Synthesis, characterization and liver targeting evaluation of self-assembled hyaluronic acid nanoparticles functionalized with glycyrrhetinic acid

Article abstract of DOI:10.1016/j.ejps.2016.09.036

Recently, polymeric materials with multiple functions have drawn great attention as the carrier for drug delivery system design. In this study, a series of multifunctional drug delivery carriers, hyaluronic acid (HA)-glycyrrhetinic acid (GA) succinate (HSG) copolymers were synthesized via hydroxyl group modification of hyaluronic acid. It was shown that the HSG nanoparticles had sub-spherical shape, and the particle size was in the range of 152.6–260.7?nm depending on GA graft ratio. HSG nanoparticles presented good short term and dilution stability. MTT assay demonstrated all the copolymers presented no significant cytotoxicity. In vivo imaging analysis suggested HSG nanoparticles had superior liver targeting efficiency and the liver targeting capacity was GA graft ratio dependent. The accumulation of DiR (a lipophilic, NIR fluorescent cyanine dye)-loaded HSG-6, HSG-12, and HSG-20 nanoparticles in liver was 1.8-, 2.1-, and 2.9-fold higher than that of free DiR. The binding site of GA on HA may influence liver targeting efficiency. These results indicated that HSG copolymers based nanoparticles are potential drug carrier for improved liver targeting.

Full text of DOI:10.1016/j.ejps.2016.09.036

European Journal of Pharmaceutical Sciences 96 (2017) 255–262
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Synthesis, characterization and liver targeting evaluation of
self-assembled hyaluronic acid nanoparticles functionalized with
glycyrrhetinic acid
⁎
Xiaodan Wang, Xiangqin Gu, Huimin Wang, Yujiao Sun, Haiyang Wu, Shirui Mao
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2016
Received in revised form 8 September 2016
Accepted 26 September 2016
Available online 28 September 2016
Recently, polymeric materials with multiple functions have drawn great attention as the carrier for drug delivery
system design. In this study, a series of multifunctional drug delivery carriers, hyaluronic acid (HA)-glycyrrhetinic
acid (GA) succinate (HSG) copolymers were synthesized via hydroxyl group modification of hyaluronic acid. It
was shown that the HSG nanoparticles had sub-spherical shape, and the particle size was in the range of
152.6–260.7 nm depending on GA graft ratio. HSG nanoparticles presented good short term and dilution stability.
MTT assay demonstrated all the copolymers presented no significant cytotoxicity. In vivo imaging analysis sug-
gested HSG nanoparticles had superior liver targeting efficiency and the liver targeting capacity was GA graft
ratio dependent. The accumulation of DiR (a lipophilic, NIR fluorescent cyanine dye)-loaded HSG-6, HSG-12,
and HSG-20 nanoparticles in liver was 1.8-, 2.1-, and 2.9-fold higher than that of free DiR. The binding site of
GA on HA may influence liver targeting efficiency. These results indicated that HSG copolymers based nanopar-
ticles are potential drug carrier for improved liver targeting.
Keywords:
Hyaluronic acid
Glycyrrhetinic acid
Polymeric nanoparticles
Liver-targeting
© 2016 Published by Elsevier B.V.
1. Introduction
improve their therapeutic efficacy by receptor-mediated endocytosis
(Deng et al., 2012), but this makes the system even more complicated.
