A novel MPEG-PDLLA-PLL copolymer for docetaxel delivery in breast cancer therapy

Article abstract of DOI:10.7150/thno.19680

Satisfactory drug loading capacity and stability are the two main factors that determine the anti-cancer performance. In general, the stability of the micelles is reduced when the drug loading (DL) is increased. Therefore, it was a challenge to have high

Full text of DOI:10.7150/thno.19680

Theranostics 2017, Vol. 7, Issue 10
2652
Ivyspring
International Publisher
Theranostics
2
017; 7(10): 2652-2672. doi: 10.7150/thno.19680
Research Paper
A Novel MPEG-PDLLA-PLL Copolymer for Docetaxel
Delivery in Breast Cancer Therapy

Liwei Tan, Jinrong Peng, Qian Zhao, Lan Zhang, Xichuan Tang, Lijuan Chen, Minyi Lei, Zhiyong Qian
State Key Laboratory of Biotherapy and Cancer Center, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Sichuan, China

Corresponding author: Tel/Fax: +86-28-85501986, E-mail: anderson-qian@163.com
©
Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(
https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2017.02.15; Accepted: 2017.04.27; Published: 2017.07.06
Abstract
Satisfactory drug loading capacity and stability are the two main factors that determine the
anti-cancer performance. In general, the stability of the micelles is reduced when the drug loading
(DL) is increased. Therefore, it was a challenge to have high drug loading capacity and good
stability. In this study, we introduced a hydrophilic poly (L-Lysine) (PLL) segment with different
molecular-weights into the monomethoxy poly (ethylene glycol)-poly (D, L-lactide)
(MPEG-PDLLA) block copolymer to obtain a series of novel triblock MPEG-PDLLA-PLL
copolymers. We found that the micelles formed by a specific MPEG2k-PDLLA4k-PLL1k copolymer
could encapsulate docetaxel (DTX) with a satisfactory loading capacity of up to 20% (w/w) via the
thin film hydration method, while the stability of drug loaded micellar formulation was still as good
as that of micelles formed by MPEG2k-PDLLA1.7k with drug loading of 5% (w/w). The results from
computer simulation study showed that compared with MPEG2k-PDLLA1.7k, the molecular chain of
MPEG2k-PDLLA4k-PLL1k could form a more compact funnel-shaped structure when interacted with
DTX. This structure favored keeping DTX encapsulated in the copolymer molecules, which
improved the DL and stability of the nano-formulations. The in vitro and in vivo evaluation showed
that the DTX loaded MPEG2k-PDLLA4k-PLL1k (DTX/MPEG2k-PDLLA4k-PLL1k) micelles exhibited
more efficiency in tumor cell growth inhibition. In conclusion, the MPEG2k-PDLLA4k-PLL1k micelles
were much more suitable than MPEG2k-PDLLA1.7k for DTX delivery, and then the novel
nano-formulations showed better anti-tumor efficacy in breast cancer therapy.
Key words: Docetaxel micelles; polymeric micelles; interaction; anti-tumor; drug loading capacity.
Introduction
Nano-formulations have become a new kind of
drug formulations which has overcome some of the
critical problems of traditional drug forms, including
with breast, lung and gynecologic cancers [9]. As one
kind of taxanes, DTX is a semi-synthetic analog of
paclitaxel. Due to its poor solubility, low selective
distribution and fast elimination, the use of DTX has
been restricted [10]. Currently, the commercial
drug
hydrophobicity
(which
reduces
the
bioavailability), the auxiliary toxicity, etc [1-3].
Combining with enhanced permeability and retention
®
formulations of DTX (Taxotere ) have been reported
(
EPR) effect, passive targeting can be achieved in solid
to cause serious side-effects due to DTX itself or
polysorbate 80 [11]. Therefore, the studies of
nano-formulations are used to overcome these
disadvantages [12-14]. Passive targeting and active
targeting that are the two main ways that
accumulated drugs into tumor sites. To improve the
biocompatibility of nano-formulations, PEG [15, 16]
and D-α-tocopheryl polyethylene glycol 1000
tumor therapy, which also enhances the
bioavailability of the drugs [4, 5]. Up to date, more
than 40 kinds of nano-formulations, which have been
studied in clinical trial, and several of them have been
launched into the market [6-8].
As the prototype microtubule stabilizers, taxanes
has been widely applied in the treatment of patients
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succinate (TPGS) [17, 18] are often used as the surface
reduced some undesired side-effects. However, its DL
is only 5% [19, 20, 44]. And what’s more, from its
operating instruction manual, it must be used within 4
hrs after re-dissolved, which indicate this formulation
will become instable after 4 hrs. And we also proved
this assumption in our previous research [43]. In order
to further enhance the stability of the DTX micellar
nano-formulations, more suitable nano-carriers which
not only can enhance the DL but also can enhance the
stability of the DTX loaded nano-formulation are
needed to be developed.
of which. The DTX formulation based on
TM
MPEG-PDLLA copolymers micelles (Nanoxel-PM
)
have been list in South Korea [19, 20]. TPGS, a
water-soluble derivative of natural vitamin E, was
used for drug delivery as a multirole excipient. Mei et
al. modified PLGA or PLA with TPGS to enhance the
biocompatibility of the DTX nanoparticles for cancer
therapy [21-26]. As more and more nano-formulations
have been developed and evaluated in pre-clinical
trials, however, the results reveal that tumor targeting
of the nano-formulations still need to be improved
According to some reports, the introduction of
charge segment to the polymer chain may help to
enhance the stability of the nanoparticles [45, 46].
[
27]. Although numerous tumor-targeting ligands,
including antibodies, proteins ligands [28, 29], have
been introduced to the nano-systems to achieve active
targeting through receptor-mediated endocytosis
Lysine as
a
natural amino acid has good
biocompatibility [47]. Some early reports indicated
that PLL have some activity against murine tumors
[48]. PLL also exhibits amount of unique membrane
properties, which include the ability to enhance the
cellular uptake of macromolecule. It is beneficial to
the uptake of nano-formulations by tumor cells [49].
These effects might be related to the cationic property
of PLL and specific interactions on cell membrane
[50]. Nowadays, PLL has been exhibited the ability as
an efficient drug and gene carrier for tumor therapy
[51, 52]. Therefore, in this study, we intend to
introduce the biodegradable and biocompatible PLL
to the polymer chain of MPEG-PDLLA. After the
introduction of PLL, a triblock polymer which was
named as MPEG-PDLLA-PLL was achieved.
However, the introduction of PLL might change the
hydrophilic-hydrophobic property of MPEG-PDLLA.
Thus, the effect of the molecular weight of the MPEG,
PDLLA and PLL in MPEG-PDLLA-PLL molecular
weight on the DL and stability needed to be
optimized in detail.
(
RME) [30], the protein corona and ligand dissociation
still impede the targeting efficacy [31, 32]. Moreover,
the stimuli-intelligent polymeric micelles, which are
response to endogenous stimulations (pH, enzyme,
reduction and oxidation) and exogenous stimulations
(
heat, light, ultrasound and magnetic field), have been
regarded as candidate vehicles for DTX [33].
However, in the case of solid tumor, passive targeting
is still determining the targeting efficacy of
nano-formulations [34, 35]. Some previous researches
have revealed that the passive targeting efficacy is
affected by particle size, surface charge, the ratio of
hydrophobic segment/hydrophilic segment, the
stealth coating, etc [36-38]. All of these factors are
connecting
with
the
stability
of
the
nano-formulations. Therefore, the stability of the
nano-formulations has critical importance to the
passive targeting, which is directly connected with the
anti-cancer properties of the nano-formulations.
Moreover, similar to drugs, many imaging substance
are loaded into micelles as probes for biological
imaging [39, 40]. Therefore, improving the stability of
drug loaded micelles can also improve the
construction of stable imaging agents.
So, in this study, we first synthesized a series of
MPEG-PDLLA-PLL triblock polymers with different
molecular weight and composition. Then, we further
evaluated the DL and stability of DTX micelles
formed by this series polymer. After optimization, a
On the other hand, achieving high drug loading
capacity is also a challenge for the designs of efficient
special
DTX/MPEG2k-PDLLA4k-PLL1k
micellar
drug
carriers.
