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                    International Journal of Nanomedicine

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O riginal R esearch

Open Access Full Text Article

Mechanism of enhanced oral absorption
of hydrophilic drug incorporated
in hydrophobic nanoparticles
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
26 July 2013
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Liang-Zhong Lv 1
Chen-Qi Tong 1
Jia Yu 1
Min Han 2
Jian-Qing Gao 2
Department of Pharmacy, Zhejiang
Provincial People’s Hospital,
Hangzhou, People’s Republic of China;
2
Institute of Pharmaceutics, College
of Pharmaceutical Sciences, Zhejiang
University, Hangzhou, People’s
Republic of China
1

Abstract: Hydroxysafflor yellow A (HSYA) is an effective ingredient of the Chinese herb
Carthamus tinctorius L, which has high water solubility and low oral bioavailability. This
research aims to develop a hydrophobic nanoparticle that can enhance the oral absorption of
HSYA. Transmission electron microscopy and freeze-fracture replication transmission election
microscopy showed that the HSYA nanoparticles have an irregular shape and a narrow size
distribution. Zonula occludens 1 protein (ZO–1) labeling showed that the nanoparticles with
different dilutions produced an opening in the tight junctions of Caco-2 cells without inducing cytotoxicity to the cells. Both enhanced uptake in Caco-2 cells monolayer and increased
bioavailability in rats for HSYA nanoparticles indicated that the formulation could improve
bioavailability of HSYA significantly after oral administration both in vitro and in vivo.
Keywords: hydroxysafflor yellow A, nanoparticles, Caco-2 cells, bioavailability, absorption

Introduction

Correspondence: Min Han
Institute of Pharmaceutics,
College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058,
People’s Republic of China
Email hanmin2@zju.edu.cn
Jian-Qing Gao
Institute of Pharmaceutics,
College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058,
People’s Republic of China
Tel/Fax +86 571 88208437
Email gaojianqing@zju.edu.cn

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http://dx.doi.org/10.2147/IJN.S47400

Improving the high water solubility and low intestinal permeability of Biopharmaceutics Classification System (BCS) Class III drugs has been a focus of study in pharmaceutical research. Generally, BCS Class III drugs are not adapted to oral formulations.
Thus, the addition of an absorption enhancer1 and chemical modification2 is necessary
to improve the means of drug delivery3,4 and enhance drug bioavailability (BA), respectively. The flower of the safflower plant, Carthamus tinctorius L, has been widely used
in traditional Chinese medicine for treatment of cerebrovascular and cardiovascular
diseases.1 Hydroxysafflor yellow A (HSYA) is extracted from the flower of the safflower plant. As the main active component extracted from the safflower plant, it has
been demonstrated to have a strong antagonistic effect on the platelet-activating factor
receptor5 as well as outstanding neuroprotective action in vivo and in vitro.6 According
to recent studies, HSYA is a BCS Class III1,7 hydrophilic drug with low oral bioavailability, which means that it is only administered in clinical therapy via injections.8,9
Water-in-oil microemulsion and the self-double-emulsifying drug delivery system have
shown great potential in improving the oral absorption of HSYA.10,11
In this study, hydrophobic nanoparticle oil solutions were prepared to increase the
oral absorption of HSYA. Hydrophobic nanoparticle oil solutions are composed of
caprylic/capric triglyceride (GTCC), bean phospholipids, and organic solvents. The
nanoparticle oil solutions were formed after the organic solvents were volatilized.
Through these procedures, small-sized nanoparticles in a stable, oily environment
were prepared, and a formulation that is more stable than emulsions was obtained.

International Journal of Nanomedicine 2013:8 2709–2717
© 2013 Lv et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
which permits unrestricted noncommercial use, provided the original work is properly cited.

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This study investigated the possible enhancing mechanism
of hydrophobic nanoparticles to HSYA.