Liver cancer is one of the most prevalent fatal diseases and the mor-
bidity is increasing annually (He et al., 2016). The main drawback of tra-
ditional chemotherapy is the high cytotoxicity and indiscriminate
distribution in various tissues. Therefore, significant efforts have been
exerted towards the design and construction of novel nano-drug
delivery systems for better therapy of hepatocytes (Wang et al., 2007;
Zhang et al., 2012). Self-assembled nanoparticles, based on polymeric
amphiphiles, have generated considerable interests as promising liver-
targeted drug delivery carriers because they can solubilize various hy-
drophobic drugs, improve the in vivo stability, prolong drug circulation
time in the bloodstream, and meanwhile passively target to tumor
tissues by the enhanced permeability and retention (EPR) effect
(Elsabahy and Wooley, 2012; Tian et al., 2015). However, passive trap-
ping of nanoparticles still cannot guarantee sufficient drug concentra-
tion within the cells, which may lead to inefficient cellular uptake
(Choi et al., 2010). To overcome the limitations of passive targeting, a
variety of liver targeting moieties such as folic acid (Liu et al., 2011), pro-
tein (Krishna et al., 2009), and saccharides (Jiang et al., 2009; Jiang et al.,
2011), have been installed on the surface of nanoparticles to further
Recently, polymeric materials with multiple functions have drawn
great attention as the carrier for nanoparticle drug delivery system
design (Arpicco et al., 2014; Mahmoudzadeh et al., 2013). It is highly
desirable if carrier and active targeting vector can be combined in one
material for system simplicity. Hyaluronic acid (HA), composed of re-
peating disaccharides of N-acetyl-D-glucosamine and D-glucuronic
acid, is a promising constituent of nanoparticles due to its high hydro-
philicity and targeting ability (Cho et al., 2012; Zou et al., 2013). HA is
found in the extracellular matrix and synovial fluids of most human tis-
sues, thus presenting excellent biological properties such as biocompat-
ibility, biodegradability and low toxicity (Arpicco et al., 2014). It is well
known HA-binding receptors such as cluster determinant 44 (CD44)
(Arpicco et al., 2014; Tripodo et al., 2015), receptor for hyaluronic
acid-mediated motility (RAHMM) (Schiffelers et al., 2004) and lym-
phatic vessel endothelial receptor-1 (LYVE-1) (Bhang et al., 2009) are
overexpressed in malignant cells. HA can specifically bind to cancer
cells to increase cellular uptake of drugs by receptor-mediated endocy-
tosis and then enhance targeting therefore therapeutic efficacy (Choi et
al., 2010).
Glycyrrhetinic acid (GA) is an active aglycone of glycyrrhizin and
possesses several beneficial pharmacological activities, such as anti-in-
flammatory, antiviral activity and antiulcerative effect (Lu et al., 2008).
GA-mediated drug delivery systems have emerged as novel liver
targeting platforms since GA molecules could provide hydrophobic
⁎
Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical University,
103 Wenhua Road, 110016 Shenyang, China.
E-mail address: maoshirui@syphu.edu.cn (S. Mao).
http://dx.doi.org/10.1016/j.ejps.2016.09.036
0928-0987/© 2016 Published by Elsevier B.V.

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X. Wang et al. / European Journal of Pharmaceutical Sciences 96 (2017) 255–262
section and liver targeting ligand in combination (Cai et al., 2016; Guo et
al., 2013; Tian et al., 2010a). It has been reported that carriers modified
with GA have higher accumulation in the liver with superior targeting
efficiency to hepatocytes, contributed to the abundant GA receptors
on hepatocyte membranes (Tian et al., 2012; Tian et al., 2010b). Further-
more, GA-modified nanoparticles might have the ability to discriminate
the normal liver tissue and hepatoma tissue (Tian et al., 2012; Zhang et
al., 2012), leading to high therapeutic profile with improved safety.
Therefore, by combining HA and GA in one material via appropriate
bridge, using HA as the hydrophilic part and GA as the hydrophobic part,
not only the nanoparticles can be prepared by self-assembly process,
liver targeting can also be enhanced based on the active targeting capac-
ity originated from both HA and GA. However, although GA could be
conjugated on HA by modifying its carboxyl groups with the help of dif-
ferent bridging groups such as ethylenediamine (Zhang et al., 2013a),
cystamine (Mezghrani et al., 2015), and adipic dihydrazide (Han et al.,
2016), it is realized that the carboxyl groups modification might affect
the targeting property of HA because the carboxyl groups are the recog-
nition sites for the enzyme and the receptors (Banerji et al., 2007;
Schante et al., 2011). Besides, Tian et al. confirmed that the C3-hydroxyl
group in GA has little influence on the targeting ability (Tian et al.,
2010a). Thus, in this paper, our hypothesis is that, conjugating GA to
HA via its hydroxyl group modification might achieve better targeting
effect. Moreover, considering that GA presents two functions, as the hy-
drophobic group and meanwhile as the liver targeting ligand, its con-
tent might greatly affect the fate of nanoparticles at different stages.
How will the GA graft ratio on HA influence the liver targeting efficiency
has not been reported so far.
