Enhanced delivery of doxorubicin to the liver through self-assembled nanoparticles formed via conjugation of glycyrrhetinic acid to the hydroxyl group of hyaluronic acid

Article abstract of DOI:10.1016/j.carbpol.2018.04.052

Liver-targeted nanoparticles is highly desired for better therapy of liver cancer. In this study, enhanced delivery of doxorubicin (DOX) to the liver cells through self-assembled nanoparticles formed via conjugation of glycyrrhetinic acid (GA) to the hydroxyl group of hyaluronic acid (HA) was investigated. The DOX loaded hyaluronic acid-glycyrrhetinic acid succinate (HSG) conjugates based nanoparticles (HSG/DOX nanoparticles) were sub-spherical in shape with particle size in the range of 180–280 nm, the drug loading was drug-to-carrier ratio and GA graft ratio dependent. In vitro release study suggested that the release of DOX from HSG nanoparticles was sustained and the release rate was pH and GA graft ratio dependent. MTT assay indicated the HSG/DOX nanoparticles presented a GA-dependent cytotoxicity to HepG2 cells. Pharmacokinetics study demonstrated the HSG/DOX nanoparticles could prolong blood circulation time of DOX and had a higher AUC value than that of DOX solution. Furthermore, tissue distribution study revealed the HSG/DOX nanoparticles significantly increased the accumulation of DOX in the liver and meanwhile decreased the cardiotoxicity and nephrotoxicity of DOX. Moreover, the liver targeting enhancing capacity was HSG conjugate structure dependent. The accumulation of HSG-20/DOX, HSG-12/DOX, and HSG-6/DOX nanoparticles in the liver was 4.0-, 3.1-, and 2.6-fold higher than that of DOX solution. In vivo imaging analysis further demonstrated HSG nanoparticles not only had better liver targeting effect, but also presented superior tumor targeting efficiency, and the tumor targeting capacity was also GA-dependent. These results indicated that HSG conjugates prepared via modifying the hydroxyl groups of HA have promising potential as a liver-targeting nanocarrier for the delivery of hydrophobic anti-tumor drugs.

Full text of DOI:10.1016/j.carbpol.2018.04.052

Accepted Manuscript
Title: Enhanced delivery of doxorubicin to the liver through
self-assembled nanoparticles formed via conjugation of
glycyrrhetinic acid to the hydroxyl group of hyaluronic acid
Authors: Xiaodan Wang, Xiangqin Gu, Huimin Wang, Jingyu
Yang, Shirui Mao
PII:
S0144-8617(18)30438-7
DOI:
Reference:
https://doi.org/10.1016/j.carbpol.2018.04.052
CARP 13506
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Revised date:
Accepted date:
5-12-2017
13-3-2018
11-4-2018
Please cite this article as: Wang, Xiaodan., Gu, Xiangqin., Wang, Huimin.,
Yang, Jingyu., & Mao, Shirui., Enhanced delivery of doxorubicin to the liver
through self-assembled nanoparticles formed via conjugation of glycyrrhetinic
acid to the hydroxyl group of hyaluronic acid.Carbohydrate Polymers
https://doi.org/10.1016/j.carbpol.2018.04.052
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Enhanced delivery of doxorubicin to the liver through self-assembled
nanoparticles formed via conjugation of glycyrrhetinic acid to the
hydroxyl group of hyaluronic acid
1
1
1
2
1,*
Xiaodan Wang , Xiangqin Gu , Huimin Wang , Jingyu Yang , Shirui Mao
1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
2Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang,
1
10016, China
*
To whom correspondence should be addressed.
School of Pharmacy
Shenyang Pharmaceutical University
Shenyang, China
Tel and Fax: +86-24-43520523
E-mail: maoshirui@syphu.edu.cn; maoshirui@vip.sina.com
1

Graphic abstract
Highlights



Enhanced delivery of doxorubicin to the liver through self-assembled
nanoparticles formed via conjugation of glycyrrhetinic acid to the hydroxyl
group of hyaluronic acid was demonstrated for better therapy of hepatocytes.
Doxorubicin-loaded nanoparticles presented higher liver and tumor targeting
efficiency, and the higher glycyrrhetinic acid graft ratio on hyaluronic acid,
the better liver and tumor targeting property.
The binding site of glycyrrhetinic acid on hyaluronic acid has significant
influence on the liver and tumor targeting efficiency.
2

Abstract
Liver-targeted nanoparticles is highly desired for better therapy of liver cancer.
In this study, enhanced delivery of doxorubicin (DOX) to the liver cells through
self-assembled nanoparticles formed via conjugation of glycyrrhetinic acid (GA) to
the hydroxyl group of hyaluronic acid (HA) was investigated. The DOX loaded
hyaluronic acid-glycyrrhetinic acid succinate (HSG) conjugates based nanoparticles
(HSG/DOX nanoparticles) were sub-spherical in shape with particle size in the range
of 180-280 nm, the drug loading was drug-to-carrier ratio and GA graft ratio
dependent. In vitro release study suggested that the release of DOX from HSG
nanoparticles was sustained and the release rate was pH and GA graft ratio dependent.
MTT assay indicated the HSG/DOX nanoparticles presented a GA-dependent
cytotoxicity to HepG2 cells. Pharmacokinetics study demonstrated the HSG/DOX
nanoparticles could prolong blood circulation time of DOX and had a higher AUC
value than that of DOX solution. Furthermore, tissue distribution study revealed the
HSG/DOX nanoparticles significantly increased the accumulation of DOX in the liver
and meanwhile decreased the cardiotoxicity and nephrotoxicity of DOX. Moreover,
the liver targeting enhancing capacity was HSG conjugate structure dependent. The
accumulation of HSG-20/DOX, HSG-12/DOX, and HSG-6/DOX nanoparticles in the
liver was 4.0-, 3.1-, and 2.6-fold higher than that of DOX solution. In vivo imaging
analysis further demonstrated HSG nanoparticles not only had better liver targeting
effect, but also presented superior tumor targeting efficiency, and the tumor targeting
capacity was also GA-dependent. These results indicated that HSG conjugates
prepared via modifying the hydroxyl groups of HA have promising potential as a
liver-targeting nanocarrier for the delivery of hydrophobic anti-tumor drugs.
Keywords: Hyaluronic acid; Glycyrrhetinic acid; Nanoparticles; Doxorubicin;
Liver-targeting
3