Material and methods
Material
HSYA (98% purity) was purchased from Chengdu Herb
purity Company, Ltd (Chengdu, People’s Republic of China),
while GTCC (caprylic/capric triglyceride) was obtained
from Gattefosse (Saint-Priest, France). Bean phospholipids
(Lipoid S 100) were purchased from Lipoid (Ludwigshafen,
Germany), and Sephadex G-100 was supplied by the Beijing
Investor Science and Technology Development Company, Ltd
(Beijing, People’s Republic of China). Methyl-β-cyclodextrin
was obtained from the Shanghai Ding Jie Biological Technology Company, Ltd (Shanghai, People’s Republic of China).
NaN3, amiloride, rhodamine-123, N-acetyl-L-cysteine,
polyoxyethylene (10) octylphenyl ether (Triton X-100)
and 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium
bromide (MTT) were purchased from Sigma-Aldrich
(St Louis, MO, USA). The rabbit anti-ZO-1 antibody
was purchased from Abcam (Cambridge, UK), while the
FITC-labeled goat anti-rabbit IgG antibody was purchased
from Wuhan Boster Biological Engineering Company, Ltd
(Wuhan, People’s Republic of China). Dulbecco’s modified
Eagle’s medium (DMEM) and fetal bovine serum (FBS)
were purchased from Thermo-Fisher Biochemical Products
(Beijing, People’s Republic of China).

Animals

Male Sprague-Dawley rats (200 g ± 20 g) were obtained
from the Animal Center of Zhejiang University (Hangzhou,
People’s Republic of China). The animal experiment was
approved by the Animal Ethics Committee of Zhejiang
University. The animals were housed in a normal laboratory
environment with access to food and water.

Preparation of HSYA-loaded
nanoparticles
Initially, 500 mg of soybean phospholipids were dissolved in
2 mL methanol. Then, 50 mg of HSYA was added and ultrasonicated to obtain a clarified mixed solution (A). Afterwards,
another 500 mg of soybean phospholipids were dissolved in
0.5 mL ethanol and ultrasonicated, then 10 mL GTCC was
added to obtain a mixture (B). The A and B mixtures were
mixed and homogenized at 9,500 rpm for 2 minutes (FJ-200
High-Speed Dispersion Homogenizer; Jiangsujintan Jincheng
Instruments Co, Jiangsu, People’s Republic of China) until a

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clear and transparent formulation was obtained. The organic
solvents were removed by using a rotary evaporator (Shanghaijiapeng Technology Company Ltd, Shanghai, People’s
Republic of China) for 30 minutes at 40°C.

Characterization of HSYA-loaded
nanoparticles
Droplet size and morphology observation

The newly prepared nanoparticles were filtered through
a 0.22 µm organic membrane, and the droplet size was
measured by using dynamic light scattering (Nano-S 90;
Malvern, Worcestershire, UK). The morphology of the
nanoparticle was observed through transmission electron
microscopy (TEM) (Philips Tecnai 10; Philips, Amsterdam,
Netherlands). The particle size, particle size distribution, and
aggregation state of the nanoparticles were characterized
by freeze-etching (BAF 060; Leica, Germany) replication
TEM (FERTEM).

Encapsulation efficiency, drug loading capacity,
and in vitro drug release
The encapsulation efficiency (EE%) and drug loading
capacity (LC%) were determined by using the Sephadex
column method, and calculated using the formulas below.11
HSYA in nanoparticles
× 100% (1)
Total amount of HSYA in dispersion
		
HSYA in nanoparticles
(2)
LC % =
×100%
Nanoparticles weight
EE % =

Briefly, 20 µL nanoparticles were dissolved in 180 µL
methanol to release HSYA as the total amount of HSYA.
Another 20 µL nanoparticles was mixed with water (1:4) to
form emulsions, then passed through a Sephadex column.
The eluted emulsions from the column were further treated
by methanol to release HSYA as the content of HSYA in
nanoparticles.
In vitro drug release was performed by using the dialysis
bag method.12,13 The dialysis bag allowed the transfer of
the released drug molecules into the release media while
intercepting the nanoparticles. The dialysis bag (molecular
weight range: 8,000 to 14,000; Sigma-Aldrich) was boiled
for more than 30 minutes and soaked in the release media
overnight prior to the experiment; the release media was
double-distilled water. One end of the dialysis bag was tied
tightly, and 10 mL release media was placed in the bag.