In
generally,
the
DL
of
nano-formulation was chosen and be further
evaluated the association of DL or micellar stability
and anticancer performance, shown in figure 1. Drug
loading capacity of the DTX/MPEG2k-PDLLA4k-PLL1k
was evaluated by the determination of DL and drug
release behavior in different environments. And in
order to theoretical reveal the connection between
polymer chemical composition-drug interaction and
the stability of the drug loaded micelles; three
dimensional (3D) model simulation was also
introduced to simulative reproduce the formation
process of the DTX/MPEG2k-PDLLA4k-PLL1k micelles.
The interaction energy between DTX and
nano-formulations is not exceeded 10%, which may
lead the patients to absorb abundant carrier materials
[
41]. So, satisfactory drug loading capacity and
stability were the two main factors for an ideal
nano-formulation. In our previous study, we have
constructed
DTX/MPEG-PDLLA) micellar nano-formulation
which was used to inhibit the tumor growth [42, 43].
The DTX nano-formulation based on
polymer micelles,
a
DTX loaded MPEG2k-PDLLA1.7k
(
MPEG2k-PDLLA1.7k
block
TM
Nanoxel-PM , (which is launched into the South
Korea market) has solved its hydrophobicity and
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Theranostics 2017, Vol. 7, Issue 10
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MPEG2k-PDLLA4k-PLL1k molecules could be obtained.
Finally, we further evaluated the therapeutic effect of
the DTX/MPEG2k-PDLLA4k-PLL1k on breast cancer in
vitro and in vivo in detail. All the results demonstrated
that introduction of PLL could regulate the stability of
acetonitrile (Sigma, USA), Roswell Park Memorial
Institute 1640 medium (RPMI 1640, Gibco, USA)
(1640), Dulbecco’s modified Eagle’s medium (DMEM,
Sigma,
USA)
(DMEM)
and
3-(4,
5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium
DTX
micellar
nano-formulations,
and
bromide (MTT, Sigma, USA) (MTT) were used
without any further purification. Annexin V-FITC
Apoptosis Detection Kit was obtained from Key Gen
Biotech (Nanjing, China). 1, 1’-dioctadecyl-3, 3, 3’,
MPEG2k-PDLLA4k-PLL1k was also
candidate for DTX delivery.
a
promising
Materials and Methods
3
’-tetramethylindotricarbocyanine
(DiD)
was
purchased from Biotium (Hayward, CA). Ki-67 Rabbit
Monoclonal Antibody was obtained from Labvision
Materials
MPEG-PDLLA copolymers were synthesized
(
Minneapolis, USA). DAPI and Coumarin-6 (C6) were
following the previous work of our group [42, 53].
obtained from Sigma-Aldrich (Saint Louis, USA). All
the materials used in this article were analytical grade
and used as received.
MCF-7 cell line, 4T1 cell line, HEK293 cells and
HUVEC cells were obtained from the American Type
Culture Collection (ATCC; Rockville, MD), and
grown in DMEM media and 1640 media, respectively.
The cell culture was maintained in a 37 °C incubator
ε
N -carbonybenzoxy-L-lysine (H-Lys (Z)-OH) and
N-t-butoxycarbonyl-L-phenylalanine (Boc-L-Phe) and
triphosgene were purchased from GL Biochem Co.,
Ltd (Shanghai, China). DTX was purchased from
Sichuan Xieli Pharmaceutical Co., Ltd (Chengdu,
China). DTX injection, as the free DTX group in this
study, was supplied by Hengrui Medicine Co, Ltd
(
Jiangsu, China). Dehydrated alcohol (Aladdin,
with a humidified 5% CO
atmosphere.
2
Shanghai, China), triphosgene (Sigma, USA)
Figure 1. Structure and passive targeting mechanism of the DTX/MPEG2k-PDLLA4k-PLL1k. The micelle was consisted of MPEG2k-PDLLA4k-PLL1k and DTX. DTX and
PDLLA as the hydrophobic part formed the core of micelles, and the shell of micelle was formed by hydrophilic segments MPEG and PLL. From the 3D model of
DTX/MPEG2k-PDLLA4k-PLL1k, DTX was completely wrapped in core of the copolymer. The accumulation of DTX/MPEG2k-PDLLA4k-PLL1k in tumor tissue was
through EPR effect.
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Fifty-two Balb/c-nu mice and fifty Balb/c mice
were used for antitumor tests, purchased from the
HFK Bio-Technology. Co., LTD (Beijing, China). All
animal care and experimental procedures were
conducted following the guidelines (IACUC-S200904-
P001) approved by the Institutional Animal Care and
Treatment Committee of Sichuan University
solution was evaporated in a rotary evaporator at 60
C, and the homogenous co-evaporation was
o
obtained. Subsequently, the mixture was dissolved in
o
double distilled water at 60 C to self-assemble into
micelles. Next the micelles were filtered through a
0.22 μm syringe filter (Millex-LG, Millipore Co., USA).
Finally the micellar solution was freeze-dried to
receive DTX micelle powder [56].
(
Chengdu, P.R. China). All the mice were treated
humanely throughout the experimental period.
The particle size distribution and the
zetapotential of the DTX micelles were determined by
dynamic light scattering (DLS) (Malvern Nano-ZS 90,
Malvern, UK). The morphology of the micelles could
be detected by transmission electron microscopy
Synthesis of Lys (Z)-NCA
Synthesis of N-carboxyanhydride of 6-
(
benzyloxycarbonyl)-L-lysine (Lys (Z)-NCA) was
carried out by the Fuchs-Farthing method using
triphosgene as in the previous description [54].
Firstly, one dose of H-Lys (Z)-OH and half one dose of
triphosgene were added to tetrahydrofuran (THF),
(
TEM). Crystallographic assay was performed on free
DTX powder, blank
copolymer and DTX/MPEG2k-PDLLA4k-PLL1k
MPEG2k-PDLLA4k-PLL1k
micelles though an X-ray diffractometer (XRD) (X'Pert
Pro, Philips, Netherlands) using CuKα radiation. The
data was assembled from step-scan mode from 5 to
5
o
and then the reaction mixture was heated to 50 C
while being stirred. The solvent was evaporated to
terminate the reaction when the mixture became
transparent. The product was purified in cold
n-hexane three times. The structure of the Lys
o
o
0 with a step size of 0.03.
Drug loading capability of MPEG-PDLLA-PLL
(
Z)-NCA was characterized by Fourier Transform
Infrared (FTIR) recorded on Nicolet 200SXV meter
Nicolet, USA).
The DL and entrapment efficiency (EE) of the
micelles were determined by High Performance
Liquid Chromatography (HPLC) (HPLC 1260,
Agilent, US) with a C18 column (4.6 mm150 mm5
μm, Grace Analusis column). The compositions of the
mobile phase were Acetonitrile/ammonium acetate
solution (45/55, v/v) at a flow rate of 1 ml/min.
Detection was taken on a diode array detector (1260
DAD VL) at a wavelength of 232 nm. Before the
measurement, the samples were diluted with
acetonitrile [57]. The results were calculated using the
following equations:
(
Synthesis of MPEG-PDLLA-PLL
Firstly, MPEG-PDLLA was synthesized from the
D, L-lactide, and MPEG by ring opening
polymerization (ROP) in an environment of nitrogen
o
with 130 C oil bath for 8 hrs, and purified by
precipitation in petroleum ether [53]. By connecting
MPEG-PDLLA and Boc-Phe-OH, the amino
end-group of MPEG-PDLLA was obtained after
removing the t-butoxycarbonyl end group from
Boc-L-Phe end-capped MPEG-PDLLA. And then, the
MPEG-PDLLA-PLL was synthesized by the ROP with
DL% = Amount of DTX determined in micelle /
(
Amount of DTX determined + copolymer) 100%
Lys
MPEG-PDLLA, which was stirred in chloroform for
2 hrs. Finally, to get the MPEG-PDLLA-PLL triblock
(Z)-NCA
through
amino
terminated
EE% = Amount of DTX determined in micelle /
Amount of DTX in feed  100%
7
Evaluation of stability
copolymer, the deprotection reaction of the
copolymer MPEG-PDLLA-PLL was carried out in
HBr/HAc solution. Purified MPEG-PDLLA-PLL was
obtained by dialysis and freeze drying [55]. The
structure of the polymer was characterized by nuclear
To investigate the stability of the DTX micelles
solution, the solution was stored at room temperature
without lyophilization. The stability status was
evaluated by macroscopic observation [58]. The DTX
micelles solution was unstable while a precipitate
could be seen clearly. This method was according to
Chinese Pharmacopoeia 2010 (ChP 2010).
