Showcasing a study on RES encapsulation in peptide

liposomes for anti-breast cancer by Dr Yinan Zhao and As featured in:

Prof. Shubiao Zhang at Dalian Minzu University.
Volume 8
Number 1
7 January 2020
Pages 1–178

Anti-breast cancer activity of resveratrol encapsulated Journal of

in liposomes Materials Chemistry B
Materials for biology and medicine
rsc.li/materials-b

RES encapsulated in tri-peptide liposomes led to obvious

apoptosis of tumor cells and great inhibition of tumors at
low doses, and significantly decreased the toxicity of RES
to mice.

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Anti-breast cancer activity of resveratrol

encapsulated in liposomes†
Cite this: J. Mater. Chem. B, 2020,
8, 27
Y. N. Zhao,a Y. N. Cao,a J. Sun,a Z. Liang,b Q. Wu,a S. H. Cui,a D. F. Zhi,a S. T. Guo,*c
Y. H. Zhen*b and S. B. Zhang *a
Published on 14 November 2019. Downloaded on 3/9/2023 3:01:19 PM.

Received 20th September 2019,
Accepted 5th November 2019
DOI: 10.1039/c9tb02051a
rsc.li/materials-b
Resveratrol (RES) is a naturally occurring and effective drug for tumor prevention and treatment.
However, its low levels of aqueous solubility, stability, and poor bioavailability limit its application,
especially when used as a free drug. In this study, RES was loaded into peptide and sucrose liposomes
(PSL) to enhance the physico-chemical properties of RES and exploit RES delivery mediated by
liposomes to effectively treat breast cancer. RES loaded PSL (the complex: PSL@RES) were stable, had a
good RES encapsulation efficiency, and prolonged RES-release in vitro. PSL@RES was exceptionally
efficient for inhibiting the growth of cancer cells, as the IC50 of PSL@RES in MCF-7 cells was found to
be only 20.89 mmol L1. The therapeutic efficacy of PSL@RES was evaluated in mice bearing breast
cancer. The results showed that PSL@RES at a dosage of 5 mg kg1 was more effective than 10 mg kg1
free RES, and PSL@RES inhibited tumor growth completely at a dosage of 10 mg kg1. PSL@RES induced
apoptosis in breast tumor by upregulation of p53 expression. This then downregulated Bcl-2 and
upregulated Bax, thereby inducing Caspase-3 activation. More importantly, encapsulation of RES within
peptide liposomes greatly reduced the toxicity of free RES to mice. Overall, the simple formulation of
liposomal nanocarriers of RES developed in this study produces satisfactory outcomes to encourage
further applications of liposomal carriers for the treatment of breast cancer.

Introduction including initiation, promotion and progression.4,5 Several

Recently, there is increased interest in the use of agents derived
from natural products for cancer therapy. Resveratrol (3,5,4 0 -
trihydroxystilbene; RES) is a naturally occurring polyphenol
and phytoalexin compound (Fig. 1) found in large quantities
in red wine, berries, peanuts and soybeans. RES has attracted
considerable attention in the last two decades for its purported
health-promoting benefits in humans, such as antioxidant,
anti-inflammatory, and putative benefits for the cardiovascular
system, including cardio-protective activities.1–3 The interest in
anti-cancer properties of RES is heightened after its chemo
preventive effects were demonstrated at all stages of cancer
a
Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of
Education, Dalian Minzu University, Dalian, Liaoning 116600, China.
E-mail: zsb@dlnu.edu.cn; Tel: +86 411 87656141
b
College of Pharmacy, Dalian Medical University, Dalian, Liaoning 116044, China.
E-mail: zhenyhwaner@aliyun.com; Tel: +86 411 86110414
c
Key Laboratory of Functional Polymer Materials of Ministry of Education and
State Key Laboratory of Medicinal Chemical Biology and Institute of Polymer
Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, China.
E-mail: stguo@nankai.edu.cn
† Electronic supplementary information (ESI) available: The encapsulation
efficiency (EE) of PSL@RES; the particle size and zeta potential of PSL@RES
and PSL; the Bcl-2/Bax ratio. See DOI: 10.1039/c9tb02051a
studies propose that RES could have a strong anti-tumor
activity by inducing apoptosis and/or cell cycle arrest in various
mammary cancer cell lines.6–9 In particular, breast cancer is
inhibited by the use of RES, and several animal experiments have
revealed a decreased incidence of mammary tumor formation
and delayed tumor onset following treatment with RES.10–12
However, like many polyphenolic compounds, RES is char-
acterized by poor bioavailability, as the solubility of RES in
water and physiological fluid is less than 0.1 mg mL1.13
Moreover, a high dosage of RES is required to reach significant
beneficial effects.14 Many studies show that greater than
200 mmol L1 RES should be used to inhibit the growth of cancer
cells,15 and usually only dosage over 50 mg kg1 shows anti-
tumor activity in animals. This dosage leads to a high number of
side effects.16 A strategy to circumvent the above-mentioned
limitations is to load RES into water soluble carriers, which offers
chemical and biological protection. Liposomes are one of the
promising nanocarriers that have drawn considerable attention
in the pharmaceutical field for delivering genes, proteins, and
drugs.17–20 The widely acclaimed advantage of liposomes has
been the encapsulation of both hydrophilic compounds (in the
core) and hydrophobic compounds (in the lipidic-bilayers). They
are remarkably cheap, non-toxic and bio-compatible in nature.
However, the major limitation of utilizing liposomes for delivery

