Liposomal nanohybrid cerasomes for mitochondria-targeted drug delivery

Article abstract of DOI:10.1039/c5tb01197c

Mitochondrial dysfunctions cause numerous human disorders and the development of mitochondria-targeted nanocarriers for drug delivery has aroused great attention. Herein, we report the synthesis of a liposomal nanohybrid cerasome modified with triphosphonium (TPP) for drug delivery to the mitochondrial matrix. The cerasomes were observed to possess an average size of about 38 nm in diameter, and the theoretical simulation of GBEMP mapping demonstrated that the amphiphilic organotrialkoxysilanes were stable as a bilayer equilibrium conformation after self-assembly. The cerasomes showed good stability, excellent biocompatibility and sustainable drug release behavior. Moreover, the TPP-targeted cerasomes resulted in greater drug accumulation in mitochondria, thus leading to a greater antitumor effect as compared to non-targeted cerasomes by using doxorubicin as a modal drug. The specific accumulation of TPP-targeted cerasomes within mitochondria was also confirmed by using JC-1 as the fluorescent probe to analyze the mitochondrial transmembrane potential change.

Full text of DOI:10.1039/c5tb01197c

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Liposomal nanohybrid cerasomes for mitochondria-targeted drug
delivery
Yanfang Wang,ab Beibei Wang,ab Han Liao,ab Xiaojie Song,ab Hao Wu,ab Huihui Wang,ab Hujun
Shen,d Xiaojun Ma a and Mingqian Tan ac*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Mitochondrial dysfunctions cause numerous human disorders and the development of mitochondria-targted nanocarrier
for drug delivery has aroused great attention. Herein, we report the synthesis of a liposomal nanohybrid cerasome
modified with triphosphonium (TPP) for drug delivery to the mitochondrial matrix. The cerasomes were observed to
possess an average size about 38 nm in diamater and the theoretical simulation of GBEMP mapping demonstrated the
amphiphilic organotrialkoxysilanes was stable as a bilayer equilibrium conformation after self-assembly. The cerasomes
showed good stability, excellent biocompatibility and sustainable drug release behavior. Moreover, the TPP-targeted
cerasomes resulted in greater drug accumulation in mitochondria, thus leading to greater antitumor effect as compared
to non-targeted cerasomes by using doxorubicin as a modal drug. The specific accumulation of TPP-targeted cerasomes
within mitochondria was also confirmed by using JC-1 as fluorescent probe to analyze the mitochondrial transmembrane
potential change.
www.rsc.org/
named the organic-inorganic hybrid cerasomes (CER) were
recently introduced by Kiyofumi Katagiri15. Cerasomes are a
kind of bio-inspired colloidal particles with a bilayer vesicular
Introduction
Mitochondrial dysfunction plays an important role in various
human disorders, such as Alzheimer’s disease (AD)1,
Parkinson’s disease2, diabetes3, obesity4, 5, and even cancer6, 7
structure consisted of a polyorganosiloxane surface, which
imparts cerasome much higher stability than conventional
liposome. Cerasome was firstly used as drug delivery system
for paclitaxel delivery, which exhibited controlled drug release
and remarkably high stability, demonstrating that liposomal
nanohybrid cerasomes can be a new promising drug delivery
system14. Later on, doxorubicin (DOX)-loaded cerasomes were
also reported to have the ability of controlled-drug release16.
The sustained release of drugs from cerasomes can be
modulated by alteration the vesicle composition17.
.
There are already many kinds of drugs ranging from
antioxidants for Alzheimer’s disease8 to chemotherapeutics for
cancer and uncouplers of antioxidants phosphorylation for
obesity9,
10
. However, the key problem to develop the
treatment of mitochondrial dysfunction disorders is how to
delivery these drugs to the cell mitochondria safely and in
sufficient amounts11.
Among numerous drug delivery systems, liposomes
attract particularly attention due to their good
biocompatibility, capable of encapsulating hydrophilic drugs in
In the development of theranostic drug delivery system to
mitochondrial dysfunction, the most challenging problem is
how to target the drug carriers to the mitochondria of the cells
directly and to realize a sustained drug release. To solve this
problem, a cation, called lipophilic triphenylphosphonium (TPP)
cation, has been used to permeate the mitochondria
membrane and accumulate in mitochondria matrix by
changing mitochondria membrane potential18-21. TPP is able to
permeate lipid bilayer easily and to accumulate several
hundred fold within mitochondria because of its positive
charge.22 Therefore, introduction of mitochondria targeting
moiety to the cerasomes is of great importance for the design
and preparation of mitochondria-targeted nanocarriers for
subcellular theranostics.
the inner aqueous core and lipophilic drugs in the lipid
13
. However, so far the liposome-based drug
bilayers12,
formulations have not widely used in clinic due to the poor
stability, aggregation by proteins in blood14. In response to
these limitations, liposomes based on Si-O-Si framework
a.Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy
of Sciences, Dalian 116023, China. E-mail: mqtan@dicp.ac.cn; Fax: +86-411-
82463027; Tel: +86-411-82463027
b.University of the Chinese Academy of Sciences, Beijing 100049, China
c. School of Food Science and Technology, Dalian Polytechnic University, Dalian
116034, China
d.State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
† Electronic Supplementary Informaꢀon (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
In this article, we developed an approach for the synthesis
of TPP modified cerasome nanocarrier for mitochondrial
targeted delivery of therapeutics. The cerasome-forming lipid
(CFL) were synthesized through several steps. When dispersed
† Yanfang Wang and Beibei Wang contributed equally to this work.