Secondly, to activate its carboxyl group, suc-GA was reacted with
DCC and DMAP in 20 mL of dimethylformamide (DMF) at 0 °C for 3 h.
The molar ratio of DCC:DMAP:suc-GA was 4:1.33:1. Briefly, HA
(200 mg) was dissolved in 10 mL of formamide, followed by addition
of different amounts of activated suc-GA. After reacting at 40 °C for
36 h, the solution was dialyzed against dimethylsulfoxide (DMSO) for
2 d and distilled water for 3 d using a dialysis membrane (MWCO:
8000–14,000). The dialyzed solution was filtered and lyophilized to ob-
tain the white, sponge-like HSG copolymers.
The structure of HSG was confirmed by 1H NMR and FT-IR. 1H NMR
spectra was performed on an AV-600 spectrometer (Bruker, Germany)
at room temperature. HA and HSG were dissolved in D2O and
D2O/DMSO-d6 (1/4, v/v), respectively, whereas GA and suc-GA were
dissolved in CDCl3. FTIR spectra were recorded in the range of 4000
and 400 cm−1 with an IFS-55 spectrometer (Bruker, Switzerland)
using KBr pellets. The degree of substitution (DS), defined as the num-
ber of GA groups per 100 disaccharide units of HA, was determined
by UV–Vis spectrophotometer (UV-2000, Unico, Shanghai, China) at
250 nm (Zhang et al., 2013a). The DS was calculated with the following
equation:
Concentration of GA=Molecular mass of GA
DSð%Þ ¼
ðConcentration of HSG‐Concentration of GAÞ=Molecular mass of unit of HA
ꢀ 100
Thus, in this study, first of all, hyaluronic acid-glycyrrhetinic acid
succinate (HSG) with different graft ratios were synthesized and char-
acterized using 1H NMR and FT-IR, the physicochemical properties of
the self-assembled nanoparticles were characterized using dynamic
light scattering (DLS) and transmission electron microscopy (TEM).
The cytotoxicity of HSG nanoparticles against HepG2 cells were
evaluated using MTT assay. By using DiR as an indicator, liver targeting
efficiency of nanoparticles with different GA graft ratio was investigated
using a non-invasive near infrared optical imaging technique in mice. To
the best of our knowledge, this is the first time that GA was conjugated
to HA via hydroxyl group, which may provide better targeting efficiency
compared to carboxyl group modification.
2.3. Determination of critical aggregation concentration (CAC) of HSG
The critical aggregation concentration (CAC) of HSG was determined
by fluorescence spectroscopy with pyrene as a probe (Li et al., 2012).
Briefly, a known amount of pyrene in acetone was added to a series of
10 mL vials, and acetone was removed by evaporation under nitrogen
stream. Then 6 mL of HSG solution in the concentration range from
1 × 10−4 to 1.0 mg/mL, was added to each vial to achieve a final pyrene
concentration of 6 × 10−7 M. The solution was sonicated for 30 min and
left overnight to equilibrate the pyrene and the nanoparticles. Thereaf-
ter, the samples were analyzed by a multimode microplate reader
(SpectraMax M3, Molecular Devices, US), with an emission wavelength
of 390 nm. The relative excitation fluorescence intensity ratio (I338/I334
)
2. Materials and methods
was calculated.
2.1. Materials
2.4. Preparation HSG self-aggregated nanoparticles and DiR-loaded HSG
nanoparticles
Hyaluronic acid (HA, 100 kDa) was obtained by oxidative depoly-
merization (Hokputsa et al., 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 Chemi-
cal Holding Co. Ltd. (Tianjin, China). N,N-dicyclohexyl carbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP) were from Shanghai
Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other
chemicals were of analytical grade and were used without further
purification.
HSG nanoparticles were prepared by self-assembly in aqueous me-
dium (Yu et al., 2008). Briefly, 10 mg of lyophilized HSG copolymers
was dispersed in 10 mL of water (or pH 7.4 PBS to evaluate stability of
the nanoparticles) under gentle shaking for 3 h, followed by sonication
using a probe-type sonicator (JY92-II, Scientz, Ningbo, China) at 100 W
for 10 min under ice bath. Solutions with a concentration of 1 mg/mL
were used in the experiment.