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Fig. 1 Structure of trans-3,5,4 0 -trihydroxystilbene (a) and cis-3,5,4 0 -trihydroxystilbene (b). The trans isomer is the biologically active form of the drug.
of hydrophobic drugs is their stability and low drug loading rates.
They are suspected to be capable of causing unquantifiable harm
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to normal tissues.21–23 Although a few studies show that lipo-
somes with specific properties improve the stabilization of
drug-liposomes and drug encapsulation efficiency, the problem
of implementing high dosage utilization of RES remains unresolved.
In one study, RES-loaded and YHWYGYTPQNVI-conjugated lipo-
somes shows a half maximal inhibitory concentration in head
and neck cancer cells of 151.59 mmol L1 RES.24 In addition, the
reported delivery systems mediated by liposomes are too complex,
causing difficulties in implementing the clinical applications
of RES.
In this study, we constructed novel and simple RES liposomes
(PSL@RES) through the assembly of the tri-peptide lipid CDO (1,2-
bis-myristyloxyamidopropyl ornithine), sucrose laurate L126, and
RES. The formulation inhibited the growth of breast cancer at very
low dosage (10 mg kg1 RES) and also exhibited controlled release
of RES in cells due to the presence of a hydrolyzable carbamate
linker in the lipid structure. As we used peptide lipid and sucrose
laurate to form the RES liposome, it exhibited very low toxicity to
mice. To the best of our knowledge, this is the first study to report
the delivery of RES by a peptide liposomal carrier for inhibition of
breast cancer in vitro and in vivo.
Fig. 2 Particle size (a), zeta potential (b), morphology of PSL (c), and PSL@RES (d). Transmission electron microscopy (TEM) images of PSL and PSL@RES
at 8000 magnification (scale bar 100 nm).
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Results and discussion
Blank liposomes were prepared using the peptide lipid CDO
and sucrose laurate L126 at the weight ratio of 1 : 1. RES was
loaded into the liposome to obtain PSL@RES liposomes using
the thin-film hydration method at the lipid/drug weight ratios
of 1 : 1, 5 : 1 and 10 : 1. The encapsulation efficiency (EE) of RES
in PSL@RES at the lipid/drug ratio of 1 : 1 was less than 70%,
but over 90% at the ratios of 5 : 1 and 10 : 1 (Fig. S1, ESI†). To
reduce the toxicity of lipids, we select relatively less lipid
content under the same drug loading. So we chose PSL@RES
with the lipid/drug ratio of 5 : 1 to use for the current study.
PSL@RES was measured for particle size, zeta potential and
morphology, as shown in Fig. 2. The average particle size of the
peptide liposome (PSL) was about 120 nm with a narrow size
distribution (Fig. 2a). Addition of RES increased the average
PSL@RES size by about 20 nm, and these particles still maintained
a homogeneous distribution. The particles within the size range of
100–200 nm can escape systemic clearance by Kupffer cells and
remain in the blood circulation for longer times.25,26 This may
enhance the bioavailability of RES. The zeta potential of blank
liposome PSL was 42.3 mV (Fig. 2b), and that of PSL@RES
decreased slightly after RES encapsulation. TEM images indicate
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that both types of PSL particles were spherical, and RES was visibly
encapsulated in PSL@RES particles as shown in Fig. 2c and d.
The PSL@RES liposome exhibited excellent stability for three
months at 4 1C and it was also stable at 37 1C in 48 h, as evidenced
from measurements of the particle size, zeta potential changes
(Fig. S2, ESI†) and the TEM images of PSL@RES at 4 1C and 37 1C
(Fig. S3, ESI†).
To find out the protection of liposome PSL to RES in the
blood circulation system and tumor micro-environment, and
the controlled release of the drug at the target site, we tested
RES release from the liposome complex in the simulated acidic
tumor micro-environment (pH 6.8) and endosomes/lysosomes
(pH 5.5) using a dialysis membrane sleeve. Fig. 3 showed that
molecular RES reached the highest concentration in a very
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short time, whereas the liposome complex PSL@RES released
RES very slowly. Only 30% RES was released at pH 6.8. At the same
time, under the endosomes/lysosomes environment (pH 5.5), the
RES release rate of PSL@RES increased significantly compared
with RES release from PSL@RES at pH 6.8. Nearly 96% RES was
released in approximately 50 h. These results demonstrated that the
peptide liposomal carrier was stable under neutral pH conditions
and inhibited RES from being released into the blood circulation. In
the acidic environment, the breakage of carbamate linkages in the
lipid structure and the proton sponge effect afforded the PSL@RES
complex efficient RES sustained release.
Fig. 3 Accumulative RES release in vitro. All values are expressed as mean
 SD (n = 3), significance level: **p o 0.01.
Fig. 4 Effects of liposomes PSL@RES on the cell viability of MCF-7 cancer cells (a) and MCR-5 non-malignant cells (b). Data represent the mean  SD of
three independent experiments.
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To evaluate in vitro anticancer activity of RES liposomes, we
first investigated the effects of the RES liposome on the viability
of human breast cancer MCF-7 cells by employing the MTT
assay and using free RES as a control. As shown in Fig. 4a,
both PSL@RES and RES produced attrition of MCF-7 cells in a
dose-dependent manner in the RES concentration range of
2–64 mmol L1. PSL@RES was more efficient in inhibiting the
growth of cancer cells than RES, as the IC50 of PSL@RES and
RES in MCF-7 cells were 20.89 mmol L1 and 25.67 mmol L1,
respectively. To investigate the cytotoxicity of PSL@RES on non-
malignant cells, a viability assay was performed in MCR-5 cells,
which were a variety of human lung fibroblasts. The results in