1
. Introduction
Treatment of liver cancer has become a major problem because of its high
morbidity and high mortality (Torre, Bray, Siegel, Ferlay, Lortet-Tieulent & Jemal,
015). In recent years, significant efforts have been exerted towards the design and
2
construction of liver-targeted nanoparticles for better therapy of hepatocytes (Jiang et
al., 2009; Krishna, Mandraju, Kishore & Kondapi, 2009; Liu et al., 2011). However,
the introduction of liver targeting moieties often complicate the preparation of
nanoparticles. In order to simplify the system, hydrophilic or hydrophobic materials
with active targeting have generated considerable interests as the carrier for
nanoparticle drug delivery system design (Arpicco, Milla, Stella & Dosio, 2014;
Schante, Zuber, Herlin & Vandamme, 2011; Tian, Wang, Wang, Zhang, Wang & Yuan,
2
012).
Hyaluronic acid (HA) is a promising hydrophilic material in the area of
nanoparticle system design (Choi et al., 2010; Choi, Saravanakumar, Park & Park,
2
2
012), because it can specifically bind to the CD44 (Arpicco, Milla, Stella & Dosio,
014; Tripodo, Trapani, Torre, Giammona, Trapani & Mandracchia, 2015), RAHMM
(Schiffelers et al., 2004) and LYVE-1 receptors (Bhang et al., 2009), which are
over-expressed in various kinds of malignant cells including the hepatoma cell line.
Son et al. prepared hyaluronic acid-poly(D,L-lactide-co-glycolide) polymeric micelles
and confirmed that the HA micelles could enter HepG2 cells via CD44
receptor-mediated endocytosis, and thus delivered DOX into HepG2 cells, leading to
strong cytotoxicity (Son et al., 2014).
Glycyrrhetinic acid (GA), one of the main bioactive compounds of licorice, has
been widely used in pharmaceutical field for the treatment of many pathologies (Lu,
Li & Wang, 2008). Due to its remarkable physicochemical characteristics, such as its
hydrophobicity (Wang, Tian, Wang, Zhang, Wang & Yuan, 2012) and targeting
properties (Tian et al., 2010), GA has been applied in nano-drug delivery systems
especially for chemotherapy of hepatocyte (Cai, Xu, Chan, Fang, He & Chen, 2016;
Guo et al., 2013). Due to the abundant GA receptors on hepatocyte membranes,
4

nanoparticles containing GA presented higher accumulation in the liver, and thereby
have better therapy of hepatocytes (Tian, Wang, Wang, Zhang, Wang & Yuan, 2012;
Tian et al., 2010). Moreover, Tian et al. found that GA-modified micelles could
discriminate the normal liver cells and the liver cancer cells, and there was 2.18-fold
improvement in uptake by HepG2 cells than that by normal liver cells (Tian, Wang,
Wang, Zhang, Wang & Yuan, 2012). And the ability of GA-modified micelles to
selectively kill liver malignant cells has been demonstrated by Zhang et al (Zhang et
al., 2012), which resulted in higher therapeutic efficacy with improved safety.
Therefore, based on the active liver targeting capacity originated from both HA
and GA, it is assumed that HA-GA conjugation as drug carrier may lead to synergistic
liver targeting effect (Han et al., 2016; Mezghrani et al., 2015; Zhang, Yao, Zhou,
Wang & Zhang, 2013). And the two reactive groups (the carboxyl and hydroxyl group)
in the backbone of HA (Choi et al., 2010; Schante, Zuber, Herlin & Vandamme, 2011),
make it flexible to modify GA molecule as required (Han et al., 2016; Mezghrani et
al., 2015; Zhang, Yao, Zhou, Wang & Zhang, 2013). Our previous studies (Wang, Gu,
Wang, Sun, Wu & Mao, 2017) demonstrated that hyaluronic acid-glycyrrhetinic acid
succinate (HSG) synthesized by modifying hydroxyl groups of HA could provide
superior liver targeting efficiency compared to carboxyl group modification. Thus, it
is reasonable to assume that by using this conjugate as the drug nanocarrier, which
can be prepared by self-assemble using HA as the hydrophilic part and GA as the
hydrophobic part
effect.
,
better liver targeting efficiency can be achieved with decreased side
In this study, doxorubicin (DOX) was chosen as a model drug because it has
been extensively used in clinic for liver cancer therapy (Li & Wallace, 2008) and the
DOX nano-delivery systems have been widely concerned (Kataoka. et al., 2000;
Matsumura & Kataoka, 2009; Shuai, Ai, Nasongkla, Kim & Gao, 2004). DOX-loaded
HSG nanoparticles were prepared and their physicochemical properties were
characterized including particle size, morphology, drug loading, and in vitro release
performance. The cytotoxicity, pharmacokinetics and the liver and tumor targeting
ability of DOX-loaded HSG nanoparticles were also investigated. Moreover,
5