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Absorption of hydrophilic drug in hydrophobic nanoparticles

The other end of the dialysis bag was closed with a dialysis
clip, and the dialysis bag was then placed in a beaker that
contained 200 mL release media. The beaker was placed on
a magnetic stirrer (Hangzhou Instrument Electric Co, Ltd)
at a rotational speed of 100 rpm at 4°C. Afterwards, 1 mL
of the freshly prepared HSYA nanoparticles (3.88 mg/mL)
was added to the release media outside the dialysis bag. At
the preset time points (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60,
72, 96, 120, 144, 168, and 192 hours), 1 mL of the release
sample was withdrawn from the media in the dialysis bag,
and equal amounts of fresh release media was immediately
added into the dialysis bag to maintain the sink condition.
The in vitro release of the HSYA solution was carried out
by adding the drug solution to the release media and withdrawing the release sample from the release media in the
beaker directly and not from the dialysis bag. All samples
were analyzed by high-performance liquid chromatography (HPLC).

Caco-2 cell culture
The Caco-2 cells were cultured in a cell incubator with an
atmosphere of 5% CO2 at 37°C. The culture medium was
DMEM, which contained 4,500 mg/L D-glucose, 584 mg/L
L-glutamine, 3.7 g/L NaHCO3, supplemented with 10%
(volume [v]/v) heat-inactivated FBS, 1% (v/v) penicillin, and
1% (v/v) streptomycin. The culture medium was replaced
every 3 to 4 days. Cell subculturing was performed when
the cells reached 80% to 90% coverage.

Cell viability assay
Caco-2 cells were seeded in 96-well plates at a density of
10,000 cells/well and cultured for 24 hours with DMEM
culture medium. When the experiment was started, the
medium was replaced with 200 µL serial dilutions of
blank nanoparticles or the control (culture medium). The
cells were exposed to the nanoparticles for 2 hours at
37°C, after which 20 µL of MTT reagent (5 mg/mL) in
Phosphate Buffer Solution (PBS) was added to each well,
and the mixtures were incubated for 4 hours. Then, 180 µL
Dimethyl sulfoxide (DMSO) was added to each well, and
the mixture was agitated gently to dissolve the crystals
completely. The absorbance values were detected by using
a KHB ST-360 microplate reader (KHB, Shanghai, People’s
Republic of China) at 570 nm. Cell viability was calculated
as follows:
Cell viability (%) = Atext/Acontrol × 100%

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(3)

Transepithelial electrical resistance
measurement in Caco-2 cells

Cells were seeded at a density of 2.5 × 105 cells/well in a
12-well polycarbonate membrane transwell (12 mm, 1 µm
pore size, 0.3 cm2 growth area) obtained from Costar (Silicon
Valley, USA). The cells were cultured for 21 days under the
previously described culture conditions. The volumes of the
culture medium were 0.5 mL and 1.5 mL on the apical and
basolateral side, respectively. At 21 days, the cell transepithelial electrical resistance (TEER) was measured using Millicell
ERS-2 Volt-Ohm Meter (Millipore, Massachusetts, USA)
to evaluate cellular integrity, as previously reported.14,15 To
evaluate the impact of HSYA solutions, HSYA-nanoparticles
and blank nanoparticles on cells integrity, 3.88 mg/mL of
HSYA solutions and the same concentration of HSYA-nanoparticles and blank nanoparticles were all diluted 10-fold to
incubate with Caco-2 cells for 2 hours. The TEER was also
measured when the formulation was removed after 2 hours
of incubation, and the culture was continued up to 24 hours
and 48 hours to evaluate if the impact of the formulation on
cell integrity was reversible.