Fig. 4b indicate that PSL@RES had much lower toxicity to MCR-5
cells than to MCF-7 cells. For example, after treatment with
PSL@RES containing a RES concentration of 64 mmol L1, cell
viability of MCR-5 was 85%, whereas that of MCF-7 cells was only
23%. Furthermore, RES induced more MCR-5 cell death than
PSL@RES. We also found that the blank liposome PSL offered no
impediment to the survival of the two cell lines. The above results
confirmed that PSL@RES liposome was selective in killing cancer
cells over normal cells, and PSL could lower the cytotoxicity of
RES to normal cells. Since the lipid in PSL liposome was
composed of acid-responsive carbamate bond, RES could release
from the liposome after the endocytosis of PSL@RES under the
endosomes/lysosomes environment of tumor cells. In addition,
we used an ornithine head lipid and sucrose ester to prepare
liposomes that are more biocompatible than other components,
such as quaternary ammonium lipids, we could increase the
tolerance of cells to the liposome complexes. Therefore, PSL@
RES appeared to be superior to RES alone, as it inhibited
the growth of tumor cells but had significantly less toxicity to
normal cells.
As apoptosis is one of the most common causes of cell
death, the ability of PSL@RES liposomes to initiate apoptosis
was studied in MCF-7 cells by fluorescence microscopy and flow
cytometry. As shown in Fig. 5a, the population of apoptotic cells
increased with increasing RES concentration. PSL@RES was
capable of inducing greater cell apoptosis than RES alone when
applied at lower RES concentrations. For applications of
PSL@RES, the rate of cell apoptosis was over 60% when the
concentration of RES was 16 mM, but only 45% for RES was
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Fig. 5 MCF-7 cells were treated with PSL@RES for 48 h before staining with AnV-Alexa Fluor 488s and PI and analyzed by imaging under an inverted
fluorescence microscope (a: 200) and quantified by flow cytometry (b: PSL@RES, c: RES, d: PSL). Cells labeled with one or both compounds were
analyzed using a quadrant plot. Significance level: **p o 0.01 and ***p o 0.001.
alone applied under the same conditions. We found that there
was no significant difference between the two groups at a RES
concentration of 64 mM (Fig. 5b and c). In addition, few necrotic
cells were observed for either PSL@RES or RES groups in the
range of all RES concentrations. For the blank liposome PSL
group, apoptotic cells remained at low levels, although they
slightly increased upon raising the PSL concentration (Fig. 5d).
These data also demonstrated that the cytotoxicity of PSL
liposomes was low.
Intracellular reactive oxygen species (ROS) plays an important
role in apoptosis and accumulating experimental evidence has
demonstrated involvement of ROS in RES-induced apoptosis in a
variety of cancer cells.27–29 To produce more information about
the role of ROS in this study, we measured the production of
intracellular ROS by the 2 0 ,7 0 -dichlorofluorescein diacetate
(DCFH-DA) assay. As shown in Fig. 6a, the level of ROS genera-
tion gradually increased with increasing RES concentration.
Significant levels of ROS were generated in MCF-7 cells at a RES
concentration of 64 mM by treatment with both PSL@RES and
free RES. The average value of the fluorescence intensity in cells
were 4 and 3.4 folds higher than those in untreated cells for
applications of PSL@RES and RES, respectively. These results
suggest that the apoptosis induced by RES could occur by
means of its pro-oxidant effects on cancer cells, as observed
in other studies.30–32 Caspases are key effectors of apoptosis in
mammalian cells. Active caspases participate in a cascade of
cleavage events that disable key homeostatic and repair enzymes
and initiate apoptosis. Higher Caspase-8, Caspase-9 and Caspase-3
activities were observed upon addition of PSL@RES and RES
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compared to the untreated control group. Fig. 6b–d showed that
the levels of caspases noticeably increased with the increase in
RES concentration. PSL@RES treated cells had slightly higher
caspase activities compared to the RES group, although there was
no significant difference between the two groups. We concluded
that cells treated with PSL@RES generated more ROS, and
consequently induced greater apoptosis than RES.
In vivo therapeutic efficacy of RES liposome complexes on
tumor-bearing mice was studied as well. BALB/c mice bearing
MCF-7 breast cancer with tumor volumes of approximately
100 mm3 in the right flanks were treated with PSL@RES-10
(10 mg kg1 RES), PSL@RES-5 (5 mg kg1 RES), PSL@RES-2.5
(2.5 mg kg1 RES) and RES-10 (10 mg kg1 RES) with six
consecutive tail intravenous injections on days 1, 3, 5, 7, 9 and 11.
Photos of the tumors in Fig. 7a demonstrated that the growth of all
tumors in the drug treatment groups was inhibited compared with
the control group. The PSL@RES-10 treatment group showed the
greatest growth inhibition. The curves of tumor growth (Fig. 7b)
were used to further analyze the inhibition rates. Compared with
the RES-10 group, the PSL@RES-5 group exhibited slightly greater
efficiency in the reduction of tumor volume, although only half
(5 mg kg1) of the RES concentration was used. More importantly,
PSL@RES-10 was nearly three times more effective than RES-10 at
inhibiting tumor growth, demonstrating a significant difference
over time (p o 0.01). The dosages used here were much lower than
those reported in other studies.33,34 In addition, TUNEL and DAPI
staining of the tumors confirmed that the PSL@RES-10 and
PSL@RES-5 doses resulted in greater apoptosis in tumor cells as
compared to the RES-10 dose (Fig. 7c).
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Fig. 6 Intracellular ROS levels determined by CM-DCHF-DA staining combined with flow cytometry (a). Protein levels of Caspase-8 (b), Caspase-9 (c)
and Caspase-3 (d). Significance levels: *p o 0.05 and **p o 0.01.