The DiR-loaded HSG nanoparticles were prepared by dialysis meth-
od (Huo et al., 2012). Briefly, 20 mg of lyophilized HSG was dissolved in
2 mL of formamide and 250 μg of DiR in 250 μL of DMF was added to the
above polymer solution. After stirring at room temperature in dark for
24 h, the solution was dialyzed against distilled water for 24 h using a
dialysis membrane with a molecular weight cut-off of 8000–14,000.
The outer solution was exchanged at 3-h intervals. Subsequently, the di-
alyzed solution was filtered through a 0.8 μm millipore membrane and
then lyophilized.
2.2. Synthesis and characterization of hyaluronic acid-glycyrrhetinic acid
succinate (HSG) copolymers
Hyaluronic acid-glycyrrhetinic acid succinate (HSG) copolymers
were synthesized via two steps. Firstly, GA (5.0 mmol), succinic anhy-
dride (20.0 mmol) and DMAP (5.0 mmol) were dissolved in 60 mL of di-
chloromethane (DCM). The mixture was refluxed at 40 °C for 12 h, then
the DCM was removed by evaporation. The precipitate was washed
with water, then filtered and dried. The white powder of 3-O-hemi-
succinate GA (suc-GA) was obtained by recrystallization in ethanol.
The amount of DiR in nanoparticles was determined by dissolving
the lyophilized nanoparticles in H2O/DMSO (1/9, v/v) and measuring
the absorbance at excitation 748 nm, emission 780 nm using a multi-
mode microplate reader (SpectraMax M3, Molecular Devices, US). The

X. Wang et al. / European Journal of Pharmaceutical Sciences 96 (2017) 255–262
257
DiR-loading content (LC) in the nanoparticles was calculated using the
following equation:
concentrations of the HSG blank nanoparticles for 24 h. Then, 20 μL of
MTT solution (5.0 mg/mL) was added, and the cells were further incu-
bated for an additional 4 h. Thereafter, the MTT medium was removed
from each well, and 150 μL of DMSO was added to dissolve the formazan
crystals. Absorbance at 570 nm was measured with a multimode micro-
plate reader (SpectraMax M3, Molecular Devices, US). The untreated
cells were taken as a control. Cell viability (%) was calculated as (absor-
bance of test group/ absorbance of control group) × 100.
The amount of DiR in nanoparticles
Total amount of DiR‐loaded nanoparticles
LCð%Þ ¼
ꢀ 100
2.5. Characterization of HSG self-aggregated nanoparticles and DiR-loaded
HSG nanoparticles
The particle size of the HSG nanoparticles and DiR-loaded HSG
nanoparticles were measured using the Zetasizer (NANOZS90, Malvern
Instruments, UK) at 25 °C with a scattering angle of 90°. The zeta poten-
tial of the HSG nanoparticles were measured by laser doppler anemom-
etry with the Zetasizer at 25 °C.
Morphology of HSG 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 grid was taped with
a filter paper to remove surface water and air-dried. Before visualiza-
tion, the sample was negatively stained with 2% phosphotungstic acid.
Stability of the HSG nanoparticles upon incubation in PBS (pH 7.4)
was studied by monitoring the change of particle size at various incuba-
tion times. Similarly stability of the nanoparticles upon dilution was also
characterized.
2.7. Evaluation of liver targeting effect
For in vivo imaging analysis, near infrared fluorescent dye, DiR was
incorporated into HSG nanoparticles. All animal studies were approved
by the University Ethics Committee of Shenyang Pharmaceutical Uni-
versity and were carried out in accordance with the Principle of Labora-
tory Animal Care. Twelve hairless Kun Ming (KM) mice were randomly
divided into four groups. DiR solution and DiR-loaded HSG nanoparti-
cles were injected into the mice via tail vein at a dose of 100 μg
DiR/kg. NIRF imaging was taken at 1, 3, 6, 12, and 24 h after injection
using an in vivo Imaging System (FX, Kodak, USA), in which the excita-
tion and emission wavelengths were 720 nm and 790 nm, respectively.
After living imaging, mice were sacrificed and the major organs includ-
ing liver, lung, spleen, kidney, and heart, were dissected from the mice.
And the fluorescence intensity was determined again with the same
system as described above.
