Resveratrol solid lipid nanoparticles to trigger credible inhibition of doxorubicin cardiotoxicity

Abstract

Background
Doxorubicin (DOX), a broad-spectrum chemotherapy drug, is clinically employed to treat cancers especially for breast cancer and lung cancer. But its clinical applications are limited by the dose-dependent cardiac toxicity. Resveratrol (Res), a polyphenolic antitoxin, has been proved to be capable of improving the cardiomyocyte calcium cycling by up-regulating SIRT-1-mediated deacetylation to inhibit DOX-induced cardiotoxicity.

Purpose
The objective of this study was to develop a solid lipid nanoparticle (SLN) loaded with Res to trigger inhibition of DOX-induced cardiotoxicity.

Methods
Res-SLN was prepared by emulsification-diffusion method followed by sonication and optimized using central composite design/response surface method. The Res-SLN was further evaluated by dynamic light scattering, transmission electron microscopy for morphology and high performance liquid chromatography for drug loading and release profile. And the Res distribution in vivo was determined on rats while the effect of inhibit DOX-induced cardiotoxicity was investigated on mice.

Results
Res-SLN with homogeneous particle size of 271.13 nm was successfully formulated and optimized. The prepared Res-SLN showed stable under storage and sustained release profile, improving the poor solubility of Res. Heart rate, ejection fractions and fractional shortening of Res-SLN treating mice were found higher than those on mice with cardiac toxicity induced by single high-dose intraperitoneal injection of DOX. And the degree of myocardial ultrastructural lesions on mice was also observed.

Conclusion
Res-SLN has a certain therapeutic effect for protecting the myocardium and reducing DOX-induced cardiotoxicity in mice.

Full text

ORIGINAL RESEARCH
Resveratrol solid lipid nanoparticles to trigger
credible inhibition of doxorubicin cardiotoxicity
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Lili Zhang
1
Kexin Zhu
1
Hairong Zeng
2
Jiaxin Zhang
1
Yiqiong Pu
3
Zhicheng Wang
4
To n g Z h a n g
3
Bing Wang
1,5
1
School of Pharmacy, Shanghai University
of Traditional Chinese Medicine,
Shanghai, People’s Republic of China;
2
Department of Pharmacy, Putuo
Hospital, Shanghai University of
Traditional Chinese Medicine, Shanghai,
People’s Republic of China;
3
Experiment
Center for Teaching and Learning,
Shanghai University of Traditional
Chinese Medicine, Shanghai, People’s
Republic of China;
4
Department of
Laboratory Medicine, Huashan Hospital,
Shanghai Medical College, Fudan
University, Shanghai, People’s Republic of
China;
5
Center for Pharmaceutics
Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences,
Shanghai, People’s Republic of China
Background: Doxorubicin (DOX), a broad-spectrum chemotherapy drug, is clinically
employed to treat cancers especially for breast cancer and lung cancer. But its clinical
applications are limited by the dose-dependent cardiac toxicity. Resveratrol (Res),
a polyphenolic antitoxin, has been proved to be capable of improving the cardiomyocyte
calcium cycling by up-regulating SIRT-1-mediated deacetylation to inhibit DOX-induced
cardiotoxicity.
Purpose: The objective of this study was to develop a solid lipid nanoparticle (SLN) loaded
with Res to trigger inhibition of DOX-induced cardiotoxicity.
Methods: Res-SLN was prepared by emulsification-diffusion method followed by sonica-
tion and optimized using central composite design/response surface method. The Res-SLN
was further evaluated by dynamic light scattering, transmission electron microscopy for
morphology and high performance liquid chromatography for drug loading and release
profile. And the Res distribution in vivo was determined on rats while the effect of inhibit
DOX-induced cardiotoxicity was investigated on mice.
Results: Res-SLN with homogeneous particle size of 271.13 nm was successfully formu-
lated and optimized. The prepared Res-SLN showed stable under storage and sustained
release profile, improving the poor solubility of Res. Heart rate, ejection fractions and
fractional shortening of Res-SLN treating mice were found higher than those on mice with
cardiac toxicity induced by single high-dose intraperitoneal injection of DOX. And the
degree of myocardial ultrastructural lesions on mice was also observed.
Conclusion: Res-SLN has a certain therapeutic effect for protecting the myocardium and
reducing DOX-induced cardiotoxicity in mice.
