enterocyte as whole and independent of P-gp. Local biodistribution evaluation showed
that fluorescence-labeled CR PMs were absorbed into the intestinal villi. In vivo bioimaging of tumor-bearing
mice verified a significant portion of CR PMs were intactly absorbed through the intestine, then distributed and
accumulated at the tumor site. For their significant intestinal permeation enhancement, CR PMs might be
considered as promising oral delivery carriers for PTX and other water-insoluble drugs.

1. Introduction
In recent years, nanoscale drug delivery systems have gained im-
pressive momentum for enhancing the oral bioavailability of poorly
water-soluble drugs. These drug delivery systems show great success by
improving the solubility and permeability of drugs in the gastro-
intestinal (GI) tract (Hou et al., 2017; Pooja et al., 2016; Wan et al.,
2015). Among these nanotechnology-based drug delivery systems,
polymeric micelles (PMs) have got more concerns because of their re-
markable properties, such as a low critical micelle concentration, high
stability, higher drug loading capacity, and flexible modified materials
(Jena and Sangamwar, 2016).
PMs are self-assembled by amphiphilic molecules in an aqueous
medium and form a nano-structure consisting of an inner hydrophobic
core and an outer hydrophilic shell (Ke et al., 2017; Qin et al., 2013).
PMs act as effective drug delivery vehicles by entrapping poorly water-
soluble drugs into their cores, which exhibits a subsequent sustained-
release character. Because of their thermodynamics, kinetic stability
and low critical micelle concentration, orally administered PMs can
resist the dilution of GI fluid and still maintain structural rigidity in the
GI environment. These properties are also advantageous for drug de-
livery to the target site (Torchilin, 2011). Orally administered PMs also
can avoid toxicity of solubilizing excipients of intravenous insoluble
drug formulations (Garrido-Siles et al., 2015; Gelderblom et al., 2001).
The mechanisms by which PMs enhance oral bioavailability of drugs
are various, such as improving drug solubility in water and biological
fluids, protecting the encapsulated drug from inactivation in the GI
environment, controlling release of loaded drugs, increasing residence
time in the intestinal tract by mucoadhesion, bypassing the efflux
pump, or selectively targeting the site of action (Banerjee et al., 2016;
Pathak and Raghuvanshi, 2015). Thus, the state of PMs during the drug
delivery process and the properties of their materials are key factors for
their oral absorption enhancement.
Paclitaxel (PTX) is a broad-spectrum chemotherapeutic drug used to
treat many cancer types, such as ovarian and breast cancer (Ahmed
et al., 2011; Bernabeu et al., 2016). However, oral administration of
PTX faces many hurdles, because of its poor solubility, poor intestinal
permeability, the first pass effect, and the efflux function of P-gp in the
GI tract (Malingre et al., 2001; Singla et al., 2002), as well as potential
cytotoxic side-effects. To mitigate these problems, we previously

⁎
Corresponding authors.
E-mail addresses: 2000017@fjtcm.edu.cn (W. Xu), kedanchu@163.com (K. Chu).

https://doi.org/10.1016/j.ijpharm.2019.118840
Received 12 July 2019; Received in revised form 22 October 2019; Accepted 30 October 2019
Available online 09 November 2019
0378-5173/ © 2019 Elsevier B.V. All rights reserved.
X. Wang, et al.
synthesized an amphiphilic carboxymethyl chitosan-rhein (CR) con-
jugate to encapsulate PTX to improve oral bioavailability of PTX by
increasing its water solubility. Carboxymethyl chitosan (CMCS), a hy-
drosoluble chitosan derivative, as the backbone of CR conjugate, has a
good biocompatibility, biodegradability, mucoadhesiveness, and per-
meation-enhancing effect (Feng et al., 2014; Liu et al., 2016; Wang
et al., 2014). Rhein (Rh), as the hydrophobic part of CR conjugate, has
an anti-proliferative, anti-angiogenesis, and apoptosis inducing action
against human carcinoma cells (Du et al., 2013; He et al., 2011;
KoraMagazi et al., 2016). CR PMs can be self-assembled by CR con-
jugate. These CR PMs displayed a good drug-loading and sustained-
release character, resulting in a better antitumor effect (Wang et al.,
2019). In our earlier report, we proposed that PTX-loaded CR PMs could
significantly enhance the oral bioavailability of PTX and display a time-
dependent cytotoxic effect in Caco-2 cells. Indeed, a synergistic anti-
tumor effect between CR conjugate and PTX was demonstrated in MCF-
7 cells in low concentrations at 48 h and 72 h, which contributed to
their antitumor efficacy. Nevertheless, the fate of CR PMs in the body
need to be further investigated.