2.6. In vitro cytotoxicity assay
The cytotoxicity of HSG blank nanoparticles against HepG2 cells
were evaluated using the MTT (3-(4,5-Dimethylthiazol)-2-yl)-2,5-
diphenyltetrazolium bromide) assay (Wang et al., 2014). Briefly,
HepG2 cells (5.0 × 103) harvested in a logarithmic growth phase were
seeded in 96-well plate and were incubated for 24 h at 37 °C in 5%
CO2. After cell attachment, the cells were incubated with various
2.8. Statistical analysis
All data were expressed as means SD from at least three separate
measurements. A two-tail paired Student's t-test was used to compare
the difference. Probability value p b 0.05 was considered statistically
significant.
Fig. 1. Synthesis scheme of HSG copolymers.

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X. Wang et al. / European Journal of Pharmaceutical Sciences 96 (2017) 255–262
Table 1
Properties of blank HSG nanoparticles (n = 3).
Samplea
Feed ratio (suc-GA/HA)
DS (%)
Size (nm)
PDI
Zeta (mV)
CAC (mg/mL)
HSG-6
HSG-12
HSG-20
1/1
1.5/1
2/1
6.0 0.9
11.8 1.3
20.4 0.7
260.7 5.4
213.4 3.5
152.6 11.1
0.102 0.050
0.098 0.022
0.324 0.029
−33.2 1.4
−34.9 0.6
−37.6 0.7
0.047
0.028
0.020
a
HSG-A, in which A represents the degree of substitution (DS).
3. Results and discussion
3.3. Preparation and characterization of blank HSG self-aggregated
nanoparticles
3.1. Synthesis and characterization of HSG
The blank HSG nanoparticles were prepared by self-assembly pro-
cess in aqueous medium. The particle sizes and the zeta potentials of
various blank HSG nanoparticles are listed in Table 1. It was noted that
In this study, in order to retain the carboxyl groups of HA, succinic
anhydride was selected as a bridge to couple HA with GA by modifying
the hydroxyl group and the synthesis scheme is presented in Fig. 1.
Firstly, a carboxyl group was introduced to the C3-hydroxyl group in
GA with the help of succinic anhydride, then the carboxyl group of
suc-GA was covalently coupled with the hydroxyl group of HA in the
presence of DCC and DMAP. Based on the different feed ratio of GA
and HA, three HSG copolymers with different GA graft ratio was synthe-
sized, and were coded as HSG-6, HSG-12 and HSG-20, respectively,
where the number represents the degree of GA substitution, as present-
ed in Table 1.
The successful synthesis of HSG copolymer was confirmed by 1H
NMR and FT-IR. Fig. 2A exemplifies the 1H NMR spectra of GA, suc-GA,
HA and HSG-12. Compared with GA, a new multiplet at 2.65 ppm ob-
served 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 character-
istic peaks of GA at 0.8–1.7 ppm, corresponding to the methyl and meth-
ylene groups, appeared in the spectrum of HSG-12. It indicated that GA
was successfully modified to the structure of HA.
The FITR spectra of HA and HSG-12 are shown in Fig. 2. Compared
with HA, in the spectrum of HSG-12, a new absorption peak emerged
at 1729 cm−1, belonging to the new formation of the ester carbonyl
group. Simultaneously, the intensity at 2930–2850 cm−1 was enhanced
sharply in the spectrum of HSG-12, which can be attributed to the car-
bon-to-hydrogen stretching vibrations from the large number of methyl
and methylene groups of GA moiety of HSG-12, further suggesting that
GA has been attached to HA backbone successfully.
3.2. Influence of GA graft ratio on the critical aggregation concentration
of HSG
A remarkably low CAC is crucial for nanoparticles since lower CAC
value can provide better resistance to particle dissociation upon ex-
treme dilution in blood, thus allowing prolonged circulation time before
reaching the targeting site (Mahmoudzadeh et al., 2013). In this study,
the CAC of HSG copolymer was determined by fluorescence spectrosco-
py and pyrene was chosen as a fluorescent probe because it can prefer-
ably locate inside the hydrophobic inner core of nanoparticles (Yu et al.,
2008). When the nanoparticles were formed, the intensity ratio I338/I334
of the pyrene excitation spectra varied substantially, reflecting the
transfer of pyrene from the aqueous environment to the hydrophobic
domains (Xiangyang et al., 2007). As shown in Table 1, the CAC value
of HSG copolymers was GA substitution dependent, since higher hydro-
phobicity could form the inner core of nanoparticles more readily. It de-
creased remarkably when increasing GA content from 6% to 12%, with
the CAC value 0.047, and 0.028 mg/mL respectively, and further increas-
ing GA graft ratio to 20% caused a slight decrease of CAC value, from
0.028 mg/mL for HSG-12 to 0.020 mg/mL for HSG-20, implying a good
hydrophile and lipophile balance has been achieved at this ratio. This re-
sult implies that HSG nanoparticles with the relatively low CAC value
may have good stability under highly diluted condition after intrave-
nous injection.