1
magnetic resonance spectroscopy ( H-NMR) (Varian
4
00 spectrometer, Varian, USA) and FTIR, the
molecular weight was characterized by Gel
Permeation Chromatography (GPC) (THF, 0.6
ml/min, Agilent 110 HPLC, America).
Molecular modeling study
The initial structures of MPEG2k-PDLLA1.7k and
MPEG2k-PDLLA4k-PLL1k were constructed using
Moltemplate, which was a general cross-platform
Preparation and characterization of the DTX
micelles
text-based
molecule
builder
for
LAMMPS
The micelles were prepared by a thin film
hydration method. Primarily, DTX and copolymers
were co-dissolved in dichloromethane. Then the
(
Large-scale Atomic/Molecular Massively Parallel
Simulator) [59], and optimized by the force field
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Theranostics 2017, Vol. 7, Issue 10
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method. Then the DTX molecule was docked to the
simulated copolymers to construct the micelle,
randomly. The molecular dynamic (MD) simulation
was calculated in a Compass force field [60]. The
temperature coupling and pressure coupling of the
MD simulation were determined by Nose-Hoover
and Berendsen, respectively. The MD simulation
process was as follows: The initial temperature of the
system was set from 0 K to 600 K, the step size was 0.5
fs and the step number was 200000. The simulated
annealing was performed while the system
temperature reached the set value, and then the
system temperature was cooled to 300 K within 100
ps. The equilibrium molecular dynamics simulation of
isothermal-isobaricensemble (NPT) was performed
after the annealing treatment [61]. The PyMol was
used for visual analysis [62].
different concentrations were added to 2.5 ml of the
RBCs suspension. Distilled water and normal saline
were added to the RBC suspension as positive and
negative controls, respectively. All samples were
incubated at 37 °C for 3 hrs and then centrifuged. The
absorbance of supernatant was determined at 545 nm
by UV/VIS Spectrometer (Lambda 35, Perkin Elmer).
When this value was greater than 5%, the sample
possessed hemolytic potential. The hemolytic rate was
calculated according to the following equations:
Hemolytic Rate= (OD sample-OD negative) / (OD positive
OD negative) 100%
-
Delivery of the micelles in vitro
DTX was replaced by a hydrophobic fluorescent
probe C6 to investigate the cellular uptake of micelles.
For fluorescence microscopy observation, MCF-7 cells
and 4T1 cells were incubated for 24 hrs in 6-well
In order to explore the mechanism of the
copolymers entrapping DTX, the interaction between
DTX and the copolymers was investigated. The
interaction energy was calculated according to the
following equation. The results were listed in Table 2
4
o
plates (210 cells per well) at 37 C. The
C6/MPEG2k-PDLLA4k-PLL1k micelles, C6/MPEG2k
-
PDLLA1.7k micelles and free C6 were added to the
plates at the final concentration of C6 was 100 ng/ml,
respectively. And then the cells were washed with
PBS after fixing with 4% paraformaldehyde in PBS.
Subsequently, the fixed cells were treated with DAPI
for 5 min, and then the fluorescence images of the
micelles cellular uptake was observed under a
fluorescence microscope; (Olympus, China) with
excitation at 488 nm. For flow cytometry analysis, the
[
63, 64].
ΔEinteraction =Ecomplex - Ecopolymer - Edocetaxel
Drug release behavior of the micelles in vitro
The drug release behaviors of the DTX micelles
in different pH environments were evaluated by
using the dialysis method. Briefly, free DTX,
5
DTX/MPEG2k-PDLLA1.7k micelles and DTX/MPEG2k
-
cells (310 cells per well) incubated for 24 hrs in
o
PDLLA4k-PLL1k micelles were separately suspended
in a dialysis bag (molecular weight cutoff: 14k Da)
and were then immersed into different pH PBS
6-well plates at 37 C, and then were treated with the
normal saline (NS) as control, C6/MPEG2k
-
PDLLA4k-PLL1k micelles, C6/MPEG2k-PDLLA1.7k
o
solution at 37 C under horizontal shaking (100
micelles and free C6 in 6-well plates as described
above. The cells were harvested in PBS after treated
with Trypsin-EDTA, and the fluorescence intensity of
rpm/min). At predetermined intervals, the aliquots (2
ml) were withdrawn and replaced by the same
amount of PBS. The amount of DTX released at
designated time points were measured by HPLC. The
chromatographic conditions and the methods
followed determination of DL.
TM
the cells was determined by an ACEA NovoCyte
TM
Flow Cytometer (NovoExpress , ACEA Bioscience.
Inc., USA)
Evaluation of cytotoxicity in vitro
To
investigate
the
stability
of
the
DTX/MPEG2k-PDLLA4k-PLL1k
micelles,
the
MTT assay
DTX/MPEG2k-PDLLA4k-PLL1k micelles were stored at
room temperature. The sample was filtered to remove
the leaked DTX at predetermined time points, and the
content of DTX remaining in the micelles solution was
measured by HPLC according to United States
Pharmacopeia (USP).
3
Firstly, the cells (310 cells/well) were cultured
o
in 96-well plates and incubated for 24 hrs at 37 C.
Micelles of different concentrations were added and
cultivation continued for 24 hrs. 20 μl MTT solution (5
mg/ml) was added into each well, and the MTT
solution was removed and replaced with DMSO (160
μl/well) after 3 hrs. The absorbance was measured at
a wavelength of 490 nm using a 680 model microplate
reader from an infinite M200 microplate reader
Hemolysis test
The
hemolytic
potential
of
the
MPEG2k-PDLLA4k-PLL1k micelles in vitro was tested as
follows: Rabbit blood without fibrinogen was washed
and diluted with normal saline to get a suspension of
(
Tecan, Durham, USA).
Cell apoptosis analysis
2
% red blood cells (RBCs). 2.5 ml of micelles with
Cells were seeded into 6-well plates at 2105
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cells/well. The different DTX formulations were
added at the dosage of 10 μg DTX per ml and
incubated for 24 hrs. The cells were collected and
washed three times in normal saline. The cells were
resuspended in a binding buffer, and Annexin V-FITC
and PI were added in sequence. The cells were
analyzed by the flow cytometry
DTX/MPEG2k-PDLLA4k-PLL1k (dose of DTX: 10
mg/kg) and blank MPEG-PDLLA-PLL micelles (dose
of copolymer: 90 mg/kg) for three consecutive days.
Tumor size and body weight were measured every
other day during the experimental period and the
tumor volumes were calculated from the formula:
volume (mm ) = length  width  0.5. For MCF-7
model, there were eight mice per group. When the
tumor diameter was reached 20 mm, five mice in each
group were sacrificed, and the tissues were excised
for further analysis. The tumor growth trend would
be monitored on the other three mice. For 4T1 model,
there were ten mice per group. Five mice in each
group were sacrificed until the mice began to die, and
the ex vivo tissues were harvested for further analysis.
The survival time of 4T1 tumor-bearing mice was
observed on the other five mice in each group.
3
2
Delivery of the micelles in vivo
In order to eliminate the artifact signal caused by
the fur of the Balb/c-nu mice during bioluminescence
imaging, the Balb/c-nu mice were chosen as the
6
experimental subject. MCF-7 cells (210 /0.1 ml)
were incubated in the right flank of the athymic
female BALB/c-nu mice by a subcutaneous injection.
The near-infrared fluorescence dye DID was used to
replace DTX as the fluorescence probe to be
encapsulated in the micelles to evaluate the
bio-distribution in vivo. The mice were divided into
To investigate the therapeutic effects further, the
apoptosis of the tumor cells was detected by terminal
control group, free DID group, DID/MPEG2k
-
deoxynucleotidyl
transferasemediated
nick-end
PDLLA1.7k group and DID/MPEG2k-PDLLA4k-PLL1k
group, which was treated with NS as control, free
labeling (TUNEL) (TUNEL, Promega, Madison, WI,
USA) staining assay, and the proliferation of the
tumor cells was analyzed by immune histochemical
staining of Ki-67 (Ki-67) (LabVision, MA, USA). On
the seventh day after administration, we killed the
mice and harvested the tumor tissues. The tumor
tissues were fixed in the 15%wt formaldehyde once
the mice were killed. The TUNEL method was used
according to the manufacturer’s instructions and the
Ki-67 staining was processed by using the labeled
streptavidin-biotin method. The apoptosis and the
proliferation of the tumor cells were detected by the
fluorescence microscope (Olympus, China).