considering that GA presented two functions, as the hydrophobic group and
meanwhile as the liver targeting ligand, its amount might greatly affect the
physicochemical properties and fate of nanoparticles, thus, influence of GA graft ratio
on the properties of HSG nanoparticles was further elucidated.
2
. Materials and methods
2
.1. Materials
Hyaluronic acid (HA, 100 kDa) was obtained by oxidative depolymerization
(Hokputsa, Jumel, Alexander & Harding, 2003) of HA (200 kDa) supplied by Xian
Rongsheng Biotechnology Co. Ltd. (Shanxi, China). Glycyrrhetinic acid (GA) was
purchased from Nanjing Zelang Medicine Technology Co. Ltd. (Jiangsu, China).
Succinic anhydride was from Tianjin Bodi Chemical Holding Co. Ltd. (Tianjin,
China). N, N-dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) were from Shanghai Sinopharm Chemical Reagent Co. Ltd. (Shanghai,
China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing Huafeng
Lianbo Technology Co. Ltd. (Beijing, China). All other chemicals were of analytical
grade and were used without further purification.
2
.2. Synthesis and characterization of hyaluronic acid-glycyrrhetinic acid succinate
conjugates
Hyaluronic acid-glycyrrhetinic acid succinate (HSG) conjugates were
synthesized via two steps as reported previously (Wang, Gu, Wang, Sun, Wu & Mao,
017). Firstly, GA (5.0 mmol), succinic anhydride (20.0 mmol) and DMAP (5.0 mmol)
2
in 60 mL of DCM was refluxed at 40℃ for 12 h, then the DCM was removed by
evaporation. The precipitate was washed with water, then filtered and dried. Pure
3
-O-hemi-succinate GA (suc-GA) was obtained by recrystallization in ethanol.
Secondly, suc-GA was reacted with DCC and DMAP in 20 mL of DMF at 0℃ for 3 h
and the molar ratio of DCC:DMAP:suc-GA was 4:1.33:1. Briefly, HA (200 mg) in 10
mL of formamide was reacted with different amounts of activated suc-GA at 40℃ for
3
6 h. The solution was dialyzed against DMSO for 2 days and distilled water for 3
days using a dialysis membrane (MWCO: 8,000-14,000). The dialyzed solution was
6

lyophilized to obtain the white, sponge-like HSG conjugates.
1
The structure of HSG was confirmed by H-NMR (AV-600, Bruker, Germany)
and FT-IR (IFS-55, Bruker, Switzerland). The degree of substitution (DS), defined as
the number of GA groups per 100 disaccharide units of HA, was estimated by UV-Vis
spectrophotometer (UV-2000, Unico, Shanghai, China) at 250 nm (Zhang, Yao, Zhou,
Wang & Zhang, 2013). The DS was calculated with the following equation:
Concentration of GA/ Molecular mass of GA
DS(%) =
×100
(
Concentration of HSG - Concentration of GA)/ Molecular mass of unit of HA
2
.3. Preparation of DOX-loaded HSG nanoparticles
The DOX-loaded HSG nanoparticles were prepared by dialysis method (Wu, Liu,
Wang & Huang, 2012). Briefly, 20 mg of lyophilized HSG was dissolved in 2 mL of
formamide with stirring for 3 h at room temperature. Different amounts of DOX·HCl
were stirred in 1 mL of dimethylformamide overnight in the presence of triethylamine
(3 times molar quantity of DOX·HCl). The above polymer and DOX solution were
mixed and stirred for 24 h, followed by dialysis against distilled water for 24 h using a
dialysis membrane (MWCO: 8,000-14,000). The outer solution was exchanged at 3-h
intervals. Subsequently, the dialyzed solution was filtered through a 0.8 μm millipore
membrane and then lyophilized.
2
2
.4. Characterization of DOX-loaded HSG nanoparticles
.4.1. Determination of the drug loading and encapsulation yields
Approximately 2mg of lyophilized DOX-loaded HSG nanoparticles were
dissolved in 2 mL formamide, and the amount of DOX in nanoparticles was
determined by UV-Vis spectrophotometer (UV-2000, Unico, Shanghai, China) at 480
nm (Wu, Liu, Wang & Huang, 2012). The drug loading (DL) and encapsulation yields
(EY) were calculated as follow:
Weight of DOX in nanoparticles
DL(%) =
× 100
Weight of HSG/DOX nanoparticles
Weight of DOX in nanoparticles
EY(%) =
× 100
Weight of DOX used for nanoparticles preparation
7

2
.4.2. Particle size and zeta potential
The particle size and the zeta potential of the HSG/DOX nanoparticles or the
blank nanoparticles were measured by dynamic light scattering and laser doppler
anemometry, respectively, using the Zetasizer (NANOZS90, Malvern Instruments,
UK) at 25℃.
2
.4.3. Morphological observation
The morphology of HSG/DOX nanoparticles was observed using transmission
electron microscopy (TEM, JEM-2100, Japan) with an accelerating voltage of 200 kV.
A drop of the sample solution was placed onto a 300-mesh copper grid with carbon.
And then the sample was taped with a filter paper to remove surface water and
air-dried. The sample was negatively stained with 2% phosphotungstic acid before
observation.
2
.4.4. Differential scanning calorimetry analysis
The existing state of DOX in nanoparticles was determined by differential
scanning calorimetry (DSC). Analysis of DOX, blank HSG nanoparticles, HSG/DOX
nanoparticles, physical mixture of DOX and blank HSG nanoparticles was carried out
using DSC equipment (Zürich, Switzerland), respectively. The temperature ranged
from 30℃to 300℃ and the heating rate was 10℃/min.
2
.5. In vitro release
The in vitro release behavior of DOX from the nanoparticles was evaluated in
phosphate-buffered saline (PBS) solutions of various pH (pH 5.5, 6.5 and 7.4), using a
dialysis method (MCWO: 8,000-14,000) (Wu, Liu, Wang & Huang, 2012). Briefly, 2
mL of HSG/DOX nanoparticles was transferred in dialysis bags and then dialyzed
against 50 mL of PBS at 37℃ in an air-bath shaker under stirring at 100 rpm. The
system was protected from light. At predetermined time intervals (0, 1, 2, 4, 6, 8, 12,
2
4, 36, 48 h), 2 mL of the medium were taken out and replaced with an equal volume
of fresh medium. The amount of released DOX was determined by UV-Vis
8