Fig. 2. (A) 1H NMR spectra of GA (a), suc-GA (b), HA (c) and HSG-12 (d), and (B) FTIR
spectra of HA (a) and HSG-12(b).

X. Wang et al. / European Journal of Pharmaceutical Sciences 96 (2017) 255–262
259
Fig. 3. Particle size distribution measured by dynamic light scattering (DLS) (A) and TEM image (B) of the HSG-12 nanoparticles.
the size of HSG nanoparticles decreased with the increase of GA graft
ratio and it decreased from 260 nm to 152 nm when GA ratio increased
from 6% to 20%. This can probably be explained by the enhanced hydro-
phobic interaction among glycyrrhetinic acid groups, making the inner
core more compacted, therefore smaller particle size. A similar tendency
was reported previously (Li et al., 2012). As for particle size distribution,
unimodal size distribution was found for HSG-6 and HSG-12 nanoparti-
cles (Fig. 5A) and it has a tendency to be broader for HSG-20 nanoparti-
cles due to the smaller size. All the HSG nanoparticles were negatively
charged with zeta potential value in the range of −33.2 to −37.6 mV,
which may attribute to the presence of ionized carboxylic groups of
HA on the surface of the nanoparticles. Such high surface charge may
provide an electrostatic repelling force among the particles, therefore
increasing the nanoparticle stability. In addition, negatively charged
nanoparticles are reported to be able to contribute to efficacious evasion
of the renal filtration (Elsabahy and Wooley, 2012).
extracellular matrix and synovial fluids of most human tissues, has ex-
cellent biological properties (Arpicco et al., 2014; Tripodo et al., 2015),
but the toxicity of novel HSG carriers is not clear. Therefore, MTT assays
were performed to test the effect of self-assembled HSG polymeric
nanoparticles on the metabolic activity of cells. The cellular viability of
HSG nanoparticles against HepG2 is shown in Fig. 5. It was found that
the average cell viability of blank HSG nanoparticles was N89%, indicat-
ing all the copolymers present no significant cytotoxicity and have good
biocompatibility to the cells.
Transmittance scanning electron microscopy (TEM) was used to
directly visualize the size and morphology of HSG nanoparticles. As
shown in Fig. 3B, HSG-12 nanoparticles had an almost spherical shape
and the observed particle size was approximately 100 nm, which was
smaller than the hydrodynamic diameter obtained by DLS. This discrep-
ancy might be due to the different sample preparation technologies. DLS
measurements were carried out under aqueous condition, but TEM im-
ages were obtained for dried samples with the shrinkage of hydrophilic
shell of nanoparticles (Han et al., 2011).
3.4. Stability of HSG self-aggregated nanoparticles
Dilution of polymeric nanoparticles upon in vivo administration may
lead to premature dissociation, drug release, and therefore low tumor-
targeting ability (Deng et al., 2012; Elsabahy and Wooley, 2012). There-
fore, for clinical application, the stability of nanoparticles is of special
importance for long term blood circulation to guarantee better thera-
peutic effect. First of all, short term stability of the nanoparticles in
vitro was investigated using evolution of particle size as a criterion. As
shown in Fig. 4A, HSG nanoparticles could maintain their size un-
changed in PBS (pH 7.4) for at least 6 days, indicating good thermody-
namic stability of the nanoparticles in aqueous media. And then the
stability of the nanoparticles upon dilution was characterized and
shown in Fig. 4B. It was found the particle size had no significant change
towards 10-times dilution compared to the original nanoparticles
(p N 0.05). These facts indicated that HSG nanoparticles had a good sta-
bility, probably attributed to its low CAC and high surface charge.