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in acidic aqueous solution, CFL formed cerasomes via sol-gel Tianjing Jingteng Experiment Equipment Co., Ltd. (Tianjin
self-assembly. Molecular dynamic simulation was considered China). Dynamic light scattering (Nano-ZS 90
as a complementary tool to experiment, which can provide an Cerasome preparation and characterization
insightful understanding of the functions of biomaterials at a
，Malvern, UK).
CFL was synthesized as shown in Supporting Information
(Scheme S1, Figure S1, S2 and S3, ESI). Cerasomes were
prepared using a combined thin-film hydration and extrusion
method according to the literature40. Briefly, chloroform
solution containing N-[N-(3-Triethoxysilyl)propylsuccinamoyl]
dihexadecylamine (compound 3, ESI) was evaporated out by a
rotary evaporators to form a lipid film. The film was dried in a
vacuum desiccator for 12 h to remove the residual methylene
chloroform, and then hydrated in pH 3.0 HCl solution to form
multi lamellar cerasome by vortexing for 15 min. The
cerasomes were sonicated by a ultrasonic probe of 30W for 15
min to form vesicular membranes. The solution was placed at
room temperature for 24 h to obtain Si-O-Si frame on the
surface via the sol-gel processes. The size and size distribution
of cerasomes were measured by transmission electron
microscope (TEM) and dynamic light scattering (DLS).
molecular and/or atomic level23-25. Herein, a coarse-grained
(CG) modeling was employed for cerasome molecular dynamic
simulation because the atomistic resolution of a biomolecular
system can be properly reduced and the large-scale motions
can be nicely preserved26-30. Gay-Berne (GB) potential31-33, a
well-known anisotropic potential, was also used for describing
the van der Waals (VDW) interactions between the CG
particles. The framework, by combining GB potential with
point electric multipole (EMP) potentials34-39, was adopted to
model the silane bilayer system. Moreover, 3-aminopropyl
triethoxysilane (APS) as a linker was conjugated with TPP (APS-
TPP) to introduce TPP moiety on the surface of the cerasomes
via the hydrolytic condensation of Si-OH between the
nanoparticles and APS-TPP. The biocompatibility, time stability
and structure stability of the resulted TPP-CER were
investigated. Taken DOX as the model medicine and
fluorochrome, TPP modified DOX-loaded cerasomes (TPP-CER-
DOX) was studied for mitochondria-targeted drug delivery.
Molecular dynamic simulation
The Gay-Berne electric multipole (GBEMP) parameters for
the bond stretching, angle bending and torsional potentials
were obtained by fitting to the atomistic profiles of the
potentials of mean force (PMFs) constructed from atomic
configurations (Optimized Potentials for Liquid Simulations,
OPLS force field)41 of liquid organics. All bonded parameters
are given in Supporting Information. The GBEMP MD
simulation protocol used in this study is described as follows.
The atomistic structure of each system was converted into its
CG representation in Euler coordinates containing three
Cartesian coordinates (x, y and z) and three Euler angles (φ, θ
and ψ) of the centers of mass. All GBEMP simulations were
carried out in the generalized Kirkwood (GK) implicit solvent in
the TINKER-based “GBEMP” program and the equation of
motion was integrated using the Euler’s rigid body integrator
with an integration step of 10 fs42. The non-bonded interaction
cutoff was set to 16 Å with a truncate scheme, and the Gay-
Berne and electrostatic interactions of 1-2 and 1-3 neighbors
were scaled with a factor of 0.003 and 1.0 respectively. Each
nucleic acid system was minimized and subsequent runs were
carried out under the temperature of 300 K for 20 ns.
