Novel glycyrrhetinic acid conjugated pH-sensitive liposomes for the delivery of doxorubicin and its antitumor activities

Article abstract of DOI:10.1039/c6ra01580h

Over the last few decades pH-sensitive drug delivery systems have been successfully developed for the treatment of cancers by improving the therapeutical effect. In this study, a novel pH-sensitive conjugate glycyrrhetinic acid-polyethylene glycol-Schiff

Full text of DOI:10.1039/c6ra01580h

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Novel glycyrrhetinic acid conjugated pH-sensitive liposomes for the delivery
of doxorubicin and its antitumor activities
Qing Chena, Huaiwei Dingb, Jinxing Zhoua, Xiufeng Zhaoc, Jiulong Zhanga, Chunrong Yangd,
Kexin Lia, Mingxi Qiaoa, Haiyang Hua, Pingtian Dinga, Xiuli Zhaoa*
Received 00th January 20xx,
Accepted 00th January 20xx
Over the last few decades pHꢀsensitive drug delivery system has been successfully developed for the treatment of cancers by
improving the therapeutical effect. In this study, a novel pHꢀsensitive conjugates glycyrrhetinic acidꢀpolyethylene glycolꢀ
Schiff bondꢀcholesterol (GPSC) has been synthesized successfully and use it to constract Doxorubicin (DOX)ꢀloaded
liposoms (GPSLP/DOX) with both pHꢀsensitive feature and active targeting ability. DOX was incorporated into liposomes
using thinꢀfilm hydration method with a relatively high encapsulation efficiency (EE %) and drug loading content (LC %).
Physicochemical characteristics, in vitro release behavior, cellular toxicity and cellular uptake, in vivo biodistribution as
well as in vivo antitumor activities of GPSLP/DOX were investigated. GPSLP/DOX showed significantly pHꢀsensitive
feature in the in vitro release assay. All the blank liposomes were nontoxic in vitro cytotoxicity assay. In MTT assay,
GPSLP/DOX showed the highest cell cytotoxicity among all the groups. Cellular uptake study revealed that GPSLP/DOX
could be taken up efficiently via receptorꢀmediated endocytosis and pHꢀresponsive drug release of DOX into cytoplasm,
which resulted in a higher cytotoxicity and therapeutical effect. In vivo NIR fluorescence image study showed that
GPSLP/DOX could specifically accumulative in liver and tumor sites via receptor mediated endocytosis. In vivo antitumor
activities results showed that this novel GPSLP/DOX could significantly inhibit tumor growth and prolong survival time due
to its pHꢀresponsive behaviour and GAꢀmediated endocytosis, resulting in a lower systematic toxicity and higher
therapeutical effect. All these results confirm that this GAꢀmediated pHꢀsensitive GPSLP/DOX is a novel nanocarrier for
the delivery of antitumor agent DOX to reach higher toxicity effect against tumor tissue.
DOI: 10.1039/x0xx00000x
www.rsc.org/
has attracted considerable attention13ꢀ15. As is known to us all, The
1
Introduction
membrane of liposome is consist of phosphatidylcholine (PC) and
cholesterol. Due to the structure of liposomes is similar to the cell
membrane, it is easy to be untaken by cells. However, easily
recognized by the reticuloendothelial system (RES) in blood system
is becoming the major barrier in clinical application of liposomes16,
17. To solve this problem, polyethylene glycol (PEG) modified
liposomes has been widely utilized to reach long circulation and
avoid the elimination by the RES18, 19. This long circulating effect
enables these smallꢀsized carriers to “passively” accumulate in tumor
tissue through the enhanced permeation and retention (EPR) effect.
However, the PEG shell of the liposomes seriously hindered the
interactions between the DDS and cells membrane after arriving at
tumor tissues20, 21. Ideally, a stable PEG coating is required for
longer blood circulation to avoid being recognized by the RES. After
Cancer is the leading cause of death with an estimated 8.2 million
deaths worldwide1. Hepatocellular carcinoma (HCC) is the fifth
most common cancer in the world and the third most common cause
of cancer mortality2ꢀ4. Despite various anticancer strategies have
been used in clinical, chemotherapy continues to be an important
therapeutical option for different malignancies, especially for
primary advanced and metastatic tumours5ꢀ7. However, severe side
effects and systematic toxicity limit the tolerable dose of many
antitumor drugs8, 9, including doxorubicin (DOX). Targeted tumor
therapy is one of the most ideal option for patients undergoing
chemotherapy as it could increase therapeutical efficacy and reduce
adverse effects10ꢀ12. Therefore, it is extremely important to seek new
therapeutic strategies to treat liver cancer.
