European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

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

Kidney-targeted astaxanthin natural antioxidant nanosystem for diabetic

nephropathy therapy
Zhong Chen, Wenhua Li, Liwang Shi, Lei Jiang, Minghui Li, Changmei Zhang *, Haisheng Peng *
Department of Pharmaceutics, Daqing Campus of Harbin Medical University, 1 Xinyang Rd, Daqing 163319, China

A R T I C L E I N F O A B S T R A C T

Keywords: Diabetic nephropathy (DN) is a frequent and severe microvascular complication associated with oxidative stress
Astaxanthin of diabetes mellitus. A novel astaxanthin-based natural antioxidant nanosystem, namely AST-GLU-LIP, with
Liposome preferential renal uptake and bioavailability were prepared and applied for treatment of diabetic nephropathy in
Kidney-targeted
rats. Our results of kidney-targeted evaluation showed that glucose-PEG600-DSPE ligand modified AST liposomes
Diabetic nephropathy
Oxidative stress
could be specifically transported by overexpressed GLUT1 on the membrane of glomerular mesangial cells and
GLUT1 achieved excellent kidney-targeted drug delivery. In addition, the results of pharmacodynamics and therapeutics
in DN rats demonstrated that AST-GLU-LIP could improve the bioavailability and antioxidant capacity of AST to
scavenge redundant ROS induced by oxidative stress. AST-GLU-LIP could also significantly improve the renal
pathological morphology to protect the kidney as a therapeutic drug for diabetic nephropathy.

1. Introduction mesangial cells, resulting in oxidative stress to promote the development

Diabetic nephropathy (DN) is a frequent and severe longstanding
microvascular complication of both type 1 and type 2 diabetes mellitus
resulting from lesions in the glomeruli and renal tubule [1]. There are
about 25% to 40% of patients with type 1 diabetes and 5% to 40% of
patients with type 2 diabetes to ultimately develop DN [2]. The main
pathological characteristics of DN are decreased glomerular filtration
rate, glomerulosclerosis, tubulointerstitial fibrosis, and renal tubular
epithelial cells damage [3,4]. DN is difficult to be detected and diag­
nosed at the early stage due to its occult onset. Renal lesions are irre­
versible and eventually develop into renal failure when some symptoms
appear. Therefore, early detection and treatment of DN, especially tar­
geted therapy of DN, is of great significance.
Glucose uptake by the kidney is mainly achieved through a type of
glucose transporter embedded in cell membrane. Glucose Transporter 1
(GLUT1) is one of glucose transporter families rich in the kidney to
transport glucose. It has been demonstrated that GLUT1 is the major
glucose transporter in the glomerular mesangial cells [5]. GLUT1 is
overexpressed by high blood glucose in glomeruli of diabetic animal
models [6–8]. GLUT1 expression is consistently high in the glomerular
mesangial cells of DN patients, and the expression intensity is positively
correlated with the degree of nephropathy lesions [5]. The over­
expression of GLUT1 will induce hyperglycemia increase in glomerular
of diabetic nephropathy. The up-regulation of GLUT1 expression will
lead to less glucose intake in peripheral tissue, resulting in intracellular
glucose metabolism disorders and hyperglycemia [9,10]. Further
structure-activity relationship studies have shown that GLUT1 has the
strongest recognition ability when the drug is attached to the sixth
carbon of glucose. Therefore, drug molecules modified by glucose ligand
exist good targeted properties [11,12].
Oxidative stress is an important pathological mechanism of DN.
Hyperglycemia is the major risk factor to promote the generation of
redundant reactive oxygen species (ROS) in oxidative stress [13,14].
Excessive toxic ROS are a class of oxygen-derived molecules with un­
paired electrons or atoms, including superoxide anions, hydrogen
peroxide, nitrogen dioxide and nitric oxide free radicals [15]. ROS
stimulate the increased accumulation of extracellular matrix proteins
including fibronectin and collagen IV in DN, which can cause glomer­
ulosclerosis and tubulointerstitial fibrosis [16,17]. Excessive ROS are
aberrantly generated by renal-infiltrating or endogenous cells, then
react with biomolecules to trigger kidney injury and renal dysfunction
[18,19]. However, many clinical researches have failed to establish
antioxidant therapy for DN. Therefore, discovery of natural antioxidant
for scavenging redundant ROS is considered an effective approach for
DN therapy.
Astaxanthin (AST), red fat-soluble substance, is a natural non-toxic

* Corresponding author.
E-mail addresses: 710752984@qq.com (C. Zhang), fisher1688@163.com (H. Peng).

https://doi.org/10.1016/j.ejpb.2020.09.005
Received 11 June 2020; Received in revised form 28 August 2020; Accepted 7 September 2020
Available online 13 September 2020
0939-6411/© 2020 Published by Elsevier B.V.
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Fig. 1. Schematic design of glucose ligand modified AST liposomes and target glomerular mesangial cells based on GLUT1 on cell membrane.