In oral administration, intestinal epithelial cells serve as drug bar-
riers to enter systemic circulation. The intestinal epithelium is com-
posed of villi, which largely increase the absorptive surface area of the
GI tract (Ensign et al., 2012). Endocytosis by enterocytes which cover
the villi is a key transport pathway for nano-sized drug delivery systems
across this barrier. It was reported that PMs are transported through
enterocytes by circumventing P-gp efflux and/or are absorbed via
lymphatic transport through M cells in Peyer’s patches of the intestine
(Ni et al., 2016). Nowadays, various biological models and methods
have been used to investigate the absorption route of PMs in the in-
testine and evaluated the intestinal enhancement ability of PMs, such as
the in situ single-pass perfusion method in rats (Li et al., 2013; Lian
et al., 2017), visualization by confocal laser scanning microscopy
(Wang et al., 2015), and bioimaging (Mao et al., 2017; Yang et al.,
2017). Caco-2 cells, an in vitro model of intestinal epithelium, are
usually utilized to investigate transport mechanism of PMs in the in-
testinal tract (Ke et al., 2017; Yin et al., 2018). These biological models
and methods provide feasibility to study the absorption of PMs, the fate
of PMs in the body and the mechanisms underlying PMs for oral de-
livery of poorly water-soluble drugs.
In this study, the absorption evaluation of PTX-loaded CR PMs in the
intestine was performed by the in situ single-pass intestinal perfusion
method. The in vivo absorption and distribution of CR PMs in tumor-
bearing mice after the oral administration were studied by bioimaging
using environment-responsive aggregation-caused quenching (ACQ)
probes. ACQ fluorophores (P2 or P4) emit intense fluorescence when
dispersed in a hydrophobic environment of nanoparticles or micelles
but become instantaneously and absolutely quenched when they leave
the hydrophobic environment and self-aggregate in water (He et al.,
2016; Ma et al., 2017; Tian et al., 2016). Thus, detecting fluorescence of
P2-loaded micelles may indicate the in situ integrity of drug-loaded
PMs. In addition, the mechanism of promoting absorption of CR con-
jugates was investigated by the in situ single-pass intestinal perfusion
and fluorescent-labeled CR PMs in the intestinal tract and in Caco-2
cells under confocal laser scanning microscope (CLSM). The behavior of
PTX-loaded CR PMs in the intestinal tract and the drug absorption and
distribution processes in the body are shown in Fig. 1.
2. Materials and methods
2.1. Materials and animals
Carboxymethyl chitosan-rhein (CR) conjugate was synthesized
using carboxymethyl chitosan and rhein according to our published
protocol (Wang et al., 2019). The molar substitution of rhein degree
were 7.80%. Paclitaxel (PTX) was purchased from Shanghai Sanwei
Pharmaceuticals Co., Ltd. (Shanghai, China). Verapamil was purchased
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International Journal of Pharmaceutics 573 (2020) 118840
from Wuhan yuanchenggongchuang Technology Co., Ltd. (Wuhan,
China). Hematoxylin, eosin and phenol red were purchased from Si-
nopharm Chemical Reagent Co., Ltd.(Shanghai, China). P4 and P2
fluorescence probe were provided by Fudan University. Methanol of
high performance liquid chromatography (HPLC) grade was obtained
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All
other chemicals were analytical grade and used without further pur-
ification.
All experiments performed on animals were in accordance with the
Institutional Animal Care and were approved by the Fujian University
of TCM Animal Care and Use Committee. Male Sprague-Dawley (SD)
rats (weight 300 ± 20 g) and ICR mice (18 ± 2 g) were purchased
from Fujian Medical University. Rats and mice were maintained at the
animal care facility, acclimatized for at least 3 days, and fasted over-
night before experiments.
2.2. Preparation of PTX-loaded CR PMs
PTX-loaded CR PMs were prepared using our published protocol
(Guo, 2017; Wang et al., 2019). Briefly, 13 mg of PTX was dissolved in
430 μL of anhydrous ethanol, dropped slowly into 2.6 mL of CR PMs
solution (7 mg/mL) under stirring at room temperature, and then dis-
persed by a probe-type ultrasonicator for 30 min in ice bath and dia-
lyzed by a dialysis bag (MWCO 14,000 Da) in 2000 mL DI water for
12 h. After dispersed by sonication, centrifuged at 3000 rpm for 10 min
and filtered using a 0.8 μm filter membrane to remove unentrapped
PTX, PTX-loaded CR PMs were then used in the following experiments.
The drug loading capacity (DL) and entrapment efficiency (EE) of
PTX in CR PMs were detected by high-performance liquid chromato-
graphy (HPLC, Shimadzu LC-20AT, Kyoto, Japan) with a C18 column
(Inertsil ODS-SP, 4.6 × 250 mm, 5 µm). The mobile phase consisted of
methanol and DI water (65:35, v/v), and the flow rate was 1.0 mL/min.