To investigate the damage caused by DTX
formulations in vivo, the ex vivo normal organs and
tumor were obtained. The tissue samples were
preserved in 10% buffered formaldehyde for 2 days.
The damage of the tissue was visualized by
hematoxylin and eosin staining (H&E stain).
DID, DID/MPEG2k-PDLLA1.7k or DID/MPEG2k
-
PDLLA4k-PLL1k via intravenous injection until the
tumor diameter was about 8 mm, respectively,. Each
group had three mice, and the final injected dose of
DID in each mouse was 100 μg/kg [65]. The mice
were anesthetized by 10% chloralic hydras solution
(
300 μg/kg, chloralic hydras) before near-infrared
imaging at predetermined. The real-time monitoring
was detected by a fluorescence imaging system (IVIS
Lumina Series III, PerkinElmer, USA; excitation = 645
nm, emission = 715 nm long pass) at the time interval
of 2 hrs, 6 hrs and 24 hrs. At the 24 hrs post-injection,
the mice were sacrificed, and the heart, liver, spleen,
lung, kidney and tumor were harvested to evaluate
the ex vivo organs’ fluorescence intensity. The
quantitative fluorescence intensity was acquired
using the onboard software.
Anti-tumor effect in vivo
Statistical analysis
The anti-tumor activity of the DTX micelles was
investigated in a subcutaneous MCF-7 model and 4T1
model. The process of the subcutaneous tumor model
establishment was the same as section tumor
targeting of the micelles. The mice were randomized
into 5 groups, and injected subcutaneously with 0.1ml
of cell suspension containing 110 cells per mouse.
When the tumor diameter was approximately 8 mm,
the mice were double-blind randomly divided into
The comparison of each group was evaluated by
the statistical analysis with one-way analysis of
variance (ANOVA) using SPSS software. All the data
were expressed as the average value±SD, and a P
value <0.05 was considered statistically significant.
6
Results and Discussion
Characterization of the MPEG-PDLLA-PLL
copolymers
control group, free DID group, DID/MPEG2k
-
PDLLA1.7k group and DID/MPEG2k-PDLLA4k-PLL1k
group which were intravenously injected with NS
MPEG-PDLLA was synthesized by our group
through ROP between MPEG and D, L-lactide [42, 53].
MPEG-PDLLA-PLL was synthesized by conjugating
PLL with the MPEG-PDLLA. The structures of the
(
control), free DTX (dose of DTX: 10 mg/kg),
DTX/MPEG2k-PDLLA1.7k (dose of DTX: 10 mg/kg),
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Theranostics 2017, Vol. 7, Issue 10
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1
copolymers were characterized by H-NMR and FTIR
figure S1 and figure S2). In figure S1, the
characteristic peaks of PLL were h (-CH-, 4.62 ppm), i
biocompatibility and extensive application in
pharmaceutical field. Nowadays, due to MPEG with
macro molecular weight (over 3.5k Da) might lead to
metabolism or other immune-genetic associated
problem, PEG2k was extensively used in micelle
preparation [66, 67]. Therefore, in this study, the
PEG2k as a certain hydrophilic segment to form the
shell structure of micelle. Particle size as a basic
characteristic of micelles was determined by DLS.
(
(
-CH
2
-, 1.35 ppm), j (-CH
2
-, 2.74 ppm), which could be
found in the H-NMR spectrum of MPEG-PDLLA-
PLL. The molecular weights of the copolymers were
1
calculated using an integral ratio of CH O- (a, at 3.33
3
ppm) to PDLLA and PLL, respectively. The
copolymers with different molecular weights are
displayed in Table 1. These results indicated that the
From
figure
2A,
the
particle
size
of
MPEG-PDLLA-PLL
successfully.
had
been
synthesized
MPEG2k-PDLLA1.7k-PLL0.5k could not be determined
by its extremely instable, and sizes of the other
copolymer micelles were less than 200 nm, which was
contributed to concentrate in tumor site through EPR
effect (figure 1) [4, 5]. Furthermore, stability and
entrapment efficiency of micelles were affected by the
drug feeding and molecular weight of copolymer, and
they were two important indicators for evaluation of
micellar drug loading capacity.
Preparation and Optimization of the DTX
loaded polymeric micelles
DTX micelles were prepared by thin film
hydration method. The properties of DTX micelles,
prepared
from
different
molecular
weight
copolymers, were investigated in detail and the
results were listed in Table 1. MPEG was regarded as
the most common hydrophilic segments owing to its
Table 1. The drug loading, entrapment efficiency, stability and sizes of micelles with different block ratio copolymers.
MPEG -PDLLA -PLL
Mw (Da)
3758
Drug Feeding (wt %)
DL (%)
EE (%)
Stability (25
o
C)
Size (nm)
x
y
z
Self-assemble (hr) Resolved (hr)
2
k-1.7k
5
8
4.90±0.23
7.66±0.2
97.91±4.51
95.97±2.47
92.88±0.64
85.26±7.2
*
4
1
0.5
0.5
*
0.5
0.5
2h
0.5
0.5
0.5
24
12
4
1
1
1
1
1
0.5
*
0.5
0.5
*
4
1
20±2
22±3
23±3
*
1
5
1
5
1
5
1
2
3
5
1
2
3
4
5
5
1
2
3
5
1
2
3
5
1
2
3
2
0
0
11.15±0.08
4.28±0.46
8.95±0.32
4.36±0.34
9.26±0.41
4.76±0.26
9.51±0.64
12.65±1.28
16.24±0.94
4.83±0.17
10.09±0.73
19.68±0.82
26.70±1.18
29.43±6.74
8.55±2.63
4.85±0.21
9.78±0.76
17.49±0.84
21.1±4.42
4.77±0.18
9.37±0.32
5.18±0.95
3.78±1.23
4.69±0.24
9.21±0.36
12.43±2.52
14.69±1.17
0.5
0.25
*
0.25
0.25
2
0.5
0.25
0.25
24
12
4
1
1
1
1
1
0.5
*
0.25
0.25
*
2
2
2
k-1.7k-0.5k
k-4k
4236
6042
6517
*
87.20±6.8
93.15±7.21
95.20±5.02
93.24±6.23
63.25±6.40
54.13±3.13
96.64±3.50
100.25±7.28
95.53±3.98
90.18±3.99
73.51±16.84
16.78±5.23
97.42±3.78
97.82±7.58
90.01±1.64
70.35±14.72
95.42±3.58
93.39±3.51
25.78±4.87
12.63±4.07
93.81±0.95
90.44±2.02
62.89±11.86
48.97±3.9
40±10
*
42±3
44±5
*
k-4k-0.5k
0
0
0
*
2
k-4k-1k
7069
43±2
45±3
48±5
126±4
186±50
*
104±5
110±8
*
0
0
0
0
0
2
2
5
k-4k-2k
k-4k-4k
k-3k-2k
8071
0
0
0
*
10112
10063
85±6
92±5
*
0
0
0
*
*
*
0.5
0.5
*
0.25
0.25
*
49±4
51±3
*
0
0
0
*
*
*
*
was indicated the property of micelles could not be determined.
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Theranostics 2017, Vol. 7, Issue 10
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Figure 2. The particle sizes of the DTX loaded micelles (the drug feeding was 5%) based on different molecular weight of copolymers (A); the maintaining period of
DTX loaded micelles based on the MPEG2k-PDLLA4k, MPEG2k-PDLLA4k-PLL0.5k, MPEG2k-PDLLA4k-PLL1k, MPEG2k-PDLLA4k-PLL2k and MPEG2k-PDLLA4k-PLL4k (B); the
MPEG2k-PDLLA1.7k-PLL0.5k and MPEG2k-PDLLA4k-PLL0.5k (C) with the 5% of drug feeding; the entrapment efficiency change (D) and the maintaining period (E) of
DTX/MPEG2k-PDLLA4k-PLL1k micelles with the increased of drug feeding, respectively. (“*” was indicated the particle size of micelle could not be determined.)