xanthophyll carotenoid [20], and its antioxidant ability is reportedly (NovoRapid, Denmark); DiO dye Beijing Fanbo Science & Technology,
800 times stronger than that of coenzyme Q10, 6000 times stronger than Co., Ltd. (Beijing, China); Malvern Zetasizer Nano ZS 90 (Malvern In­
that of vitamin C, and 100 times stronger than that of glutathione [21], struments Ltd., UK); Fluorescence Spectrometer RF-5301PC (Shimadzu
which is due to its conjugated double bond and hydroxyl group [22,23]. Company, Japan); Beckman Coulter CytoFlex S (Beckman Coulter, Brea,
AST has been found in most of marine organisms, and possesses CA, USA); Kodak multi model imaging system (CarestreamHealth Inc.,
remarkable antioxidative ability for anti-diabetes, anti-inflammation, USA); Leica AF6000 Modular Systems (Leica AF6000, Wetzlar, Ger­
anticancer, cardiovascular disease prevention, and vision improvement many); Synergy HTX Multi-Reader (BioTek, USA); Transmission elec­
[24,25]. However, the low stability and solubility of AST result in poor tron microscope (TEM, Tecnai G2 F20 S-TWIN, FEI, Hillsboro, OR, USA);
bioavailability and antioxidant capacity, which significantly restricts its Blood glucose meter (Sinocare Inc., Changsha, China)
application in drug therapy [26–28]. Therefore, our research will pre­
pare glucose ligand to modify liposomes encapsulating AST, and target
2.2. Methods
glomerular mesangial cells based on GLUT1 on cell membrane, as shown
in Fig. 1. It not only improves the bioavailability of AST, but also ach­
2.2.1. Preparation and characterization of liposomes
ieves efficient targeted drug delivery for DN therapy.
Size, polydispersity index, zeta potential measurement: Yolk
lecithin, cholesterol, Glucose-PEG600-DSPE, astaxanthin (Mole ratio =
2. Materials and methods
95: 20: 5: 6.35) were dissolved in chloroform in a round bottom flask.
The mixture was evaporated to form a thin lipid membrane under
2.1. Materials
reduced pressure, then the liposome solution was formed by ultrasonic
hydration after adding normal saline. The liposomes were prepared
Human Renal Mesangial Cells (HRMCs) were donated by the Second
through 100 nm polycarbonate membrane extrusion. Sephadex G50 was
Affiliated Hospital of Harbin Medical University. Astaxanthin, 1%
used to remove the unencapsulated astaxanthin. The particle size,
streptozocin (STZ) were purchased from Innochem (Beijing, China).
polydispersity index (PDI), and zeta potential were measured using a
PBS, FBS, DMEM were purchased from Gibco (Shanghai, China). GLU-
Malvem Zetasizer Nano ZS 90.
PEG600-DSPE compound was synthesized by our previous experiment.
Morphology measurement: The prepared liposome solution was
Yolk lecithin (EPC), cholesterol (CHO) were purchased from AVT
diluted with ultra-pure water and dropped on the copper grid, then
(Shanghai) Pharmaceutical Tech Co., Ltd. Male SD rats were purchased
stained with phosphotungstic acid (5%, w/v) and air-dried at room
from Changchun yisi laboratory animal technology company (Chang­
temperature. The morphology of liposomes was characterized on H7600
chun, China). The operating protocols for rats were completely obeyed
Transmission Electron Microscopy.
the regulations of the Ethics Committee of Harbin Medical University.
Encapsulation efficiency and loading efficiency measurement:
Assay kits of malondialdehyde (MDA), superoxide dismutase (SOD),
Astaxanthin was dissolved in DMSO and prepared into astaxanthin so­
glutathione peroxidase (GSH-Px), urine protein (UP), serum creatinine
lutions with concentrations of 1, 2, 5, 10, 20, and 40 μg/mL, respec­
(SCR), blood urea nitrogen (BUN), HE, PAS, Masson were purchased
tively. Ultraviolet absorption was recorded at 474 nm using a Metash
from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). DiR:
UV-5100B spectrometer to draw a standard curve. Sephadex G50 was
HEDE Biotech-nology Co. Ltd. (Beijing,China); aspart insulin
used to remove the unencapsulated astaxanthin. 400 μL chloroform and