In recent years, various drug delivery systems (DDS) have been reaching target tissue, PEG chain becomes unnecessary or even
developed, which could provide targeted delivery anticancer drugs to undesirable. Removal of the PEG shielding can allow for more
tumor tissue. Liposomes based delivery system, as an appealing efficient cellular association of the carrier. Furthermore, hydrophilic
candidate with decreased side effects and enhanced tumor delivery, shell of PEG will make a weak exclusion between liposomes and the
cell membrane, thus decrease the cellular uptake and showed lower
cytotoxicity22, 23. There are multiple design strategies that can be
implemented to overcome the barriers associated with the use of
PEGylation, including the conjugation of PEG on the surface of
liposomes via pH sensitive18, matrix metalloprotease (MMP)
27
sensitive24, 25, thermo sensitive26, and redox sensitive28. Among
these approaches, pHꢀsensitive chemical bonds have been most
widely used to design sensitive nanoꢀsystems for drug delivery in
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cancer therapy. Tumours have been demonstrated to exhibit acidic cholesterolꢀSchiff baseꢀPEG derivative also contained an aminoꢀ
pH ranging from 5.7 to 7.0, while the pH of normal tissue is 7.429, 30
group moiety to provide the possibility of coupling receptor ligands
.
Significant pH difference could also be found at the subcellular such as GA. We hypothesized that PEGylated liposomes accumulate
level: pH value of early endosomes and late endosomes were 5.5 and in target sites via the GA (“active” targeting). Within the “acidified”
4.5, respectively31. As a result, the design of pHꢀsensitive acidꢀlabile of the tumour microenvironment, the carriers will lose their PEG
liposome may be a good strategy for cancer treatment, owing to the coating by hydrolysis of a Schiff base pHꢀsensitive bond, and
existence of mildly acidic microenvironment in both the interstitium facilitate the tumor cellular uptake. Once internalized in target cells,
of solid tumours and endosomes of tumor cells. Schiff base bond is DOX was expected to release at the acidic pH due to escape from
an excellent pHꢀsensitive candidate bond which is easy to construct endosomes with the help of the DOPE, allowing cytoplasmic and
and can be hydrolyzed by the acidic pH of tumor nuclear accumulation of released DOX.
microenvironment32, 33
.
2
Materials and methods
In order to ensure the anticancer drug could efficiently accumulate in
2.1 Materials and animals
GA (purity >98% by HPLC) was purchased from Fujie
Pharmaceutical Co., Ltd. (Xi'an, China). DOX·HCl was obtained
from Huafeng United Technology Co., Ltd. (Beijing, China). 1ꢀ
ethylꢀ3ꢀ(3ꢀdimethylaminopropyl)
carbodiimide
hydrochloride
(EDC·HCl) and Nꢀhydroxylꢀsuccinimide (NHS) were obtained from
GL Biochem., Ltd. Soybean phospholipid (SPC, No.20140728)
was manufactured by Shanghai Advanced Vehicle Technology
(AVT) Pharmaceutical Co., Ltd.; cholesterol (Chol, No.20140908)
was purchased from Anhui Tianqi Chemical Technology Co., Ltd.;
AminoꢀPolyethylene glycolꢀcarboxyl group (NH2ꢀPEG2000ꢀCOOH,
MW, with 2kDa of PEG chains) was purchased from Seebio
Biotech, Inc. (Shanghai, PR, China). 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,
5ꢀ diphenyltetrazolium bromide (MTT) was also obtained from
Sigma Aldrich. Cholesteryl chloroformate were purchased from J&K
Scientific LTD. Dichloromethane (DCM, 99%, kelong chemical,
Chengdu, China) was distilled over anhydrous sodium sulfate.
Tetrahydrofuran (THF, 99%, Kelong chemical, Chengdu, China)
was freshly distilled over sodium prior to use. All other reagents
were of commercial special grade and used without further
purification.
Scheme 1: schematic diagram of GPSLP/DOX and mechanism of
GPSLP/DOX to enhance antitumor activities.
Nude mice (20 ± 2 g) were used in the biodistribution and antitumor
assay. All care and handing of animals were performed with the
approval of Institutional Authority for Laboratory Animal Care of
Shenyang Pharmaceutical University.
tumour cells, it is necessary to design an effective delivery system
which could not only incorporate the therapeutic agents but could
also be efficiently uptaken by the tumour cells. Glycyrrhetinic acid
(GA) is one of the main bioactive compounds of licorice and is
2.2 Synthesis and characterization of GA-modified PEG2000
SIB-Chol
-
widely used in medicine for the treatment of much pathology34ꢀ36
.
Recent reports also showed that carriers modified with GA could
reach higher accumulation in the hepatocytes because of abundant
GA receptors on hepatocyte membranes, suggesting that vehicles
modified with functional group–GA could improve the tumor
To synthesis the GAꢀpolyethylene glycol (PEG)ꢀschiff baseꢀ
cholesterol (GPSC) conjugates, paraꢀhydroxyl benzaldehyde and
paraꢀphenylenediamine were used to constitute pH sensitive Schiff
base bond. The whole process can be divided into two steps:
targeting efficiency for hepatocellular carcinoma chemotherapy37, 38
.
(i) Synthesis of NH2ꢀPEGꢀSIBꢀChol. Cholesteryl chloroformꢀmate
was reacted with paraꢀhydroxyl benzaldehyde (molar ratio = 2:1) in
dry DCM at room temperature under argon in the presence of Nꢀ
Ethyldiisopropylamine (DIPEA) for about 4 h. After thin layer
chromatography (TLC) showed the disappearance of paraꢀhydroxyl
benzaldehyde, the reaction mixture was poured into distilled water
and used DCM to extract. The extraction was evaporated under
vacuum. The residue was purified on a silicaꢀgel chromatography
column (DCM: MeOH = 1:1) to get CholꢀCHO. Then, CholꢀCHO
and paraꢀphenylenediamine (molar ratio=1:2) were reacted in
methylbenzene with gentle stirring at 120℃ in oil bath overnight.