We have first investigated the effect of the
molecular weight of the hydrophilic and hydrophobic
segment in MPEG-PDLLA on the stability of DTX
micelles. And the results revealed that the DTX
micelles formed by MPEG-PDLLA consist of mPEG2k
and PDLL1.7k was more stable than the others. PLL
MPEG2k-PDLLA1.7k-PLL0.5k
,
however, had been
dramatically decreased, as listed in Table 1. It might
ascribe to the increase of hydrophilic percentage of
the polymer chain. In order to solve the problem, we
further increased the molecular weight of PDLLA
segments. And the effect of PLL segments on the
stability of the DTX micelles formed by
MPEG-PDLLA-PLL triblock copolymer was studied
in detail and discussed as following:
was
added
to
the
polymer
chain
of
MPEG2k-PDLLA1.7k to evaluate the effect of PLL
segment to the stability of MPEG-PDLLA micelles.
The stability of DTX micelles formed by
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Theranostics 2017, Vol. 7, Issue 10
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Nanoxel-PMTM (DL was 5%). It indicated that
DTX/MPEG2k-PDLLA4k-PLL4k micelle was stable
with high drug feeding. Moreover, the EE of the
micelle remained above 95% when the drug feeding
was increased from 5% to 20% (figure 2D). Hence
modifying 1k Da of the PLL onto MPEG2k-PDLLA4k
could achieve a high drug loading capacity of DTX
micellar carrier. In this study, in consideration of DL
and stability, the DTX/MPEG2k-PDLLA4k-PLL1k
micelle with 10% DL was used as a novel DTX
formulation for exploration in detail.
Effect of PLL’s molecular weight on micellar
stability
To improve the hydrophilic of the copolymer,
PLL was modified to MPEG2k-PDLLA4k. Under the
fixed drug feeding and molecular weight of MPEG
and PDLLA, we optimized the effect of PLL molecular
weight on the stability of DTX micelles and the results
were displayed in figure 2B. As the increase of
molecular weight of PLL from 0k Da to 1k Da, the
maintaining period of DTX/MPEG-PDLLA-PLL was
increased from 15 min to 24 hrs. It indicated that the
introduction of PLL can affect the stability of
DTX/MPEG-PDLLA-PLL micelles. While further
increase the PLL molecular weight from 1k Da to 4k
Da, however, the maintaining period had decreased
from 24 hrs to 30 min. it could conclude that we could
obtain the DTX/MPEG2k-PDLLA4k-PLL1k micelles
with the longest maintaining period while the
molecular weight of PLL was 1k Da. It might be
ascribed to the molecular weight of the PLL was
above 2k Da, which resulting in the hydrophilicity of
the copolymer was too strong to form stable core-shell
structure.
Characterization of
DTX/MPEG2K-PDLLA4K-PLL1K micelle
The critical micelle concentration (CMC) of the
MPEG2k-PDLLA4k-PLL1k micelles and MPEG2k
PDLLA1.7k micelles was determined by fluorophoto-
meter (LS55, Perkin Elmer). The CMC of the MPEG2k
-
-
-3
PDLLA4k-PLL1k micelles was 10 mg/ml, as same as
the MPEG2k-PDLLA4k-PLL1k micelles (10 mg/ml),
which could remain stable in blood (figure S3).
-3
Morphological
study
of
the
DTX/MPEG2k-PDLLA4k-PLL1k was determined by
DLS and TEM. The size distribution of the
DTX/MPEG2k-PDLLA4k-PLL1k (the DL was 10%)
micelles was around 50 nm, which was slightly larger
than the blank micelles (48 nm) (figure 3). Moreover,
as shown in figure 3A, the particle size did not
increase after the DTX/MPEG2k-PDLLA4k-PLL1k
micelles were re-dissolved. The result was similar
with the formulation which appeared as compact
spheres by transmission electron microscopy (figure
Effect of PDLLA’s molecular weight on
micellar stability
PDLLA was the only hydrophobic segment in
this triblock copolymer, which determined the
oil-water balance of the copolymer. From figure 2C, it
was easy to find that the stability of
MPEG2k-PDLLA4k-PLL1k (2 hrs, DL 5%) was superior
to MPEG2k-PDLLA1.7k-PLL0.5k (15 min, DL 5%). For the
diblock copolymers, however, the maintain period of
DTX/MPEG2k-PDLLA1.7k micelles was decreased
from 4 hrs to 15 min, while the molecular weight of
PDLLA was increased from 1.7k Da to 4k Da. So,
3
C).
Zeta potential of DTX/MPEG2k-PDLLA4k-PLL1k
was around 28±2.14 mV, as shown in figure 3B,
lyophilized did not change this property. The positive
charges on micelle surface were derived from the
amino of PLL.
Crystallographic assay was performed by XRD
and the result was shown in Figure S4. In comparison
with blank MPEG2k-PDLLA4k-PLL1k and DTX, the
graph of DTX/MPEG2k-PDLLA4k-PLL1k lacked the
characteristic diffraction peaks, indicating that DTX
was completely wrapped in the micelles. These results
suggested that the DTX/MPEG2k-PDLLA4k-PLL1k
micelles were produced successfully.
when the hydrophilic segment was
a certain
molecular weight, adjusting the molecular weight of
hydrophobic segment was also a way to increase the
micellar stability. In this study, in consideration of the
investigation on effect of the molecular weight on
micellar stability in previous section, PDLLA 4k Da
was used as the hydrophobic segment of
MPEG-PDLLA-PLL for further exploration.
Effect of drug feeding on the micellar stability
According to the results of the comparison of
drug loading capacities of copolymers with different
molecular weight, MPEG2k-PDLLA4k-PLL4k micelle
was selected for the further investigation. As shown
in figure 2E, it was easy to find that the maintaining
period has decreased from 24 hrs to 1hr during the
drug feeding increased from 5% to 50%. When drug
feeding was 20%, however, the maintaining period
was 4 hrs, as long as the list DTX formulation
Molecular modeling study
In order to further study the mechanism of the
copolymer loaded DTX, the dynamics simulation
technique was used for simulating its variation of
conformations. The 3D structure of the DTX micelles
is shown in figure 4. The self-assembly processes of
the
DTX/MPEG2k-PDLLA1.7k
micelles
and
DTX/MPEG2k-PDLLA4k-PLL1k micelles in annealing
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Theranostics 2017, Vol. 7, Issue 10
2661
dynamics simulation were presented in figure 5.
Initially, the DTX was close to PDLLA, while the
hydrophilic segments formed the shell. Then the DTX
number of the hydrogen bonds between DTX and
MPEG2k-PDLLA4k-PLL1k promoted the micellar
stability. Furthermore, the interactions between
copolymers and DTX molecules were investigated
and the results were shown in table 2. The Einteraction of
DTX/MPEG2k-PDLLA4k-PLL1k micelles (-107.0920
was
entrapped
by
the
polymers.
For
MPEG2k-PDLLA1.7k, the PDLLA was not long enough
to entrap DTX molecules completely, and the DTX
molecule was only on the concave zone of the surface
kJ/mol) was lower than Einteraction of DTX/MPEG2k
-
of the micellar structure. For MPEG2k-PDLLA4k-PLL1k
,
PDLLA1.7k micelles (-40.9464 kJ/mol), which indicated
that the DTX/MPEG2k-PDLLA4k-PLL1k micelles were
more stable. Therefore, both the non-bonded
interaction and the complete drug cover contributed
to the high DL and stability.
however, the molecular weight of PDLLA was 4k Da
and the degree of polymerization was 45, which had
enough space to touch the DTX. The PLL was one of
the hydrophilic segments in MPEG2k-PDLLA4k-PLL1k
,
which could form the shell structure with MPEG to
package DTX molecules completely in the core. It
indicated that the molecular structure of
MPEG2k-PDLLA4k-PLL1k was definitely conducive for
DTX inclusion. In addition, due to the existence of the
amino groups on PLL segment (figure 6), a large
Table 2. Interaction Energy and Energies of components
contributed to it (kJ/mol)
System
E
complex
E
copolymer
Edocetaxel ΔEinteraction
DTX/MPEG2k-PDLLA1.7k
-1441.0494 -1356.4659 -43.6371 -40.9464
DTX/MPEG2k-PDLLA4k-PLL1k -4125.8341 -3975.1050 -43.6371 -107.0920
Figure 3. Characterization of DTX/MPEG2k-PDLLA4k-PLL1k. The particle size distribution (A) and zeta potential (B) of the micelles were measured by DLS; TEM
images of blank micelles 1, DTX/MPEG2k-PDLLA4k-PLL1k micelles (Self-assembled 2 and Re-dissolved 3) (C).