Fig. 7 In vivo antitumor efficacy of RES liposomes in MCF-7 tumor-bearing mice. Photos of tumor growth in mice (a). Tumor growth as a function of
time; results are presented as the means  SD. **p o 0.01, ***p o 0.001 (b). Evaluation of tumor cell apoptosis in vivo by TUNEL assay (nuclei were
stained blue with DAPI, apoptotic cells were shown in green) (c). Mice received injections of PSL@RES-10 (10 mg kg1), PSL@RES-5 (5 mg kg1) and
PSL@RES-2.5 (2.5 mg kg1) as indicated by the arrows. Mice with RES-10 (10 mg kg1) and saline treatment were used as controls.

The main causes of breast cell apoptosis may be associated
with changes in the tumor suppression gene and apoptosis
related to protein expression.35,36 We measured the expression
of p53, Bax, Bcl-2 and caspase-3 proteins to characterize the
signaling pathways involved in apoptosis induced by PSL@RES
(Fig. 8a–d). The results of Western Blot experiments demon-
strated that PSL@RES significantly enhanced the expression of
p53 in a concentration-dependent manner. For the treatment
groups of PSL@RES-2.5, PSL@RES-5, and PSL@RES-10, the
expression of p53 was significantly higher than the control group.

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Fig. 8 Protein expression of Bcl-2, Bax, p53 and Caspase-3 in MCF-7 tumor cells by Western blot. Anti-b-actin was used as a loading control to
normalize the data. Tumor-bearing mice were treated with PSL@RES and RES for 13 days, and saline was used as control. (a) Representative
autoradiographs of Bax, Bcl-2, p53 and Caspase-3 expression. Relative protein expression level of p53 (b), Bcl-2 (c), Bax (c) and Caspase-3 (d) were
quantified by densitometry traces. Results are expressed as mean  SD from three-independent experiments.

The PSL@RES-10 group increased their p53 expression by 3.13
fold compared to the control, the RES-10 group increased about
2.01 fold over the same control (Fig. 8a and b). P53 is recognized
as an important suppression gene that subsequently promotes
apoptosis by its ability to control the transcription of pro-
apoptotic members of the Bcl-2 family, such as Bcl-2 and Bax.
In this work, the expression of Bax increased significantly after
the treatment with PSL@RES-10. In contrast, the expression of
the Bcl-2 protein decreased prominently under the same con-
ditions (Fig. 8a and c). The Bcl-2/Bax ratio was found to
diminish 45% in the PSL@RES-10 group over the RES-10 group
(Fig. S4, ESI†). This ratio reflects favorable apoptosis capability
of the liposome complex. Finally, we investigated the Caspase-3
activity in breast tumors and obtained the results shown in
Fig. 8a and d. The treatment group with PSL@RES-10 showed
significantly higher Caspase-3 activity compared to the saline
control and the RES treatment groups. These results confirmed
that RES liposomes induced apoptosis in breast tumors, accom-
panied by the activation of Caspase-3. Based on our data, we
demonstrated that PSL@RES increases the expression of p53 in
breast tumors, downregulated Bcl-2, and upregulated Bax, thus
inducing Caspase-3 activation.37–39
Since toxicity of drug carriers is a major obstacle for their
application in clinical trials, we evaluated general organ toxicity
of the PSL@RES and RES in the in vivo mice model. Compared with
the control group, no apparent signs of dehydration, locomotor
impairment, anorexia, or other symptoms associated with animal
toxicity were observed during the treatment, and no behavioral
abnormalities occurred. Furthermore, the body weight of the
mice after the treatment under three dosages of PSL@RES did
not present evidence of obvious loss during the entire experi-
ment in vivo. For the RES group, however, body weight dropped
noticeably at day 13 after the treatment (Fig. 9a). The body
weight measurements confirmed that the toxicity of RES was
reduced after the encapsulation in the PSL liposome. Major
organs including the heart, liver, spleen, lung and kidney were
stained with hematoxylin and eosin (H&E) for histological
analysis (Fig. 9b). The histological slides of these organs showed
no significant damage or inflammatory lesions after the treatment
with any of the three dosages of PSL@RES. However, RES-10
treatment produced observable heart damage, as myocardial fibers
were disordered, with instances of cellular breaks and note-
worthy infiltration of inflammatory cells. Moreover, liver tissue
was seriously damaged after the treatment with RES-10. Hepatic
peri-portal inflammatory cell infiltration and liver cell diffuse
vacuolar degeneration were observed. The results confirmed that
RES remained encapsulated in PSL liposomes under non-acidic
conditions, leading to the protection of the normal organs.
In order to further investigate in vivo toxicity of PSL@RES,
renal indicators and liver enzymes were also measured. As shown
in Fig. 10, we found that PSL@RES did not produce significant
changes in renal indicators and liver enzymes, including blood
urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST)
and alanine aminotransferase (ALT). Although the BUN values
were slightly low in the PSL@RES-10 and PSL@RES-5 treatment
groups as compared with the saline group, all values were within