3.5. In vitro cytotoxicity of HSG nanoparticles
For clinical application, ideally, carriers developed for drug delivery
should exhibit low toxicity and high biocompatibility (Zhang et al.,
2013b). It is well known that hyaluronic acid, present in the
Fig. 4. The stability of HSG nanoparticles at various incubation time (A) and towards
dilution (B) (n = 3).

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X. Wang et al. / European Journal of Pharmaceutical Sciences 96 (2017) 255–262
3 h and started to decrease gradually thereafter, and nothing is visible
after 24 h. In contrast, much stronger fluorescence signal in the liver re-
gion was observed for various HSG nanoparticles tested and the highest
intensity level was achieved at 12 h. Moreover, it was found that the
liver targeting efficiency was significantly affected by the DS of GA,
and HSG-20 nanoparticle had considerable strong fluorescence signal
even at 24 h after injection, compared to only 12 h for HSG-6 based
nanoparticles. As shown in Fig. 6C, the ex vivo fluorescent image of ex-
cised organs further confirmed that DiR-labeled HSG nanoparticles
showed higher fluorescence accumulation in the liver compared with
free DiR and the GA content had significant effect on the liver targeting
ability. Quantitative analyses (Fig. 6B) indicated that the fluorescence
intensity of HSG-6, HSG-12, and HSG-20 nanoparticles in liver was
1.8-, 2.1-, and 2.9-fold higher than that of free DiR, respectively.
It is well known that the particle size may influence the
biodistribution of nanoparticles in body as well (Hickey et al., 2015).
In the liver, the size of the fenestrations in the hepatic endothelium is
100–150 nm in diameter (Gaumet et al., 2008; Moghimi et al., 2001),
and carrier systems with diameters of 150–300 nm can induce nonspe-
cific RES uptake (Gaumet et al., 2008). In our work, three DiR-labeled
HSG nanoparticles (160–260 nm) could be taken up by the RES cells
present in the liver. But it seems that the particle size was not the
main reason for the different accumulation in liver. Tian et al. prepared
glycyrrhetinic acid-modified chitosan/poly(ethylene glycol) nanoparti-
cles with the similar particle size as we reported here (Tian et al.,
2010a), and the nanoparticles also presented an increasing liver
targeting ability as the GA content increased, further indicating that
the distinct difference in body distribution is not related to the differ-
ence in particle size.
Fig. 5. In vitro cytotoxicity of HSG nanoparticles against HepG2 cells after incubation for
24 h (n = 6).
3.6. Liver-targeting properties of nanoparticle observed by in vivo imaging
To evaluate in vivo tissue distribution and liver-targeting ability of
HSG nanoparticles, DiR was selected as a NIR fluorescent dye and was
investigated using a NIR fluorescence imaging technique. 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). After loading the
DiR, the particle sizes showed no significant change compared to
those of corresponding blank nanoparticles (p N 0.05).
Glycyrrhetinic acid, the hydrophobic group, is anticipated to exist in-
side the inner core when nanoparticles are formed. However, Park et al.
prepared folate-mediated MPEG/PCL nanospheres and theoretically, fo-
late groups should be included into the inner cores, because folate was
coupled with the terminal hydrophobic PCL block. But the XPS results
evidenced that some folate existed on the surface of the nanospheres
(Park et al., 2005). Chiu et al. also confirmed that palmitoyl groups
existed both on the surface and inside of the N-palmitoyl chitosan
The real-time images of free DiR and DiR-labeled HSG nanoparticles
are shown in Fig. 6A. For the free DiR group, a weak fluorescence signal
was detected 1 h after drug delivery, the intensity was the strongest at
Fig. 6. Fluorescence imaging of KM mice after 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).

X. Wang et al. / European Journal of Pharmaceutical Sciences 96 (2017) 255–262
261
Table 2
Properties comparison of hyaluronic acid-graft-glycyrrhetinic acid nanoparticles with different bridging groups.