The detection wavelength was 227 nm and column temperature was set
at 30 °C. The amount of PTX was determined by comparing the peak
areas with a PTX standard curve. DL and EE of PTX were calculated
with the following equation:
amount of PTX entrapped in PMs
DL(%) = × 100
amount of PTX − loaded CR PMs
amount of PTX entrapped in PMs
EE(%) = × 100
amount of PTX dissolved in anhydrous ethanol
P4/P2 and PTX co-loaded CR PMs were prepared as the methods
aforementioned with modification. In brief, 18 mg of CR conjugate
were dissolved in 2.6 mL of distilled water. 12 mg of PTX and 40 μg of
P4/P2 were dissolved in 200 μL methanol, then added into CR con-
jugate solution under stirring, followed by sonication and dialysis. After
dialysis for 12 h, the solution was dispersed by probe-type ultra-
sonicator and centrifuged at 3500 r/min for 5 min. The supernatant was
P4/P2 and PTX co-loaded CR PMs.
2.3. Characterization of PTX-loaded CR PMs
2.3.1. Size, size distribution and zeta-potential measurement
The size (nm) and size distribution (polydispersity index, PDI) of
PTX-loaded CR PMs were measured by the dynamic laser light scat-
tering (DLS) method. PTX-loaded CR PMs were dispersed in DI water
with a concentration of 1 mg/mL. The size, size distribution, and mi-
celle surface charge analysis (zeta potential, mV) were detected using a
laser particle size analyser (NicompTM 380ZLS, California, USA) at
room temperature.
2.3.2. AFM
The morphology of PTX-loaded CR PMs was observed by atomic
force microscopy (AFM, 5500, Agilent, USA). The PTX-loaded CR PMs
solution was deposited on a mica plate and air-dried. AFM images were
X. Wang, et al.
Fig. 1. Scheme of predicted effects of CR PMs to improve oral absorption of PTX: (A) Transcellular pathway by pinocytosis bypass P-glycoprotein (P-gp) efflux. (B)
Transcytosis by M cell.
obtained using an intermittent contact (tapping) mode with a constant
stiffness of 40 N⋅m−1.
2.3.3. TEM
PTX-loaded CR PMs were observed by transmission electron mi-
croscopy (TEM, H7650, Hitachi, Japan). The sample was prepared ac-
cording to the methods described previously (Wang et al., 2019). A
droplet of PTX-loaded CR PMs solution was placed on a carbon coated
copper grid and the excess fluid was removed by a filter paper. Then the
sample was stained by a droplet of 2 wt% phosphotungstic acid for 30 s
and naturally dried before TEM observation.
2.4. In situ intestinal absorption assay
In situ single-pass perfusion experiments were carried out based on
the reported methods (Ni et al., 2016; Wang et al., 2014). Each healthy
SD rat (180–200 g) fasted for 12 h and had free access to water before
the experiment. Rats were anesthetized with an intraperitoneal injec-
tion of 20% (v/v) urethane solution. A heating lamp was used to
maintain body temperature during the following procedure. After the
abdomen of the rat was opened along the midline, about 10 cm of the
duodenum, jejunum, ileum, and colon segments were selected, and then
a tiny “V” incision was cut at the beginning and the end of each selected
segment. Each perfusion tube was cannulated at both ends of each
segment from the “V” incision and fixed with cotton thread. A pad
wetted with isotonic saline covered the surgical area to keep the in-
testine segments wet. After being flushed with 0.9% (w/v) saline
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International Journal of Pharmaceutics 573 (2020) 118840
solution, each intestinal segment was perfused with Krebs-Ringer’s
buffer (K-R buffer) solution for 0.5 h. Then, the drug perfusion solution
was utilized for perfusion at a flow rate of 0.2 mL/min. The drug per-
fusion solution was prepared with K-R buffer solution containing
30 mg/mL PTX and 60 mg/mL phenol red. All solutions were main-
tained at 37 °C. Every 15 min the perfusion was collected up to 120 min.
After perfusion, rats were sacrificed. The length and perimeter of each
segment was measured, and PTX and phenol red in the collected per-
fusate solution samples were detected.
UV and HPLC were utilized to determine the concentration of
phenol red at 559 nm and PTX at 227 nm, respectively. In brief, 0.2 mL
samples were mixed with 4.8 mL NaOH solution (0.1 M) for detecting
the phenol red concentration using UV spectrophotometer (UV-9600,
Ruili, Beijing, China). Meanwhile, 0.2 mL of all samples were diluted
with 0.8 mL methanol, vortex mixed, and centrifuged at 10,000 rpm for
10 min to obtain the supernatant for detection of PTX content by HPLC
according to the above method. The absorption rate constant (Ka) and
the effective permeability coefficients (Peff) and the fraction of ab-
sorption (Fa) were evaluated using the following equations.
Cpout C ν
K a = ⎜⎛1− × PRin ⎟⎞ ×
⎝ C pin C PRout ⎠ π r 2l
ν Cpout C
Peff = − ln ⎛⎜ × PRin ⎞⎟
2π rl ⎝ Cpin CPRout ⎠
X. Wang, et al.
Fig. 2. Characterization of PTX-loaded CR PMs. (A) Size distribution by DLS.
(B) AFM image of PTX-loaded CR PMs. (C) TEM image of PTX-loaded CR PMs.