Keywords: resveratrol, solid lipid nanoparticles, doxorubicin, heart failure
Introduction
Doxorubicin (DOX) is an anthraquinonoid broad-spectrum chemotherapy drug against
lymphoma, breast cancer and other malignant tumors. Actually, DOX is restricted on
clinical applications due to its severe cardiac toxicity.
1
The certain pathogenesis of
DOX-induced cardiotoxicity is still unclear, but intracellular calcium disorders, p53-
mediated cardiomyocyte apoptosis, oxidative stress and mitochondrial function dis-
orders are concerned.
2–5
Resveratrol (Res) is a polyphenolic antitoxin widely found in
natural plants, possessing the properties of anti-inflammatory, anti-tumor, and protect-
ing cardiovascular pharmacological activity.
6,7
It is certain that Res can improve the
cardiomyocyte calcium cycling by up-regulating silent information regulator-1 (SIRT-
1)-mediated deacetylation, thereby inhibiting the production of reactive oxygen species
to protect the myocardium and improve DOX-induced cardiotoxicity.
8,9
Correspondence: Tong Zhang
Experiment Center for Teaching and
Learning, Shanghai University of
Traditional Chinese Medicine, 1200 Cailun
Road, Pudong New District, Shanghai
201203, People’s Republic of China
Email zhangtdmj@hotmail.com
Bing Wang
Center for Pharmaceutics Research,
Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 501 Haike
Road, Pudong New District, Shanghai
201203, People’s Republic of China
Email bwang@simm.ac.cn
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http://doi.org/10.2147/IJN.S211130

Although Res possesses the effect of attenuating DOX-
induced cardiotoxicity, Res is poorly soluble in water,
which makes it hard to achieve a satisfactory effect after
orally taken. To overcome this obstacle, solid lipid nano-
particle (SLN) is employed. SLN, drew up in the early
1990s, is a drug delivery system made up of skeleton
materials and drugs and can improve drug’s solubility
and bioavailability.
10
The drugs are wrapped or embedded
in the lipid nucleus, consisting of physiological compat-
ibility, biodegradable natural or synthetic solid lipid with
high-melting point like lecithin or glycerol, and made into
particles with the size of about 50–1000 nm.
11
The reported Res-SLNs were prepared by the probe
ultrasonication method,
12
emulsification and solidification
method,
13,14
solvent injection method
15
or homogenization
method.
16
They were dividedly utilized for transdermal
drug delivery, treating breast cancer, anti-inflammatory,
enhancing hepatoprotection and brain delivery, but nothing
about Res-SLN inhibiting DOX-induced cardiotoxicity
was discussed. In this study, we interrogated SLN as
a drug delivery carrier of Res for the potential treatment
of Dox-induced cardiotoxicity. The Res-SLN was prepared
by emulsification and sonication method, followed with
optimation of the formulations and pharmacodynamic eva-
luation. The prepared Res-SLN was able to improve the
bioavailability of Res for better protecting the myocardium
and inhibiting the DOX-induced cardiac toxicity
(Figure 1).
Materials and methods
Materials
Res was purchased from ZELANG (Nanjing, China) and
Res reference substance (M28F-Q3FG, purity ≥98.513%)
was procured from National Institutes for Food and Drug
Control of China. PC-98T egg yolk lecithin (AL15018, purity
98%) was obtained from A.V.T. Pharmaceutical Co., Ltd
(Shanghai, China). Glycerol monostearate and glycerol tris-
tearate were purchased from Sinopharm Chemical Reagent
Co., Ltd (Shanghai, China), while poloxamer 188 and sodium
dichromate were from Yuanye Bio. (Shanghai, China).
Fifty male Kunming mice (20–25 g), 10 male and female
SpragueDawley(SD)rats(180–220 g) were purchased from
Shanghai Slac Laboratory Animal Company and raised in
Laboratory Animal Center, Shanghai University of
Traditional Chinese Medicine. Experiments were performed
in compliance with the requirements of the animal ethics
committee of Shanghai University of Traditional Chinese
Medicine (Ethical Accreditation No. SZY201507002).
Preparation of Res solid lipid
nanoparticles
Res-loaded SLNs were prepared using emulsification-
diffusion method followed by sonication.