After TLC showed the disappearance of CholꢀCHO, the mixture was
Here, we report the design of multifunctional liposomal
a
preparation acting GA as a targeting moiety for enhanced cellular
delivery ability and anticancer activity. This drug delivery system
was relatively stable in blood system, while degraded in tumour
microenvironment via pHꢀsensitive Schiff base bond between a long
shielding PEG chains and cholesterol (PEG2kꢀSIBꢀChol, PSC)
(Scheme 1). As another important component of liposome
membrane, cholesterol is more chemically stable and easy to be
modified compared with phospholipids39ꢀ41. Instead of coupling PEG
to phospholipids as described previously42, we coupled functional
PEG via Schiff base linkage to cholesterol as lipidic anchor. This
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evaporated under vacuum, the residue was dissolved in CH2Cl2, and exchange resinꢀmini column centrifugation method. DOX was
the insoluble material was purified on a silicaꢀgel chromatography determined using a UV–Vis spectrophotometer at 481 nm. Samples
column. The collected solution was evaporated under vacuum, the
residue (NH2ꢀSIBꢀChol) was evaporated again under vacuum and
stored under −20 ◦C until use.
(100ꢁL) were loaded onto a miniꢀcolumn (56 mm×8mm i.d.) and
eluted with DI water to separate unloaded DOX. The EE% and LC%
could be calculated as follows:
EE%= the amount of DOX in the liposomes ×100
For the synthesis of the PEGꢀSIBꢀChol conjugate, COOHꢀPEG2000
ꢀ
the total amount of DOX
NH2 and NH2ꢀSIBꢀChol (molar ratio = 1:1.5) were reacted in DCM
with gentle stirring at room temperature in the presence of DIPEA
and EDC over night. After TLC showed the disappearance of
COOH–PEG2000ꢀNH2, the mixture was extracted by distilled water
and DCM. After evaporated the DCM under vacuum. The residue
was dissolved in chloroform and filtered again to purify production.
DL%= mass of DOX in the liposomes ×100
mass of DOX/liposomes
2.5 Drug release studies
In vitro release behaviour of DOX from liposomes was measured by
a dialysis (MWCO 3.5 KDa) method. The release profiles of DOX
from different liposomes were studied at 37 °C under pH 7.4
(normal pH) and pH 6.0 (tumor microenvironment pH). In brief, 2.0
ml of liposomes were added into the dialysis bag and placed it into a
conical flask with 100 ml PBS solution in different pH. In order to
meet sink condition, 0.5% Tween 80 was added into the release
medium. All the flask were placed into a shaking incubator and the
stirring speed was adjusted to 100 rpm. At different time point, 1.0
ml of the release medium was taken and equal volume of flash buffer
was added to the flask. The amount of released DOX was measured
using a UV spectrophotometer at 481 nm and cumulative amount of
DOX release was calculated according to a standard calibration
curve (data were not shown). All the release experiments were
conducted in triplicate.
(ⅱ) Synthesis of GAꢀPEGꢀSIBꢀChol (GPSC). For the synthesis of
the GPSC conjugate, PEGꢀSIBꢀChol and GA (molar ratio = 1:1)
were reacted in DCM with gentle stirring in the presence of DIPEA,
HATU and EDC at room temperature for 48 hours. The GAꢀPEGꢀ
SIBꢀChol conjugate was extracted same as the method of PEGꢀSIBꢀ
Chol conjugate.
2.3 Preparation of DOX-loaded liposome
DOX·HCl was stirred with quintuple the number of mole of TEA in
12.5% methanol/chloroform mixture overnight to obtain the DOX
base31. Liposomes were made by the established thin film hydration
method followed by extrusion. GA modified PEGylated pHꢀ
responsive liposome (GPSLP/DOX) was prepared using thin film
hydration method. DOX: SPC: DOPE: Chol: GPSC=4:40:40:5:5
(total lipid content: 3 mol mlꢀ1) were distilled in ethanol, dried into a
thin film on a rotary evaporator, and then hydrated with PBS buffer
(pH 7.4), followed by 1h (37 ℃) incubation and 2 min bathꢀtype
sonication. Then it was sonicated with a probeꢀtype sonicator at
200W power for 3min. The resulting liposomes were filtered through
polycarbonate membranes of gradually decreasing pore sizes (0.45
and 0.22 µm). Unloaded DOX was removed using cation exchange
resinꢀmini column centrifugation method. DOX Lip (LP/DOX) was
prepared using the same way except GPSC was replaced by
cholesterol. The prescription of DOX loaded PEGꢀLip (PLP/DOX)
was DOX: SPC: DOPE: Chol: mPEG2000ꢀChol = 4:40:40:5:5 and
prepared using the same way. Different blank liposomes were
prepared in the same method without adding any DOX.