Cpout C
Fa (%) = ⎜⎛1− × PRin ⎟⎞ × 100
⎝ C pin C PRout ⎠
where v is the flow rate (mL/min), r and l are the radius and the length
of the perfused intestinal segment (cm), Cpin and Cpout are the con-
centrations of PTX in the perfusion solution and collected samples
(respectively), and CPRin and CPRout are the concentrations of phenol red
in the perfusion solution and collected samples (respectively). Phenol
red was used to correct the appreciable influence of the secretions or
absorption of water during the experiment due to its nonabsorbable
property in the intestine.
The composition of the K-R buffer was glucose (7.78 mM), NaCl
(133 mM), KCl (4.56 mM), NaH2PO4 (1.50 mM), MgCl2 (0.20 mM),
NaHCO3 (16 mM), CaCl2 (3.33 mM). The pH was maintained at 7.4.
PTX in Taxol® or PTX-loaded CR PMs, phenol red and verapamil were
dissolved in K-R buffer solution at pH 7.4 to yield a final concentration
of 30 µg/mL, 60 µg/mL, and 400 µg/mL, respectively. K-R buffer was
employed as control.
After the procedures of in situ intestinal absorption, the segments of
the duodenum, jejunum, ileum and colon were carefully removed,
rinsed and fixed with 4% paraformaldehyde for 24 h. Then the seg-
ments were embedded in paraffin media for sectioning (5 μm) with a
paraffin slicing machine (RM2235, Leica, Germany). The sections were
stained with hematoxylin and eosin and observed using a microscope
(MDL, Leica, Germany).
4
International Journal of Pharmaceutics 573 (2020) 118840
Fig. 3. The absorption rate constant (Ka) and the effective permeability coef-
ficients (Peff) of PTX in different formulations (Mean ± SD, n = 3, * p < 0.05,
**p < 0.01 over Taxol®). Ver: Verapamil.
2.5. Intestinal fluorescence labeling procedures
SD rats (180–200 g) fasted 12 h before the experiment with free
access to water. In order to track CR PMs in the intestine, the fluor-
escent dye nile red (Sigma, America) was entrapped into the CR PMs
using the method as the previously described preparation of PTX-
loaded CR PMs. Nile-red-loaded CR PMs solution (2 mL/kg) was orally
administrated to these rats. After 1 h, the rats were euthanized, and
segments of the duodenum, jejunum, ileum and colon were carefully
removed, rinsed, and frozen in embedding media (OCT, SAKURA,
Japan) for cryostat sectioning (Leica, Germany, 30 μm). The sections
were fixed with 4% paraformaldehyde for 10 min, then stained with
FITC-phalloidin (Enzo, America) for 90 min followed by DAPI (ROCHE,
China) for 5 min. The sections were sealed with anti-fluorescence
quenching solution (Beyotime, China) and observed under confocal
laser scanning microscope (CLSM, TCS SP5II, Leica, Germany).
2.6. Caco-2 cell uptake assay
2.6.1. Caco-2 cells culture
Caco-2 cells (obtained from the cell bank of Chinese Academy of
X. Wang, et al.
Table 1
Fa of different formulations in different intestinal segments (Mean ± SD, n = 3, *p < 0.05, **p < 0.01 over Taxol®).
Formulations
duodenum
Taxol® 8.11 ± 2.14
Taxol®+Verapamil 20.23 ± 6.68**
Taxol®+CR conjugate 18.36 ± 2.28*
PTX-loaded CR PMs 31.31 ± 5.19**
PTX-loaded CR PMs+Verapamil. 25.86 ± 4.32**
Sciences) were cultured in MEM with 20% FBS, 1% Glutamax, 1% pe-
nicillin and streptomycin, 1% non-essential amino acids and 1% sodium
pyruvate solution (100 mM) in an incubator (Thermo 311, MA, USA) at
37 °C, 5% CO2 and 95% relative humidity. The cells were grown in cell
culture flasks at 80% confluence, were detached from the dishes using
trypsin and transferred into cell culture dishes.
2.6.2. Cellular uptake
The cells were seeded at a density of 1 × 105 cells/well in cell
culture dishes. After culture for 4 days, the culture medium was re-
moved. After gently washing with PBS at 37 °C for three times, 0.5 mL
of P4 and PTX co-loaded CR PMs was added into the dishes. After 2 h,
the preparation solutions were removed. PBS at 4 °C was added to the
dishes to end the uptake. The cells were washed for 3 times and fixed
with 4% paraformaldehyde, then washed with cold PBS for 5 times to
remove residual preparations. 1 mL of 10 µg/mL Hoechst33342 (for
dyeing cell nucleus) was added into the dishes and incubated for
15 min. After washed with cold PBS for 3 times, 0.2 mL PBS was added
for CLSM observation (λex/λem of 651/662 nm). To investigate the P-gp
inhibition during transportation of CR PMs, P4 and PTX co-loaded CR
PMs with 100 μM verapamil was prepared and used in the uptake assay.