Experimental
Materials and Instruments
Hexadecylamine, bromohexadecane, dicyclohexylcarbodiimide
(DCC),
N-Hydroxysuccinimide
(NHS),
(3-
aminopropyl)triethoxysilane (APS), 6-Bromohexanoic acid,
triphenylphosphine, phosphotungstic acid and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
were all purchased from Aladdin Reagents Co., Ltd. (Shanghai,
China) and used without further treatment. Succinic anhydride
was purchased from Institute of Guangfu Fine Chemical
Engineering (Tianjin, China). The doxorubicin hydrochloride
(DOX·HCl) was purchased from Meilun Biotech. Co. (Dalian,
China). Curcumin (CUR) was purchased from Zelang Medical
Technology Co., Ltd. (Nanjing, China). Mito-Tracker Deep Red
FM was bought from Life Technologies Co., Ltd. (New York,
USA). The solvents were all purchased from Beijing Chemical
Works (Beijing, China) and all dried with molecular sieve
before used. De-ionized water was prepared by a Mili-Q-Plus
system (18.2 MΩ). Transmission electron microscopy (TEM)
Synthesis of CER-DOX and TPP-CER-DOX
The TPP-CER was synthesized by using APS as a linker to
conjugate TPP and CER via the hydrolytic condensation of Si-
OH of the cerasomes and siloxane of APS-TPP(Scheme S2, ESI).
Detailed characterization of TPP-APS was given in Figure S4
and S5, ESI. Breifly, 1 mL APS-TPP in a syringe was pumped to 5
mL CER solution at a speed of 6.36 mL/h under stirring. The
reaction was carried out at room temperature for 24 h to fully
hydrolytic condensation. Sephadex G-50 chromatography was
used to purify the TPP-CER and the filtrate was examined by
UV-visible spectrophotometer and DLS.
was performed using
a JEOL JEM-2000EX Transmission
electron microscope with a beam voltage of 120 kV in Dalian
Medical University, China. The 200-230 mesh holey support
films used for TEM were purchased from Zhongjingkeyi
Technology Co., Ltd. (Beijing, China). The fluorescence images
were acquired by laser scanning confocal microscope (LSCM).
The amount of DOX and curcumin was measured by UV-visible
spectrophotometer (UV 2550). Human cervical cancer Hela cell
lines were purchased from Nanjing KeyGen Biotech. Co. Ltd.
(Nanjing, China). JC-1(5, 59, 6, 69-tetrachloro-1, 19, 3, 39-tetra
ethylbenzimidazol-carbocyanine iodide) (Beyotime Institute of
Biotechnology, China). Syringe filter was purchased from
As for drug encapsulation, a lipid film containing CFL was
dried via rotary evaporator and vacuum desiccator, and then
hydrated in the pH 3.0 HCl aqueous solution of DOX
hydrochloride (0.575 mM). The modification of TPP on the
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surface of CER-DOX was same as mentioned above. The medium (containing 10% fetal bovine serum) to different
resulted TPP-CER-DOX was dialyzed using a membrane with concentrations, respectively. L929 cells were inoculated into
the cut-off limit 10 kDa in PBS. According to the literature43, 96-wells plates at a density of 5×103 cells per well in 200 μL of
the fluorescence intensity of DOX encapsulated in liposomal RPMI-1640 medium (containing 10% FBS) and incubated at 37
structure was of 1/3 compare to that of equal concentration of oC in CO2 incubator for 48 h. Then the medium was replaced
free DOX. The concentration of DOX was determined by UV- with 200 μL of CER and TPP-CER solutions aforementioned and
visible spectrophotometer at 498 nm, while the loading of DOX the cells were incubated for an additional 24 and 48 h. The
was determined by fluorescent spectrophotometer at an medium was replaced with 200 μL of culture medium
excitation wavelength of 470 nm and the spectra was collected containing 0.5 mg/mL MTT and the plates were incubated for 4
at the range of 515 to 800nm. For hydrophobic CUR h. After that, the medium was removed and 200 μL of DMSO
encapsulation, the TPP-CER-CUR was prepared in a pH 3.0 HCl was added to each well to dissolve the formazan crystal. The
aqueous solution (containing 30 % (v/v) CH3CH2OH) of CUR dissolved solutions were transferred to another 96-well plate,
(5.43mM) followed the same procedure of TPP-CER-DOX.
and the optical density (OD) of the cells was measured by a
microplate reader (Wellscan MK 3, Labsystem Dragon, USA) at
570 nm by using 630 nm as a reference. Cell viability was
calculated through comparison with the control, which was
the cells without any treatment by the CER.
In Vitro Release
The hydrophilic DOX release from cerasomes in pH 7.4
phosphate buffer saline (PBS) was analyzed using a dialysis bag.
Firstly, 5 mL of CER-DOX solution at a DOX encapsulated
concentration of 0.293 mM was placed in a dialysis bag with a
Anti-tumor experiments
molecular weight cutoff of 10 kDa. Then the dialysis bag was In vitro biological efficacy of CER-DOX and TPP-CER-DOX was
immersed in a 100mL beaker containing 50 mL of pH 7.4 PBS. also evaluated by the MTT assay. These cerasomes were
o
The beaker was incubated at 37 C shaking tables. At the set diluted with RPMI-1640 medium (containing 10% FBS) to
time point, 2 mL of release medium was withdrawn from the different DOX concentrations and free DOX solutions at the
beaker and another 2 mL fresh PBS was added to the beaker. same concentration were used as control. We chose two types
The concentration of the DOX released from CER-DOX was of cells: Hela cells and NCI cells, which were inoculated into 96-
measure by ultraviolet spectrophotometer. For hydrophobic wells plates at a density of 5×103 cells per well in 200 μL of
curcumin, the dialysis medium was pH 3.0 HCl (containing 30% RPMI-1640 medium (containing 10% fetal bovine serum) and
(v/v) CH3CH2OH) aqueous solution. To prevent biological incubated at 37 oC in CO2 incubator for 48 h. Then the cell
contamination, 0.1% sodium azide was added to the dialysis medium was replaced with 200 μL of the DOX
、CER-DOX and
medium.