spectrophotometer (UV-2000, Unico, Shanghai, China) at 480 nm.
2
.6. In vitro cytotoxicity assay
The cytotoxicity of the HSG/DOX nanoparticles and DOX solution against
HepG2
cells
were
determined
using
the
MTT
(3-(4,
5
-Dimethylthiazol)-2-yl)-2,5-diphenyltetrazolium bromide) assay (Wang et al., 2014).
Briefly, HepG2 cells (5.0 × 103) were seeded in each well of a 96-well plate and were
incubated for 24 h at 37℃to allow cell attachment. Then, the cells were incubated
with the HSG/DOX nanoparticles or DOX solution (final concentrations of DOX
were 0.03, 0.1, 0.3, 1 and 3μg/mL) for 24 h. Following incubation, 20 μL of MTT
solution (5.0 mg/mL) was added, and the cells were further incubated for another 4 h.
Thereafter, the MTT medium was removed from each well, and 150 μL of DMSO was
added to dissolve the formazan crystals. Absorbance was measured at 570 nm with a
multimode microplate reader (SpectraMax M3, Molecular Devices, US). The
untreated cells were taken as control. The relative cell viability (%) was calculated as
(absorbance of test group/ absorbance of control group) × 100.
2
.7. Pharmacokinetic study in rats
Sprague–Dawley (SD) rats were used to evaluate the pharmacokinetics of the
HSG/DOX nanoparticle. All animal studies were approved by the University Ethics
Committee and were carried out in accordance with the Principle of Laboratory
Animal Care. Twenty SD rats (male, 200 ± 20 g) were randomly divided into four
groups. HSG/DOX nanoparticles and DOX solution were intravenously administered
via a tail vein at a single dose of 5 mg DOX/kg. Blood samples (about 500 μL) were
collected from retro-orbital plexus into heparinized tubes at 0.083, 0.25, 0.5, 1, 2, 3, 4,
6
, 8, 10, 12, and 24 h and then centrifuged at 10,000 rpm for 10 min. The plasma
samples were stored at -20℃ until analysis.
Extraction of doxorubicin from the plasma was carried out as described in the
previous report with slight modifications (Guo et al., 2014). Briefly, plasma sample
(
200 μL) was added to 2.8 mL of extraction solvent (0.3 M HCl/EtOH=3/7, v/v) and
9

the mixture was vortex mixed for 5 min. After centrifugation at 10,000 rpm for 10
min, the supernatant was transferred and used for fluorescence measurement by a
multimode microplate reader (SpectraMax M3, Molecular Devices, US) at
excitation/emission wavelength of 500/588 nm.
2
.8. Tissues Distribution Study
Kun Ming (KM) mice were used to assess the effect of different formulations on
tissue distribution of DOX. One hundred and twenty KM mice (male, 20 ± 2 g) were
randomly divided into four groups. HSG/DOX nanoparticles and DOX solution were
intravenously administered via a tail vein at a single dose of 5 mg DOX/kg. At
different time points (0.5, 1, 3, 6, 12, and 24 h) post injection, the mice were
sacrificed and the major organs (heart, liver, spleen, lung, and kidney) were collected.
All the tissues samples were stored at -20℃ until analysis.
The isolated tissue samples were weighed accurately and homogenized with
0
.9%NaCl solution (tissue samples/0.9%NaCl=1/2, g/mL). Thereafter, the tissue
homogenates were processed using the same method for plasma samples as described
above and drug content in each tissue was determined.
2
.9 Evaluation of tumor targeting effect by in vivo imaging
For in vivo imaging analysis, near infrared fluorescent dye, DiR was
incorporated into HSG nanoparticles as reported previously (Wang, Gu, Wang, Sun,
Wu & Mao, 2017). The amount of DiR in HSG-6, HSG-12, and HSG-20
nanoparticles was calculated to be 0.87%, 0.95%, and 1.03%, respectively, and the
particle sizes were 257.6, 231.1, and 168.9 nm, respectively (n=3). There was no
significant difference between the size of HSG/DiR nanoparticles and HSG/DOX
nanoparticles (p> 0.05).
To evaluate the tumor targeting ability of HSG nanoparticles, BALB/c nude mice
(six weeks old, 20-25 g) were used to generate HeGp2 tumor-xenografted mouse
model (Huo et al., 2012). Twelve tumor-bearing mice were randomly divided into
four groups. HSG/DiR nanoparticles and DiR solution were injected into the
10

tumor-bearing mice via a tail vein at a dose of 100 μg DiR/kg, respectively. The near
infrared fluorescent imaging were performed at 1, 3, 6, 12, and 24 h after injection
using an in vivo Imaging System (FX, Kodak, USA), and the excitation and emission
wavelengths were 720 nm and 790 nm, respectively. After living imaging, the
tumor-bearing mice were sacrificed. The major organs including liver, lung, spleen,
kidney, heart, as well as the tumor, were dissected from the mice, and the fluorescence
intensity was determined again with the same system as described above.
2
.10. Statistical analysis
All data were expressed as means ± SD from at least three different experiments.
The pharmacokinetic parameters were calculated using DAS (Drug and Statistics) 2.0
software. A two-tail paired Student's t-test was used to compare the difference.
Probability value p<0.05 was considered statistically significant.
3
. Results and discussion
3
.1. Synthesis and characterization of HSG conjugates
HSG conjugates were synthesized by modifying the hydroxyl group with the
help of succinic anhydride (Supplementary Materials Fig. S1) and the conjugates
were coded as HSG-X, where X represents the GA graft ratio. The successful
1
synthesis of HSG was confirmed by H-NMR and FT-IR (Supplementary Materials
Fig. S2, Fig.S3). A new multiplet at 2.65 ppm observed in the spectrum of suc-GA can
be attributed to the two adjacent methylene groups of the succinyl moiety, suggesting
that succinyl group was attached to GA successfully. In comparison with HA, the
characteristic peaks of GA at 0.8-1.7 ppm, corresponding to the methyl and methylene
groups, appeared in the spectrum of HSG-20, indicating GA was successfully
conjugated to the backbone of HA. Compared with HA, in the spectrum of HSG-20, a
−
1
new absorption peak emerged at 1729 cm , which can be attributed to the newly
−
1
formed ester carbonyl group. Simultaneously, the intensity at 2930~2850 cm was
enhanced sharply in the spectrum of HSG-20, which can be attributed to the
carbon-to-hydrogen stretching vibrations from the large number of methyl and
11