Cellular uptake of HSYA

Cells were seeded at a density of 2.5 × 105 cells/well onto
six-well plates and cultured for 14 days for the uptake
experiments. Cells were treated with different concentrations of HSYA solution and HSYA nanoparticles for 2 hours.
The cells were preincubated with a number of endocytotic
inhibitors, such as NaN3 (1.32 mg/mL), chlorpromazine (10 µg/mL), methyl-β-cyclodextrin (methyl-β-CD)
(13.3 mg/mL), and amiloride (50 µM), for 30 minutes to
understand the endocytotic mechanism of the formulations.
Blank nanoparticles were also added to investigate whether
they have an effect on Caco-2 cellular uptake of HSYA. At
the end of the experiments, the cells were washed three times
using ice-cold PBS, then frozen and thawed three times. One
mL of water was added to each well. The wells were probesonicated 25 times to obtain the cell lysates. The lysates were
centrifuged at 13,000 rpm for 5 minutes, and the supernatant
liquid was analyzed using HPLC.

Cellular transport of HSYA
Caco-2 cells were seeded onto the 12-well transwell at a
density of 2.5 × 105 cells/well and cultured at a condition
similar to that during TEER measurements. At 21 days, the
transmembrane resistance met the requirements,16,17 and the
cell model was ready for transport experiments. One mg/mL

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of the HSYA solutions was added to the apical side of the
cells, and 1.5 mL DMEM without FBS was added to the
basolateral side. At 15, 30, 45, 60, 90, 120, 180, 240, 360,
and 480 minutes, 500 µL of the sample from the basolateral
side was collected and replaced with the same volume of fresh
DMEM without FBS. Another group of cells was preincubated with 150 µg/mL of cyclosporin A (CsA, a recognized
p-gly-coprotein [p-gp]) for 30 minutes. The cells were preincubated with 0.5 mL methanol, added to the samples of the
nanoparticle group, and then probe-sonicated 25 times (4°C,
150 W, active every 2 seconds within a 3-second duration).
All samples were centrifuged at 13,000 rpm for 5 minutes.
Then, 50 µL of the supernatant was injected into the liquid
chromatography system (Agilent 1,200 system; Agilent,
Santa Clara, CA, USA) for investigation.

Effect of HSYA nanoparticles
on ZO-1 distribution
The cells were seeded in a 12-well polycarbonate membrane
transwell at a density of 2.5 × 105 cells/well and cultured for
21 days before the experiment, followed by incubation with
10-fold dilution HSYA nanoparticles for 2 hours at 37°C.
Then, the cells were washed three times with ice-cold PBS
and fixed with 4% paraformaldehyde solution for 30 minutes, followed by rinsing with ice-cold PBS two times and
permeabilization in 0.2% Triton X-100/PBS for 5 minutes.
Non-specific binding was blocked with 5% Bovine serum
albumin (BSA)/PBS for 30 minutes. Primary antibody
(rabbit anti-ZO-1 antibody) at a concentration of 1:100 was
applied and incubated at room temperature for 2 hours. The
cells were washed three times with PBS, and the secondary
antibody (FITC-conjugated goat anti-rabbit IgG) was added
at a concentration of 1:48 and incubated at room temperature
for 1-hour. Finally, the cells were washed three times with
PBS and observed under two-photon confocal microscopy
(BX61 W1-FV1000; Olympus, Japan).

In vivo pharmacokinetic studies
Pharmacokinetic studies were carried out by dividing the rats
into two groups, with three rats in each group. The animals in
the HSYA group were given a dose of 25 mg/kg, which was
also the dose given in our previous study.11 Another group
received the oral HSYA nanoparticles at a dose of 25 mg/kg.
After the administration of HSYA solution and HSYA formulations to the rats through oral gavage, blood samples
were collected from the eye ground vein into heparinized
tubes at preset time points of 5, 15, 30, 45, 60, 90, 120, 180,
240, 360, 480, and 1,440 minutes. The blood samples were

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centrifuged at 4,000 rpm for 10 minutes, and the supernatant
was transferred into another plastic tube and stored at −20°C
until further analysis.