TPP-CER-DOX solutions aforementioned and the cells were
incubated for an additional 24 and 48 h. The medium was
replaced with 200 μL culture medium containing 0.5 mg/mL
MTT and the plates were incubated for 4 h. After that, the
medium was removed and 200 μL DMSO was added to each
well to dissolve the formazan crystal. The dissolved solutions
were transferred to another 96-well plate, and the cell OD
values were measured by a microplate reader (Wellscan MK 3,
Labsystem Dragon, USA) at 570 nm by using 630 nm as a
reference. Cell viability was calculated through comparison
with the control cells without any treatment by the CER.
Quantification of TPP-CER in Subcellular Compartments
Time Stability and Structure Stability
The time stability of CER was estimated by measuring the
average size of CER solution using DLS. CER solution was hold
at room temperature for 100 d. To determine the structure
stability of CER, different volume of 50 mM Triton X-100
aqueous solution was added to 2 mL CER solution at room
temperature and the average size of the solution was
measured by DLS.
Drug Loading Capacity and Encapsulation Efficiency
The drug loading capacity (DLC) and encapsulation efficiency
(EE) of drug-loaded CER were determined by microfiltration.
Briefly, 5 mL of drug-loaded CER was placed in a syringe with a
polyethersulfone filter, then the solution was pushed through
the filter. The absorbance of the drug-loaded CER solution and
filtrate was measured by ultraviolet spectrophotometer. Using
the standard calibration curve, the amount of drug
encapsulated in CER cerasomes was calculated.
Hela cells were inoculated into 35-mm dishes at a density of
5×105 cells per dish in 1 mL of RPMI-1640 medium (containing
10% fetal bovine serum) and incubated at 37 oC in CO2
incubator for 24 h. The cells were treated with TPP-CER-DOX
or CER-DOX for 3.5 h, then Mito-Tracker Deep Red FM was
added to the medium and the cells were incubated for another
0.5 h. After the treatment, the cells were washed thrice with
pH 7.4 PBS. The quantification of TPP-CER in subcellular
compartments was then acquired by LSCM. Oil inserted 60x
and 100x objective lens were used in xy and xyz mode with
The DLS and EE were evaluated by the following formulas17, 44,
45
.
800×800 pixel resolution. DOX was excited with a 488 nm line
and emission was collected between 520-600 nm, while Mito-
Tracker Deep Red FM was excited with a 633 nm laser and
emission was collected between 666-698 nm.
Cytotoxicity Evaluation
Isolation of Mitochondria from cytosolic Extracts
In vitro cytotoxicity of CER and TPP-CER was evaluated by a
MTT assay. The CER and TPP-CER were diluted with RPMI-1640
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Quantification of TPP-CER in subcellular compartments was realize the therapy and imaging of mitochondrial at
further carried out by isolating mitochondria from cytosolic subcellular level.
extracts through differential centrifugation. Briefly, Hela cells
were inoculated into 90-mm dishes at a density of 2×106 cells
per dish in 5 mL of RPMI-1640 medium (containing 10% fetal
bovine serum) and incubated at 37 oC in CO2 incubator for 48 h.
The cells were treated with TPP-CER-DOX or CER-DOX for 4 h,
and then the cells were washed thrice with pH 7.4 PBS. The
cells were collected and put into a glass homogenizer for
homogenizing. Until more than 50% cells were broken, the
solution was centrifuged at 1000G for 10 min to precipitate
the cell debris, and then the supernatant was centrifuged at 15
000 G for 10 min to isolate mitochondria from cytosolic
extracts. Mitochondria were quantified with cytosolic extracts
at the wavelength of 488 nm by UV-visible spectrophotometer
(UV 2550).
a
Detection of the mitochondrial transmembrane potential
Mitochondrial transmembrane potential was evaluated by a
mitochondrial membrane potential assay kit with JC-1. Hela
cells were inoculated into 35-mm dishes at a density of 5×105
cells per dish in 1 mL RPMI-1640 medium (containing 10% fetal
bovine serum) and incubated at 37 oC in CO2 incubator for 24 h.
Fig.1: Schematic illustration of the formation process of the Doxorubicin-loaded
cerasomes (CER-DOX) and TPP modified cerasomes (TPP-CER-DOX). CFL: cerasome-
forming lipid.