methylene groups of GA moiety in HSG-20, further suggesting that GA has been
attached to HA backbone successfully.
3
.2. Influence of drug-to-carrier ratio on the characteristics of DOX-loaded HSG
nanoparticles
DOX-loaded HSG nanoparticles were prepared via self-assembly and DOX was
loaded into the nanoparticles based on its hydrophobic and electronic interaction with
the HSG conjugate. First of all, taking HSG-12/DOX nanoparticles as an example,
influence of drug-to-carrier ratio on the characteristics of DOX-loaded HSG
nanoparticles was investigated. As shown in Table 1, drug-to-carrier ratio could
significantly affect properties of the nanoparticles. As the drug-to-carrier ratio
increased from 5% to 20%, the drug loading increased from 5.01% to 12.48%,
however, the encapsulation yields decreased from 95.47% to 65.04%. When further
increasing drug-to-carrier ratio from 20% to 30%, the drug loading only increased
slightly (from 12.48% to 13.24%), but the encapsulation yields decreased
significantly (from 65.04% to 45.01%). As to the influence on particle size, when
drug-to-carrier ratio was 5%, the particle size was smaller than the blank
nanoparticles (p<0.05). The result suggested that hydrophobic DOX might make a
tightly packed hydrophobic core at low drug loading (Lao, Zhang, Xu & Jiang, 2010).
But when drug-to-carrier ratio was increased up to 10%, the addition of higher
amount of drugs could enlarge the inner core volume, compromising the impact of
hydrophobic interaction, thus no significant change in particle size was found
(
p>0.05). Further increasing drug-to-carrier ratio to 20% and 30% caused significant
increase in particle size (p<0.05), this can probably be explained by the fact that at
specific GA graft ratio, the hydrophobic interaction between drug and GA will be
saturated at higher drug loading, thus the additional drug incorporation will lead to
core volume increase (Xiangyang et al., 2007), therefore increased particle size.
Moreover, it was noticed that compared with the blank nanoparticles, zeta potentials
of all the HSG/DOX nanoparticles decreased significantly (p<0.05), indicating the
presence of electrostatic interaction between the drug and nanoparticles, but the
12

influence of drug loading amount on zeta potential was insignificant (p>0.05).
Therefore, it is reasonably to conclude that hydrophobic interactions are the major
binding force to load DOX into HSG/DOX nanoparticles. Taking both drug loading
and encapsulation yield into account, unless otherwise specified, HSG/DOX
nanoparticles prepared at drug-to-carrier ratio 20% were used in the following studies.
3
.3. Influence of GA graft ratio on the characteristics of DOX-loaded HSG
nanoparticles
By keeping drug-to-carrier ratio 20%, influence of GA graft ratio on the
characteristics of DOX-loaded HSG nanoparticles was investigated. As shown in
Table 1, by increasing GA graft ratio from 6% to 20%, the drug loading and
encapsulation yields increased significantly (p<0.05)，which could be attributed to the
stronger hydrophobic interactions, therefore increased DOX loading. Meanwhile,
particle size of HSG/DOX nanoparticles decreased with the increase of GA graft ratio
and it decreased from 281.7 nm at GA ratio 6% to 184.8nm at GA ratio 20%, with
sharp particle size distribution (Fig. 1A). This can probably be explained by the
enhanced hydrophobic interaction between GA groups and DOX, making the inner
core more compacted, therefore smaller particle size.
Table 1. Effect of drug-to-carrier ratio and GA graft ratio on the properties of
HSG/DOX nanoparticles (n=3).
Drug/carrier Size
Zeta
(mV)
DL
(wt%)
EY
Sample
PDI
(w/w, %)
(nm)
(wt%)
HSG-6/DOX
HSG-12/DOX
20
0
281.7±6.7
213.4±3.5
204.4±4.2
212.4±2.3
235.9±4.1
248.6±3.8
184.8±3.3
0.035±0.028
0.098±0.048
0.109±0.031
0.154±0.016
0.110±0.042
0.066±0.048
0.188±0.042
-27.2±1.0
-34.9±0.6
-30.4±1.6
-29.5±1.9
-28.2±0.8
-26.4±1.1
-31.3±0.6
9.66±0.44
-
50.12±2.39
-
5
5.01±0.19
7.62±0.32
12.48±0.74
13.24±0.61
13.94±0.65
95.47±3.69
80.25±3.41
65.04±3.82
45.01±2.06
72.90±3.41
1
2
3
0
0
0
HSG-20/DOX
20
13

Fig. 1. Particle size distribution measured by dynamic light scattering (DLS) (A) and
TEM image (B) of the HSG-12/DOX nanoparticles prepared at drug-to-carrier ratio of
2
0%.
Morphology of HSG / DOX nanoparticles was visualized by TEM. Fig. 1B
exemplifies that HSG-12/DOX nanoparticles were sub-spherical in shape and the
observed particle size was approximately 50 nm, which was smaller than the
hydrodynamic diameter obtained from DLS measurement (Table 1). This discrepancy
might be due to the shrinkage of hydrophilic shell of nanoparticles during the drying
process of TEM samples preparation (Han, Han, Lee, Kang, Kim & Park, 2011).
To confirm the existing state of DOX in the HSG nanoparticles, DSC analyses
were performed for DOX, blank HSG nanoparticles, HSG-12/DOX nanoparticles,
corresponding physical mixture of DOX and blank HSG nanoparticles, respectively,
and the thermograms are presented in Fig. 2. Obviously, the DSC thermogram of
DOX exhibit an endothermic peak at 205℃. The characteristic peaks of DOX did not
occur in the thermogram of HSG-12/DOX nanoparticles, but appeared in the physical
mixture of DOX and blank HSG nanoparticles, indicating that DOX existed in
amorphous state in the HSG nanoparticles (Huo et al., 2012).
14

Fig. 2. DSC spectra of DOX, HSG-12 blank nanoparticles, physical mixture of DOX
and blank HSG nanoparticles, and HSG-12/DOX nanoparticles.
3
.4. Release behavior of doxorubicin from the nanoparticles
First of all, influence of system pH on the in vitro release of DOX from HSG
nanoparticles was investigated. As shown in Fig. 3, HSG/DOX nanoparticles
exhibited a pH-dependent DOX release pattern, which can be explained by the
pH-dependent solubility of DOX (Guo et al., 2014). For instance, HSG-20/DOX
released nearly 51% of DOX in PBS at pH 5.5 after 48 h, which was significantly
higher than that at pH 6.5 (35%) (p<0.05) and at pH 7.4 (29%) (p< 0.01). This
pH-dependent release behavior is advantages for liver targeting drug delivery system
design while it can result in a sustained release of DOX in the bloodstream (pH 7.4) to
prolong drug circulation time and lead to a faster release into the extracellular matrix
of tumor (pH 6.5) or inside the endosome/lysosome of tumor cells (pH 5.5) to achieve
better tumor-targeted drug delivery (Egusquiaguirre, Igartua, Hernandez & Pedraz,
2
012). A similar trend was found for HSG-12/DOX and HSG-6/DOX nanoparticles.
15