144
Z. Chen et al.
500 μL methanol were added into 100 μL liposomes for ultrasound 10
min, then the astaxanthin content was detected at 474 nm.
Encapsulation efficiency (%, w/w) = AST encapsulated content/ASTtotal
content;
Loading efficiency (%, w/w) = AST encapsulated content/AST encapsulated
content + nanoparticles content.
Liposomes release in vitro: 1.0 mL astaxanthin liposome solution
(500 μg/mL) was placed into the dialysis bag (Mw cut-off 3 kDa) and
was immersed into a phosphate-buffered saline (PBS) solution (pH =
7.4, containing 10% FBS, 0.1% Tween 80) or PBS solution (pH = 5.5,
containing 0.1% Tween 80) in a 37 ◦ C shaker (60 r/min). The astax­
anthin content in the dialysis solution was detected at different times (4,
8, 12, 24, 36, 48 h) and the dialysis bag was placed into the newly
configured dialysate. After 48 h, the release concentration of astax­
anthin was calculated according to OD value, and the release curves
were drawn.
2.2.2. Properties assessment of liposomes in vitro
Antioxidant capacity of astaxanthin: 100 μL astaxanthin (46.88
μM) was placed into different volumes of 30% H2O2 (10 M) (10, 15, 20,
25, 30 μL). All the reaction volumes were supplemented with DMSO to
130 μL and reacted for 30 min at room temperature. The residual
astaxanthin was detected by ultraviolet to calculate the anti-hydrogen
peroxide ability of astaxanthin.
Cell viability assay: HRMCs (5 × 104 mL− 1) were seeded on each
well of 96-well plate overnight. The same concentration gradient of AST,
GLU-LIP and AST-GLU-LIP (20, 50, 100, 200, 400, 600, 800, 1000 μg/
mL) were added into each well for incubating 24 h at 37 ◦ C. 10 μL MTT
solution (5 mg/mL) was added into each well for continually incubating
for an additional 4 h. The liquid in wells was discarded completely and
DMSO was added to shake for 10 min. Finally, the absorption values of
each well at 490 nm were measured with Microplate Reader.
Cellular uptake: HRMCs were cultured with high glucose medium
(30 mM) to prepare diabetic cell model (HG). HRMCs were cultured
with low glucose medium (5.6 mM) to prepare normal cell model (NC).
The cells were cultured in 12-well plate for 12 h, then DiO-labeled
different liposomes (20 μL) were added and incubated for an addi­
tional 4 h. The culture media was removed, and the cells were washed
with PBS for 3 times, digested, harvested and analyzed using flow
cytometry.
Scavenge ROS in vitro: HRMCs were cultured with high glucose
medium (30 mM) in 12-well plate. The same concentration of AST, GLU-
LIP and AST-GLU-LIP (10 μM) were added into each well for incubating
24 h at 37 ◦ C, then DCFH-DA fluorescent probe (10 μM) were added and
incubated for an additional 20 min. The culture media was removed, and
the cells were washed with PBS for 3 times, digested, harvested and
analyzed using flow cytometry.
Cell apoptosis: Normal HRMCs were cultured with AST, GLU-LIP
and AST-GLU-LIP (10 μM) in 12-well plate for 12 h, then hydrogen
peroxide (100 μM) were added to incubate for an additional 4 h to
induce cell apoptosis. The culture media was removed, and the cells
were digested, harvested, and stained with apoptotic assay kit, then
analyzed using flow cytometry.
2.2.3. Establishment and validation of diabetic rat model
DN model procedures: Male SD rats were allowed to acclimatize for
1 week, and were provided high sugar and fat fodder to feed for 6 weeks.
Insulin resistance model was successfully established after the insulin
tolerance test (ITT) and the oral glucose tolerance test (OGTT) being
detected standard. Rats were provided water but no food for 12 h, and
then intraperitoneally injected with 1% STZ (35 mg/kg). The rats were
continued to fast for additional 2 h, and were fed with high sugar and fat
fodder. During 1 to 7 day, insulin was injected to reduce blood glucose to
prevent death. Fasting Blood Glucose (FBG), Random Blood Glucose
145
European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154
(RBG), Urine Output (UO) were measured at 8 week. AST, AST-LIP, AST-
GLU-LIP (10 mg/kg) preparations were given to DN rats after 8 weeks.
Some relative indicators were detected at 16 week.
OGTT and ITT measurement: OGTT: All rats were fasted overnight,
and were given a concentration of 25% glucose solution (2.0 g/kg) by
gavage. Tail tip blood was collected at 0, 30, 60, 90 and 120 min to
detect the blood glucose of the rats. ITT: Rats were injected with insulin
aspart (1.0 U/kg) by tail vein, and tail tip blood was collected at 0, 30,
60, 90 and 120 min to detect the blood glucose of the rats.
FBG, RBG and UO measurement: FBG and RBG were measured
using the blood glucose meter, and the UO was collected by the meta­
bolic cage for 24 h.
2.2.4. Renal targeting evaluation of liposomes
2.2.4.1. GLUT1 expression of immunofluorescence assay. Frozen kidney
tissue of the rats were sectioned with a thickness of 5 μm. All sections of
tissues were washed with PBS for three times, 3% Triton drilled for 20
min, and 5% BSA closed for 30 min at room temperature. The primary
antibody (diluted 1:100) was added. After removing the primary anti­
body, the membranes were incubated with the appropriate rhodamine-
labeled secondary antibody (diluted: 1:200) for incubating 2 h at room
temperature. The membranes were washed with TBST for three times to
remove secondary antibody. DAPI was used to stain nuclear for 1 min,
then washed and sealed. All sections were obtained using fluorescence
microscope (200×).
2.2.4.2. Renal imaging of DN rats after treatment in vivo. The rats were
respectively injected DiR labeled LIP, GLU-LIP, Insulin + GLU-LIP and
free DiR (40 μg/kg) by tail vein. After 6 h, saline was given by perfusion
for 10 min to remove the interference of fluorescence in blood. The
kidneys of each group were photographed by Carestream in vivo FX
Professional Imaging System equipped with an excitation-pass filter at
720 nm and emission-pass filter at 790 nm. The protocol of exposure: X-
ray exposure time was 30 s. The data were evaluated and statistically
analyzed using the Carestream imaging system.
2.2.4.3. Renal fluorescent location of liposomes. The rats were respec­
tively injected DiO labeled LIP, GLU-LIP, Insulin + GLU-LIP and free DiO
(100 μg/kg) by tail vein. After 6 h, saline was given by gavage for 10 min
to remove the interference of fluorescence in blood. The rats were killed
and kidney tissues were sectioned with a thickness of 5 μm, then were
washed with PBS for three times to remove OTC. DAPI was used to stain
nuclear for 1 min, then washed, sealed. All sections were obtained using
fluorescence microscope (200×).
2.2.5. Pharmacodynamics and therapeutics of liposomes
2.2.5.1. Antioxidative ability of liposomes in vivo. Small pieces of kidney
tissue were taken to prepare 10% tissue homogenate, then were
centrifuged at 3000 r/min for 10 min. The MDA, SOD, GSH-PX levels of
supernatant were measured by kits.
2.2.5.2. General physiological indicators. At 16 week, all rats were
weighed and kept in metabolic cages to collect 24-h urinary output and
measure water intake.
2.2.5.3. Renal evaluation index. Kidney index: The rats were sacrificed
and the kidneys were dissected and weighed. The ratios of kidney weight
and body weight (kidney index) were calculated. 24-h urinary protein,
urinary creatinine, and blood urea nitrogen were measured by kits. One
kidney was fixed in 4% paraformaldehyde and embedded in paraffin for
hematoxylin and eosin staining (H&E), periodic acid-schiff staining
(PAS) and Masson staining to observe the renal lesions under an optical
microscope and evluate the pathological morphology quantitatively.
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Table 1 encapsulation efficiency (EE) and loading efficiency (LE) of AST-GLU-

Size, polydispersity index (PDI), zeta potential (ZP), encapsulation efficiency LIP (Glucose-PEG600-DSPE modified astaxanthin liposomes) and AST-
(EE) and loading efficiency (LE) of AST-GLU-LIP and AST-LIP (n = 3). LIP (Astaxanthin liposomes) were summarized in Table 1 respectively,
Samples Size (nm) ZP (mV) PDI EE (%) LE (%) and the size distribution, zeta potential distribution were presented in
AST-LIP 116.5 ± − 27.2 ± 0.189 ± 82.25 ± 6.95 ±
Fig. 2a and b. Glucose-PEG600-DSPE was synthesized by our previous
1.2 0.98 0.05 3.74 0.56 research. Lipophilic DSPE was embedded in liposome and hydrophilic
AST-GLU- 121.2 ± − 31.3 ± 0.213 ± 80.36 ± 6.79 ± glucose was outside, which could be specifically transported by over­
LIP 3.5 0.53 0.08 3.26 0.49 expressed GLUT1 on the glomerular mesangial cell membrane and
achieved excellent renal targeted drug delivery. There are no obvious
changes in the parameters after glucose ligand modification. Moreover,
2.3. Data analysis
the results of zeta potential indicated that there are large of negative
charges on the surface of liposomes, which is beneficial to be stable in
Each measurement was repeated at least three times and the data
the whole system [17].
were expressed as mean ± standard deviation (S.D.). All results were
Encapsulation efficiency (EE) and loading efficiency (LE) of AST-LIP
analyzed with one-way analysis of variance (ANOVA). A statistical test
and AST-GLU-LIP were measured according to the standard curve of
with P < 0.05 was considered to indicate a statistically significant
AST, as illustrated in Fig. S1. All results indicated that these indexes of
difference.
AST-LIP and AST-GLU-LIP were basically consistent.
The release curves of AST-GLU-LIP and AST-LIP in different solution
3. Results and discussion
are shown in Fig. 2c. PBS + 10%FBS (pH = 7.4) is selected to simulate
the blood environment, while pH = 5.5 is selected to simulate the
3.1. Preparation and characterization of liposomes
lysosomal acidic environment. As shown in the results, no burst release
was observed on the four formulations, and the release of liposomes is
The average particle size, zeta potential, polydispersity index (PDI),
faster in pH 5.5 than that in pH 7.4. The sustained release behavior of

Fig. 2. Characterization of liposomes. (a) Size distribution of AST-GLU-LIP and AST-LIP using Malvern Zetasizer Nano ZS 90. (b) The zeta potential distribution of
AST-GLU-LIP and AST-LIP. (c) Accumulative release rate of AST-GLU-LIP and AST-LIP in different pH. (d) The morphology analysis of AST-LIP by applying TEM. (e)
The morphology analysis of AST-GLU-LIP by applying TEM. Scale bar: 500 nm.