Determination of HSYA concentrations
in plasma
The plasma concentrations of HSYA were determined
through HPLC, as we described in our preliminary work.11
Two hundred ul plasma samples were collected in plastic
centrifuge tubes with 100 µL of 6% perchloric acid added
to precipitate protein. The mixture was vortexed for 2 minutes. After centrifugation at 13,000 rpm for 10 minutes, the
supernatant was detected using HPLC.

HPLC analysis of samples
Chromatographic conditions were performed according
to the literature:18 a C18 column (Agilent SRB- C18, 5µm,
4.6 mm × 250 mm, Agilent) was used at 40°C. The mobile
phase was 32% methanol, 2% acetonitrile, and 66% phosphate solution (0.1% concentration). The injection volume
was 50 µL, and the flow rate was 0.8 mL/minute. The detection signal was 403 nm.

Pharmacokinetic analysis
The maximum plasma concentration was defined as Cmax,
while the time to reach Cmax was defined as tmax. The area under
the plasma concentration time curve (AUC) and mean retention time (MRT) were calculated from 0 hours to 12 hours.
All parameters were calculated by using Kinetica 4.4 software
(Thermo Fisher Scientific Inc., MA, USA).

Statistical analysis

All values are expressed as mean ± standard deviation (SD).
Statistical significance was assessed using one-way analysis
of variance (ANOVA) among more than three groups, with
P,0.05 considered statistically significant. The means of
the two groups were compared by performing two-tailed
student’s t-tests.

Results
Characterization of HSYA-loaded
nanoparticles
Figure 1A and B show that the freshly prepared HSYA nanoparticles were diluted with 10-fold GTCC. The morphology
of the diluted nanoparticles was characterized by TEM and
FERTEM. Figure 1A shows that the formed nanoparticles
exhibited a spherical structure with a relatively uniform
size. FERTEM was performed to further characterize the

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Absorption of hydrophilic drug in hydrophobic nanoparticles

A

nanoparticles, and to evaluate the impact of nanostructures
on drug release. In the case of the HSYA solution, complete
release was obtained after 0.5 hours, whereas in the case of
the HSYA nanoparticles, HSYA was totally released from the
nanoparticles after 192 hours (approximately 50% was totally
released), and the release was better sustained in vitro.

B

0.2 µm

Cell viability assay

0.5 µm

C

Figure 3A shows that the blank nanoparticles diluted 10-, 15-,
30-, and 50-fold showed almost no cytotoxicity after incubation for 2 hours.

Statistics graph (1 measurements)
Number (%)

30
20

TEER measurement in Caco-2 cells

10
0

1

10

100

1000

Size (d·nm)

10000

Mean with +/− 1 standard deviation error bar

Figure 1 (A) TEM image of HSYA nanoparticles, (B) FERTEM image of HSYA
nanoparticles, and (C) Size distribution of freshly prepared HSYA nanoparticles.
Abbreviations: FER, freeze-etching replication; HSYA, hydroxysafflor yellow A;
TEM, transmission electron microscopy.

HSYA accumulative
release rate (%)

A

120
100

HSYA-NPs

Cellular uptake and transport studies
of HSYA
Figure 4B shows that the apparent permeability coefficient
(Papp) of 1 mg/mL HSYA was (5.38 ± 1.58) × 10−7, which
A
Cell viability (%)

surface morphology of the HSYA nanoparticles. The HSYA
nanoparticles exhibited irregular morphology. The cross section of the nanoparticles was not flat and had a similar layer- or
shell-like structure (Figure 1B). Dynamic light scattering studies (Figure 1C) showed that the mean diameter of the HSYA
nanoparticles without dilution was 49.2 nm ± 5.8 nm, and the
polydispersity was 0.38. The encapsulating efficiency and
drug loading capacity was 71.68% and 0.71%, respectively.
The equilibrium dialysis method was used to determine
the in vitro release behavior of the HSYA solution and HSYA

Figure 3B shows that after the 2-hour incubation of HSYA
solution and HSYA nanoparticles, the TEER of Caco-2 cells
decreased. The TEER did not significantly increase after
the HSYA solution or the HSYA-loaded nanoparticles were
removed and culture continued for 24 hours and 48 hours.
When the blank nanoparticles were incubated for 2 hours, the
TEER did not decrease. The TEER increased with continued
cell culture. From these results, we hypothesize that HSYA
is somewhat toxic to Caco-2 cells, thereby destroying the
integrity of the cells. The blank nanoparticles showed almost
no effect on the integrity of the cells.