Figure 4. The surface binding state of DTX/MPEG2k-PDLLA1.7k (A), DTX/MPEG2k-PDLLA4k-PLL1k (B), respectively. DTX was represented by the thin stick, the
copolymers were depicted with solid surface.
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Theranostics 2017, Vol. 7, Issue 10
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Figure 5. The dynamics simulation annealing process of the DTX/MPEG2k-PDLLA1.7k and DTX/MPEG2k-PDLLA4k-PLL1k, respectively.
Figure 6. Interaction models between the MPEG2k-PDLLA1.7k (A), MPEG2k-PDLLA4k-PLL1k (B) and DTX. DTX molecule, hydrophobic block and hydrophilic block
was represented by the red stick, cyan stick and green stick, respectively. Shot dash line was the hydrogen-bond, the number was length (Å).
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Theranostics 2017, Vol. 7, Issue 10
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The study of drug release behavior in vitro
MPEG2k-PDLLA1.7k micelles, while 76.4% of DTX was
released from the MPEG2k-PDLLA4k-PLL1k micelles
within 120 hrs. So the DTX/MPEG2k-PDLLA4k-PLL1k
micelles could provide extended sustained release of
The stability of DTX/MPEG2k-PDLLA1.7k and
DTX/MPEG2k-PDLLA4k-PLL1k was quantified by the
content of DTX remaining in the micelles aqueous
solution. As shown in figure 7A, the DTX content in
micelles decreased with the extension of time. At 120
hrs, 74.7% DTX leaked from the MPEG2k-PDLLA1.7k
micelles, and 25.2% DTX leaked from the
MPEG2k-PDLLA4k-PLL1k micelles. It indicated that the
stability of DTX/MPEG2k-PDLLA4k-PLL1k micelles in
DTX,
compared
with
the
behavior
of
DTX/MPEG2k-PDLLA1.7k micelles.
Furthermore, the drug release behavior of the
DTX/MPEG2k-PDLLA4k-PLL1k micelles in different
pH media was investigated, and the results were
exhibited in figure 7C. In the first 12 hrs, 80% of DTX
was released in the acid medium; only 40% of DTX
was released in the basic medium. Finally, the drug
was released completely in the medium with pH5.
Therefore, MPEG2k-PDLLA4k-PLL1k might prevent
drug release in the basic environment while
promoting drug release in the acidic environment,
and the mechanism of this phenomenal would be
researched in detail in the future. This property of
DTX/MPEG2k-PDLLA4k-PLL1k might enable the
specific release of DTX at the acidic tumor site.
pH7.4
PBS
solution
was
superior
to
DTX/MPEG2k-PDLLA1.7k that of micelles.
The in vitro release behavior of free DTX and
DTX micelles based on MPEG2k-PDLLA1.7k and
MPEG2k-PDLLA4k-PLL1k are shown in figure 7B. In
the free DTX group, 94.9% DTX was released into the
media within 24 hrs, and the micelle groups released
7
3.8% and 56.2%, respectively. It suggested that the
micelles could prevent drug release quickly. Similarly,
6.6% of DTX was released from the
9
Figure 7. The release profile of DTX formulations. The content of DTX remaining in the micelles aqueous solution (A); the DTX formulations release profiles in PBS
.4 (B); In vitro release profiles of DTX from DTX/MPEG2k-PDLLA4k-PLL1k system in different pH media (C); the first 12 hrs of the in vitro release profiles in different
pH media (D). Statistical difference from the free DTX group (“*” p˂0.05)
7
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Theranostics 2017, Vol. 7, Issue 10
2664
PDLLA4k-PLL1k micelles were safe for using in breast
cancer therapy.
Hemolysis test and biocompatibility evaluation
of the micelles
Since the cationic polymer easily caused
Delivery of the micelles in vitro
hemolysis,
the
hemolytic
test
of
the
To investigate the transport ability of
MPEG2k-PDLLA4k-PLL1k micelles should be carried
out. From figure S5, the hemolysis did not happen at
any concentration of MPEG2k-PDLLA4k-PLL1k
micelles. The hemolytic rate (0 to 3.10%) of
MPEG2k-PDLLA4k-PLL1k, shown in table S1, was
under the international standard (5%), which did not
cause hemolysis. To trace toxicity caused by the
formulations accurately, the normal organs in each
group were checked. From figure S7 and S8, no
obvious damage appeared in the each group. These
DTX/MPEG2k-PDLLA4k-PLL1k
in
vitro,
the
fluorescence microscope and flow cytometry were
used to analyze the cellular uptake. In this study, C6
was used as a fluorescence indicator to be loaded into
the micelles. From figure 8A and B, the fluorescence of
the C6 can be observed in the cytoplasm, which
indicated that both MPEG2k-PDLLA1.7k
MPEG2k-PDLLA4k-PLL1k could transfer drugs into
and
tumor cells.
results
indicated
that
the
DTX/MPEG2k
-
Figure 8. The cellular uptake of micelles. The fluorescence images of micelles by MCF-7 cell lines (A) and 4T1 cell lines (B); Flow cytometer analysis of C6 cellular
binding in the MCF-7 cells(C) and 4T1 cells (D), respectively. The quantitative analysis of C6 cellular binding in the MCF-7 cells (E) and 4T1 cells (F), respective. 1, 2,
3
, 4 represented control, free C6, C6/MPEG2k-PDLLA1.7k and C6/MPEG2k-PDLLA4k-PLL1k, respectively. Statistical difference between the groups (“**” p˂0.01)
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Theranostics 2017, Vol. 7, Issue 10
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Furthermore, to quantify the cellular uptake of
the DTX micelles, the intensity of the tumor cells,
treated with different formulations, was detected by
flow cytometer, and the results are shown in figure 8C
and D, respectively. The fluorescence signal of the free
C6 group was stronger than the control group, but
much weaker than the micelle groups. Moreover, as
shown in figure 8E and F, the fluorescence signal
intensity of the C6/MPEG2k-PDLLA4k-PLL1k group
was three times that of the C6/MPEG2k-PDLLA1.7k
group. In 4T1 cell lines, the fluorescence intensity of
the C6/MPEG2k-PDLLA4k-PLL1k group and the
C6/MPEG2k-PDLLA1.7k group was consistent with
that of MCF-7 cells. Therefore, these results indicated
that the micelles would be an effective way to
promote cell uptake for hydrophobic drugs, and
MPEG2k-PDLLA4k-PLL1k was suitable to cargo
hydrophobic drugs into cells.
of 4T1 cells and MCF-7 cells in a dose-dependent
manner, and the blank MPEG-PDLLA-PLL micelles
were little toxicity for the tumor cells. Moreover, the
half maximal inhibitory concentration (IC50) values
(figure 9C) of the DTX/MPEG2k-PDLLA4k-PLL1k
group on MCF-7 (7.16 μg/ml) and 4T1 (6.35 μg/ml)
were much lower than the other groups. Additionally,
cell apoptosis experiments were used for MTT assay
further certification. The proportion of apoptotic cells
in each group was shown in figure 9D. The MCF-7 cell
and
4T1
cell
apoptosis
rate
of
the
DTX/MPEG2k-PDLLA4k-PLL1k group was 45% and
41%, respectively, much higher than the other groups.
The results of the cell apoptosis experiments were
consistent with the MTT assays. In previous research,
micelles were proved to prevent the drug efflux
mediated by P-glycoproteins [68]. So compared with
free DTX, the cellular uptake enhancement and
intracellular retention prolongation of DTX-loaded
micelles improved the cytotoxicity for tumor cells.
In vitro cytotoxicity of the micelles
The MTT assay and the apoptosis experiments
were used to evaluate cytotoxicity of DTX
formulations. The results of MTT assay on HEK293
cells and HUVEC cells (figure S6) indicated that low
toxicity of the MPEG2k-PDLLA4k-PLL1k micelles for
normal cells. From figure 9A and 9B, it can be seen
that all the DTX formulations can inhibit the growth
Also,
the
anti-tumor
effect
of
DTX/MPEG2k-PDLLA4k-PLL1k was superior to
DTX/MPEG2k-PDLLA1.7k micelles, which would
contribute to the stability and the micellar affinity of
the MPEG2k-PDLLA4k-PLL1k micelles to cells [50].