17
Briefly,
a certain amount of egg yolk lecithin and Res were dis-
solved completely in 30 mL absolute alcohol and then
mixed with 120 mg molten glycerol monostearate (GMS)
i) Emulsification diffusion
Sustained release Res
Lipids
Res-SLNs protect myocardium from doxorubicin
Doxorubicin
Doxorubicin
20 mg/kg Res
1 g/kg Res-SLN
60 mg/kg digoxin
Res-SLNs treated
Res-SLN
Improved solubility
Better efficiency
ii) Sonication
270 nm
O
OOH
OH
OH
OH N+OO
OO
O
O
RR
R
OO-
P
HO
Glycerol monostearate (GMS)
Egg yolk lecithin
0
Control
Model
Res
Res-SLN
Digoxin
Control
Model
Res
Res-SLN
Digoxin
Control
Model
Res
Res-SLN
Digoxin
Ejection fractions (%)
Fractional shortening (%)
Heart rate (bpm)
20
40
60
80
00
200
400
600
20
40
60
80
100
Resveratrol (Res)
20 mg/kg
Figure 1 Schematic illustration of the preparation of Res-SLN and its effects against doxorubicin cardiotoxicity.
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to form an oil phase. The organic solvent was removed by
vacuum rotary evaporation (Yarong, Shanghai, China) to
obtain a layer of lipid film. The water phase containing
poloxamer 188 was added to the lipid film in 40
mins through a needle under sustained ultrasound. The
Res-SLN was collected after intermittently sonicated by
a probe sonicator (Xinzhi, Ningbo, China) at 400 W for 12
mins. The amount of egg yolk lecithin, Res and the
volume of the water phase were optimized using central
composite design/response surface method (Table 1).
Morphological characterization of
Res-SLN
The Res-SLN was diluted by pure water. Particle size dis-
tribution and ζ-potential of Res-SLN were determined by
Nano ZS 90 Dynamic Light Scattering (Malvern
Instruments, Malvern, UK). The mean diameter of Res-
SLN stored hermetically at different temperatures on the
5th and 10th day was also determined to evaluate its stabi-
lity. After that, the Res-SLN was imaged under transmission
electron microscopy (TEM) to investigate its morphology
by the approach of a reported procedure.
18
Res-SLN was
diluted at 0.5 mg/mL and placed on a carbon-coated copper
grid, followed by being stained with phosphotungstic acid
solution (1%, w/v) for 1 min. The prepared samples were
imaged under TEM (FEI, Columbus, OH, USA).
Determination of drug loading and
entrapment efficiency
To calculate the entrapment efficiency and drug loading
of Res-SLN, the unencapsulated Res was isolated from
Res-SLN through 10 KD ultrafiltration centrifuge tube
(Millipore, Billerica, MA, USA) by centrifugation at
15,000 rpm for 30 mins (Anting, Shanghai, China) and
detected by HPLC. The HPLC system (Agilent, Santa
Clara, CA, USA) was under the following condition:
Topsil C
18
column (4.6×150 mm, 5 μm, Welch,
Shanghai, China); injection temperature, 25°C; mobile
phase, acetonitrile–water (25:75, v/v, 1.0 mL/min); detec-
tion wavelength, 305 nm; sample size, 10 μL.
Stability of Res-SLN
The effect of storage temperature on the stability of Res-SLN
was studied. Optimized Res-SLNs were prepared repeatedly
and stored at 4°C or 25°C. Samples were taken to determine
the amount of Res by HPLC on the 1st, 5th and 10th day.
In vitro release of Res-SLN
In vitro release study of Res from Res-SLN was performed by
the dialysis method; 30 mL Res-SLN injected into a dialysis
bag (8000–14,000 Da, molecular weight cut-off) was sunk
into 400 mL simulated gastric fluid, stirred at the speed of
50 rpm and maintained at 37°C. At regular intervals, 2 mL
Res containing simulated gastric fluid was taken out and
replaced by 2 mL fresh-simulated gastric fluid. The aliquots
were filtrated with 0.45 μm Millipore filtration before injected
into the HPLC system.
In vivo pharmacokinetic evaluation
Pharmacodynamic study was assessed in SD rats. Ten rats
were randomly divided into two groups and received
intragastric administration (10 mL/kg weight) of Res-
SLN or Res-lipid physical mixture at 10.2 mg/mL doses.
At regular intervals, 0.5 mL blood was collected through
orbit at 0, 5, 10, 20, 30, 60, 90, 120, 180, 360, 720 and
1440 mins after administration into centrifuge tubes con-
taining heparin sodium. The blood samples were subse-
quently centrifuged at 3000 rpm for 10 min to obtain
plasma.