P4 water solution with or without verapamil was prepared as controls.
For flow cytometry, Caco-2 cells (4 × 105 cells/mL) were seeded in
a 6-well plate and incubated for 4 days, then washed for 2 times with
PBS. 1 mL of different preparations were added into the plate, respec-
tively. After incubation for 2 h, the residual preparations were removed,
and cold PBS at 4 °C was added to end the uptake. Subsequently, cells
were washed with PBS, digested by trypsin, collected, washed and
centrifugated for three times, then stored in 0.5 mL PBS for flow cyto-
metry study.
2.7. In vivo bioimaging
In vivo distribution of PTX-loaded CR PMs was detected by IVIS
using an ACQ fluorophore, P2, whose fluorescence becomes quenched
in water but not in a hydrophobic environment (Lv et al., 2018; Wang
et al., 2018). Oral administration of CR PMs loaded with both P2 and
PTX (1.7 μg/mL of P2 and 0.7 mg/mL of PTX in P2 and PTX co-loaded
CR PMs solution) was conducted in murine hepatoma H22 cells tumor
xenograft ICR mice (n = 3) with P2 solution as control. The tumor
model was established by subcutaneous inoculation of H22 cells
(2 × 106 cells per mouse) at the right axilla of male ICR mice (weighing
16–20 g). The xenograft mice was used in the bioimaging experiment
when the tumor volume reached approximately 200–400 mm3. At 3, 6,
9, 12 and 24 h upon oral administration, respectively, mice were an-
esthetized by isoflurane using XGI-8 Gas Anesthesia System (Perki-
nElmer, Waltham, USA) and imaged using IVIS Spectrum (PerkinElmer,
waltham, USA) with excitation/emission wavelengths set to 710/
760 nm. Another fifteen tumor-bearing mice were sacrificed at 3, 6, 9,
12 and 24 h after oral administration of CR PMs loaded with P2 and
PTX, respectively. GI tracts, major organs and tumors were removed
and imaged by IVIS.
International Journal of Pharmaceutics 573 (2020) 118840
Fa (%)
jejunum ileum colon
7.93 ± 4.9 12.69 ± 2.63 8.25 ± 1.63
17.39 ± 4.45 23.97 ± 5.70 20.01 ± 4.67*
15.36 ± 1.97 22.55 ± 6.07 22.45 ± 7.20*
28.91 ± 10.56** 30.75 ± 11.82** 24.45 ± 9.72**
24.84 ± 3.06** 27.75 ± 4.44* 23.63 ± 2.38*
2.8. Statistical analysis
All data are reported as mean ± standard deviation (S.D.).
Statistical significance was tested by one-way analysis of variance
(ANOVA) using GraphPad Prism 5.0 software (La Jolla, CA, USA).
Significant difference was set at *p < 0.05 and very significant as
**p < 0.01.
3. Results and discussion
3.1. Characterization of PTX-loaded CR PMs
The content of PTX in PTX-loaded CR PMs was verified by HPLC. DL
and EE were calculated as 35.46 ± 1.07% and 80.12 ± 3.37%
(n = 5), respectively. The sufficient drug-loading capability contributes
to the delivery of CR PMs for PTX. The size and distribution profile
(Fig. 2A) of PTX-loaded CR PMs revealed an average diameter of
180.6 ± 6.0 nm with a PDI of 0.12 ± 0.02 (n = 5), which indicated a
small particle size and a narrow range of particle size distribution. The
small, uniform size distribution at less than 200 nm allowed these PMs
to be easily absorbed in the intestine, thereby more likely affecting
tumor tissues through the enhanced permeability and retention (EPR)
effect. The spherical and homogeneous morphology of PTX-loaded CR
PMs was confirmed by AFM micrographs (Fig. 2B) and TEM images
(Fig. 2C). Zeta potential of PTX-loaded CR PMs was −26.2 ± 0.7 mV
(n = 5).
3.2. In situ intestinal absorption
The intestinal absorption of PTX in the various formulations was
investigated quantitatively using in situ single-pass perfusion experi-
ments, in which the absorption rate constant (Ka) and the effective
permeability coefficients (Peff) were calculated to evaluate absorption
of PTX in situ. In situ single-pass perfusion assays provided a functional
intestinal barrier and an intact blood supply for oral administration
study using a rat model. Since transporters and intestinal enzymes exist
in this in vivo model, many studies examining the role of transporters in
oral absorption were carried out using this approach (Escribano et al.,
2012). The correlation of effective permeability coefficients between
rat and human intestinal absorption was verified in previous studies,
especially that Peff of passively absorbed substances in rat jejunum is
highly correlated with human jejunum (Fagerholm et al., 1996; Zhang
et al., 2012). Therefore, in situ single-pass perfusion can provide sig-
nificative data for clinical studies of oral administration.