2.6 In vitro cytotoxicity assay
2.6.1
Cell culture
Human hepatocellular carcinoma cell line (HepG2 cells) from the
Cell Culture Centre of Peking Union Medical College (Beijing,
China) was grown in RPMIꢀ1640 medium (Gibco) supplemented
with 10% FBS (Gibco), 100 IU mlꢀ1 penicillin, and 100mg mlꢀ1
streptomycin. The cells were maintained at 37℃ in a humidified
incubator with 5% CO2 atmosphere. Cells in the exponential phase
of growth were used in the experiments.
2.6.2
In vitro cytotoxicity
Cytotoxicity of free DOX and different liposomes were evaluated by
MTT assay. Cells were seeded in a 96ꢀwell plate (NUNC, Roskilde,
Denmark) at a density of 7000 cells per well, and incubated for 24 h.
The medium was then replaced with the DOXꢀloaded liposomes or
free DOX solution at equivalent drug concentrations ranging from
0.1 ꢁg mlꢀ1 to 20 ꢁg mlꢀ1 and further incubated for 24h and 72 h,
respectively. At designated time intervals, 20µL MTT reagent (5 mg
mlꢀ1) was added to each well and the cells were incubated for another
4 h at 37℃. The medium was removed and then 200ꢁL DMSO were
added to each well to dissolve the formazan crystals formed by the
living cells. Cells without treatment were used as control. The
absorbance at 490 nm of the solution in each well was recorded
using a Microplate Reader (Tecan Spectra, Wetzlar, Germany/
Austria). Cell viability was calculated using this formula:
2.4 Characterization of DOX-loaded liposomes
The particle size distribution and zeta potential of different
liposomes were determined. Briefly, different formulations were
dissolved in distilled water to reach a proper concentration and
detected using a laser light scattering technique (LLS; Nanoꢀ2S90
Zetasizer, Malvern, UK).
The morphology of the DOXꢀloaded liposomes were observed using
transmission electron microscopy (TEM) (Jeol JEMꢀ2000EX,
Tokyo, Japan). In order to avoid multiscattering phenomenon,
liposome dispersions were diluted properly with purified water. The
samples were negatively stained by 4% phosphotungstic acid and
dried on carbonꢀcoated grids for observation.
Asample ꢀAblank
=
Cell viability (%)
×100
Acontrol ꢀAblank
The drug encapsulation efficiency (EE %) and drug loading content
(LC %) of DOXꢀloaded liposomes were determinated by the cation
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2.7 Cellular uptake studies
The antitumor activity was evaluated in terms of tumor weight (g).
The tumor growth inhibition rate (IR) was calculated according to
Eq.:
2.7.1
Confocal laser scanning microscopy (CLSM)
HepG2 cells were seeded in a 6ꢀwell plate at a density of 5×105 cells
per well. After 24 h incubation, the medium was replaced with
serumꢀfree RPMI 1640 medium and different formulation were
added to each well to make the final concentration of DOX was
10ꢁg mlꢀ1 and further incubated for 0.5, 2 and 4h. Then the cells
were washed with cold PBS for three times and fixed with 4%
paraformaldehyde at room temperature for 15 min, following by
nuclei staining with Hoechst 33258 for 15 min. The fixed cells were
finally rinsed twice with cold PBS and then imaged by a laser
scanning confocal microscope (Olympus FV1000ꢀIX81, Tokyo,
Japan).
IR (%) = 1 ꢀ weight of tumor in the experimental group ×100%
weight of tumor in the control group
At day of 17, half of the mice were sacrificed and the tumor were
harvested and other six mice were used to measure the survival
curves. The survival time of all mice were recorded each day until
all mice’ death.
2.9 Statistical analysis
All experiments were performed at least three times. Quantitative
data are presented as the mean ± SD. Statistical comparisons were
determined by the analysis of variance (ANOVA) among at least
three groups or Student’s tꢀtest between two groups. Pꢀvalues<0.05
and Pꢀvalues <0.01 were considered statistically significant.
2.7.2
Flow cytometry analysis
Flow cytometry was used to quantitative analysed the cellular uptake
of various DOXꢀloaded formulations. DOX serves as a fluorescence
probe to examine the uptake of drugꢀloaded liposomes owing to its
intrinsic fluorescence property. Briefly, cells were seeded into 6ꢀwell
plates at a density of 1×106 cells per well and cultured at 37℃ in a
5% CO2 for 24 h to allow attachment. The medium was then
replaced with serumꢀfree RPMI 1640 medium and different DOX
formulation was added into each well (equivalent DOX
concentration, 10 ꢁg mlꢀ1). After incubation for different times, the
cells were washed using PBS for three times, harvested and
resuspended in 0.5 ml of PBS for the flow cytometrix analysis
(Becton Dickinson，San Jose, CA).