HSYA solution

80
60

100

40

B

20
0
100

150

200

250

TEER (Ω · cm2)

50

0

T (hours)

B
HSYA accumulative
release rate (%)

120

HSYA solution

120
100

HSYA-NPs

80
60
40

80
60
40
20
0
NPs-10 fold

NPs-15 fold

NPs-30 fold

NPs-50 fold

600
500

HSYA solution

HSYA-NPs

Blank-NPs

400
300
200
100
0

20

Control

0
0

2

4

6

8

10

12

14

2 hours incubation
of NPs

Removed NPs Removed NPs and
and continued continued culture
culture 24 hours
48 hours

T (hours)
Figure 2 HSYA release from nanoparticles in double-distilled water at 4°C
(n = 3).
Note: Figure 2B is an enlarged version of Figure 2A.
Abbreviations: HSYA, hydroxysafflor yellow A; NP, nanoparticle.

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Figure 3 (A) Cytotoxicity of blank nanoparticles on Caco-2 cells after incubation
for 2 hours; (B) Cellular integrity studies of incubation with nanoparticles and
removal of nanoparticles (n = 3).
Abbreviations: HSYA, hydroxysafflor yellow A; NP, nanoparticle; TEER, transepit­
helial electrical resistance.

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increased to (1.27 ± 0.64) × 10−6 when the Caco-2 cells
were preincubated with CsA (p-glycoprotein inhibitors)
for 30 minutes. This result indicates the presence of p-glycoprotein efflux, which may be one of the reasons behind
poor HSYA absorption. Figure 4A shows that both high and
low concentrations of HSYA nanoparticles can significantly
promote HSYA absorption.
Endocytotic inhibitor experiments were conducted
(Figure 4C) to clarify how the nanoparticles promote HSYA
absorption. Prior to the experiments, Caco-2 cells were
preincubated for 30 minutes with three kinds of inhibitors,
namely, chlorpromazine (10 µg/mL) to inhibit clathrin
vesicles, methyl-β-CD (13.3 mg/mL) to inhibit caveolae,
and amiloride (50 µM) to inhibit pinocytosis. However, the
results revealed that the inhibitors did not have any significant
effect on HSYA uptake. Blank nanoparticles also have no
significant effect on HSYA absorption.

Effect of HSYA-loaded nanoparticles
on ZO-1 distribution
ZO-1 is a tight junction-associated protein that is localized
on the cytoplasmic surface just beneath the membrane.
Figure 5A shows that the ZO-1 of the control group was
uniformly distributed in the tight connection region of the

The plasma concentration-time profiles of HSYA after
intravenous or oral administration of HSYA solution and
HSYA nanoparticles to rats are shown in Figure 6, and the
pharmacokinetic parameters are summarized in Table 1. The
AUC of HSYA nanoparticles was 23.3-fold greater than that
of the HSYA solution.

Discussion
HSYA is the main component of safflower yellow pigments,
which is the aqueous extract of safflower florets. Recent studies investigated the effect of HSYA on lipopolysaccharideinduced inflammatory signal transduction in human alveolar
epithelial A549 cells,19 which includes inhibition of protein
oxidation/nitration, 12/15-lipoxygenase,20 exertion of therapeutic actives on cerebral ischemia induced by thrombosis,21
and promotion of blood circulation by influencing hemorheology, plasma coagulation, and platelet aggregation,22 among
others. HSYA is a hydrophilic drug with poor oral bioavail-

**
HSYA-NPs

HSYA

1

*

0.5
0
0.1

1

12

1 mg/mL HSYA

10

1 mg/mL HSYA + CSA

8
6
4
2
0
15

HSYA (mg/mL)