The cells were treated with or without of TPP-CER-DOX or CER-
DOX for 4 h. After the treatment, the cells were incubated in JC-
1 solution (5 μg/mL) for another 0.5 h. The cells were washed
thrice with pH 7.4 PBS and quantified by laser LSCM. Oil
inserted 60x and 100x objective lens were used in xy and xyz
Synthesis of cerasomes
The detailed synthesis of CFL was described in the electronic
supplementary information (ESI). The resulted CFL was then
hydrated in the pH 3.0 HCl aqueous solution in the absence or
presence of DOX hydrochloride to form cerasomes or CER-DOX,
respectively, through the self-assembly approach. The TEM
image of the obtained cerasomes in Fig. 2A revealed that they
owned spherical morphology with an average size about 38
nm and a narrow size distribution (Fig. 2B), while the
hydrodynamic diameter of cerasomes measured by DLS was
about 210 nm. As we know, by TEM we image single particle,
while DLS gives an average size estimation, which is biased
toward the larger-size end of the population distribution
because large particles scatter the light much more intensely
than smaller particles. These results were also reported in
previous literature.46
mode with 800
×800 pixel resolution. J-aggregates (J-Aggr)
were excited with a 543 nm laser and emission was collected
between 550-650 nm, while J-monomer was excited with a
488 nm laser and emission was collected between 500-540 nm.
Results and discussion
The use of both TPP and cerasomes has tremendous potential
to make an effective mitochondria-targeted nanocarrier due to
several advantages. Firstly, the cerasomes have much higher
stability than conventional liposome, which exhibited
controlled drug release property. Secondly, the surface of the
cerasomes can also be modified to provide further
functionality, such as targeting agents, or fluorescent agents,
to make targeted or multimodal imaging agents, respectively.
Thirdly, as a targeting agent, the TPP with positive charge is
able to easily permeate lipid bilayers and to accumulate within
the mitochondria because of the large membrane potential (-
150 to -70 mV, negative inside), resulting in highly efficient
mitochondrial uptake.22 Herein, the formation of TPP-targeted
drug-loaded cerasomes (TPP-CER-DOX) is illustrated in Fig. 1.
Taken DOX as the model drug and fluorescent agent, DOX-
The TPP-CER nanoparticles were prepared by surface
modification of CER with TPP-APS. The characteristic
absorption peak of TPP-COOH was at 262, 268 and 274 nm (Fig.
2C). After the purification of Sephadex G-50 chromatography,
the TPP-CER not only shows the TPP absorbance as illustrated
in Fig.2 C, but also can be detected with DLS (Fig.2 D), while
the small molecule TPP only has the absorption peak and can
not be detected by DLS. The results further demonstrated that
TPP-CER nanoparticles were successfully prepared. The
number of TPP molecules per cerasome was calculated to be
415. The DLS data showed that the average size of CER-DOX
and TPP-CER-DOX nanoparticles was about 226 and 220 nm
(Fig. S6), respectively. The CER-DOX and TPP-CER-DOX had a
very close size and size distribution. The encapsulation of DOX
and the modification of TPP on the surface of cerasome didn’t
loaded cerasomes (CER-DOX) were prepared through
a
combined film hydration and self-assembly process.
Mitochondria-targeted molecules TPP was conjugated to CER-
DOX by using APS and 6-bromohexanoic acid as linkers. The
TPP-CER-DOX can permeate lipid bilayers and enter the cells by
clathrin-mediated endocytosis43. This results in the
mitochondria targeting and the encapsulated drug release to
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affect their hydrophilcity. The Zeta potential of the CER, TPP- particles were derived through fitting to the corresponding
CER and CER-DOX was shown in Table S1.The zeta-potential of atomistic energy profiles in gas phase using genetic
a
CER and TPP-CER in neutral condition is –18.61±6.41 mV and algorithm. The potential energies have been monitored during
+15.37±0.68 mV, respectively. As for CER-DOX, the zeta- the GBEMP simulation. For the system, the energy landscape
potential is –13.49±2.17 mV and the loaded DOX didn’t change became flat after 10 ns simulation, indicating the systems
the surface charge of CER significantly, which is consistent with reach its equilibrium. From Fig. 3 C, one can see that the
the previous report.16
bilayer structure is supposed to be stable as an equilibrium
conformation. The structural properties of the final
equilibrium conformation are given in Table 1. The thickness
of the membrane of CER measured from the TEM images was
5.81±0.96 nm (Fig. 2 A, inset), which is close to the thickness
obtained from GMEMP simulation.
Fig. 2: (A)TEM image of cerasomes, inset shows the enlarged TEM image of
single cerasome; inset shows the enlarged single particle. Scale bar= 100 nm. (B)
Size distribution of cerasomes measured by TEM. (C) The characteristic
absorption peak of TPP-COOH and TPP-CER. (D) DLS size distribution of the
filtrate containing TPP-CER that has the same characteristic absorption peak with
TPP-COOH, while that containing small molecule TPP-COOH was not detected by
the DLS.