For determination of Res content in plasma, 50 μL genis-
tein as internal standard (IS) was added into 100 μL plasma
before mixed with 850 μL. The mixture was vortexed for 5
mins and centrifuged at 12,000 rpm for 10 mins. The super-
natant was collected and quantified by ultra-HPLC (UPLC)
in tandem with mass spectrometry (MS).
The Ultimate 3000 UPLC system (Thermo, Waltham,
MA, USA) was applied and chromatographic separation
was achieved on a Syncronis C
18
column (2.1×50 mm,
1.7 μm; Thermo). The LC gradient elution was pro-
grammed as 70% water:30% methanol (0 min), 5%
water:95% methanol (3 mins), and 70% water:30%
methanol (8 mins). The flow rate was 0.3 mL/min.
Injection volume was 10 μL. The MS condition for
Res and IS quantification of TSQ triple quadrupole
mass spectrometry (Thermo) with electrospray ioniza-
tion is shown in Table 2.
Table 1 Facts and levels for central composite design-response
surface method
Factors Levels and code values
-α−1 0 +1 +α
Lecithin (mg) 24 67.78 132 196.22 240
Res (mg) 20 40.27 70 99.73 120
Water volume (mL) 100 140.54 200 259.46 300
Abbreviation: Res, resveratrol.
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Pharmacodynamic study of Res-SLN
Animal experiments
Male KM mice (20–25 g) were kept in an experimental animal
barrier environment for 1 week adaptive feeding. Fifty mice
were randomly divided into 5 groups: control group (normal
saline), model group (normal saline), Res group (20 mg/kg
free Res solution), Res-SLN group (1 g/kg Res-SLN lyophi-
lized powder suspension, equivalent to 20 mg/kg free Res),
and positive control group (60 mg/kg Digoxin solution). After
3 days of administration, except the control group, all mice
were treated with single intraperitoneal injection of 20 mg/kg
DOX solution.
19
The control group was injected with the same
volume of normal saline. Thereafter, mice in each group
received the corresponding drug for another 5 days according
to the above-mentioned dose.
Measurement of heart functions
Echocardiography was applied to the measurement of heart
functions. Every mouse was treated with isoflurane for
anesthesia and placed in a supine position with abdominal
hair removed on a Philips Sonos 5500 color Doppler ultra-
sound system (Philips, Amsterdam, Netherlands). Heart rate
(HR), left ventricular ejection fractions (EF) and left ventri-
cular fractional shortening (FS) were recorded and analyzed
to figure the degree of heart failure out.
Hematoxylin and Eosin staining
After the treatment, the mice were sacrificed and the hearts
were removed. The myocardial tissue was circumcised at 1/4
of the apex of the left ventricle and fixed with 10% formal-
dehyde. After dehydration, it was embedded in conventional-
dehydrated paraffin, cut into 5-μm slices, and stained with
Hematoxylin and Eosin. After that, it was made into
a pathological section and microscopically observed under
an optical microscope. Arrangement of cardiomyocytes,
interstitial cells, cell necrosis and inflammatory cell infiltra-
tion were observed under 200-fold microscopy.
Statistical analysis method
Statistical analysis was performed by SPSS22.0 statistical
software (SPSS Inc., Chicago, IL, USA). The data were
expressed as mean ± standard deviation (x ± s). If the data
is in a normal distribution and the variance is homogeneous,
the LSD method is used for comparison between the groups.
P<0.05 indicates that the difference is statistically significant.
Results
Optimization and characterization of
Res-SLN
Central composite design-response surface method
As shown in Tab le 3,theinfluence of three factors (A: lecithin
dosage; B: Res drug dosage, and C: water volume) and five
levels on the response variables (Y
1
: drug loading and Y
2
:
particle size) were studied. The quadratic polynomial regres-
sion equation was fitted by Design Export 7.0 software (Stat-
Ease Inc., Minneapolis, MN, USA). Regression analysis was
performed for determining the optimal region for response
studies (Figure 2A). The model equation is:
Y1¼þ18:30 4:13 Aþ8:61 B0:052 C
1:46 ABþ0:19 ACþ0:17 BC
þ1:10 A20:65 B21:83 C2
;
ðR2¼0:9767;P<0:001Þ
Y2¼þ293:91 45:85 Aþ95:58 B2:61 C
30:41 ABþ9:13 ACþ4:59 BC
þ0:2:A213:96 B22:66 C2
;
ðR2¼0:9599;P<0:001Þ:
The R
2
values are closer to 1 (>0.95), showing that the model
fits well. And, P-values are<0.001, which indicates that the
equation model is significant and has better correlation.