Fig. 3. Release profiles of doxorubicin from HSG nanoparticles under different pH:
HSG-6/DOX (A), HSG-12/DOX (B), and HSG-20/DOX (C) (n=3).
The release of DOX from HSG nanoparticles was also dependent on the GA
graft ratio. The higher GA graft ratio, the slower the drug release rate. For instance,
the fractional release of the HSG-20/DOX, HSG-12/DOX and HSG-6/DOX
nanoparticles reached about 29%, 36% and 45% at pH 7.4 after 48 h, respectively.
This phenomenon suggests that higher GA content resulted in stronger hydrophobic
interaction between the hydrophobic core and drug, which may act as a barrier to
16

restrict the outward diffusion of DOX from the nanoparticles (Xiangyang et al.,
2
007).
3
.5. In vitro cytotoxicity assay
The cytotoxicity of HSG/DOX nanoparticle was investigated in HepG2 cells
using an MTT assay. As shown in Fig. 4, free DOX and HSG/DOX nanoparticles
exhibited a dose-dependent cytotoxicity to HepG2 cells. And the IC50 of DOX
solution, HSG-20/DOX, HSG-12/DOX and HSG-6/DOX nanoparticles after 24-h
incubation was 0.452, 0.450, 0.518, and 1.169 μg/mL, respectively. It was found that
only HSG-20/DOX nanoparticles showed cytotoxicity equivalent to that of DOX
solution. This result might be explained by the fact that DOX release from the
HSG/DOX nanoparticles was incomplete within 24-h incubation period. As evidenced
by in vitro release profile (Fig. 3), even under acid condition, DOX released from
HSG-20/DOX, HSG-12/DOX, and HSG-6/DOX in 24 h were 46%, 53% and 57 %,
respectively. Therefore, it is reasonable to deduce that if HSG/DOX nanoparticles are
incubated with HepG2 cells for a longer period, they could be more cytotoxic against
cancer cells.
Fig. 4. In vitro cytotoxicity of DOX-loaded HSG nanoparticles against HepG2 cells
after incubation for 24 h (n=6).
In addition, HSG/DOX nanoparticles exhibited a GA-dependent cytotoxicity to
HepG2 cells. When GA ratio was increased from 6% to 12%, the IC50 decreased
17

remarkably, but no statistical difference in cytotoxicity was found at GA ratio 12%
and 20%. This could probably be explained by two reasons. First of all, GA ratio can
affect the particle size of HSG nanoparticles (Table 1). As reported previously, the
smaller size HA micelles facilitated the transport across the cell membrane, therefore
increasing the cellular uptake (Liu et al., 2011). Likewise, due to the smaller size of
HSG/DOX nanoparticles at higher GA graft ratio, they could be more efficiently
taken up by HepG2 cells via an endocytotic process, thus leading to higher
cytotoxicity. Secondly, GA can affect the surface properties of nanoparticles. A large
number of studies have evidenced that the hydrophobic groups existed both on the
surface and inside of the nanoparticles (Park, Lee & Lee, 2005; Zhang, Yao, Zhou,
Wang & Zhang, 2013). Chiu et al. confirmed that with the increase of palmitoyl graft
ratio, there were more palmitoyl groups exposed on the surface of chitosan
nanoparticles (Chiu et al., 2010). Therefore, it is reasonable to assume that some GA
molecules also existed on the surface of HSG/DOX nanoparticles, and the larger the
GA content, the higher density of GA on the surfaces of the nanoparticles, which
could deliver more DOX into HepG2 cells by GA receptor-mediated endocytosis until
a saturation was achieved, as observed in the case of GA 12% and 20% samples. This
assumption was further demonstrated by the followed pharmacokinetic and tissue
distribution studies.
3
.6. Pharmacokinetic study in rats
Pharmacokinetics of the HSG/DOX nanoparticles were studied in rats. The
plasma concentration-time curves are shown in Fig. 5, and the selected
pharmacokinetic parameters are listed in Table 2. As expected, DOX solution showed
relatively short halftime and was quickly removed from the plasma. In contrast, the
HSG/DOX nanoparticles exhibited a longer and GA-dependent circulation time in
plasma. The HSG-20/DOX, HSG-12/DOX, and HSG-6/DOX nanoparticles had
significantly higher AUC(0-∞) (22.8, 17.4, and 13.3-fold, respectively), longer t1/2
(5.1, 3.7, and 2.4-fold, respectively) and MRT(0-∞) (9.2, 7.4, and 4.5-fold, respectively),
and lower CL (24.6, 18.0, and 14.5-fold, respectively) than that of DOX solution. This
18

phenomenon might be due to that the hydrophobic inner core of the nanoparticles can
serve as a reservoir for DOX to provide sustained release of drug, resulting in
prolonged residence of DOX in systemic blood circulation, and this sustained release
ability was GA ratio-dependent. This is in good agreement with the in vitro release
results (Fig. 3). By simulating the bloodstream (pH 7.4) environment, DOX released
from HSG-20/DOX, HSG-12/DOX, and HSG-6/DOX in 48 h was 29%, 35% and
4
5%, respectively. Thus, the nanoparticles with higher GA graft ratio had longer
circulation time in plasma. And meanwhile, it is well known that the protein corona
formed around nanoparticles upon entering a biological fluid can significantly affect
the in vivo fate of nanoparticles (Lundqvist, Stigler, Elia, Lynch, Cedervall & Dawson,
2
008; Lundqvist. et al., 2011). Contributed to the hydrophilic outer shell and the
negative charge, the HSG/DOX nanoparticles were conducive to prevent the
unwanted protein adsorption, and thereby avoid the uptake by the reticuloendothelial
system (RES) (Choi, Saravanakumar, Park & Park, 2012), leading to reduced
clearance rate, thus, GA ratio dependent liver targeting effect was achieved.
Fig. 5. Mean plasma concentration-time curves after intravenous administration of
DOX solution and DOX-loaded HSG nanoparticles at a dose of 5 mg/kg in rats (n=5).
19