3
3.1
Results and discussion
Synthesis and characteristic of GPSC
To prepare the GA modified PEG cholesterol derivate (GPSC), we
first synthesized CholꢀSIBꢀNH2 by two step reactions as shown in
Fig.1. The first step consisted of introducing paraꢀhydroxy
benzaldehyde to the cholesterol molecule (CholꢀCHO). In the second
step, ꢀSIBꢀ group was synthesized by conjugating paraꢀ
phenylenediamine to the terminal group of CholꢀCHO (CholꢀSIBꢀ
NH2). Multifunctional PEG (NH2ꢀPEG2000ꢀCOOH) was used to
modify the CholꢀSIBꢀNH2 via amidation reaction. The –NH2 group
2.8 Animal studies
2.8.1
Animal culture
In this study, H22 bearing nude mice were chosen for the in vivo
assay. In brief, H22 cells (0.2 ml) were carefully injected
subcutaneously into the right limb armpit of the mice at 5×106 H22
cells in 200ꢁL. When the tumor was reached approximately 100
mm3, the mice were used for the different experiments.
2.8.2
In vivo biodistribution assay
In the biodistribution assays，the H22 tumor model was used to
evaluate the biodistribution of different formulation. The
fluorescence probe 1, 1’ꢀdioctadecylꢀ3, 3, 3’, 3’ꢀ tetramethyl
indotricarbocyanine Iodide (DiR, Invitrogen, USA) was chosen to
load into the different liposomes through via the tail veins. The
preparation method of different DiR liposomes were in the same way
as DOX loaded liposomes. At various time point the mice were
anesthetized and imaged using an in vivo image system (Carestream,
USA).
2.8.3
In vivo antitumor activity
In vivo antitumor activities of different formulation were assessed in
nude mice bearing H22 model. Mice were randomly separated into
five groups (n=12) as followed: saline, DOX solution, LP/DOX,
PLP/DOX, GPSLP/DOX at a dose of 5 mg/kg, respectively.
Different formulation were given through tail vein every 2 days for 4
times. Tumor volume and body weight were measured every 2 days.
The tumor size was determined with a calliper in two dimensions
and calculated using the following Eq.:
Fig.1: Synthesis pathway of GPSC.
was permitted to react with GA via amidation reaction in the end. As
shown in Fig.2, The formation of GPSC was confirmed by the H
1
NMR spectrum and chemical shift values were described as follows:
peaks of 1.07ꢀ2.03 ppm corresponding to the protons of cholesterol.
The peaks at 0.7ꢀ1.59 ppm corresponding to the protons of GA unit.
The chemical shift of PEG2000 was 3.70 ppm and the chemical shift
of Schiff base bond of the pHꢀsensitive GPSC was 8.50 ppm. All
the longest diameter of tumor×(the shortest diameter of tumor)2
V
=
2
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these data confirmed that the novel pHꢀsensitive biomaterial GPSC sufficient time in vivo, which could reach accumulation in tumor
was synthesized successfully.
sites. Therefore, in this study, the liposomes with anionic charge
could efficiently escape from RES and accumulated to the tumor.
Table 1: characterization of different liposomes.
Particle size
(nm)
Polydispersity
index
Zeta potential
(mV)
EE %
LC %
Blank Lip
LP/DOX
PLP/DOX
GPSLP/DOX
101.5±2.3
110.2±4.5
120.9±1.8
135.5±2.6
0.103±0.03
0.212±0.06
0.254±0.04
0.243±0.08
ꢀ10.23±0.68
ꢀ7.45±1.12
ꢀ4.59±0.39
ꢀ5.19±0.73
ꢀ
58.45±1.5
56.41±2.3
53.83±5.8
2.62±0.31
2.51±0.24
2.39±0.26
The morphology of the liposomes were observed through
transmission electron microscope (TEM) as shown in Fig.3. Results
showed that the liposomes were homogeneously distributed
spheroids with narrow size distribution. The particle size determined
from transmission electron microscope was in line with the size
measured by dynamic light scattering (Table 1). The particle size
plays a vital role in in vivo circulation and tissue targeting based on
receptorꢀmediated endocytosis.
3.3
In vitro release assay
Fig.2: Typical 1HꢀNMR spectrum of GPSC in CDCl3
As we all known, after reaching to target site, the nanoꢀvectors
would face in an acidic condition (pH value range from 6.8 to 5.5)
during the process of endocytosis46, 47. In order to improve the
endocytosis efficiency of the GPSLP/DOX, PEG was conjugated to
the cholesterol by Schiff base bonds, which showed pHꢀresponsive
in such weak acidic environment. In vitro drug release profiles of
DOX were studied using dialysis method in pH 7.4 (blood
circulation pH) and pH 6.0 (tumor microenvironment pH). The in
vitro accumulative release percentage of DOX from DOX solution
and liposomes were shown in Fig.4, DOX solution rapidly released
up to nearly 100% in 10 hours in PBS (pH7.4). In addition, there
was no significant difference between the release profiles of
3.2
Characterization of liposomes
In this section, particle size, zeta potential, encapsulation efficiency
(EE %) of different types of liposomes were analysed and presented
in Table 1. There was no significant difference among mean
encapsulation efficiency of different liposomes within the range of
53.8%ꢀ58.5%. Although the EE% of PLP/DOX tended to be higher
than GAꢀPEGꢀSIBꢀDOX liposomes, the difference was not
statistically significant (p>0.05). Compared with LP/DOX, there was
a narrow increase in the mean particle size, which were 120.9nm and
PLP/DOX
in
pH
7.4
and
Fig.3: Size distribution and transmission electron microscopy of
GPSLP/DOX. Scale bar represents 100 nm.