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Fig. 9 (a) Body weights of mice were determined every two days, significance level: **p o 0.01. (b) Histological analysis of organs extracted from
tumor-bearing mice (200).
the normal range. For the RES-10 group, however, the ALT value
was greatly elevated compared with the saline group. This further
confirmed that free RES can lead to serious inflammation of the
liver. These data were consistent with our organ histological analysis
(Fig. 8b), further confirming that encapsulation of RES within
peptide liposomes reduces the systematic toxicity of RES to mice.
Conclusions
In summary, we successfully developed PSL@RES liposomes
with impressive biocompatibility and therapeutic benefits for
RES delivery. PSL@RES complexes exhibited improved aqueous
solubility and stability of RES. In addition, the PSL@RES
liposome was inferred to enter tumor cells and tissues by
enhanced permeability and retention. This was followed by
controlled RES release at the target sites over a prolonged
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period of time, leading to the induction of apoptosis of tumor
cells. Importantly, PSL@RES inhibited the growth of tumors at
a significantly reduced dosage of RES with no evident toxicity to
normal tissues in a mouse model. These unique properties
indicate that the PSL@RES liposome delivery nanocarriers may
become a promising new tool for future clinical applications
of RES.
Materials
RES was purchased from Meilun Biotechnology (Dalian, China).
Sucrose laurate L195 (1% monoester, HLB: 1) and sucrose laurate
L1695 (80% monoester, hydrophilic/lipophilic balance, HLB: 16)
were purchased from the Shineroad Company (Shanghai, China).
Tri-peptide lipid CDO was synthesized by our laboratory.
RPMI1640 and fetal bovine serum (FBS) were purchased from
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Fig. 10 Toxicity assessments of PSL@RES to kidney and liver functions. The normal range of ALT, AST, BUN and CREA were 62–120 IU L1, 134–199 IU L1,
4.78–9.52 mmol L1 and 44.20–91.05 mmol L1, respectively. Values are expressed as mean  SD of 3 animals in each group.
Gibco (Grand Island, USA). MCF-7 and MCR-5 cells were obtained
from the Institute of Biochemistry and Cell Biology (Nanjing,
China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) was purchased from Sangon Biotech Co., Ltd (Shanghai,
China). Apo-ONE homogeneous caspase-3, caspase-8 and caspase-9
assay kits were purchased from Beyotime Biotechnology (Shanghai,
China). The ANNEXIN V-FITC-PI apoptosis kit was purchased
from Beyotime Biotechnology (Shanghai, China). All other
chemicals were of reagent grade. All materials were used as
received. All water used was purified using a Milli-Q Plus 185 water
purification system (Millipore, Massachusetts, USA), giving a
resistivity greater than 18 MO cm.
Experimental
Preparation of RES liposomes
Liposomes were prepared using the thin film hydration method.40
To prepare RES liposome (PSL@RES), chloroform solutions of
CDO and sucrose laurate L126 (HLB = 6) were added to a round-
bottomed flask (1 : 1 at weight ratio), along with a solution of trans-
RES in methanol at different lipid–drug weight ratios (1 : 1, 5 : 1
and 10 : 1). The methanol was then evaporated to form a thin film,
followed by high-vacuum desiccation. The dry lipid film was
resuspended in 1 mL distilled water to obtain liposomes in a
concentration of approximately 1 mg mL1. The liposomes were
extruded through a 200 nm aperture in a liposome extruder (FL-1
Avestin, Canada). The extruded liposomes were filtered through
0.22 mm filters to remove the unincorporated drug and to sterilize
them for subsequent studies. Plain liposomes (PSL) were made
without adding RES. For in vivo studies, the lipid concentration
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was scaled up to 2–3 mg mL1 and the hydration time was
increased to improve the incorporation efficiency of RES.