146
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Fig. 3. Characteristics evaluation of liposomes in vitro. (a) Antioxidative activities of AST. (b) AST surplus rate in scavenging H2O2. (c) Cell viability of HRMCs after
incubated with GLU-LIP, AST and AST-GLU-LIP at different concentrations for 24 h. (d) Cellular uptake of different DiO-labeled liposomes by HRMCs. (e) ROS level of
different preparations in HRMCs. (f) HRMCs apoptosis level of different preparations. Data are shown as mean ± S.D. (n = 3 independent experiments). *P < 0.05,
**
P < 0.01, ***P < 0.001, compared with HG.

two kinds of liposomes may give continuous protection of the drug and that high glucose stimulates GLUT1 overexpressed in mesangial cells
lead to the persistent therapeutic effect [29]. The morphology of AST- [31]. GLUT1 is responsible for the transportation of glucose into
LIP and AST-GLU-LIP were observed to be spherical or oval by Trans­ mesangial cells, and overexpressed GLUT1 promotes the entry of more
mission Electron Microscopy (TEM) images (Fig. 2d and e). glucose into mesangial cells [32,33]. Therefore, the glucose ligand
modified liposomes can be specifically transported by the overexpressed
GLUT1 on mesangial cells to enhance the drug’s targeted therapeutic
3.2. Properties assessment of liposomes in vitro
effect.
The scavenge ROS activity of different liposomes was detected by
100 μL AST (46.88 μM) was select to react with different volumes of
ROS assay kit with DCFH-DA (2,7-Dichlorofuorescin Diacetate) probe.
hydrogen peroxide (10 M), and the results were shown in Fig. 3a, b. It
DCFH-DA can freely pass through the cell membrane and be hydrolyzed
indicates that AST has strong antioxidant capacity in vitro, and exhibits
to DCFH by some relevant esterase. DCFH can be oxidized to DCF
a good linear relationship with concentration. It has been reported that
(Dichlorofluorescein) by ROS, which has robust green fluorescence and
AST has a renoprotective effect on DN, increases the activity of SOD and
its fluorescence intensity processes a linear with ROS concentration
decreases malondialdehyde generation in the serum of diabetic rats
[34]. The results of the anti-ROS activity of different liposomes were
[20,25]. However, the drawbacks of poor aqueous solubility and low
shown in Fig. 3e. In HRMCs, HG group significantly increased the levels
bioavailability of AST have limited its utility [30]. AST has been
of intracellular ROS, but the level of ROS was decreased after AST, AST-
encapsulated into glucose ligand modified liposomes by us for the first
LIP, AST-GLU-LIP drug administration. Among of them, AST-GLU-LIP
time, which not only improves the bioavailability of AST, but also ac­
was demonstrated to have the most robust ability of scavenging ROS
quires targeted delivery of therapeutic agents.
compared with AST and AST-LIP group. The difference was statistically
The viability results of GLU-LIP, AST and AST-GLU-LIP at different
significant (***P < 0.001).
concentrations were shown in Fig. 3c, indicating that GLU-LIP, AST and
HRMCs apoptosis results of different preparations were shown in
AST-GLU-LIP all were nontoxic for HRMCs. Cellular uptake results of
Fig. 3f. The H2O2-induced group significantly promoted cell apoptosis in
different DiO-labeled liposomes by HRMCs were evaluated using Flow
comparison to control group⋅H2O2 tended to increase the generation of
Cytometric Analysis in Fig. 3d. Both normal cells and high-glucose
ROS, which correlated with ATP production and apoptosis in mesangial
cultured cells showed no difference in the cellular uptake of DiO-
cells [35,36]. Our research results showed that H2O2-induced HRMCs
labeled liposomes. However, the fluorescence intensity of GLU-LIP in
apoptosis was markedly attenuated by the antioxidants AST, especially
cells was obviously higher than that of LIP (*P < 0.05). It demonstrates
AST-GLU-LIP. AST-GLU-LIP plays an critical role in redox regulation,
that glucose ligand modified liposomes can be taken in more by HRMCs,
and effectively reduces HRMCs apoptosis induced by oxidative stress.
especially high glucose induced HRMCs. In the GLU-LIP group, the up­
take ability of liposomes in cells cultured with high glucose was signif­
icantly higher than that in normal cells (*P < 0.05). The main reason is

147
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Fig. 4. Establishment and validation of DN rat model. (a) Schematic design of DN rat model after liposomes administration. (b) The body weight of feeding high
sugar and fat fodder for 6 weeks. **P < 0.01. (c) The oral glucose tolerance test (OGTT). *P < 0.05. (d) The insulin tolerance test (ITT). *P < 0.05. (e) The blood
glucose curve after injecting STZ at 6 weeks. Data are shown as mean ± S.D. (n = 6 independent experiments).

3.3. Establishment and validation of DN rat model
The DN model of rats was built in Fig. 4a and the occurrence of
serious complications was a sign of diabetes developing into early DN
after streptozocin (STZ) injection for 6 weeks [37–39]. Male SD rats
were feed with high sugar and high fat fodder for 6 weeks to build in­
sulin resistance model. The purpose of insulin resistance model is to
improve molding rate and survival rate of type 2 diabetes model after
injecting STZ. Low dose of STZ cannot form insulin resistance, but high
dose is more likely to lead to rat die or become type 1 diabetes model.
After 6 weeks, the weight of rat in model group were significantly
heavier than that of the normal group (Fig. 4b). OGTT in Fig. 4c showed
that the insulin resistance group had a faster rise in blood glucose and a
lower glucose utilization rate than the control group. ITT in Fig. 4d
showed that the insulin resistance group had a slower decline in blood
glucose than the normal group, which indicated that insulin resistance
model had been successfully established.
After insulin resistance model successful establishment, STZ was
intraperitoneally injected into rat on 6th week, and the blood glucose
curve after STZ injection was shown in Fig. 4e. It could be seen that
pancreatic β-cells were damaged and blood glucose was enhanced in
1–2 h, while pancreatic β-cells were destroyed to lead more insulin
release and blood glucose decrease in 6–10 h. After 24 h, the function of
the islet was destroyed, and the blood glucose was continuously high.
The diabetes model was successful built, and the fasting blood glucose
(FBG) ≥ 7.8 mmol/L or random blood glucose (RBG) ≥ 16.7 mmol/L,
urine output (UO) ≥ 50% original UO, which were in line with the
standard of the diabetes model [40]. FBG, RBG and UO were detected in