Table 2. Pharmacokinetic parameters after intravenous administration of DOX
solution and DOX-loaded HSG nanoparticles in rats at a dose of 5 mg/kg (n=5).
Parameter
DOX solution
HSG-6/DOX
29.195±4.600
13.114±1.882
11.122±1.991
0.175±0.030
15.357±1.663
HSG-12/DOX
38.208±11.399
21.701±8.623
16.984±8.956
0.141±0.041
HSG-20/DOX
49.843±10.062
27.009±9.210
23.570±7.610
0.103±0.018
AUC(0-∞)(mg/L*h) 2.190±0.916
MRT(0-∞)(h)
t1/2(h)
2.934±0.900
4.640±3.194
2.537±0.782
1.840±0.053
CL(L/h/kg)
Cmax(mg/L)
15.831±0.977
19.615±3.373
Besides, it is well known that particle size can affect the in vivo behavior of
nanoparticles (Gaumet, Vargas, Gurny & Delie, 2008; Moghimi, Hunter & Murray,
2
001), and there are some difference in the particle size of three HSG/DOX
nanoparticles (160-260 nm, as shown in Table 1). However, it is reported that carrier
systems with diameters of 150-300 nm can be uptaken by RES and then be
transported to the liver and spleen (Hickey, Santos, Williford & Mao, 2015). On the
other hand, the hydrophilic surface with highly negative charge of the nanoparticles is
not conducive to the recognition and phagocytosis of macrophage (Choi,
Saravanakumar, Park & Park, 2012), counteracting some of the effect of particle size
on the in vivo behavior. Therefore, the particle size was not the main reason for the
different pharmacokinetic behavior.
3
.7. Tissues distribution study
The HSG/DOX nanoparticles were injected intravenously into KM mice to
evaluate their tissue distribution. From Fig. 6 and Table 3, it was found that the
nanoparticles had superior liver targeting efficiency and the liver targeting capacity
was GA ratio dependent. In the liver, the AUC of HSG-20/DOX, HSG-12/DOX, and
HSG-6/DOX nanoparticles was 4.0, 3.1 and 2.6-fold higher than that of DOX solution,
respectively. As mentioned above, the particle size was not the main reason for the
20

biodistribution of nanoparticles in the body, because all of these three HSG/DOX
nanoparticles (160-260 nm) can induce nonspecific RES uptake of liver (Gaumet,
Vargas, Gurny & Delie, 2008) and the hydrophilic surface with highly negative charge
could offset partial effect of particle size on hepatic passive targeting. And Tian et al.
showed that nanoparticles with similar particle size also presented an increasing liver
targeting ability as the GA content increased (Tian, Wang, Wang, Zhang, Liu & Yuan,
2
010), which further indicates that the discrepancy of accumulation in the liver is not
related to the difference in particle size. Therefore, the GA-dependent liver targeting
capacity of the HSG/DOX nanoparticles may be explained as follows: with the
increase of GA graft ratio, the density of GA on the surface of the nanoparticles
increased, leading to higher binding affinity to the liver region by GA
receptor-mediated endocytosis.
Fig. 6. Tissue distribution of DOX after intravenous administration of DOX solution
and DOX-loaded HSG nanoparticles at
p<0.05.**p<0.01.***p<0.001.
a
dose of
5
mg/kg (n=5).
*
21

Additionally, the HSG/DOX nanoparticles also could reduce significant amounts
of DOX distribution into the heart and kidney. The AUC of HSG-20/DOX,
HSG-12/DOX, and HSG-6/DOX nanoparticles was 2.1, 1.7, 1.5-fold, and 2.6, 2.4,
1
.9-fold lower than that of DOX solution in the heart and kidney, respectively. It is
well known that the major limitation of DOX for successful cancer treatment is
primarily ascribed to its indiscriminate distribution in various tissues, thereby result in
serious side effects including cardiotoxicity and nephrotoxicity (Tacar, Sriamornsak &
Dass, 2013). Therefore, the ability of the HSG/DOX nanoparticles to reduce DOX
distribution in the heart and kidney, has important clinical significance for decreasing
the side effects of DOX.
Table 3. The AUC after intravenous administration of DOX solution and DOX-loaded
HSG nanoparticles in various tissues of mice at a dose of 5 mg/kg (n=5).
AUC(0-24) (μg/g*h)
Tissue
DOX solution
HSG-6/DOX
58.957±8.713
HSG-12/DOX
52.748±4.242
HSG-20/DOX
42.678±6.332
Heart
Liver
90.954±21.251
123.831±6.361
65.866±9.990
62.463±8.846
236.058±23.632
322.324±41.949 387.628±45.788
497.536±22.354
73.797±10.856
78.037±21.296
90.896±16.277
Spleen
Lung
78.407±16.464
90.447±18.532
74.833±15.396
65.514±7.466
Kidney
122.394±24.371 99.286±15.855
3
.8 Evaluation of tumor targeting effect by in vivo imaging
Tissues distribution study only demonstrated the liver-targeting property of
HSG/DOX nanoparticles. To evaluate the tumor targeting ability, in vivo imaging
technique was performed using HepG2 tumor-xenografted mice, and the liver
targeting property was further validated.
As shown in Fig. 7A, for the DiR solution group, almost no fluorescence signal
in the liver region was observed at 1 h, and the intensity was the strongest at 6 h and
then decreased rapidly. Compared with DiR solution, the fluorescence intensity of
22