30

45

60

T (min)

90

120

Ps

e

N
k
an
Bl

ilo

rid

D
l-β
hy
M
et

az
pr
om
C

hl
or

-C

in

Ps

l
tro
C

on

e

*

0.3
0.25
0.2
0.15
0.1
0.05
0

Am

1.5

HSYA transport (µg)

2

C

HSYA uptake
(µg HSYA/mg protein)

In vivo pharmacokinetic studies

B

N

HSYA uptake
(µg mg/protein)

A

cell. However, in comparison to the control cell layers, the
individual regions exhibited discontinuous distribution in
the HSYA nanoparticle group, and part of the cells exhibited
extrusion and deformation.

Figure 4 (A) HSYA solution and HSYA nanoparticle uptake by Caco-2 monolayers after incubation for 2 hours; (B) HSYA transport across Caco-2 cells (surface area of
monolayer = 1.12 cm2); (C) Endocytotic inhibitor studies on Caco-2 cells of HSYA-NPs.
Notes: *P,0.05; **P,0.01, compared to control.
Abbreviations: CSA, cyclosporin A; HSYA, hydroxysafflor yellow A; Methyl-β-CD, methyl-β-cyclodextrin; NP, nanoparticle; T, time.

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Absorption of hydrophilic drug in hydrophobic nanoparticles

A

Table 1 Pharmacokinetic parameters after intravenous or oral
administration of HSYA formulations to rats

B

Intravenous
HSYA

Control

NPs

Figure 5 (A) ZO-1 staining in control cell layers not subjected to HSYA
nanoparticles; (B) ZO-1 staining in cells incubated with 10-fold dilution HSYA-NPs
for 2 hours at 37°C.
Abbreviations: HSYA, hydroxysafflor yellow A; NP, nanoparticle.

ability,1,10 which limits its application in clinical practice
despite its many prominent pharmacological effects. In our
previous studies,11 we developed a self-double-emulsifying
drug delivery system (SDEDDS), which was composed of
water in oil emulsions and hydrophilic surfactants that can
self-emulsify into water-in-oil-in-water (w/o/w) double
emulsions in the aqueous gastrointestinal environment.
This formulation increased the oral bioavailability of HSYA
2.17-fold compared to HSYA solution.

Concentration (µg/mL)

A
90
80
70
60
50
40

HSYA solution

30
20
10
0
0

1

2

Concentration (µg/mL)

B

5

6

7

8

10

12

14

8

10

12

14

4

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

HSYA solution

0

2

4

C
Concentration (µg/mL)

3
T (hours)

8
7
6
5
4
3
2
1
0

6
T (hours)

HSYA NPs

0

2

4

6
T (hours)

Figure 6 (A) Plasma concentration-time profiles of HSYA after intravenous
administration of HSYA solution, (B) oral administration of HSYA solution, and
(C) HSYA-NPs to rats at a dose of 25 mg/kg.
Abbreviations: HSYA, hydroxysafflor yellow A; NP, nanoparticle.

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Oral
HSYA
solution

HSYA-NPs

Tmax (min)

5

Cmax(µg/mL)
AUC0–12
(µg minute/mL)
MRT (minute)
Thalf (minute)
Frel (%)
Fab (%)

45 ± 15
56.51 ± 29.05
0.33 ± 0.05
2,477.68 ± 221.11 39.92 ± 6.20

90.00 ± 51.96
4.80 ± 2.77*
931.53 ± 406.34**

77.47 ± 6.74
353.32 ± 64.29
_
100

311.12 ± 26.52**
579.97 ± 216.47
2,333
38

227.20 ± 12.29
407.61 ± 241.36
100
1.61

Notes: The values represent mean ± standard deviation (n = 3); **P,0.01; **P,0.05
compared to control.
Abbreviations: AUC, areas under the concentration time curve; Cmax, maximum
plasma concentration; Frel, relative bioavailability; Fab, absolute bioavailability; HSYA,
hydroxysafflor yellow A; MRT, mean retention time; NPs, nanoparticles; Thalf, halflife; Tmax, time to maximum plasma concentration.