Particle size, zeta potential and drug loading
efficiency
The optimal prescription of Res-SLN was as follows: the
concentration of lecithin was 47.17%, the concentration of
Res was 23.98%, and the volume ratio of water phase and oil
phase was 6.174. The average diameter of Res-SLN was
271.13 nm with a ζ-potential of −25.8±0.33 mV (Figure 2B
and C). The TEM observations showed that the Res-SLN
Table 2 The MS condition for Res and IS quantification
MS condition Parameter settings
Res IS
Spray voltage 3500 3500
Vaporizer temperature 90 90
Sheath gas pressure 25 25
Aux. gas pressure 5 5
Capillary temperature 350 350
Tube lens 61 68
Collision pressure 2.0 2.0
Collision energy 28 35
Protonated molecular ions [M+H]
+
(m/z) 227.0 269.1
Main fragment ion peak (m/z) 143.0 133.1
Abbreviations: MS, mass spectrometry; Res, resveratrol; IS, internal standard.
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was spherical solid particles with uniform size and essen-
tially no adhesion between nanoparticles (Figure 2B).
Stability
To quantitatively detect Res in non-biological samples,
HPLC was introduced. Res was in good linearity over the
concentration range of 1.08–54.00 μg/mL and the linear
equation was Y ¼77:013X 1:1707 R ¼0:9999ðÞ.
The long-term storage experiment showed that the
particle size of Res-SLN would grow up from 271.1 to
389.8 nm as time increases at 25°C, and the drug loading
would be reduced slightly (Figure 2D). While both particle
size and drug loading were stable at 4°C (Figure 2D).
Obviously, the prepared Res-SLN is more stable at 4°C
which indicated that it is more suitable for storing at 4°C
rather than room temperature.
In vitro release of Res-SLN
The in vitro release of Res-SLN was investigated by
dialysis in simulated gastric juice. The release of opti-
mized Res-SLN in simulated gastric juice was relatively
stable. As shown in Figure 2E, over 80% of Res was
released into the medium after 30 hrs. The Res-SLN
release profile revealed a sustained property.
In vivo pharmacokinetics evaluation
UPLC-MS/MS
UPLC in tandem with MS was introduced to detect Res in rat
plasma. Res in rat plasma was in good linearity over the
concentration range of 1.368–684 ng/mL with a linear equa-
tion of y=0.02083x-0.1167 (r=0.9996). The UPLC-MS/MS
method developed for Res was specific(Figure 3A)andin
good precision, accuracy, recovery and matrix effect which
listed in Tab le 4. Meanwhile, Res was stable in rat plasma at
room temperature, −20°C and under freezing and thawing
conditions. (Table 5)
Pharmacokinetics of Res-SLN in rats
The pharmacokinetics study was performed in rats follow-
ing intragastric administration of Res-SLN and Res-lipid
physical mixture, respectively. Concentration–time profiles
of Res are shown in Figure 3B, and the pharmacokinetics
parameters are presented in Table 6. Compared with the
Res-lipid physical mixture, C
max
and AUC of Res-SLN
were significantly increased and T
max
was significantly
shortened (P<0.05), suggesting that entrapping of Res in
the Res-SLN may promote the oral absorption of Res and
SLN as a carrier may promote oral bioavailability of
insoluble drugs.
Table 3 The factors, levels and results of central composite design-response surface method
Number A: lecithin (mg) B: resveratrol (mg) C: water volume (mL) Y
1
: drug loading (%) Y
2
: particle size (nm)
1 132.00 70.00 200.00 18.74 309.37
2 67.78 40.27 259.46 8.76 169.93
3 196.22 99.73 259.46 21.20 334.03
4 132.00 70.00 200.00 18.88 309.37
5 196.22 40.27 140.54 6.72 165.77
6 132.00 120.00 200.00 28.97 372.30
7 196.22 99.73 140.54 21.65 307.70
8 24.00 70.00 200.00 29.67 372.93
9 132.00 20.00 200.00 3.65 121.13
10 132.00 70.00 200.00 18.67 301.23
11 132.00 70.00 200.00 18.83 301.23
12 67.78 40.27 140.54 10.66 198.47
13 132.00 70.00 200.00 18.74 274.97
14 67.78 99.73 259.46 30.65 451.87
15 67.78 99.73 140.54 32.13 479.63
16 240.00 70.00 200.00 12.90 200.57
17 132.00 70.00 300.00 14.33 279.83
18 132.00 70.00 100.00 11.65 277.50
19 132.00 70.00 200.00 16.01 269.97
20 196.22 40.27 259.46 4.88 156.17
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Pharmacodynamic study of Res-SLN
Behavior, weight and survival rate
Fifty mice were divided equally into five groups.