Molecular dynamic simulation was adopted herein as a
complementary tool to further understand the formation of
cerasomes at a molecular and/or atomic level. We constructed
a starting structure containing 72 CFL molecules to simulate
the formation of cerasomes (Fig. 3A and 3B). Gay-Berne
electric multipole (GBEMP) mapping for silane is depicted in
Fig. S8. The CG model is composed of three elliptic rigid bodies
and one spherical rigid body. The hydrophobic tail of the silane
model is represented by two elliptic rigid bodies in each of
which contains one Gay-Berne site and one non-interacting
EMP site. The non-interacting EMP site (denoted by orange
filled circle) would not involve in electrostatic interaction but
serve just as the connection purpose. As for the hydrophilic
head group, the elliptic rigid body contains one Gay-Berne site
and two EMP interacting sites while the spherical rigid body
Fig. 3: (A) CFL structure model. (B) The bilayer structure of cerasomes system. (C)
Potential energies of the system evolving with MD simulation time. The starting
structure (t = 0 ns) of the system was constructed as a form of bilayer.
Table 1. Structural parameters of the 72 silane bilayer system, obtained from
GBEMP simulation.
Structural Parameters
Thickness DHH (Å)
Area per lipid A L (Å2)
Volume per lipid VL (Å3)
GBEMP Model
53.0
68.5
1825.0
In vitro drug release and cytotoxicity
contains one Gay-Berne site and one EMP site which both Like liposome, cerasome is composed with an internal aqueous
share a same location shown by a red filled circle. GBEMP compartment and a hydrophobic interval of the bilayer
energy function can be seen in the ESI. To parametrize the membrane. The internal aqueous compartment can
Gay-Berne potentials for the CG models (Fig. S8), the atomistic encapsulate hydrophilic drugs, while the hydrophobic interval
energy profiles (using OPLS atomistic force field) were of the bilayer membrane can carry hydrophobic drugs. Taken
constructed for the intermolecular van der Waals (VDW) DOX and CUR as the models of hydrophilic and hydrophobic
interactions between two identical molecular fragments drugs, respectively, drug-loaded cerasomes were prepared and
(homodimer), illustrated in Fig. S9. In each case, diverse purified. The encapsulation efficiency (EE) was evaluated to be
configurations were generated with different orientations 3.34 % and 33.29 % and the drug loading capacity (DLC) was
(such as, cross, end-to-end, face-to-face, etc.) as well as at 53.40 % and 92.15 %, respectively. Fig. 4A shows the
various separations. The initial Gay-Berne parameters for CG accumulative DOX and CUR release profile. Dialysis bags
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containing CER-DOX and CER-CUR were placed in PBS and and TPP-CER-DOX decreased to 82% and 77% with the increase
PBS/CH3CH2OH solutions, respectively, while both CER-DOX of DOX concentration. The result indicated that the cell
o
and CER-CUR had a sustained release within 24 h at 37 C. As viability decreased with the increase of incubating time. As for
shown in Fig. 4A, 36.85 % and 46.02 % of the encapsulated NCI cells, the cell viability of free DOX decreased to 45% and
DOX and CUR released from cerasomes within 24 h, indicating 14% with the increase of DOX concentration from 50 to 2500
a remarkable controlled-release of both hydrophilic and nM at 24 h and 48 h, respectively (Fig. 5C and 5D). The
hydrophobic drugs.
tendency of cell viability of CER-DOX and TPP-CER-DOX
incubating with NCI cells was similar to that of Hela cells. The
cell viability of CER-DOX and TPP-CER-DOX was also higher
than that of free DOX. Moreover, the cell viability of CER-DOX
and TPP-CER-DOX decreased with the increase of DOX
concentration. The concentration of free DOX was equal to
that of DOX encapsulated in the cerasomes, while the cell
viability of encapsulated DOX was lower than that of free DOX,
due to the shielding effects of the cerasomes. It should be
pointed out that the TPP-CER-DOX resulted greater
cytotoxicity than CER-DOX probably because it targeted the
mitochondria of the cells and led to stronger cytotoxicity.
However, the efficacy of the TPP-CER-DOX does not look so
great even at 48 h due to the sustained release of DOX from
the nanocarriers. Although more than 30% DOX can be
released from CER-DOX in 24 h (Figure 4A), the results
revealed that released DOX can not kill the cells so effectively
as those free DOX. Similar result was also reported in previous
work of monodisperse hollow mesoporous silica (HMS)
nanocages as nanocages for drug delivery48. Further animal
treatment study will be conducted to evaluate the potential of
the nanodrug
Fig. 4: (A) Release of DOX from CER-DOX and Curcumin from CER-CUR in 24 h. (B) In
vitro cytotoxicity of cerasomes (CER) and TPP-modified cerasomes (TPP-CER) (n=3) at
24 h and 48 h, respectively, for L929 cells.