Sample
Binding site
Bridging group
DS (%)
CAC (mg/mL)
Size (nm)
liver targeting ability
HA-Suc-GA (HSG)
HA-Etda-GA (HGA)
HA-Cyst-GA
Hydroxyl groups
Carboxyl groups
Carboxyl groups
Succinic acid
Ethylenediamine
Cystamine
20.4
20.2
23.8
0.020
0.078
0.035
152.6 11.1
203.4 2.2
190.9 0.5
2.9-fold higher than that of free DiR at 24 h
1.8-fold higher than that of free DiR at 24 h (Zhang et al., 2013a)
1.8-fold higher than that of free DiR at 12 h (Mezghrani et al., 2015)
Chiu, Y.-L., Ho, Y.-C., Chen, Y.-M., Peng, S.-F., Ke, C.-J., Chen, K.-J., Mi, F.-L., Sung, H.-W.,
2010. The characteristics, cellular uptake and intracellular trafficking of nanoparticles
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nanoparticles and with the DS increase of palmitoyl groups, there were
more palmitoyl groups present on the surface of nanoparticles (Chiu et
al., 2010). Based on these results, it is reasonable to assume that in the
HSG nanoparticles prepared in our study, GA molecules were partially
exposed on the surface of the particles. And this is in good agreement
with our experimental data. The larger the GA content, the higher den-
sity of GA on the surfaces of the nanoparticles, which could lead to
higher binding affinity to the liver region by GA receptor-mediated
endocytosis.
Deng, C., Jiang, Y.J., Cheng, R., Meng, F.H., Zhong, Z.Y., 2012. Biodegradable polymeric mi-
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In the past, most studies have been devoted to the specific ligand-
decoration of nanoparticles for targeted drug delivery to the liver or
the treatment of hepatocellular carcinoma (Jiang et al., 2009; Liu et al.,
2011). In this study, interestingly, we found that the binding site
might have a certain effect on the targeting properties of nanoparticles.
As shown in Table 2, with the similar DS of GA, HSG nanoparticles syn-
thesized by modifying the hydroxyl groups as presented in this study
had smaller size, lower CAC value and higher liver targeting capacity
compared with that of nanoparticles synthesized by modifying carboxyl
groups (Mezghrani et al., 2015; Zhang et al., 2013a). Therefore, coupling
HA with GA by modifying hydroxyl groups might provide better physi-
cochemical properties and high targeting efficiency compared to
carboxyl group modification. Overall, HSG nanoparticles could be ex-
pected to be a high efficient drug delivery vehicle to achieve liver-
targeting.
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4. Conclusion
A multifunctional drug delivery carrier based on hyaluronic acid-
glycyrrhetinic acid succinate (HSG) nanoparticles was synthesized suc-
cessfully in this study. This novel HA derivative could self-assemble into
nanoparticles in aqueous solution. In vitro studies showed HSG nano-
particles had a good stability and presented no significant cytotoxicity.
In vivo investigation confirmed that HSG nanoparticles had superior
targeting efficiency to liver and the liver targeting capacity was signifi-
cantly affected by the DS of GA. The accumulation of DiR-loaded HSG-
6, HSG-12, and HSG-20 nanoparticles in liver was 1.8-, 2.1-, and 2.9-
fold higher than that of free DiR. These results indicated that HSG nano-
particles have great potential as liver-targeted carriers for biomedical
applications.
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Acknowledgments
Mahmoudzadeh, M., Fassihi, A., Emami, J., Davies, N.M., Dorkoosh, F., 2013. Physicochem-
ical, pharmaceutical and biological approaches toward designing optimized and effi-
cient hydrophobically modified chitosan-based polymeric micelles as a nanocarrier
system for targeted delivery of anticancer drugs. J. Drug Target. 21, 693–709.
Mezghrani, O., Tang, Y., Ke, X., Chen, Y., Hu, D., Tu, J., Zhao, L., Bourkaib, N., 2015. Hepato-
cellular carcinoma dually-targeted nanoparticles for reduction triggered intracellular
delivery of doxorubicin. Int. J. Pharm. 478, 553–568.
This project is financially supported by the National Natural Science
Foundation of China (Grant No. 81273446) and Liaoning Institutions ex-
cellent talents support plan (No. LR2013047).
Moghimi, S.M., Hunter, A.C., Murray, J.C., 2001. Long-circulating and target-specific nano-
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Park, E.K., Lee, S.B., Lee, Y.M., 2005. Preparation and characterization of methoxy
poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric
nanospheres for tumor-specific folate-mediated targeting of anticancer drugs. Bio-
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