135.5nm for PLP/DOX and GPSLP/DOX, respectively. In fact, zeta
potential influences the liposomes stability in dispersion through
electrostatic repulsion among particles. Compared with blank Lip
and LP/DOX, there was a decrease in zeta potential of PLP/DOX
and GPSLP/DOX, which were ꢀ4.59mV and ꢀ5.19mV, respectively.
This interesting result was attributed to the charge shielding of PEG
and GA outside on the surface of liposomes43, 44. Furthermore, the
neutral or anionic surface charge of nanoparticles could efficiently
escape from the renal elimination45. Reticuloendothelial system
(RES) could destroy any foreign particles through opsonisation.
Hence, the liposomes must be hidden from the RES to circulate for a
Fig.4: pHꢀsensitive release of DOX from different formulations at 37oC.
(n=3)
pH 6.0 with only about 50% of the accumulation release due to the
presence of PEG on the outer surface of liposomes, which might
hinder the release of DOX. This phenomenon also clarified the
importance of PEG chains in the surface stability of liposomes. For
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the GPSLP/DOX, it could be observed that the drug release
noticeably increased about 20% more in pH 6.0 than pH 7.4 over 72
h, indicating significantly pHꢀsensitivity of the designed liposome.
There was no significant difference between PLP/DOX and
GPSLP/DOX in pH 7.4. However, the accumulative release
percentage of GPSLP/DOX was higher than PLP/DOX in pH 6.0,
which showed significantly pHꢀsensitive feature. Overall, the release
rate of DOX from GPSLP/DOX increased as the pH decreased,
demonstrating that the introduction of GPSC on liposomes greatly
affected the release behaviour of DOX from liposomes. The
probable explanation for this phenomenon was that long PEG chains
inserted on the surface of liposomes hindered the drug releasing
from liposomes in pH 7.4, whereas, PEG gradually deviated from
the liposome surface in pH 6.0 (slightly acidic environment) and the
amount of released drugs increased as well. These results suggested
that this liposomal delivery system could retain DOX while
circulating in blood plasma and normal liver tissue, thereby reducing
the systematic toxicity. This is expected to limit the leakage of DOX
in plasma circulation and relieve the nonspecific adverse effects
caused by DOX. Moreover, GPSLP/DOX had a superior release
profile at pH 6.0 compared to conventional PEG long circulation
liposomes (PLP/DOX), indicating that DOX could release in the
acidic tumor microenvironment or endosomes in cytoplasm. This
phenomenon would ensure effective intracellular delivery and lead
to a high concentration of DOX in the tumor cells, which resulted in
a higher cytotoxicity to the tumour cells. Thus the enhanced amount
of drug release from liposomes might improve the therapeutic
potential of DOX in tumor cells.
Fig. 5: a: In vitro cytotoxicity of blank liposomes treating with HepG2
cells; b and c: MTT assay of different formulation in 24 and 72 h,
respectively, against HepG2 cells (n = 3); d: IC50 of different formulation
in 24 and 72h. *p<0.05, **p<0.01
increase in concentration, there was no significant cytotoxicity
observed for blank liposomes against HepG2 cells at the
concentrations of 100ꢀ1000 µg/mL, reflecting that blank liposomes
were biocompatible and suitable for a potential drug delivery system.
3.4
In vitro cytotoxicity assay
For the MTT studies of different DOX loaded liposomes, cell
viability decreased as the dose increased, which showed significant
doseꢀ independent feature. The cell cytotoxicity of different
formulation has been showed in the Fig5.b and c. From the
cytotoxicity data we could find that compared with DOX solution
group, LP/DOX group showed higher cytotoxicity against cells no
matter in 24h or in 72h. This phenomenon was probably because the
hydrophobic lipid membrane of liposomes could enhance the cellular
uptake to reach higher cytotoxicity. In addition, the cytotoxicity of
PLP/DOX groups showed lower cytotoxicity compared with
LP/DOX group. This was attributed to the hydrophilic shell of PEG
The cytotoxicity of free DOX solution, PLP/DOX, GPSLP/DOX and
corresponding blank liposomes was investigated in HepG2 cells
using standard MTT method. The viability of cells was measured
immediately after stopping incubation. As revealed in Fig.5.a, with
an
would form
a weak exclusion between cell membrane and
formulation, which resulted in a lower cytotoxicity. Interestingly,
there was significant difference between GPSLP/DOX group and
other groups. As shown in Fig.5.d, the IC50 of GPSLP/DOX were
2.01±0.61 and 0.80±0.53 ꢁg mlꢀ1 for 24h and 72h, respectively.
The higher cytotoxicity of GPSLP/DOX groups was probably two
reasons: (1) GA mediated cell endocytosis could enhance the cellular
uptake of the formulation. (2) Due to the pHꢀsensitive PEG shell
could increase the drug release from the preparation and a large
amount of DOX could efficiently release to the cytoplasm, which
resulted in a higher therapeutical effect.