To verify the biocompatibility of TPP-CER, the cytotoxic
effects of CER and TPP-CER were evaluated in L929 cells by
MTT assays. As shown in Fig. 4B, cell viability remained around
100 % for both CER and TPP-CER after 24 h or 48 h incubation
at the concentration less than 100 μg/mL. When the
concentration was more than 100 μg/mL, the cell viability of
TPP-CER was still around 100%, while that of CER was gradually
reduced with the increase of the concentration and the
incubation time. When L929 cells were incubated with CER of
1000 μg/mL for 24 h and 48 h, the cell viability was decreased
to 74% and 67%, respectively. Previous works demonstrated
that CER had lower cytotoxicity than silica nanoparticles,47 but
mild cytotoxicity than liposomes17. In this case, the CER
showed growth inhibition in a concentration- and time-
dependent manner in L929 cells from 1 to 1000 μg/mL, and
the modification of TPP enhanced the biocompatibility of CER.
In Vitro Anti-tumor effect
NCI and Hela cells were chosen to evaluate the in vitro
anticancer performance of free DOX、CER-DOX and TPP-CER-
DOX with equal concentration of DOX. Fig. 5 shows the results
of cell viability measured by MTT assay for 24 and 48 h,
respectively. As for the Hela cells incubated for 24 h, when the
concentration of DOX was below 100 nM, the cell viability of
free DOX was above 90%, while the cell viability decreased to
27.67% with the increase of DOX concentration (Fig. 5A). The
cell viability of CER-DOX and TPP-CER-DOX was higher than
that of free DOX. For CER-DOX, the cell viability maintained
around 100% with concentration change. The cell viability of
TPP-CER-DOX was somewhat lower than that of CER-DOX.
From 1 to 500 nM, the cell viability was more than 90%, while
the cell viability decreased to 82% at the concentration of 2500
nM. When the incubating time increased to 48 h, the cell
viability of free DOX decreased to 18% with the increase of
DOX concentration from 50 to 2500 nM (Fig. 5B). The cell
viability of CER-DOX and TPP-CER-DOX was also higher than
that of free DOX, and the cell viability of TPP-CER-DOX was
somewhat lower than CER-DOX. The cell viability of CER-DOX
Fig. 5: Cytotoxicity of free DOX, CER-DOX and TPP-CER-DOX for Hela cells and NCI cells
incubated for 24 and 48h, respectively. (A) 24h, Hela cells; (B) 48h, Hela cells; (C) 24h,
NCI cells; (D) 48h, NCI cells.
In vitro Mitochondrial Targeting effect
In order to assess the effectiveness of TPP-conjugated
cerasomes for mitochondria localization in cells, the co-
localization of TPP-CER-DOX was investigated in human
cervical cancer Hela cells by using a mitochondria-tracker
(Deep Red) as reference. Hela cells were incubated with the
CER-DOX and TPP-CER-DOX for 4 h in serum medium, followed
by washing with PBS and stained with mitochondria-tracker for
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30 min. Fluorescence micrographs of red and green stand for
the distribution of cerasomes and mitochondria-tracker,
respectively. They were overlaid and co-located in yellow by
LSCM. Fig. 6 shows the distribution of the non-targeted CER-
DOX and the targeted TPP-CER-DOX in Hela cells. Some of CER-
DOX signals were from mitochondrial, but there is no signal
from nuclei (Fig. 6A). Previous work suggested that the cellular
uptake of cerasomes occurred probably through a process of
clathrin-mediated
endocytosis,
which
resulted
in
rearrangement of the cell cytoskeleton.47 Since the CER-DOX
and TPP-CER-DOX particles were incubated with Hela cell for
only 4 h, the amount of released DOX targeted in the nuclei
was not sufficient and no fluorescence signal was seen from
nuclei area. Moreover, there existed significant co-localization
of the TPP-CER-DOX with mitochondria-tracker in the
mitochondria of cells, while the red signals of non-targeted
CER-DOX and mitochondria-tracker differed in position.
Quantitative analysis of the fluorescence images in each group,
the Pearson’s coefficient of targeted TPP-CER-DOX was 0.760 ±
0.037, while that of non-targeted CER-DOX was only 0.535 ±
0.135. The Pearson’s coefficient increased averagely by 22.5%.
The results demonstrated that the TPP modified cerasomes
TPP-CER-DOX could promote the cerasomes entering
mitochondria to release drug in the mitochondria. Such
efficient mitochondria-targeted drug delivery by the cerasome
system is promising for long-term mitochondria-targeted
therapy.
Fig. 6: Mitochondrial localization of CER-DOX and TPP-CER-DOX. Hela cells were
exposed to non-targeted CER-DOX and TPP-targeted TPP-CER-DOX for 4 h (Scale bar: 20
µm). The cells were then stained with mitochondrial marker Mito-Tracker Deep Red for
30 min. The staining solution was washed by pH 7.4 PBS and the cells were observed by
fluorescence microscopy. (A)The merge images showed that the overlap of Mito-
Tracker (Green) and CER-DOX (Red) was unobvious (Top), while the overlap of TPP-CER-
DOX was significant (Down). (B) The Pearson’s Coefficient analysis plots of CER-DOX
(left) and TPP-CER-DOX (right).