In this study, Taxol®, a commercial formulation of PTX, displayed
the lowest Ka and Peff in intestinal segments among all experimental
formulations (Fig. 3) maybe due to the unstability of Taxol® and the
efflux of the P-gp transporter to free PTX. This mechanism was further
confirmed by the enhanced absorption of PTX in the presence of ver-
apamil (a P-gp inhibitor). Compared with Taxol®, CR conjugate sig-
nificantly promoted the absorption of PTX in duodenum and colon
(p < 0.05), and the absorption enhancement result is approximated to
verapamil. The absorption enhancement effect of CR conjugate maybe
related to the mucoadhesion and permeation-enhancing property of
5
X. Wang, et al. International Journal of Pharmaceutics 573 (2020) 118840

Fig. 4. Different intestinal sections stained with hematoxylin and eosin after in situ single-pass perfusion study.

CMCS, the physiological characteristics of duodenum and colon, etc.
The detailed promotion mechanism will be further studied in the future.
PTX-loaded CR PMs improved the absorption of PTX significantly
compared to Taxol® in the duodenum, jejunum, ileum and colon
(p < 0.01 or p < 0.05). Ka of PTX-loaded CR PMs in duodenum, je-
junum, ileum and colon was 2.88, 3.13, 2.46 and 2.40 times higher than
Taxol®, respectively. Peff of PTX-loaded CR PMs in duodenum, jejunum,
ileum and colon was 3.15-, 3.82-, 2.62- and 2.52-fold over Taxol®, re-
spectively. Fa of different formulations are listed in Table 1, which
shows the absorption bioavailability of PTX in different formulations.
PTX-loaded CR PMs displayed better absorption bioavailability than
other formulations, and significantly enhanced the absorption of PTX

6
X. Wang, et al.
compared to Taxol® in all intestinal segments (p < 0.01). About 30%
(average value of Fa in different intestinal segments) of PTX in PTX-
loaded CR PMs solution passed through the GI track, while only 9% of
PTX in Taxol® solution was absorbed. The result of enhanced absorption
of CR conjugate for PTX was similar to verapamil.
In order to study the mechanism of the enhanced absorption, ver-
apamil was added into PTX-loaded CR PMs solution to inhibit the ac-
tivity of P-gp. The results showed that Ka, Peff and Fa were not sig-
nificantly different between PTX-loaded CR PMs and PTX-loaded CR
PMs with verapamil, suggesting that the transport mechanism of CR
PMs across the intestinal epithelial cells may bypass P-gp efflux and is
therefore hypothesized to proceed via endocytosis by enterocyte (Ni
et al., 2016).
After the in situ intestinal absorption assays, the intestinal segments
were extracted from the sacrificed rats, stained with hematoxylin and
eosin, then observed under the microscope. As shown in Fig. 4, the
Taxol® group exhibited obvious tissue toxicity with slight morpholo-
gical changes in the different intestinal segments, such as intestinal villi
injury, intestinal epithelial inflammatory cell infiltrations, edema and
hyperemia. The intestinal toxicity of Taxol® arose from severe cyto-
toxicity of PTX and its solubilizer, Cremophor EL (Wan et al., 2015).
Cremophor EL perturbed the cell membrane and promoted oxidative
stress as a result of intestinal epithelial cell damage (Campos et al.,
2014; Gutierrez et al., 2006). Thus, Taxol®+verapamil group and
Taxol®+CR conjugate group displayed the same damage in the in-
testinal villi and the enterocytes as the Taxol® group.
The intestinal villi of the Taxol®+CR conjugate group showed
visible injuries with damaged morphous of intestinal villi. That may be
because the adhesive character of CR conjugate prolonged the retention
time of PTX and Cremophor EL on the surface of the intestinal villi,
which induced greater injury. Compared with the Taxol® group, Taxol®
+verapamil group demonstrated enhanced intestinal toxicity, while
7
International Journal of Pharmaceutics 573 (2020) 118840
Fig. 5. Absorption of CR PMs in intestine
after oral administration under CLSM. CR
PMs were labeled with Nile red (red); actin
was stained with FITC-phalloidin (green);
nuclei were counterstained with DAPI
(blue). (For interpretation of the references
to colour in this figure legend, the reader is
referred to the web version of this article.)
the intestinal villi of PTX-loaded CR PMs + verapamil group showed
minimal damage. These results suggest inhibition of the efflux pump P-
gp by verapamil weakened self-protection in epithelial cells (Wan et al.,
2015). In contrast, the intestinal villi of PTX-loaded CR PMs group re-
mained intact and undamaged comparable to the control group. The
morphous of villi in the PTX-loaded CR PMs group was integrated
without any variation, and the cellular heteromorphosis were not ob-
served in the lamina propria of the intestine in the PTX-loaded CR PMs
group. All these indicate that PTX-loaded CR PMs did not irritate the
intestinal tract for the following reasons. First, PTX was entrapped into
the core of CR PMs, avoiding direct contact with the mucosa and in-
testinal villi that protect the intestinal tract from damage. Second, CR
conjugate had a low cytotoxicity as indicated in the cytotoxicity assay
(Wang et al., 2019). Third, the suggested endocytosis transport route of
PTX-loaded CR PMs decreased injury to the intestinal villi. Therefore,
PTX-loaded CR PMs showed the lowest intestinal toxicity and irritation
in the experimental groups. PTX-loaded CR PMs were safe for oral
administration, and could be exploited as oral drug carriers.