148
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Table 2 8th week after injecting STZ for two weeks, as shown in Table 2. It
FBG, RBG, UO in blood of DN rats after injecting STZ for two weeks. (n = 6). usually finds that greater than 90% of STZ treated rats obtain sufficient
Group FBG (mmol/L) RBG (mmol/L) UO (mL/24 h) diabetes to be used in animal model researches of diabetic nephropathy
[38,39].
Control 4.50 ± 0.41 5.53 ± 0.41 27.00 ± 2.94
Model 8.80 ± 0.54 21.83 ± 1.56 52.33 ± 7.13

Fig. 5. Immunofluorescence images of kidney tissues staining with GLUT1 antibody (200×).

Fig. 6. Targeting distribution of various DiR-labeled liposomes in kidney tissues of rats. (a) The distribution of fluorescence signal in the kidney of rats. (b) Histogram
statistically analyzed the distribution in the kidney of rats. *P＜0.05, model group compare with control group in every preparation; #P＜0.05, ##P＜0.01, every
preparation compares with LIP in model group. Data represented the mean ± S.D. (n = 3).

149
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Fig. 7. Renal fluorescent localization of different liposomes in renal tissues of rats. (a and) Fluorescent images after saline administration in control and model group.
(b and g) Fluorescent images without drug administration. (c and h) Fluorescent images after LIP administration. (d and i) Fluorescent images after GLU-LIP
administration. (e and j) Fluorescent images after GLU-LIP + insulin administration (200×).

Fig. 8. Antioxidative effect of liposomes on the kidney in vivo. (a) The MDA level. (b) The SOD level. (c) GSH-PX level. **P < 0.01, ***P < 0.001, compare with model
group. Data are shown as mean ± S.D. (n = 6 independent experiments).

3.4. Renal targeting evaluation of liposomes
3.4.1. GLUT1 expression of immunofluorescence assay
Immunofluorescence assay was used to detect the GLUT1 expression
in kidney tissue of DN rats. The results showed that GLUT1 expressed
more in renal tubules than that in glomeruli. However, GLUT1 in renal
tubules was not selected as a target point, because most liposomes with
sizes larger than 100 nm could not pass through the glomerular filtration
barrier to reach renal tubules [41–43]. Therefore, liposomes accumu­
lated in glomeruli to overcome the free small molecule drugs directly
through the glomerular filtration barrier and improved the glomerular
therapeutic effect of targeted drugs. DN model showed higher over
expression of GLUT1 in glomeruli than that of control group, which was
marked by arrow in Fig. 5. Over expression of GLUT1 could increase
excess uptake of glucose, therefore more liposomes modified by glucose
ligand will be transported into mesangial cells based on GLUT1 on renal
mesangial cells in diabetic glomeruli.
3.4.2. Biodistribution and renal imaging of liposomes in vivo
We examined targeting distribution of various DiR-labeled liposomes
in different tissues and organs of SD rats, such as brain, heart, liver,
spleen, lung, kidney, testis, skin, muscle, thyroid, thymus, pancreas and

150
Z. Chen et al. European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154

Fig. 9. Renal evaluation index of different preparations administration 8 weeks. (a) Kidney index. (b) Urine protein. (c) Serum creatinine. (d) Blood urea nitrogen.
*P < 0.05, **P < 0.01, ***P < 0.001, compare with model group. Data are shown as mean ± S.D. (n = 6 independent experiments). (e) H&E, PAS and Masson staining
of kidney vertical section (200×).