(Figure 4A) All of them were in good spirits and behaved
normally in the first 3 days of the experiments. After
injected with Dox (or saline) to induce heart failure, the
mice in the model group started losing weight and the
action of them was slower (Figure 4B). At the 7th day,
the survival rate was lower. As for the groups injected with
different drugs, the mice were better than the model group
to some extent. The mice in Res-SLN group lost least
weight (P<0.01) and the survival rate was significantly
higher than other drug groups. According to the data
showed in Figure 4C, Res owns the benefit of inhibiting
Dox-induced heart failure and entrapping Res in the SLN
was helpful for improving its efficiency.
A
BD
CE
Drug loading capacity (%)
X1 = A: lecithin (mg)
25.5
18
10.5
3
99.73
84.87
70.00
55.13
40.27
400
375
290
206
120
99.73
84.87
70.00
55.13
40.27 67.78
99.89
132.00
164.11
196.22
140.64
170.27
200.00
229.73
259.46
200
245
290
335
380
67.78
99.89
132.00
164.11
194.22
40.27
140.54
170.27
200.00
229.73
259.46
120
185
250
315
380
55.13
70.00
84.87
99.73
170.27
200.00
229.73
259.46
11
15.75
20.5
25.25
30
140.54 67.78
99.89
132.00
164.11
196.22
140.54
170.27
200.00
229.73
259.46
3
9.6
16
22.5
29
40.27
55.13
70.00
84.87
99.73
B: Res (mg) B: Res (mg)
C: Volume (mL)
C: Volume (mL)
C: Volume (mL)
B: Res (mg)
C: Volume (mL)
A: lecithin (mg)
B: Res (mg)
16
14
12
10
8
6
4
2100 nm
0
0.1
-200
0612 18 24
Time (h)
Time (days)
0510
25oC
4oC
0
10
20
30
100
80
60
40
20
0
40
30 36
0
200000
400000
600000
800000
1000000
1200000
-100 0 100 200
Zeta potential (mV)
Total counts
Resveratrol release (%) Drug loading (%)
110 100 1000 10000
Size (d.nm)
Intensity (%)
A: lecithin (mg) A: lecithin (mg)
A: lecithin (mg)
67.78
99.89
132.00
164.11
196.22
33
X2 = B: Res (mg)
Actual factor
C: Volume (mL) = 200.00
Drug loading capacity (%)
Particle size (nm)
Particle size (nm)
Particle size (nm)
Drug loading capacity (%)
Drug loading capacity (%)
X1 = A: lecithin (mg)
A: lecithin (mg) = 132.00
X2 = B: Res (mg)
Actual factor
B: Res (mg) = 70.00
32.13
3.65
Drug loading capacity (%)
32.13
3.65
X1 = B: Res (mg)
X2 = C: valume (mL)
Actual factor
Drug loading capacity (%)
32.13
3.65
Particle size (nm)
X1 = A: lecithin (mg)
X2 = B: Res (mg)
Actual factor
C: Volume (mL) = 200.00
X1 = A: lecithin (mg)
X2 = C: Volume (mL)
Actual factor Actual factor
B: Res (mg) = 70.00
479.633
121.133
Particle size (nm)
479.633
121.133
A: lecithin (mg) = 132.00
X2 = C: Volume (mL)
X1 = B: Res (mg)
Particle size (nm)
479.633
121.133
Figure 2 The preparation and characterization of Res-SLN. (A) The response surface and contour plots of the fitting equations of the particle size and drug loading. (B)
Particle size and TEM photography of the optimized Res-SLN. Bar = 100 nm. (C) Zeta-potential of the optimized Res-SLN. (D) Stability on drug loading of optimized Res-
SLN at 4°C or 25°C during 10 days. (E) Drug release from the optimized Res-SLN in simulated gastric juice (n=3).
Abbreviations: Res, resveratrol; SLN, solid lipid nanoparticles; TEM, transmission electron microscopy.