3.3. Intestinal fluorescence labeling study
The absorption of CR PMs in the intestine was visualized by CLSM.
CR PMs fluorescently labeled with hydrophobic Nile red (red) to vi-
sualize its absorption in the intestinal tract. Actin was stained with
FITC-phalloidin (green), while the nuclei were counterstained with
DAPI (blue) (Zhang et al., 2013). As shown in Fig. 5, actin filaments of
the intestinal mucosal and nuclei were localized by FITC-phalloidin
(green) and DAPI (blue), respectively. Red fluorescence of CR PMs were
found in the duodenum, jejunum, ileum and colon. Strong red fluor-
escence is particularly along the midline of the intestinal villi, espe-
cially in the duodenum and ileum, suggesting that nile-red-loaded CR
PMs was absorbed into the intestinal villi. The qualitative results by
X. Wang, et al.
Fig. 6. Images of cellular uptake by Caco-2 cells of P4 and PTX co-loaded CR PMs by CLSM (A). Red: P4 in CR PMs; Blue: Hoechst33342. Flow cytometry results of
Caco-2 cells treated with P4 and PTX co-loaded CR PMs with and without verapamil for 2 h (B and C). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
CLSM are consistent with the quantitative results from the in situ single-
pass perfusion study.
3.4. Caco-2 cell uptake
As an in vitro model of intestinal epithelium, Caco-2 cells were used
for evaluating cellular uptake of PTX-loaded CR PMs. To investigate
absorption enhancement and the state of PTX-load CR PMs during the
uptake of enterocytes, P4, an environment-responsive ACQ near in-
frared fluorescent probe, was employed in the study. P4 molecules emit
intensive red fluorescence in the hydrophobic environment, while self-
aggregation in the aqueous environment causes them to lose their
fluorescence-emitting capabilities (Hu et al., 2015; Ma et al., 2017).
Thus, when P4 molecules were entrapped into the hydrophobic cores of
CR PMs, they displayed a strong red fluorescence, and they were
quenched when they were released from CR PMs to water. Therefore,
CR PMs were tracked using P4 under CLSM. CLSM images (Fig. 6A) of
cellular uptake of P4 and PTX co-loaded CR PMs showed CR PMs were
internalized by Caco-2 cells. Cell nucleus was located by Hoechst33342
(blue), and CLSM images showed P4 and PTX co-loaded CR PMs was at
cytoplasm. This result verified that CR PMs could be taken up directly
by Caco-2 cells. In contrast to P4 solution and P4 solution with ver-
apamil, the red intensive fluorescence in P4 and PTX co-loaded CR PMs
with or without verapamil groups indicated CR PMs were intactly ab-
sorbed into enterocytes. The absorption enhancement of CR PMs for the
oral administration of PTX mainly due to the entrapment and delivery
of PTX. When verapamil was added into the P4 and PTX co-loaded CR
PMs solution, the results showed no significant difference to without
verapamil. Flow cytometry analysis indicated no significant difference
in mean fluorescence intensity between P4 and PTX co-loaded CR PMs
with verapamil and without verapamil (Fig. 6B and C). This suggests
8
International Journal of Pharmaceutics 573 (2020) 118840
the absorption route of CR PMs in the intestine maybe bypass P-gp
efflux. The Caco-2 cell uptake assay of R123-loaded CR PMs evaluated
by flow cytometry was also verified there was no significant difference
in mean fluorescence intensity between R-123-loaded CR PMs with and
without verapamil (detailed in Supplementary data). These confirmed
that CR PMs entrapped insoluble drugs in their hydrophobic cores and
disturbed the contact between the drugs and P-gp, thereby avoiding the
efflux of P-gp.
CR conjugate also can increase the fluorescence amount of free R-
123 in Caco-2 cell uptake assay. There are no significant difference in
mean fluorescence between free R-123 with verapamil group and free
R-123 with CR conjugate group (See Fig. S1). This suggested CR con-
jugate acted with a P-gp inhibition effect. The depletion of ATP in the
Caco-2 cells may be one of the reasons of the P-gp efflux inhibition of
CR PMs (detailed in Supplementary data).
3.5. In vivo bioimaging
In order to further investigate the in vivo fate of PTX-loaded CR PMs
during drug absorption, an ACQ fluorophore (P2) was co-loaded into
the micelles for imaging using IVIS. Because of its water-quenching
property, P2 may facilitate nanocarrier quantification by lighting up the
intact hydrophobic core (Ma et al., 2017; Xie et al., 2018). It was
confirmed that P2 alone became quenched in water (Fig. 7C-a) and
PTX-loaded CR PMs showed no fluorescence (Fig. 7C-b). However, CR
PMs co-loaded with P2 and PTX emitted strong red fluorescence
(Fig. 7C-c). These data suggest that P2 was entrapped into the hydro-
phobic core of CR PMs.