151
Z. Chen et al.
fat, and found no significant accumulation or difference in other organs,
except that preparations were captured by the reticuloendothelium
system in the liver and spleen (Fig. S2).
To clearly observe the drug distribution of different liposomes in the
kidney of rats, all groups of rats were dissected and photographed under
the same conditions. As shown in Fig. 6a, free DiR (red) fluorescence
probe could pass through the glomerular filtration barrier to the renal
pelvis, where they could be excreted in urine, but the DiR-labeled LIP
and DiR-labeled GLU-LIP remained and accumulated in renal cortex
including glomeruli and renal tubules. Fig. 6b showed that the accu­
mulation of GLU-LIP liposomes was significantly higher than that in the
LIP group, especially DN model group (*P < 0.05). GLU-LIP + Insulin
group was conducted to reduce blood glucose using insulin aspart (5 U/
kg) to explore the effect of hypoglycemia on cellular uptake. After in­
sulin intervention, accumulation of GLU-LIP was obviously improved in
comparison to common liposomes (##P < 0.01). It indicates that the
decrease of blood glucose can promote the uptake of liposomes in DN
model rats. The main reason for the blood glucose will be competitive
with the GLUT1 in glomeruli, while insulin can reduce blood glucose,
which promotes the GLUT1 to combine with more glucose ligand
modified liposomes.
3.4.3. Renal fluorescent location of liposomes
Fluorescent images of different DiO labeled liposomes revealed their
cellular uptake and distribution in the kidney of rats. In both control and
model groups, the free small molecule DiO was mainly accumulated in
renal tubules, while little in glomeruli (Fig. 7b). However, liposomes
could target accumulate in glomeruli, maily mesangial cells, and lipo­
somes showed robust fluorescence image in glomeruli, especially GLU-
LIP, which was maked by white arrow and red circle (Fig. 7d). More
cellular uptake of DiO labeled GLU-LIP in DN model group resulted in
presenting brighter fluorescent imaging in comparison to control group
(Fig. 7i), which were due to over expression of GLUT1 in mesangial cells
[44]. Fig. 7e showed that reduced blood glucose by insulin could
effectively enhance the uptake of GLU-LIP by mesangial cells, especially
in DN model (Fig. 7j). The results were consistent with the renal dis­
tribution and accumulation experiment in vivo above, and the locali­
zation experiment truly demonstrated that GLU-LIP accumulated in the
mesangial cells of kidney.
3.5. Pharmacodynamics and therapeutics of liposomes
3.5.1. Antioxidative ability of liposomes in vivo
Oxidative stress in diabetic kidney will induce redox imbalance and
produce a large number of ROS. ROS attack the unsaturated fatty acids
in the biological membrane, and trigger lipid peroxidation to form
malondialdehyde (MDA). MDA causes cell metabolism and dysfunction,
even causes cells death. Therefore, MDA can directly reflect the rate of
peroxidation, as well as the degree of cell damage [45]. The content of
MDA in DN model group was 5.23 ± 1.21 nmol/mg, but reduced after
drug administration (Fig. 8a). The content of MDA in AST group was
4.53 ± 0.37 nmol/mg, AST-LIP was 3.59 ± 0.67 nmol/mg, AST-GLU-LIP
was 3.02 ± 0.36 nmol/mg, which existed a statistical significant dif­
ference compared with DN model group (**P < 0.01, ***P < 0.001).
Superoxide dismutase (SOD) is a kind of free radical scavenger in
oxidative stress, which can specifically catalyze the reduction of GSH to
hydrogen peroxide. Glutathione peroxidase (GSH-Px) can remove the
lipid peroxides induced by ROS, resist the destruction of ROS produced
by oxidative stress in renal cells, and play a role in protecting the
structure of cell membrane. Therefore, both SOD and GSH-Px can be
used as indirect indicators to supervise the body’s scavenging ability of
ROS [46]. The values of SOD in AST, AST-LIP, AST-GLU-LIP group were
21.00 ± 0.84 U/mg, 27.29 ± 3.10 U/mg, 38.90 ± 1.38 U/mg, respec­
tively. The values of GSH-PX in AST, AST-LIP, AST-GLU-LIP group were
200.75 ± 98.79 U/mg, 299.13 ± 9.45 U/mg, 323.62 ± 14.02 U/mg,
respectively. In contrast, the values of SOD and GSH-PX in DN model
152
European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154
group were 14.50 ± 168 U/mg and 197.05 ± 7.90 U/mg, demonstrating
AST-GLU-LIP could effectively increase endogenous antioxidant sub­
stances and reduce the damage of oxidative stress for diabetic kidneys
(Fig. 8b and c). There was a statistically significant difference in com­
parison to DN model group (**P < 0.01, ***P < 0.001).
AST has preferable effective compared with other antioxidants such
as vitamin E and β-carotenoid, preventing lipid peroxidation in solution
and various biomembrane systems [47,48]. In addition, AST processes
renal protective effect to prevent renal fibrosis [49,50]. AST can
ameliorate oxidative stress induced by renal fibrosis, and strengthen the
cellular anti-oxidative capacity, and diminish fibronectin accumulation
in glomerular mesangial cells [51]. The mechanism of AST antioxidation
may relate to the unique structure of its terminal ring moiety [52]. AST
can trap free radicals in both conjugated polyene chain and terminal ring
moiety, in which the hydrogenatom at the C3 methine is considered to
be a radical trapping site [53]. Although the anti-diabetic nephropathy
effect of AST has been obviously proved, there are still some disadvan­
tages such as non-targeted and poor effect for DN therapy. Our research
has designed a novel targeted delivery drug, namely AST-GLU-LIP,
which not only achieves antioxidant capacity of AST, but also pre­
cisely targets mesangial cells of glomerulus with the help of high
expressed GLUT1.
3.5.2. General physiological indicators
AST-GLU-LIP would reduce weight loss, decrease water intake and
urine output of rats in ND model at 16 week (Fig. S3a–c). The body
weight of AST-GLU-LIP group was 456.83 ± 18.68 g, which was much
heavier than that of DN model group (334.33 ± 18.30 g). However, the
water intake and urine output of AST-GLU-LIP group were 103.33 ±
14.72 mL and 104.17 ± 12.43 mL, respectively. They were obviously
reduced by 54.6% and 46.0% compared with DN model group (227.67
± 24.77 mL, 192.97 ± 31.20 mL). There was a significantly statistical
difference (*P < 0.05, **P < 0.01, ***P < 0.001).
3.5.3. Renal evaluation index
Kidney index, Urine Protein (UP), Serum Creatinine (SCR), and
Blood Urea Nitrogen (BUN) are important indexes to evaluate renal
injury. Kidney index in Fig. 9a showed that the kidney index was higher
in DN model group (7.14 ± 0.53 mg/kg) than that in other groups.