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Figure 3 Pharmacokinetics of Res-SLN in rats. (A) The mass pattern of Res reference solution (black peak), rat plasma containing Res (red peaks), and blank rat plasma
(green peaks) in 8-min run. (B) Pharmacokinetic curves of Res-SLN and Res-lipid physical mixture in rats (n=5).
Abbreviations: Res, resveratrol; SLN, solid lipid nanoparticles.
Table 4 The data of methodological verification of UPLC-MS/MS for Res
Number Concentration
(ng/mL)
Precision (%) Accuracy (%) Recovery (%, mean ± SD) Matrix effect (%, mean ± SD)
Intra-
day
Inter-
day
16.84 1.4 11.3 98.1–101.4 87.1±9.3 94.7±3.5
268.4 1.9 9.9 96.4–102.0 93.5±2.5 93.2±3.6
3684 1.2 7.8 98.9–101.9 85.7±3.2 89.0±0.9
Abbreviations: UPLC, ultra-high performance liquid chromatography; MS, mass spectrometry; Res, resveratrol.
Table 5 The stability of Res in rat plasma under different
conditions (n=5)
Groups Concentration
(ng/mL)
Recovery
(%)
RSD
(%)
Room temperature 6.84 94.9±9.9 11.6
68.4 97.1±6.8 7.9
684 92.2±9.1 11.1
−20 °C 6.84 89.6±4.3 10.3
68.4 89.7±6.0 6.7
684 89.0±9.3 13.2
Freeze thawing 6.84 84.3±4.9 14.2
68.4 82.6±11.6 14.1
684 73.2±4.4 6.0
Abbreviations: Res, resveratrol; RSD, relative standard deviation.
Table 6 Pharmacokinetic parameters of Res-SLN and Res-lipid
physical mixture in rats (n=5)
Parameter Res-SLN Res-lipid physical
mixture
t
1/2
(1/h) 0.033±0.012 0.023±0.008
AUC (h × ng/mL) 264.288±26.334* 218.380±14.700
Vd (mL) 0.475±0.109 0.559±0.053
CL (mL/h) 0.014±0.003 0.013±0.003
MRT (h) 8.931±0.480* 11.015±0.389
C
max
(ng/mL) 150.544±32.120* 14.145±2.639
T
max
(h) 0.083±0.000* 3.333±2.160
Notes: *P<0.05, Res-SLN group vs Res-lipid physical mixture group.
Abbreviations: Res, resveratrol; SLN, solid lipid nanoparticles; Vd, apparent
volume of distribution; CL, clearance; MRT, mean residence time.
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Cardiac function detection
The cardiac function was examined by Doppler ultrasonic
diagnostic apparatus and HR, EF and FS were recorded in
Figure 4D–F.
Heart function and HR decreased (P<0.05) in mice in
the model group which was injected with Dox to induce
heart failure but not with therapeutic drugs. The HRs of
Res-SLN and Digoxin group were increased compared to
the model group (P<0.01) in which the HR of Res-SLN
was better. On the contrary, free Res cannot increase the
HR of Dox-induced heart failure mice (P>0.05).
The EF and FS in echocardiography are introduced as
indicators in the clinical evaluation of cardiac function.
20
EF
and FS were decreased in the model group (P<0.01), sug-
gested that the Dox had induced the heart failure in mice.
BothEFandFSinRes-SLNwereincreasedcomparedtothe
model group (P<0.01). Res-SLN can improve the EF and FS
of left ventricle, while Digoxin only improves the EF.
0123456789
25
600
400
200
80
60
40
20
80
100
60
40
20
0
80
90
100
AB
D
C
EF
70
60
50
0246810
Time (days)
0
0
Control
Model
Res
Res-SLN
Digoxin
Control
Model
Res
Res-SLN
Digoxin
Control
Model
Res
Res-SLN
Digoxin
Control
Model
Res
Res-SLN
Digoxin
Body weight (g)
Heart rate (bpm)
Fractional shortening (%)
Ejection fractions (%) Percent survival (%)
30
35
40
45
50
Figure 4 Res-SLN inhibits doxorubicin-induced cardiotoxicity. (A) Schematic representation of the reported treatment for doxorubicin-induced cardiotoxicity. (B) The body
weight of mice in different groups (n=10). (C) The survival rate of mice in different groups (n=10). (D–F) The heart rate, ejection fractions and fractional shortening of mice
in different groups. (*P<0.05, **P<0.01, ***P<0.01).