Analysis of Mitochondrial Transmembrane Potential
The mitochondrial membrane potential could be reduced due
to the increasing of proton permeability of the mitochondrial
inner membrane caused by the accumulation of lipophilic
cations, such as TPP, within the mitochondria 49. Therefore, to
further confirm the TPP-CER-DOX accumulation within
mitochondria, the mitochondrial transmembrane potential
was analyzed in Hela cells. Herein, the fluorescent probe JC-1
was used for detection of mitochondrial membrane potential.
It is well know that when the membrane potential is high, JC-1
aggregates in mitochondrial matrix to emit red light. When the
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membrane potential is low, JC-1 presents in cytoplasm in the visible spectrophotometry to determine the accumulated DOX
form of monomers to emit green light. The proportion of amount. The results showed that the proportion of CER-DOX
red/green fluorescence intensity can represent the change of accumulated in mitochondria was 51.87%, while that of TPP-
mitochondrial membrane potential. The red fluorescence of CER-DOX was 60.16% (Fig. 7C). Compared to CER-DOX, the
CER-DOX in Fig. 7A is stronger than that of TPP-CER-DOX, while proportion of TPP-CER-DOX accumulated in mitochondria
the green fluorescence is weaker than that of TPP-CER-DOX, increased about 8.29 %, further suggesting the specific
suggesting that the mitochondrial membrane potential accumulation ability of the TPP-CER-DOX in mitochondria-
became smaller for the case of TPP-CER-DOX and the JC-1 was targeted drug delivery.
more easily presented in cytoplasm in the form of monomers
to emit green light. The quantification of the red/green
Conclusions
fluorescence intensity proportion for CER-DOX was 0.620,
while it was only 0.476 for TPP-CER-DOX, which means the
proportion of red/green fluorescence intensity of CER-DOX is
greater than that of TPP-CER-DOX(Fig. 7B). Compared to CER-
DOX, the results demonstrated that the mitochondrial
In summary, we have developed a general and facile synthesis
method to fabricate TPP modified liposomal cerasome that
could be applied as nanocarrier for mitochondria-targeted
therapeutics by using DOX as model medicine. The cerasomes
were prepared via the self-assembly and sol-gel reaction of
amphiphilic organotrialkoxysilanes to form bilayer vesicles
covered with a silicate surface. The APS-TPP targeting moiety
was conjugated on the cerasomes to specifically recognize the
mitochondria. The stability of cerasome was good due to the
siloxane network formed on the cerasome surface. The
theoretical simulation of GBEMP mapping demonstrated the
amphiphilic organotrialkoxysilanes was supposed to be stable
as a bilayer equilibrium conformation after self-assembly. The
membrane
potential
decreased,
indicating
better
accumulation ability of TPP-CER-DOX within the mitochondria.
cerasomes showed excellent biocompatibility and had
a
sustained release ability within 24 h at 37 oC after loading the
drugs of DOX or CUR. Taken DOX as the model medicine and
fluorochrome, the TPP-CER-DOX resulted in greater drug
accumulation in mitochondria as compared to non-targeted
CER-DOX. The quantitative analysis of mitochondrial
transmembrane potential further confirmed the remarkable
accumulation ability of TPP-CER-DOX within the mitochondria.
We believe that the TPP modified cerasome may have
potential as nanocarrier for mitochondria-targeted
theranostics.
Acknowledgements
This work was supported by the National Nature Science
Foundation of China (91227126), National Special Fund for Key
Fig. 7: (A) Fluorescence images of Hela cells incubated with CER-DOX or TPP-CER-DOX
by using fluorescent probe JC-1 to indicate the mitochondrial transmembrane potential
change (Scale bar: 40 μm). When the membrane potential is high, JC-1 aggregated in
mitochondrial matrix as JC-1 aggregates to emit red light (585/590 nm). When the
membrane potential is low, JC-1 presented in cytoplasm in the form of monomers to
emit green light (514/529 nm). The proportion of red and green fluorescence intensity
can represent the change of mitochondrial membrane potential. (B) The proportion of
red and green fluorescence intensity of Hela cells incubated with CER-DOX and TPP-
CER-DOX for 4 h. (C) The proportion of mitochondria extracts in Hela cells incubated
with CER-DOX and TPP-CER-DOX for 4 h, respectively.
Scientific
Instrument and
Equipment Development
(2013YQ17046307) and the Nature Science Foundation of
Liaoning Province, China (2013020177).
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A general and facile synthesis method was reported to fabricate TPP modified
liposomal cerasomes as nanocarrier for mitochondria-targeted drug delivery.