Particle size, zeta potential and morphology
For the measurement of particle size and zeta potential, 20 mL
of the liposomes were diluted in distilled water (1 mL). The
particle size and zeta potential were measured by dynamic light
scattering (DLS) using a Nano-Particle Size Analyzer (HORIBA
SZ-100, Japan). The measurements for each sample were done
in triplicate. The morphology of liposome PSL and RES lipo-
some PSL@RES was determined using a JEOL 2100F transmission
electron microscope (TEM) as previously described.41
Encapsulation efficiency of PSL@RES
The amount of RES loaded in liposomes was determined using
a UV spectrophotometer (UV-2700, Shimadzu Corporation,
Kyoto, Japan). The wavelength for peak detection of RES was
measured by performing a spectral scan of RES dilutions in
methanol in a range between 200–400 nm, and a standard curve
was plotted. The PSL@RES and free RES were separated by
dialysis; the total RES concentration (Ctotal) and free RES
concentration (Cfree) were quantified according to the standard
curve. The encapsulation efficiency (EE) of PSL@RES was
calculated using the following formula:
Ctotal  Cfree
EE ð%Þ ¼  100:
Ctotal
In vitro release of RES
The in vitro release kinetics of RES was measured in a solution
(pH 6.8) composed of water and methanol (90 : 10, v/v) using a
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dialysis membrane following the method of Chinese Pharmaco-
poeia (2015) with minor modifications.42,43 Samples equivalent to
1.0 mg of RES were dispersed in 2 mL of aqueous solution and
then placed in the dialysis bag with Molecular Weight Cut Off
500. The dialysis bag was dipped with the help of a thread in a
conical flask containing 40 mL of solution (37 1C) on a shaking
bed. The dialysate aliquots were withdrawn at 0.5, 1, 2, 4, 6, 8, 10,
12, 20, 24, 28, 32, 36, 44, 48, 64, 72 and 84 h and the same
amount of fresh solution was added to the medium. The RES
released into the medium was determined using a UV spectro-
photometer at 308 nm.
Cytotoxicity of RES liposome on cancer cells
To study the inhibition of liposome complexes of PSL@RES on
Published on 14 November 2019. Downloaded on 3/9/2023 3:01:19 PM.
tumor cells, MCF-7 cells or MCR-5 cells were seeded at 5 
104 cells per well in 96-well plates for 24 h at 37 1C and 5% CO2.
The medium was replaced with media containing either free
RES, PSL@RES, or PSL at a RES concentration ranging from
2 mm to 64 mm. After 48 h, the drug-containing media was
discarded from the wells, and cells were washed twice with
complete medium, then MTT (20 mL, 5 mg mL1, pH 7.4) was
added. Cells with reagent were incubated for 4.5 h, following
which 150 mL of dimethyl sulfoxide (DMSO) was added into
each well to dissolve the substrate. The absorbance at 570 nm
was monitored by using the enzyme mark instrument (Sunrise
Tecan, Australia). Cells without lipoplexes served as controls.
Cell viability was expressed as a percentage of the control. Cell
viability was calculated as [[Abs]sample/[Abs]control  100%]. All
experiments were repeated three times, and data are presented
as the mean  standard deviation (SD). Half maximal inhibitory
concentration was calculated by the Logit method.
Apoptosis induction in vitro
To study the apoptosis induction capability of the RES liposomes,
MCF-7 cells (105 cells per well) were seeded in 24-well plates and
incubated at 37 1C under 5% CO2 for 18–24 h to produce a
confluence fraction of about 80%. They were then treated with
PSL@RES, PSL and free RES (RES concentration of 2–64 mM) for
48 h to induce apoptosis. After 48 h, the cells were washed twice
with 1 PBS, and detached using 0.25% trypsin-EDTA. The cells
were collected in tubes and centrifuged at 1500 rpm for 5 min to
obtain the cell pellets. The supernatant was discarded, and cell
pellets were rinsed twice with ice-cold PBS. The cell pellets were
resuspended in 195 mL 1 Annexin V-FITC binding buffer.