Abbreviations: Res, resveratrol; SLN, solid lipid nanoparticles.
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It can be concluded that Res-SLN can improve the
decrease of cardiac indexes (HR, EF, FS) induced by
DOX in mice.
Pathological observation
Microstructure of myocardial histopathology showed that the
myocardial fibers of the mice in the control group were well
arranged and no abnormality appeared in the myocardial
cells (Figure 5A). In the model group, the myocardial fibers
were distorted and the cells showed obvious vacuoles degen-
eration of different sizes (Figure 5B). After the mice were
treated with drugs (free Res, Res-SLN and Digoxin), the
myocardial fibers were arranged regularly and a few vacuoles
degenerated in the myocardial cells (Figure 5C–E).
Discussion
In recent years, studies on the antagonism of DOX cardiotoxi-
city by botanicals have become a hot topic,
21
such as some
important active ingredients, Astragalus polysaccharide,
22
oxymatrine,
23
baicalin,
24
rhamnosin,
25
Res,
26
etc. In this
study, we developed a Res-SLN for better absorption and
efficacy after orally taken of Res, directing against its poor
solubility. The presented results verify an important role as
Res-SLN to be a protector against Dox-induced cardiotoxicity.
A role for SLN is to be a submicron drug delivery
system for delivering Res, leading to release gradually and
distribute better in the body. Our Res-SLN made up with
egg yolk lecithin and GMS released approximately 80% of
Res within 24 hrs, which is similar to those with glyceryl
Figure 5 Res-SLN protects myocardium from doxorubicin-induced cardiotoxicity. Myocardial tissue biopsy (200X) of mice in control group (A), model group (B), Res group
(C), Res-SLN group (D), and Digoxin group (E) (the vacuoles were indicated by black arrows and circles).
Abbreviations: Res, resveratrol; SLN, solid lipid nanoparticles.
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behenate.
15,16
The findings of pharmacokinetics demon-
strated that Res-SLN resulted in better distribution in rat
than Res-lipid physical mixture. Other strategy was
reported that Res-loaded liposome was also able to
improve the efficacy of Res on stimulating the prolifera-
tion but preventing its cytotoxicity.
27
This role for Res-SLN is not limited to it, Res-SLN
triggers more credible improvement on cardiac function of
cardio-damaged mice by DOX than free Res. An estab-
lished DOX-induced cardiotoxicity model was employed
to evaluate the Res-SLN; 20 mg/kg DOX was intraperito-
neally injected into mice, which caused severe damage to
cardiac myocytes. As reported, after treated by 20 mg/kg
Dox, the left ventricular myocardium of a rat appeared
massive fragmentation and lysis of the myofibrils, which is
similar to the model mice in our study.
28
The most promis-
ing therapeutics meaning of our findings is that Res-SLN
can mitigate the collapse of the heart caused by DOX,
evidenced by restoring the HR, ES, FE of heart, which is
also proved by the myocardial tissue biopsy of mice.
Conclusion
In our study, SLN encapsulating Res was successfully pre-
pared and optimized by emulsification-diffusion method
followed by sonication. The prepared Res-SLN has
a credible therapeutic effect on DOX-induced cardiotoxicity
in mice by protecting the cardiomyocytes and improving the
HR, EF, and FS values in mice. However, Res-SLN’s
research on anti-myocardial toxicity still requires deeper
and multi-faceted mutual verification. Based on the promis-
ing evidence that we have already found, we will be able to
keep working on elucidating the correlation between multi-
ple mechanisms that Res-SLN is involved.
Abbreviation list
Dox, doxorubicin; Res, resveratrol; SLN, solid lipid nano-
particles; GMS, glycerol monostearate; TEM, transmission
electron microscopy; IS, internal standard; UPLC, ultra-
high performance liquid chromatography; HR, heart rate;
EF, ejection fractions; FS, fractional shortening.
Acknowledgments
The work was supported by grants from Shanghai
Committee of Science and Technology (17401902300 and
18401931400), the Program of Shanghai Academic/
Technology Research Leader (18XD1403700), Shanghai
Three-year Action Plan for the Development of Traditional
Chinese Medicine [ZY(2018-2020)-CCCX-2001-04] and the
National Scientific and Technological Major Special Project
of China (2018ZX09201008-002).
Disclosure
The authors report no conflicts of interest in this work.
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