In this study, we developed a hydrophobic nanoparticle
oil solution, the novel formulation of which could be a
nanocarrier for hydrophilic drugs with the drug dispersed
in the oil phase. Actually, a clear and transparent nanoparticles oil solution was formed when organic solutions A
and B were mixed, which means HSYA could be dissolved
in organic solvent with soybean phospholipids. After the
evaporation of organic solvent, the nanoparticles of HSYA/
soybean phospholipids dispersed in GTCC were formed
with the structure of a hydrophilic core and hydrophobic
surface. As a water soluble drug, HSYA was wrapped in
the core of nanoparticles. No deposition could be observed.
The formulation composition and preparation process in
the present study was determined after some experimental
optimization, as not all kinds of oil and surfactant could
form the nanoparticles of “hydrophilic drug dispersed in
hydrophobic oil”. Due to its pure anhydrous environment,
the nanoparticle was more stable for HSYA when compared
to SDEDDS. From the pharmacokinetic results (Figure 6
and Table 1), we can see that the nanoparticles increased
the absolute bioavailability and relative bioavailability to
23.6- and 23.3-fold, respectively. Figure 1 shows that the
nanoparticles exhibited an irregular structure, with a particle size of 49.2 nm ± 5.8 nm. The polydispersity of HSYA
nanoparticles was 0.38, which indicated that the particle size
distribution of nanoparticles is relatively non-uniform. In the
future, we will optimize the prescription of nanoparticles in
order to get uniform size, and to shape rounded particles. In
this article, the reversed-dialysis bag method was applied to
the in vitro release studies,23 and sustained in vitro release
was achieved (Figure 2). However, the nanoparticles did
not exhibit a sustained release effect in vivo. We speculate

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Lv et al

that there are significant differences between in vivo and
in vitro environments.
Results of uptake studies on Caco-2 cells (Figure 4A)
showed that the formulation can remarkably increase the
membrane permeability of HSYA across Caco-2 cell monolayers at low and high concentrations. However, the endocytotic mechanism of nanoparticles is complex. When cells
were pretreated with endocytosis inhibitors, such as chlorpromazine, methyl-β-CD, and amiloride, for 30 minutes, the
uptake of HSYA decreased to some extent, but did not differ
significantly (Figure 4C). Based on the endocytosis results,
we assume that the uptake process of HSYA nanoparticles
may involve a number of factors. As shown from Figure 4B,
CsA (a recognized p-glycoprotein inhibitor) could significantly increase the absorption of HSYA on Caco-2 cells,
which indicated that the presence of p- glycoprotein efflux
may be involved in HSYA absorption. Although there are no
other studies in the literature discussing the mechanisms for
the low absorption of HSYA solution, our preliminary studies
support this hypothesis.11 Figure 5 shows that the treatment
of a monolayer with HSYA nanoparticles at a 1:10 dilution
appeared to cause a redistribution of ZO-1. The results indicate that the formulation could break the tight junction of
Caco-2 cell layers to promote drug absorption through the
paracellular pathway. Meanwhile, the cells were treated with
blank-nanoparticles (NPs) for 2 hours followed by another
48-hour incubation with fresh culture medium without
blank-NPs, and cell membrane integrity was not influenced
as indicated by the TEER results (Figure 3A), suggesting
good biocompatibility of the nanoparticles.

Conclusion
In the present study, a novel oral delivery system of
HSYA was developed to improve the oral bioavailability
of HSYA. The prepared nanoparticles had a diameter of
49.2 nm ± 5.8 nm and had sustained release properties. The
results of Caco-2 monolayer uptake and pharmacokinetics
studies suggest increased HSYA absorption both in vitro
and in vivo, which indicates great potential for the future
formulation of HSYA.

Acknowledgments
This study was funded by the Natural Science Foundation
of Zhejiang Province, People’s Republic of China
(No Y2100564; No Y2110124; No Y2100645).

Disclosure
The authors report no conflicts of interest in this work.

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