Among of them, AST-GLU-LIP group (4.16 ± 0.33 mg/kg) could obvi­
ously decrease the kidney index in comparison to AST group (6.64 ±
0.98 mg/kg) and AST-LIP group (5.05 ± 0.41 mg/kg). There was a
statistically significant difference comparing with DN model group (**P
< 0.01, ***P < 0.001). Similar trends of UP, SCR, and BUN were shown in
Fig. 9b–d. The values of UP, SCR, BUN in AST-GLU-LIP group were
27.66 ± 5.48 mg/24 h, 41.69 ± 7.00 μmol/L, 4.16 ± 0.33 mmol/L,
respectively. In contrast, the UP, SCR, BUN in DN group were 82.22 ±
6.12 mg/24 h, 80.95 ± 16.59 μmol/L, 7.15 ± 0.53 mmol/L, demon­
strating the better renal function was achieved by the administration of
AST-GLU-LIP.
The results of H&E staining, PAS staining and Masson staining were
evaluated to confirm DN pathological features. As shown in Fig. 9e, HE
staining showed that the glomerular basement membrane (GBM) was
thickened, the renal tubules were expanded, the glomeruli were swell,
and some denatured proteins substances were produced and precipi­
tated in the renal tubules in the DN model group, which were typical DN
complications. The precipitation of denatured proteins in the renal tu­
bules are usually used as indicators for kidney diseases [54]. However,
after the treatment of AST-GLU-LIP, the renal pathological morphology
is significantly improved.
DN is characterized by glycogen accumulation in the kidney [55].
The glycogen in the mesangial extracellular matrix is mainly stained by
PAS. The increase of the extracellular matrix (ECM) indicates damage of
mesangial cells. The results had shown that extracellular matrix was
increased in DN model group, which was represented by light purple.
After the treatment of AST-GLU-LIP, the extracellular matrix was
Z. Chen et al.
obviously reduced and improved renal glycogen-induced damage during
diabetes.
Collagenous fiber, as a kind of collagen, played an essential role in
renal tissue fibrosis, which deteriorated DN development [56,57].
Therefore, Masson was used to stain collagenous fiber, and blue repre­
sented fibrosis in Fig. 9e. It could be seen that fibrosis in the DN model
group was severe, and glomeruli lesions were more serious in compar­
ison to control group. However, after AST-GLU-LIP treatment, the dis­
ease progression was alleviated in DN group.
4. Conclusion
A novel astaxanthin-based natural antioxidant nanosystem, namely
AST-GLU-LIP, were prepared and applied for treatment of diabetic ne­
phropathy in rats. The glucose ligand modified AST liposomes could be
specifically transported by overexpressed GLUT1 on glomerular
mesangial cell membrane and achieved excellent kidney-targeted drug
delivery. AST-GLU-LIP could improve the bioavailability and antioxi­
dant capacity of AST to scavenge redundant ROS induced by oxidative
stress. AST-GLU-LIP could also significantly improve the renal patho­
logical morphology to protect the kidney as a therapeutic drug for dia­
betic nephropathy.
Author contributions
C.M. Zhang and H.S. Peng managed the overall project and designed
the experiments with contributions from all authors. Z. Chen and W.H.
Li carried out all the experiments. Other authors participated in some
related experiments.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was financially supported by the Fundamental
Research Funds for the Heilongjiang Provincial Natural Science Foun­
dation (No. LH2019H011); the Fundamental Research Funds for the
Provincial Universities of Heilongjiang province in 2018; Foundation for
post-doctoral Research of Heilongjiang province (LBH-Q19163).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejpb.2020.09.005.
References
[1] N. Papadopoulou, et al., Diabetic nephropathy in type 1 diabetes, Minerva Med.
109 (2017).
[2] G. Tramonti, et al., Review and discussion of tubular biomarkers in the diagnosis
and management of diabetic nephropathy, Endocrine 43 (3) (2012) 494–503.
[3] E. Dounousi, et al., Improvements in the management of diabetic nephropathy,
Rev. Diabet. Stud. 12 (1–2) (2015) 119–133.
[4] G. Yang, et al., Effect of berberine on the renal tubular epithelial-to-mesenchymal
transition by inhibition of the Notch/snail pathway in diabetic nephropathy model
KKAy mice, Drug Design, Development and Therapy, vol. 11, 2017, pp.
1065–1079.
[5] H. Zhang, et al., Podocyte-specific overexpression of GLUT1 surprisingly reduces
mesangial matrix expansion in diabetic nephropathy in mice, Am. J. Physiol. Renal
Physiol. 299 (2010) F91–F98.
[6] C. Heilig, et al., D-glucose stimulates mesangial Cell GLUT1 expression and basal
and IGF-I-sensitive glucose uptake in rat mesangial cells: implications for diabetic
nephropathy, Diabetes 46 (1997) 1030–1039.
[7] C.W. Heilig, et al., Role for GLUT1 in diabetic glomerulosclerosis, Expert Rev. Mol.
Med. 8 (4) (2006) 1–18.
153
European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154
[8] F. Hajiaghaalipour, et al., Modulation of glucose transporter protein by dietary
flavonoids in type 2 diabetes mellitus, Int. J. Biol. Sci. 11 (5) (2015) 508–524.
[9] L. Gnudi, et al., GLUT-1 overexpression: Link between hemodynamic and
metabolic factors in glomerular injury? Hypertension 42 (1) (2003) 19–24.
[10] X. Chen, et al., Huangqi (astragalus) decoction ameliorates diabetic nephropathy
via IRS1-PI3K-GLUT signaling pathway, Am. J. Transl. Res. 10 (2018) 2491–2501.
[11] P. Ma, et al., Enhanced anti-hepatocarcinoma efficacy by GLUT1 targeting and
cellular microenvironment-responsive PAMAM–camptothecin conjugate, Drug
Deliv. 25 (2018) 153–165.
[12] P. Ma, et al., Overcoming multidrug resistance through the GLUT1-mediated and
enzyme-triggered mitochondrial targeting conjugate with redox-sensitive
paclitaxel release, ACS Appl. Mater. Interfaces 10 (15) (2018) 12351–12363.
[13] M. Brownlee, Biochemistry and molecular biology of diabetic complications,
Nature 414 (2001).
[14] D. Luis-Rodriguez, et al., Pathophysiological role and therapeutic implications of
inflammation in diabetic nephropathy, World J. Diabetes 3 (1) (2012) 7–18.
[15] N. Kashihara, et al., Oxidative stress in diabetic nephropathy, Curr. Med. Chem. 17
(2010) 4256–4269.
[16] S. Wang, et al., Salidroside alleviates high glucose-induced oxidative stress and
extracellular matrix accumulation in rat glomerular mesangial cells by the TXNIP-
NLRP3 inflammasome pathway, Chem. Biol. Interact. 278 (2017) 48–53.
[17] T. Ding, et al., Kidney protection effects of dihydroquercetin on diabetic
nephropathy through suppressing ROS and NLRP3 inflammasome, Phytomedicine
41 (2018).
[18] T. Sun, et al., A melanin-based natural antioxidant defense nanosystem for