Figure 9. The anti-tumor effect of the DTX formulations. Cytotoxicity studies on 4T1 cells (A) and MCF-7 cells (B); C: IC50 value of DTX formulations on 4T1and
MCF-7 cells; D: Cell apoptosis on 4T1and MCF-7 cells; Statistical difference from the free DTX group (“*” p˂0.05) (“**” p˂0.01)
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Theranostics 2017, Vol. 7, Issue 10
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PDLLA4k-PLL1k group was much higher than that the
Delivery of the micelles in vivo
DID/MPEG2k-PDLLA1.7k
group.
post-injection, the fluorescent signals in mice were
decreased. Furthermore, to evaluate the
At
24
hrs
The in vivo targeting ability of MPEG2k
-
PDLLA4k-PLL1k micelles for a MCF-7 cells tumor
engraftment was investigated on nude mice. DID was
used as the fluorescent chromogenic agent entrapped
in micelles for the real-time drug delivery efficiency
test by fluorescence imaging system. During 2 hrs
post-injection the same dose of DID, the fluorescence
images of tumors was presented in figure 10A, the
tumor fluorescence signals of mice treated with DID
micelles were significantly stronger than that treated
with free DID, and DID fluorescence of the micelle
groups began to concentrate in tumor. As the time
was prolonged, the accumulation of fluorescent
signals in tumors reached the peak levels at 6 hrs. The
biodistribution in mean organs, the mice were
sacrificed, and fluorescent signals in ex vivo mean
organs and tumors were determined (figure 10B). The
fluorescence intensity of micelle groups was stronger
in tumor and weaker in normal organs than the free
DID group. Compare with DID/MPEG2k-PDLLA1.7k
group,
the
fluorescence
intensity
of
DID/MPEG2k-PDLLA4k-PLL1k group in tumor was
stronger, which was much weaker in heart, kidney
and lung. These results indicated that drugs could be
accumulated
MPEG2k-PDLLA4k-PLL1k
into
tumor
sites
through
.
tumor fluorescence intensity of the DID/MPEG2k
-
Figure 10. In vivo biodistribution of control 1, free DID 2, DID/MPEG2k-PDLLA1.7k 3 and DID/MPEG2k-PDLLA4k-PLL1k 4. Real-time fluorescence images of the
tumor-bearing mice after intravenous injection at set time points (A); the fluorescence images of ex vivo mean organs and tumors at 24 hrs post-injection (B); the
fluorescence images of tumors (C); the quantitative analysis of fluorescence intensity in tumors at set time points post-injection (D); the quantitative analysis of
fluorescence intensity in ex vivo mean organs and tumors (E)
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Theranostics 2017, Vol. 7, Issue 10
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This experimental phenomenon was dependent
on the enhancement of micelle-mediated drug
delivery efficiency, which was due to the long
circulation time caused by surface PEGylation, the
ideal particle size and the cell affinity. The PEG
molecule on the surface of the micelles could prevent
the reticuloendothelial system (RES) recognition and
reduce the liver and spleen uptake, endowing the
micelles with a longer circulation time [69]. The
particle sizes (less than 200 nm) could effectively
target the tumor using size-dependent properties and
EPR effect of the tumor (figure 1) [4, 5]. Positive ion on
the surface of the MPEG2k-PDLLA4k-PLL1k easily
promoted the micelles’ penetration of the
figure (figure 13E). The most serious damage was
observed in DTX/MPEG2k-PDLLA4k-PLL1k group.
The
results
suggested
that
DTX/MPEG2k-PDLLA4k-PLL1k micelles could inhibit
growth of the MCF-7 tumor effectively in vivo.
In order to explore the in vivo effectiveness of the
DTX formulation on different kinds of breast cancer
tumors, the tumor growth curve and survival time of
the 4T1 tumor-bearing mice was recorded in figure
12C and D, respectively. From the 4T1 tumor growth
curve, all DTX formulations showed efficient
antitumor ability. DTX/MPEG2k-PDLLA4k-PLL1k
micelles could significantly inhibit 4T1 tumor growth
(p<0.01), and the antitumor effect could be intuitive in
figure 12A and B, which was consistent with the
results of the MCF-7 tumor-bearing mice model.
Survival time of the 4T1 tumor-bearing mice (figure
cytomembrane. Therefore, the MPEG2k-PDLLA4k
-
PLL1k micelles were concentrated in the tumor tissue.
Antitumor activity of micelles in vivo
1
2D) was observed for 35 days after administration.
To evaluate the in vivo anti-tumor active of
DTX/MPEG2k-PDLLA4k-PLL1k, MCF-7 tumor-bearing
mice and 4T1 tumor-bearing mice were treated with
different DTX formulations, respectively. Because of
the weak anti-tumor activity of the blank
MPEG2k-PDLLA4k-PLL1k micelles, the results of the
evaluation of anti-tumor activity of which were not
shown in this section. According to images of MCF-7
tumor bearing mice with different treatments on 28
days after administration (figure 11A), photographs
of MCF-7 tumors (figure 11B) and the MCF-7 tumor
Mice in the control group treated with NS were begun
to die at 21 days after administration, and the
remaining mice were dead within 28 days. Compared
to the control group, DTX/MPEG2k-PDLLA1.7k and
free DTX could extend the survival time of the 4T1
tumor-bearing mice, but all the mice were dead after
3
0
days. In contrast, 60% of mice in the
DTX/MPEG2k-PDLLA4k-PLL1k group were still alive
at 35 days. The TUNEL, Ki67 and H&E stain were
furthermore to evaluate the inhibition of 4T1 cell in
vivo. As shown in figure 14A and 14B, compared with
control group, free DTX group and the
DTX/MPEG2k-PDLLA1.7k group, weak Ki-67 positive
immune-active and the most number of apoptotic
cells were observed in DTX/MPEG2k-PDLLA4k-PLL1k
group. The damage of tumor tissue was investigated
by H&E stain (figure 14E). The intact nucleus number
of tumor tissues in DTX/MPEG2k-PDLLA4k-PLL1k
group was decreased with the comparison of control
group, free DTX and DTX/MPEG2k-PDLLA1.7k group.
It could be found that, both MCF-7 tumors and
growth
curve
(figure
11C),
DTX/MPEG2k-PDLLA4k-PLL1k micelles exhibited the
most efficient inhibition to proliferation of MCF-7
tumor, and one of the solid tumors in
DTX/MPEG2k-PDLLA4k-PLL1k group was removed
completely.
The
tumor
volume
of
DTX/MPEG2k-PDLLA1.7k group was smaller than that
of the free DTX group (p<0.05). We have monitored
the tumor growth of DTX/MPEG2k-PDLLA4k-PLL1k
group for 45 days, and the tumor start to grow at 38
days. In order to further investigate the in vivo
antitumor effect of DTX formulations, TUNEL
staining and Ki-67 staining were used for detecting
the MCF-7 tumor cellular apoptosis and proliferation,
respectively, and the results were shown in figure 13A
and B. The number of the apoptotic bodies in the
DTX/MPEG2k-PDLLA4k-PLL1k group was 9.28%
4
T1 tumors, whose growth could be inhibited by a
DTX formulation, and DTX/MPEG2k-PDLLA4k-PLL1k
micelles exhibited the maximal anti-tumor active in
vivo. Therefore, improving the drug loading capacity
of micelles could enhance the anti-tumor effect of
DTX.
Body weight change rate of the MCF-7
tumor-bearing mice and 4T1 tumor-bearing mice are
shown in figure 11D and 12E, respectively. Both in
MCF-7 tumor-bearing mice and 4T1 tumor-bearing
mice models, the body weights decreased after
treatment with the DTX formulation. From the result
of in vivo bio-distribution, the formulation could not
be metabolized completely in normal organs.
Therefore, the DTX was accumulated in normal
organs after three consecutive days’ administration,
(
figure 13D), which was higher than the other groups.