Alexa Fluors488 (5 mL) and propidium iodide (PI) solutions
(100 mg mL1, 1 mL) were added to each 100 mL cell suspension.
The cells were incubated in the dark at room temperature for
10–20 min, after which 400 mL binding buffer was added to the
tubes. The stained cells were analyzed by flow cytometry (Becton
Dickinson, Franklin, NJ, USA) at an excitation wavelength of
488 nm. The emission of Alexa Fluors488 was recorded in the
FL-1 channel while that of PI was recorded in the FL-3 channel. Cells
were gated upon acquisition using forward vs. side scatter to
eliminate the dead cells and debris, and 10 000 gated events were
collected for each sample. Analysis was performed making use of
the CellQuestt Pro software (Becton Dickinson, Franklin, NJ, USA).
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Measurement of the generation of intracellular ROS
MCF-7 cells were seeded at 105 cells per well in 24-well plates
and allowed to adhere for 18–24 h. All the treatments were
carried out in DMEM. Following 24 h, the cells were washed
once with PBS and detached using 0.25% trypsin–EDTA. The
cells were centrifuged at 1500 rpm for 5 min and treated with a
solution of the ROS-indicator dye DCFH-DA (500 mL) for 20 min.
Cells were analyzed immediately for ROS levels by flow cyto-
metry using a Becton Dickinson FACSCaliburt.
Caspase activity assay in vitro
The Apo-ONE homogeneous caspase-3, caspase-8 and caspase-9
assay kits were used to study the activation of caspases. MCF-7
cells were seeded at a density of 105 cells per well in 24-well
plates and allowed to adhere for 18–24 h. They were treated for
24 h with PSL@RES, PSL and free RES at a RES concentration of
2–64 mM. After 24 h, the cells were washed once with serum free
media. The caspase substrate was diluted in the caspase buffer
according to the Caspase Activity Assay Kit, and 50 mL was
added to the cells containing 50 mL of media. The plates were
incubated at room temperature in the dark with gentle shaking
for 2 h. The fluorescence of each well was read at a wavelength
of 405 nm using a Bio-Tek Synergy HT multi-detection micro-
plate reader (BioTek, Winooski, VT, USA).
In vivo antitumor activity
All animal experiments were performed in compliance with the
Animal Management Rules of the Ministry of Health of the
People’s Republic of China (Document No. 55, 2001) as well as
under institutional guidelines. All experiments were in accordance
with and approved by the Institutional Animal Care and Use
Committee of Dalian Medical University. Male BALB/c nude mice
of 4–6 weeks of age (body weight: 18–20 g) were purchased from
Nanjing Biomedical Research Institute of Nanjing University.
The tumor-bearing mice model was established by subcuta-
neous inoculation of MCF-7 cancer cells into the right flank of
BALB/c mice. When the tumor volumes were approximately
100 mm3, the mice were randomly divided into five groups.
Three groups were treated with PSL@RES in three dosages
(10 mg kg1, 5 mg kg1 and 2.5 mg kg1), the other groups
were treated with free RES (10 mg kg1) in DMSO aqueous
solution; normal saline solution was used in the negative control
group. All groups were injected intravenously six times in 2 day
intervals, and the body weights of mice and the tumor volumes
were measured at the same intervals. The tumor volume was
calculated using the formula for a prolate ellipsoid as follows:
tumor volume = ((width)2  length)/2. All the data of tumor
volumes and body weight were expressed as relative values, with
respect to original volume and weight. After treatment, all the
animals were euthanized and sacrificed, and major organs and
tumor were collected and washed with PBS, and then fixed in 4%
formaldehyde. Subsequently, all the organs and tumors were
inspected by histopathological techniques. Hematoxylin and eosin
(H&E) staining and the terminal deoxynucleotidyltransferase-
mediated dUTP nick end labeling (TUNEL) assay were applied to
J. Mater. Chem. B, 2020, 8, 27--37 | 35