theranostic application in acute kidney injury, Adv. Funct. Mater. 29 (48) (2019).
[19] M.S. Paller, et al., Oxygen free radicals in ischemic acute renal failure in the rat,
J. Clin. Invest. 74 (4) (1984) 1156–1164.
[20] A. Sila, et al., Astaxanthin from shrimp by-products ameliorates nephropathy in
diabetic rats, Eur. J. Nutr. 54 (2) (2015) 301–307.
[21] E. Suyono, et al., Combination of blue, red, white, and ultraviolet lights for
increasing carotenoids and biomass of microalga Haematococcus pluvialis, Proc.
Environ. Sci. 28 (2015) 399–405.
[22] Y.M.A. Naguib, Antioxidant activities of astaxanthin and related carotenoids,
J. Agric. Food Chem. 48 (4) (2000) 1150–1154.
[23] M. Gammone, et al., Marine carotenoids against oxidative stress: effects on human
health, Mar. Drugs 13 (2015) 6226–6246.
[24] R. Ambati, et al., Astaxanthin: sources, extraction, stability, biological activities
and its commercial applications—a review, Mar. Drugs 12 (1) (2014) 128–152.
[25] X. Zhu, et al., Astaxanthin promotes Nrf2/ARE signaling to alleviate renal
fibronectin and collagen IV accumulation in diabetic rats, J. Diabetes Res. 2018
(2018) 6730315.
[26] S.-A. Park, et al., The effects of particle size on the physicochemical properties of
optimized astaxanthin-rich Xanthophyllomyces dendrorhous-loaded
microparticles, LWT - Food Sci. Technol. 55 (2) (2014) 638–644.
[27] P. Kittikaiwan, et al., Encapsulation of Haematococcus pluvialis using chitosan for
astaxanthin stability enhancement, Carbohydr. Polym. 70 (4) (2007) 378–385.
[28] Q. Wang, et al., Preparation of astaxanthin-loaded DNA/chitosan nanoparticles for
improved cellular uptake and antioxidation capability, Food Chem. 227 (2017).
[29] J. Qu, et al., Nanostructured lipid carriers, solid lipid nanoparticles, and polymeric
nanoparticles: which kind of drug delivery system is better for glioblastoma
chemotherapy? Drug Deliv. 23 (2016) 1–28.
[30] C.-H. Chiu, et al., Improved hepatoprotective effect of liposome-encapsulated
astaxanthin in lipopolysaccharide-induced acute hepatotoxicity, Int. J. Mol. Sci. 17
(2016) 1128.
[31] W. Youli, et al., Transgenic overexpression of GLUT1 in mouse glomeruli produces
renal disease resembling diabetic glomerulosclerosis, Am. J. Physiol. Renal Physiol.
299 (2010) F99–F111.
[32] K. Inoki, et al., TGF-1 stimulates glucose uptake by enhancing GLUT1 expression in
mesangial cells, Kidney Int. 55 (1999) 1704–1712.
[33] J.M. Zheng, et al., Rhein reverses the diabetic phenotype of mesangial cells over-
expressing the glucose transporter (GLUT1) by inhibiting the hexosamine pathway,
British J. Pharmacol. 153 (2008) 1456–1464.
[34] B. Kramer, et al., Effect of Pulsed Light on structural and physiological properties of
Listeria innocua and Escherichia coli, J. Appl. Microbiol. 116 (2013).
[35] B. Kang, et al., High glucose promotes mesangial cell apoptosis by oxidant-
dependent Mechanism, Am. J. Physiol. Renal Physiol. 284 (2003) F455–F466.
[36] Y. Shi, et al., Knockdown of thioredoxin interacting protein attenuates high
glucose-induced apoptosis and activation of ASK1 in mouse mesangial cells, FEBS
Lett. 585 (12) (2011) 1789–1795.
[37] M. Breyer, et al., Mouse models of diabetic nephropathy, J. Am. Soc. Nephrol.:
JASN 16 (2005) 27–45.
[38] B.L. Furman, Streptozotocin-induced diabetic models in mice and rats, Curr.
Protocols Pharmacol. 70 (1) (2015).
[39] G.H. Tesch, et al., Rodent models of streptozotocin-induced diabetic nephropathy
(Methods in Renal Research), Nephrology 12 (3) (2007) 261–266.
[40] S. Tan, et al., ARF influences diabetes through promoting the proliferation and
malignant development of beta cells, Artificial Cells Nanomed. Biotechnol. 46
(sup3) (2018) S702–S706.
[41] H. Soo Choi, et al., Renal clearance of quantum dots, Nat. Biotechnol. 25 (10)
(2007) 1165–1170.
[42] J.E. Zuckerman, et al., Targeting therapeutics to the glomerulus with
nanoparticles, Adv. Chronic Kidney Dis. 20 (6) (2013) 500–507.
[43] G. Wang, et al., Kidney-targeted rhein-loaded liponanoparticles for diabetic
nephropathy therapy via size control and enhancement of renal cellular uptake,
Theranostics 9 (21) (2019) 6191–6208.
Z. Chen et al.
[44] S.Y. Yu, et al., Losartan reverses glomerular podocytes injury induced by AngII via
stabilizing the expression of GLUT1, Mol. Biol. Rep. 40 (11) (2013) 6295–6301.
[45] M. Catherwood, et al., Glucose-induced oxidative stress in mesangial cells, Kidney
Int.. 61 (2002) 599–608.
[46] M. Kitada, et al., Resveratrol improves oxidative stress and protects against
diabetic nephropathy through normalization of Mn-SOD dysfunction in AMPK/
SIRT1-independent pathway, Diabetes 60 (2011) 634–643.
[47] I. Faraone, et al., Astaxanthin anticancer effects are mediated through multiple
molecular mechanisms: a systematic review, Pharmacol. Res. 155 (2020), 104689.
[48] J. Gu, et al., Astaxanthin-loaded polymer-lipid hybrid nanoparticles (ATX-LPN):
assessment of potential otoprotective effects, J. Nanobiotechnol. 18 (1) (2020) 53.
[49] Y. Naito, et al., Prevention of diabetic nephropathy by treatment with astaxanthin
in diabetic db/db mice, BioFactors (Oxford, England) 20 (2013) 49–59.
[50] Q. Chen, et al., Astaxanthin ameliorates experimental diabetes-induced renal
oxidative stress and fibronectin by upregulating connexin43 in glomerular
mesangial cells and diabetic mice, Eur. J. Pharmacol. 840 (2018).
154
European Journal of Pharmaceutics and Biopharmaceutics 156 (2020) 143–154
[51] D. Leehey, et al., Role of angiotensin II in diabetic nephropathy, Kidney Int. 77
(Suppl.) (2000) S93–S98.
[52] S. Goto, et al., Efficient radical trapping at the surface and inside the phospholipid
membrane is responsible for highly potent antiperoxidative of the carotenoid
astaxanthin, Biochim. Biophys. Acta (BBA) – Biomembranes 1512 (2001) 251–258.
[53] W. Aoi, et al., Astaxanthin limits exercise-induced skeletal and cardiac muscle
damage in mice, Antioxidants Redox Signal. 5 (2003) 139–144.
[54] D. Ni, et al., Molybdenum-based nanoclusters act as antioxidants and ameliorate
acute kidney injury in mice, Nat. Commun. 9 (1) (2018).
[55] R. Gatica, et al., Over-expression of muscle glycogen synthase in human diabetic
nephropathy, Histochemistry Cell Biol. 143 (3) (2014) 313–324.
[56] S. Malik, et al., Apigenin ameliorates streptozotocin-induced diabetic nephropathy
in rats via MAPK-NF-kappaB-TNF-alpha and TGF-beta1-MAPK-fibronectin
pathways, Am. J. Physiol. Renal Physiol. 313 (2) (2017) F414–F422.
[57] Q. Wang, et al., MicroRNA-377 is up-regulated and can lead to increased
fibronectin production in diabetic nephropathy, FASEB J. 22 (12) (2008)
4126–4135.