3.5
Cellular uptake assay
In this study, CLSM was used to evaluate the cellular uptake of
different formulations. As shown in Fig.6.a, there was only a weak
fluorescence signal in all groups when the incubation time was
approximately 0.5 h. When the incubation time increased, red
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fluorescence intensity increased in different degrees. Compared with reagent was used to observe the biodistribution behaviour of
different
Fig.6: a: Fluorescence microscopy images of HepG2 cells incubated
with different formulations at different time point. Red and blue colors
indicate DOX and Hoechst 33258, respectively. Scale bar represents
50ꢁm. b: Flow cytometry measurement of cellular uptake of different
formulation at different time point. c: Fluorescence intensity of different
formulation at 4h. (a), (b), (c), (d), (e) were control, DOX solution,
Fig.7: In vivo whole body imaging of H22 tumorꢀbearing nude mice
after free DiR solution and DiR liposomes administration at different
time point with the same dose of DiR (20 ꢁg mlꢀ1).
LP/DOX, PLP/DOX and GPSLP/DOX, respectively.
formulations. As shown in Fig.7, during the first 12 h, the
fluorescence signal of DiR solution group showed whole body
biodistribution after admistration through tail vein. However, the
DOX solution group, there was higher fluorescence intensity among
all other groups. The intracellular DOX was increased in the
fluorescence intensity decreased dramatically and the intensity
became weaker in liver and spleen at 24 h. There was only a weak
liposomes and DOX was delivered to the cytoplasm. Among all the
fluorescence signal was observed in 72 h, indicating that most of
DiR was eliminated through RES. In comparison, the biodistribution
intensity. GA receptorꢀmediated cell endocytosis and pHꢀresponsive
behaviour of different formulations were not the same as DiR
solution group. There was strong fluorescence intensity in tumor and
result. This results indicated that the cellular uptake of different
liver for both LP/DOX group and PLP/DOX group on the first 12 h
cytoplasm and a large amount of DOX was released from the
groups, GPSLP/DOX group showed the highest fluorescence
rapid drug release were probably the main reason to explain this
formulation was timeꢀindependent.
due to the EPR effect. For LP/DOX group, the signal became weaker
in both tumor and liver in 72 h, this phenomenon was probably
Intracellular uptake efficiency of different liposomes was also
because LP/DOX was recognized by RES and eliminated in liver or
quantitatively analysed by flow cytometry. As was illustrated in
some other organs. Hopefully, PLP/DOX showed significant higher
Fig.6.b, compared with free DOX group, the cellular uptake of
intensity in tumor site, this result confirmed that PEG shell could
different liposomes significantly increased in HepG2 cells when the
avoid the reorganization of RES to reach long circulation feature. As
incubation time was 4 h. There was significant difference between
was expected, GPSLP/DOX showed the best targetability in tumor
LP/DOX and PLP/DOX group in every time point, indicating that
sites among all the groups. The exciting result was probably
the PEG shell could decrease the cellular uptake in different degree.
attributed to pHꢀsensitive PEG shell was stable in blood circulation,
However, there was a significantly increase in fluorescence intensity
which resulted in a lower drug leakage rate and rapid release in
for GPSLP/DOX group in every time point, which was 1.92ꢀ, 1.24ꢀ
acidic microenvironment. In addition, GA, modified on the surface
and 1.32 fold higher than DOX, LP/DOX, PLP/DOX group,
of the liposomes played an important role in the target the tumor sits.
respectively(Fig.6c). This result was in consistent with the CLSM,
These advantages of GPSLP/DOX attributed to the higher
indicating that the GA modified could dramatically increase the
affinity between the liposomes and the cell membrane.
targetability in the in vivo biodistribution assay.
3.7
In vivo antitumor activity
All the results of quantitative and qualitative analysis of cellular
uptake demonstrated that the GPSLP/DOX liposomes had better cell
selectivity and stronger internalization than nonꢀtargeted PLP/DOX.
The GPSLP/DOX could specifically recognize cells through
overexpressing targeting binding sites of GA receptors and mediate
efficiently internalization in HepG2 cells.
In vivo antitumor activities of the DOXꢀloaded liposomes were
assessed in mice bearing H22 liver tumor at a dose of 5 mg/kg. The
average tumor volumes and changes of body weights were
monitored during the experiment to observe the antitumor efficiency
and toxicity, respectively. Fig.8.a showed the tumour growth
inhibition of drug loaded liposomes. As expected, control group
showed a rapid increase of tumor size within experimental period,
3.6
In vivo biodistribution studies
whereas,
DOX
solution
In order to evaluate the in vivo biodistribution of different
formulations in nude mice bearing H22 tumor cells. In vivo
fluorescence imaging study was carried out in this section. DiR
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Journal Name
different formulation for several times, the survival time of control
group was 15 days. Compared with control group, DOX solution
showed a slightly higher survival time, which was 21 days. This was
probably because although DOX could inhibit tumor growth, the
nonspecific toxicity would also decrease its survival time. In
comparison, three different formulation groups showed
a
significantly higher survival time, which was 27 days, 33 days and
39 days for LP/DOX, PLP/DOX and GPSLP/DOX groups,
respectively. Among all of them, GPSLP/DOX group showed the
highest survival time, this result was because the active target
molecule GA modified on the surface of the liposomes could
increase the affinity between liposomes and the tumor sites, thus
decreased the systematic toxicity. In addition, pHꢀresponsive rapid
drug release in tumor microenvironment could also increase the
therapeutical effect and increase its survival time.