Paper
evaluate for acute toxicity and apoptosis of cancer cells.44 The
blood serum was obtained from the mice before sacrifice by
means of retro-orbital bleed and centrifugation. Hepatic and
renal damage was assessed by measuring the level of Aspartate
transaminase (AST), Alanine transaminase (ALT), Creatinine
(CREA), and Blood urea nitrogen (BUN) in the serum samples.
Apoptosis factors activity assay by Western blot
After the mice were euthanized, the tumors were removed and
collected by centrifugation to extract protein using modified
RIPA buffer and 1 tablet/10 mL of protease inhibiter cocktail
tablets (Roche, Basel, Switzerland). The lysate was centrifuged
at 12 000 rpm for 15 min at 4 1C and the protein extract was
collected in the supernatant. Protein concentration was deter-
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mined using the Pierce BCA protein assay kit from Thermo
Scientific (MA, USA). For Western blotting, 25 mg of total
cellular proteins were separated on a 10% SDS-PAGE gel and
transferred to polyvinylidene difluoride (PVDF) membranes
(Hybond-P, Amersham, USA). Non-specific binding sites were
blocked by incubating the membranes in PBS-0.1% Tween-20
with 5% skimmed milk (PBS-T) for 2 h at room temperature. The
membranes were incubated with primary antibodies overnight at
4 1C, and then washed three times with PBS-T. The membranes
were then incubated with an appropriate secondary antibody for 2 h
at room temperature and washed three times with PBS-T. We used
the primary antibodies: anti-b-actin (1 : 2000), anti-Bax (1 : 2000),
anti-Bcl-2 (1 : 2000), anti-Caspase 3 (1 : 500) and anti-p53 (1 : 1000).
The blots were developed according to the manufacturer’s chemi-
luminescence protocol (Roche, Basel, Switzerland), and analyzed
using Scion Image Software.
Statistical analysis
All data are expressed as mean  standard deviation (SD) for
three independent tests. Differences between groups were
analyzed using a one-way analysis of variance (ANOVA) followed
by LSD’s multiple comparison post hoc test. The results were
considered statistically significant when p o 0.05, *; p o 0.01, **;
p o 0.001, ***.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21606041, 21776044,
21503035), Fundamental Research Funds for the Central
Universities.
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