Furthermore, the activities of different DTX
formulations on proliferation of tumor cells were
analyzed by Ki-67 (figure 13C). Compared with
DTX/MPEG2k-PDLLA1.7k group (60.12%), Free DTX
group (67.27%) and control group (78.96%), the Ki67
positive cells in DTX/MPEG2k-PDLLA4k-PLL1k group
(
31.46%) were a significant decrease. The H&E stain of
tumor tissues in different groups were shown in
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Theranostics 2017, Vol. 7, Issue 10
2668
which might lead the body weight reduction. The
than that in DTX/MPEG2k-PDLLA1.7k. The simulation
results supported the high drug loading capacity of
MPEG2k-PDLLA4k-PLL1k micelles. The drug release
behavior of DTX micelles confirmed the sustained
DTX/MPEG2k-PDLLA4k-PLL1k
group
and
DTX/MPEG2k-PDLLA1.7k group had less body weight
reduction than the free DTX group, which indicated
that the systemic toxicity of the DTX was decreased by
loading DTX in micelles. Finally, the body weight of
the mice in each group recovered to normal.
release property of the DTX/MPEG2k-PDLLA4k-PLL1k
.
By evaluating the cellular uptake and anticancer
properties of DTX/MPEG2k-PDLLA4k-PLL1k in vitro, it
demonstrated that the DTX/MPEG2k-PDLLA4k-PLL1k
micelles maintained better anticancer performance
than that of DTX/MPEG2k-PDLLA1.7k. The in vivo
biodistribution image results of the DTX micelles
further suggested the tumor targeting effect of the
Conclusion
In this study, we designed and synthesized the
copolymer MPEG2k-PDLLA4k-PLL1k for DTX loading.
The MPEG2k-PDLLA4k-PLL1k had a high drug loading
capacity. By 3D molecular simulation results of DTX
micelles, it revealed that DTX could be completely
wrapped in the polymer (MPEG2k-PDLLA4k-PLL1k),
which was shown to be embedded in the copolymer
DTX/MPEG2k-PDLLA4k-PLL1k
.
The
good
biocompatibility of the DTX/MPEG2k-PDLLA4k-PLL1k
was exhibited in a safety evaluation, and the in vivo
anti-tumor study showed obvious effects in the
treatment of breast cancer. In conclusion,
DTX/MPEG2k-PDLLA4k-PLL1k would be a promising
candidate for breast cancer targeted therapy.
(
MPEG2k-PDLLA1.7k) surface only. The number of
hydrogen bonds between DTX and copolymer
molecules in DTX/MPEG2k-PDLLA4k-PLL1k was more
Figure 11. The micelles inhibited tumors growth in MCF-7 model, control 1, free DTX 2, DTX/MPEG2k-PDLLA1.7k 3 and DTX/MPEG2k-PDLLA4k-PLL1k 4. Images of
MCF-7 tumor bearing mice with different treatments on 28 days after administration (A); images of subcutaneous tumors in each group (B); growth curves of tumor
in the mice (C); body weight of the mice in each group (D). Statistical difference from the free DTX group (“*” p˂0.05)
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Theranostics 2017, Vol. 7, Issue 10
2669
Figure 12. The micelles inhibited tumors growth in MCF-7 model, control 1, free DTX 2, DTX/MPEG2k-PDLLA1.7k 3 and DTX/MPEG2k-PDLLA4k-PLL1k 4. Images of
MCF-7 tumor bearing mice with different treatments on 21 days after administration (A); images of subcutaneous tumors in each group (B); growth curves of tumor
in the mice (C); Survival of the 4T1 tumor-bearing mice (D); body weight of the mice in each group (E). Statistical difference between groups (“*” p˂0.05) (“**”
p˂0.01)
Figure 13. The immunofluorescence staining of tumor slices with treatment of control 1, free DTX 2, DTX/MPEG2k-PDLLA1.7k 3 and DTX/MPEG2k-PDLLA4k-PLL1k
4
in MCF-7 model. A, B and E was representative Ki-67 immunohistochemical, TUNEL immunoflourescent and H&E stain in each group. C and D was the quantitative
analysis of Ki-67 immunohistochemical, TUNEL immunoflourescent in each group, respectively. Statistical difference between groups (“*” p˂0.05) (“**” p˂0.01).
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Theranostics 2017, Vol. 7, Issue 10
2670
Figure 14. The immunofluorescence staining of tumor slices with treatment of control 1, free DTX 2, DTX/MPEG2k-PDLLA1.7k 3 and DTX/MPEG2k-PDLLA4k-PLL1k
in 4T1 model. A, B and E was representative Ki-67 immunohistochemical, TUNEL immunoflourescent and H&E stain in each group. C and D was the quantitative
4
analysis of Ki-67 immunohistochemical, TUNEL immunoflourescent in each group, respectively. Statistical difference between groups (“*” p˂0.05) (“**” p˂0.01).
bocyanine
THF: Tetrahydrofuran;
Abbreviations
PLL: Poly (L-Lysine)
MPEG-PDLLA: Monomethoxy poly (ethylene glycol)-
poly (D, L-lactide)
FTIR: Fourier Transform Infrared
ROP: Ring opening polymerization
1
H-NMR: Nuclear magnetic resonance spectroscopy
DTX: Docetaxel
DL: Drug loading
GPC: Gel Permeation Chromatography
DLS: Dynamic light scattering
EE: Entrapment efficiency
DTX/MPEG2k-PDLLA4k-PLL1k: DTX loaded MPEG2k
TEM: Transmission electron microscopy
XRD: X-ray diffractometer
-
PDLLA4k-PLL1k
DTX/MPEG-PDLLA: DTX loaded MPEG-PDLLA
EPR effect: Enhanced permeability and retention
effect
TPGS: D-α-tocopheryl polyethylene glycol 1000
succinate
RME: Receptor-mediated endocytosis
Lys (Z)-NCA: N-carboxyanhydride of 6-(benzylo-
xycarbonyl)-L-lysine
H-Lys (Z)-OH: N -carbonybenzoxy-L-lysine
Boc-L-Phe: N-t-butoxycarbonyl-L-phenylalanine
CMC: Critical micelle concentration
IC50 value: Half maximal inhibitory concentration
RES: Reticuloendothelial system
HPLC: High Performance Liquid Chromatography
MD: Molecular dynamic
NPT: Equilibrium molecular dynamics simulation of
isothermal-isobaricensemble
TUNEL: Terminal deoxynucleotidyl transferaseme-
diated nick-end labeling
ε
Ki67: Rat antimouse monoclonal antibody
H&E stain: Hematoxylin-eosin staining
DiD: 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindotricar-
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Theranostics 2017, Vol. 7, Issue 10
2671
2
2
2
1. Wang T, Zhu DW, Liu G, Tao W, Cao W, Zhang LH, et al. DTX-loaded
star-shaped TAPP-PLA-b-TPGS nanoparticles for cancer chemical and
photodynamic combination therapy. RSC Adv. 2015; 5: 50617-27.
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Acknowledgements
This work was financially supported by the
National Natural Science Foundation of China
(
31525009 and 31222023), the National Key Research
and Development Program of China
2017YFC1103502), Sichuan Innovative Research
(
2
2
2
4. Dunwan Z, Wei T, Hongling Z, Gan L, Teng W, Linhua Z, et al. Docetaxel
Team Program for Young Scientists (2016TD0004),
and Distinguished Young Scholars of Sichuan
University (2011SCU04B18).
(
DTX)-loaded polydopamine-modified TPGS-PLA nanoparticles as a targeted
drug delivery system for the treatment of liver cancer. Acta Biomater. 2016; 30:
44-54.
1
5. Zhang XD, Yang Y, Liang X, Zeng XW, Liu ZG, Tao W, et al. Enhancing
therapeutic effects of docetaxel-loaded dendritic copolymer nanoparticles by
co-Treatment with autophagy inhibitor on breast cancer. Theranostics. 2014; 4:
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Supplementary Material
Supplementary figures and tables.
http://www.thno.org/v07p2652s1.pdf
1
2
Competing Interests
The authors have declared that no competing
interest exists.
2
7. Nicolas J, Mura S, Brambilla D, Mackiewicz N, Couvreur P. Design,
functionalization strategies and biomedical applications of targeted
biodegradable/biocompatible polymer-based nanocarriers for drug delivery.
Chem Soc Rev. 2013; 42: 1147-235
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