HSG/DiR nanoparticles increased significantly at each time point, and reached the
highest at 12 h. Moreover, the higher amount of GA content, the stronger the
fluorescence signal of HSG/DiR nanoparticles in the liver. The GA-dependent liver
targeting ability was further confirmed by the ex vivo fluorescent image and
semi-quantitative analysis (Fig. 7C and 7B). The accumulation of HSG-20/DiR,
HSG-12/DiR, and HSG-6/DiR nanoparticles in the liver was 4.2, 3.2, 2.3-fold higher
than that of DiR solution, respectively. This was in good consistent with the results of
tissue distribution study.
Fig. 7. Fluorescence imaging in KM mice after intravenous administration of free DiR
and DiR-loaded HSG nanoparticles, respectively. (A) Time-dependent in vivo images
after i.v. injection. (B) Ex vivo fluorescence images of tissues at 24 h post-injection.
(
C) Quantification of the ex vivo tissue uptake characteristics after 24 h post-injection
n=3). *p<0.05.**p<0.01.***p<0.001.
(
In the tumor, for the DiR solution group, nothing is visible throughout the
detection time. In contrast, the fluorescence intensity of HSG/DiR nanoparticles was
significantly stronger, and further strengthened with GA content increase. The
23

accumulation of HSG-20/DiR, HSG-12/DiR, and HSG-6/DiR nanoparticles in the
tumor was 6.0, 4.2, 2.8-fold higher than that of DiR solution, respectively. This could
be explained by the fact that HSG nanoparticles can passively target to tumor tissues
by the enhanced permeability and retention (EPR) effect and also can actively target
the HepG2 solid tumor cells by HA and GA receptor-mediated endocytosis. And the
higher GA level of HSG nanoparticles can enhance the GA receptor-mediated
endocytosis, thus increasing the accumulation in tumor sites. Simultaneously, smaller
particles could penetrate the tumor tissue gaps more easily (Hickey, Santos, Williford
&
Mao, 2015) and further increase the EPR effect.
In our previous studies(Wang, Gu, Wang, Sun, Wu & Mao, 2017), we found that
coupling HA with GA by modifying hydroxyl groups might exhibit higher liver
targeting efficiency compared to carboxyl group modification. Encouragingly, in this
study, in vivo imaging analysis suggested that the nanoparticles synthesized by
modifying the hydroxyl groups not only had better liver targeting property, but also
presented higher tumor targeting efficiency compared with that of nanoparticles
synthesized by modifying carboxyl groups (as shown in Table 4), which further
confirmed that the binding site could influence the targeting ability of nanoparticles.
Overall, nanoparticles based on HSG conjugates synthesized via modifying hydroxyl
groups could be used as promising liver targeting carriers for hydrophobic anti-tumor
drugs.
Table 4. Properties comparison of hyaluronic acid-graft-glycyrrhetinic acid
nanoparticles with different bridging groups.
Binding
site
DS
Sample
Bridging group
Succinic acid
Liver targeting ability
Tumor targeting ability
(%)
Hydroxyl
groups
4.2-fold higher than that 6.0-fold higher than
of free DiR at 24 h that of free DiR at 24 h
HA-Suc-GA (HSG)
HA-Etda-GA
20.4
(HGA)
Carboxyl
groups
1.83-fold higher than 2.88-fold higher than
that of free DiR at 24 h that of free DiR at 24 h
(Zhang, Yao, Zhou, Wang
Ethylenediamine 20.2
&
Zhang, 2013)
HA-Cyst-GA (Mezghrani Carboxyl
et al., 2015) groups
1.8-fold higher than that 2.6-fold higher than
of free DiR at 24 h that of free DiR at 24 h
Cystamine
23.8
24

4
. Conclusion
In this study, a liver-targeting drug delivery system based on HSG/DOX
nanoparticles was prepared for better therapy of hepatocytes. In vitro studies showed
the HSG/DOX nanoparticles exhibited sustained and pH-dependent DOX release
pattern, and GA-dependent cytotoxicity to HepG2 cells. In vivo investigation
demonstrated that HSG/DOX nanoparticles could prolong blood circulation time,
enhance the liver targeting efficiency, and reduce the cardiotoxicity and
nephrotoxicity of DOX. Using DiR as a NIR fluorescent dye, the tumor-targeting
ability of HSG nanoparticles was further investigated using an in vivo imaging
technique. The better liver targeting property (4.2-, 3.2-, and 2.3-fold, respectively)
and higher tumor targeting efficiency (6.0-, 4.2-, and 2.8-fold, respectively) of
HSG-20/DiR, HSG-12/DiR, and HSG-6/DiR nanoparticles were disclosed in
comparison with DiR solution. And coupling HA with GA by modifying hydroxyl
groups presented higher liver and tumor targeting efficiency compared to carboxyl
group modification. In conclusion, HSG conjugates obtained by modifying hydroxyl
groups have promising potential as a liver targeting carrier for hydrophobic
anti-tumor drugs delivery. Meanwhile, this result also reminds us that not only
polymer type, but also the binding site may influence its biological function as a drug
carrier, and this point should be taken into consideration in polymer conjugate based
nanoparticle design.
Acknowledgments
This project is financially supported by the Natural Science Foundation of China
(Grant No. 81273446).
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Figure legends
Fig. 1. Particle size distribution measured by dynamic light scattering (DLS) (A) and
TEM image (B) of the HSG-12/DOX nanoparticles prepared at drug-to-carrier
ratio of 20%.
Fig. 2. DSC spectra of DOX, HSG-12 blank nanoparticles, physical mixture of DOX
and blank HSG nanoparticles, and HSG-12/DOX nanoparticles.
Fig. 3. Release profiles of doxorubicin from HSG nanoparticles under different pH:
HSG-6/DOX (A), HSG-12/DOX (B), and HSG-20/DOX (C) (n=3).
Fig. 4. In vitro cytotoxicity of DOX-loaded HSG nanoparticles against HepG2 cells
after incubation for 24 h (n=6).
Fig. 5. Mean plasma concentration-time curves after intravenous administration of
DOX solution and DOX-loaded HSG nanoparticles at a dose of 5 mg/kg in rats
(n=5).
Fig. 6. Tissue distribution of DOX after intravenous administration of DOX solution
and DOX-loaded HSG nanoparticles at a dose of 5 mg/kg (n=5).
*
p<0.05.**p<0.01.***p<0.001.
Fig. 7. Fluorescence imaging in KM mice after intravenous administration of free DiR
and DiR-loaded HSG nanoparticles, respectively. (A) Time-dependent in vivo
images after i.v. injection. (B) Ex vivo fluorescence images of tissues at 24 h
post-injection. (C) Quantification of the ex vivo tissue uptake characteristics
after 24 h post-injection (n=3). *p<0.05.**p<0.01.***p<0.001.
29