Whole-body imaging of H22 tumor xenograft ICR mice upon oral
administration of CR PMs co-loaded with P2 and PTX (Fig. 7A) in-
dicates that the abdominal regions of mice exhibited significant
X. Wang, et al. International Journal of Pharmaceutics 573 (2020) 118840

Fig. 7. In vivo whole-body images (A) and ex vivo images of dissected GI segments (B) of mice after oral administration of P2 and PTX co-loaded CR PMs, with
Integrated fluorescence intensities of the whole-body and dissected GI plotted (D). Samples with or without P2 are shown in (C): P2 quenched in water (a), PTX-
loaded CR PMs in water (b), and P2 and PTX co-loaded CR PMs in water (c). Ex vivo images of organs (from top to bottom: heart, liver, spleen, lung and kidney) are
shown in (E), and tumors are shown in (F), plots of their fluorescence intensities are shown in (G). Quenched P2 solution as control. For each data point, n = 3.

9
X. Wang, et al.
fluorescence at 3 h, which then decreased. The results echo fluorescence
images of dissected GI tracts (Fig. 7B), which showed obvious fluores-
cence at 3 h, 6 h, 9 h and 12 h. The intensity peaked at 3 h and declined
over time (Fig. 7D). In contrast, the control group with P2 quenched
solution showed faint fluorescence both in whole-body and in GI ima-
ging. The results suggest that CR PMs were gradually out of the GI due
to absorption and/or elimination after 3 h. These results corresponds to
in vivo investigation of PTX-loaded CR PMs using H22 tumor xenograft
mice orally administrated by a single dose (20 mg/kg). PTX of CR PMs
increased accumulation in the tumor by intestinal absorption were
2.01-, 6.59- and 9.51-times higher than Taxol® in 6 h, 9 h and 12 h,
respectively (detailed in Supplementary data).
In addition, fluorescence was observed in dissected heart, liver,
spleen, lung, kidney and tumor in P2 and PTX co-loaded CR PMs
groups, but not in the quenched P2 solution group (Fig. 7E and F),
implying possible absorption and migration of intact P2 and PTX co-
loaded CR PMs to these organs. The results suggest that CR PMs can be
absorbed as whole through the GI membrane for distribution through
the body. Stronger fluorescence in liver was observed because liver has
the abundant blood flow and is the main metabolism organ in the body.
High fluorescence intensity was observed in the tumor of one of three
mice. Fluorescence peaked in the liver at 6 h and 24 h and in the tumor
at 9 h (Fig. 7G). The second peak time in the liver at 24 h probably was
due to metabolism and enterohepatic circulation of CR PMs. Markedly,
fluorescence in the GI tract was an order of magnitude stronger than
those of other major organs and tumor. This indicated that some of CR
PMs were absorbed intact through the GI membrane, distributed
through the body for accumulation at the tumor site, while other PMs
were disrupted during migration.
4. Conclusions
In this study, we investigated the absorption of CR PMs in the in-
testinal tract. CR conjugate can enhance PTX absorption in the intes-
tine, especially in duodenum and colon. PTX-loaded CR PMs showed
significant absorption promoting effects of PTX in duodenum, jejunum,
ileum and colon compared to Taxol®. Compared with verapamil, in situ
intestinal absorption experiments and Caco-2 cell uptake assay showed
the transport mechanism of CR PMs across intestinal epithelial cells
may bypass the P-gp efflux. During the process of intestinal transport,
PTX-loaded CR PMs easily transported through the intestinal membrane
without notable injury to intestinal villi. The image captured by CLSM
in intestinal fluorescence labeling assay and Caco-2 cell uptake assay
verified the absorption of PTX-loaded CR PMs in the intestine, which is
consistent with our in situ intestinal absorption experimental results.
Moreover, in vivo bioimaging of tumor-bearing mice confirmed a sig-
nificant portion of CR PMs were absorbed through the intestine as
whole, then distributed in the body and very probably accumulated at
the tumor site. Therefore, we propose that CR PMs are expected to be
promising oral drug delivery carriers for PTX and other water-insoluble
drugs.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 81603301), the Personnel Training Funding
of Fujian Province Health Department (2016-ZQN-69), Joint National
Local Engineering Research Center of Fujian and Taiwan Chinese
medicine molecular biotechnology, Fujian Key Laboratory of Chinese
Materia Medica, Fujian University Key Laboratory for Research and
10
International Journal of Pharmaceutics 573 (2020) 118840
Development of TCM Resources, and the National Special Fund for
Chinese medicine resources Research in the Public Interest of China
(No. 2017-66). We thank Prof. Wei Wu and Prof. Weili Zhao in Fudan
University for providing P4 and P2 fluorescence probe. We also thank
Life Science Editors for editing assistance and Clairissa D. Corpstein for
the language editorial assistance.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpharm.2019.118840.
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