Based on all the in vitro and in vivo studies，it was confirmed that
the active liver targeted pH sensitive liposomes (GPSLP/DOX)
could successfully elicit the desired pharmacological and therapeutic
responsive without acute adverse effect.
Fig.8: The mean tumor volume (a), body weight (b), KaplanꢀMeier
survival curve (c) and tumor inhibition rate (d) of different formulation
in nude mice bearing H22 tumor cells on intravenous administration of
different formulation(n=6). *p<0.05, **p<0.01, # not significant.
4
Conclusions
In this study, a novel pHꢀsensitive conjugate GPSC was successfully
synthesized and applied as active targeted composition to establish
the GPSLP/DOX for targeting therapy of liver cancer. These
liposomes exhibited promise in delivering therapeutic doses of DOX
to hepatocellular carcinoma cells expressing GA receptors. In vivo
antitumor study showed that the systematic toxicity of DOX was
reduced for all the DOXꢀloaded liposomes. Furthermore,
GPSLP/DOX showed the highest tumor suppression. In conclusion,
GPSLP/DOX are promising drug carriers for liver cancer therapy.
treated group just showed a slightly inhibition in tumour growth.
Hopefully, all the DOXꢀloaded liposomal formulations suppressed
the growth of tumours effectively. Particularly, GPSLP/DOX could
inhibit the tumor growth more efficiently than other liposomal
groups with the tumour growth inhibition rate (IR) reached to
81.76%. The higher tumor growth inhibition ratio of GPSLP/DOX
was resulted from several reasons: (1) PEG shell modified on the
surface of the liposomes could reach long circulation effect and
avoid elimination by RES. (2) GAꢀmediated cell endocytosis could
increase the accumulation of DOX in tumour tissues. (3) pHꢀ
responsive drug release from liposomes could reduce systematic
toxicity and increase therapeutical effect. Therefore, we have reasons
to suppose that the higher therapeutic potential is due to the high
DOX concentration accumulated in tumours.
Acknowledgement
The authors are grateful for the financial support from the National
Natural Science Foundation of China (81202483) as well as the
program for Liaoning Natural Science Foundation (LR
2015020543), Scientific Research Foundation for the Rturuned
Overseas Chinese Scholars by State Education Ministry
(201303003), Science and Technology project of Shenyang (F15ꢀ
Fig.8b described the change of body weight after treated with saline,
DOX solution and three different liposomal groups. Body weight of
liposomal formulation groups and saline group showed a slightly
increase during treatment and no significant difference was observed 199ꢀ1ꢀ24, F15ꢀ139ꢀ9ꢀ06)
with each other (P>0.05). In contrast, significant weight loss
.
(P<0.001) was observed in DOX solution treated mice compared to
control and the liposomal groups. This result was probably free
DOX showed wholeꢀbody biodistribution behaviour, which led to
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Over the last few decades pH-sensitive drug delivery system has been successfully developed for
the treatment of cancers by improving the therapeutical effect. In this study, a novel pH-sensitive
conjugates glycyrrhetinic acid-polyethylene glycol-Schiff bond-cholesterol (GPSC) has been
synthesized successfully and use it to constract Doxorubicin (DOX)-loaded liposoms
(GPSLP/DOX) with both pH-sensitive feature and active targeting ability. DOX was incorporated
into liposomes using thin-film hydration method with a relatively high encapsulation efficiency
(EE %) and drug loading content (LC %). Physicochemical characteristics, in vitro release
behavior, cellular toxicity and cellular uptake, in vivo biodistribution as well as in vivo antitumor
activities of GPSLP/DOX were investigated. GPSLP/DOX showed significantly pH-sensitive
feature in the in vitro release assay. All the blank liposomes were nontoxic in vitro cytotoxicity
assay. In MTT assay, GPSLP/DOX showed the highest cell cytotoxicity among all the groups.
Cellular uptake study revealed that GPSLP/DOX could be taken up efficiently via
receptor-mediated endocytosis and pH-responsive drug release of DOX into cytoplasm, which
resulted in a higher cytotoxicity and therapeutical effect. In vivo NIR fluorescence image study
showed that GPSLP/DOX could specifically accumulative in liver and tumor sites via receptor
mediated endocytosis. In vivo antitumor activities results showed that this novel GPSLP/DOX
could significantly inhibit tumor growth and prolong survival time due to its pH-responsive
behaviour and GA-mediated endocytosis, resulting in a lower systematic toxicity and higher
therapeutical effect. All these results confirm that this GA-mediated pH-sensitive GPSLP/DOX is
a novel nanocarrier for the delivery of antitumor agent DOX to reach higher toxicity effect against
tumor tissue.
Schematic diagram of GPSLP/DOX and mechanism of GPSLP/DOX to
enhance antitumor activities.