Biodegradable poly(ethylene glycol)-poly(?-carprolactone) polymeric micelles with different tailored topological amphiphilies for doxorubicin (DOX) drug delivery

Article abstract of DOI:10.1039/c6ra06040d

Three poly(ethylene glycol)-poly(?-carprolactone) (PEG-PCL) copolymers with different topologies but identical molar ratio between PEG to PCL were designed. These copolymers, namely, diblock (L-PEG-PCL), triblock (B-PEG-PCL), and star shaped (S-PEG-PCL) copolymers, were extensively characterized by ¹H Nuclear Magnetic Resonance (¹H NMR), Fourier Transform Infrared Spectroscopy (FTIR), and Gel Permeation Chromatography (GPC) analyses. The effect of topology on crystallization was investigated by X-Ray Diffraction (XRD), Differential Scanning Calorimetry (DSC). Results showed that the diblock copolymer possessed the highest crystallinity, followed by triblock and star shaped copolymers. Although the topology did not affect the self-assembly behavior of copolymers, the effects on size, size distribution, drug loading content, and drug release rate of the polymeric micelles were observed. The micelles self-assembled from linear diblock copolymer achieved higher cellular uptake and lower half maximal inhibitory concentration (IC₅₀). These findings are favorable to establish the foundation to further design proper structure of amphiphilic copolymers as nanocarrier systems for efficient anticancer therapy.

Full text of DOI:10.1039/c6ra06040d

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ARTICLE
Biodegradable
Poly(ethylene
glycol)ꢀPoly(εꢀ
carprolactone) Polymeric Micelles with Different
Tailored Topological Amphiphilies for Doxorubicin
(DOX) Drug Delivery
Cite this: DOI: 10.1039/x0xx00000x
Received 00th Mar 2016,
Accepted 00th
Y. Chen,a Y.X. Zhang,a Z.F. Wu,a X.Y. Pen b, T. Su,b J. Cao b*, B. He, b S. Li, a*
DOI: 10.1039/x0xx00000x
Three poly(ethylene glycol)ꢀpoly(εꢀcarprolactone) (PEGꢀPCL) copolymers with different topologies but
identical molar ratio between PEG to PCL were designed. These copolymers, namely, diblock (LꢀPEGꢀ
PCL), triblock (BꢀPEGꢀPCL), and star shaped (SꢀPEGꢀPCL) copolymers, were extensively characterized
www.rsc.org/
1
by H Nuclear Magnetic Resonance (1H NMR), Fourier Transform Infrared Spectroscopy (FTIR), and
Gel Permeation Chromatography (GPC) analyses. The effect of topology on crystallization was
investigated by XꢀRay Diffraction (XRD), Differential Scanning Calorimetry (DSC). Results showed
that the diblock copolymer possessed the highest crystallinity, followed by triblock and star shaped
copolymers. Although the topology did not affect the selfꢀassembly behavior of copolymers, the effects
on size, size distribution, drug loading content, and drug release rate of the polymeric micelles were
observed. The micelles selfꢀassembled from linear diblock copolymer achieved higher cellular uptake
and lower half maximal inhibitory concentration (IC50). These findings are favorable to establish the
foundation to further design proper structure of amphiphilic copolymers as nanocarrier systems for
efficient anticancer therapy.
nanocarriers by monocyte phagocytic system (MPS) cells7, 8. Herein,
PEGꢀPCL copolymers are currently investigated as carriers in
1. Introduction
Polymeric micelles have been considered as excellent candidates
in the fabrication of drug delivery systems for their widely
recognized merits such as enhancement of drug solubility in water,
amelioration of side effects, prolongation of circulation time and
improvement of drug bioavailability1ꢀ4. As we know, the amphiphilic
copolymer can selfꢀassemble into coreꢀshell structured micelles in
aqueous medium, where the cores are usually constructed with the
different drug delivery systems, and the most promising application
remains in the field of anticancer drug delivery.
In recent years, much interest has been focused on designing
different architectures of PEGꢀPCL copolymers, which can be
categorized as linear, graft, block, branched, crossꢀlinked, starꢀ
shaped, dendron/dendrimer topologies9, 10. Among them, the diꢀ
block and triꢀblock polymers are the most common polymers used in
drug delivery system. Interestingly, the area of block copolymers has
been explored with new macromolecules derived of miktoarm
architecture, where a miktoarm star copolymer is formed by different
species of nonꢀlinear chains in form of star, polymers with these
characteristics in its geometric order provide the possibility of new
potential properties to conventional linear block copolymers. In this
way, some research groups have reported macromolecules with
hydrophobic polymers and the shells are hydrophilic polymers5, 6
.
Poly(εꢀcaprolactone) (PCL) has been frequently used to form the
core of selfꢀassembled amphiphilic copolymers owing to its
desirable hydrophobicity, high aggregation propensity, and low glass
transition temperature that ensures
a partial rubbery core .
Poly(ethylene glycol) (PEG) is the most widely used hydrophilic
polymer to impart structure stability and stealth properties to reduce
nonꢀspecific protein adsorption, thus reducing clearance of
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miktoarm star copolymer architecture, as for example: AB2 and AB4 influence on their properties, the information on the influence of the
where A = poly(εꢀcaprolactone) (PCL) and B = polystyrene 11, A2B motion of the molecular and biological processes exerted by
and A2BA2 where
A
=
poly(nꢀhexyl isocyanate) and
B
=
architecture are relatively limited and unsystematically.
poly(ethylene glycol) (PEG) 12, or ABC star terpolymer where A =
Prompted by these limitations, here we designed PEGꢀPCL
PEG, B = poly(2ꢀvinylpyridine) and C = PCL 13. In particular, the amphiphilic copolymers with different structures and the copolymers
unique properties of the structures that assembled from these were used to investigate the effect of topologies of PEGꢀPCL
polymers in solution have led to the application in drug delivery. As copolymers on their micellar properties and anticancer efficiency as
miktoarm polymers are branched macromolecules consisting of shown in Scheme 1. A comprehensive investigation on different
polymeric arms emanating from their core, this architecture offers topologies of three type copolymers with same molar ratio and
great opportunities for the tailoring of nanocarriers, as the chemistry molecular weight, namely LꢀPEGꢀPCL, BꢀPEGꢀPCL and SꢀPEGꢀ
of both the core and the arms can be finely tuned in order to trigger PCL, was conducted. The crystallization of three types of PEGꢀPCL
their controlled selfꢀassembly in aqueous media14. These unique copolymers were investigated by XRD, FTIR and DSC analysis. The
properties of miktoarm polymers are actively investigated, effect of topologies of copolymers on their micellar properties such
particularly in small molecule encapsulation and release14ꢀ16
.
as CMC, particle size and size distribution, drug loading capacity
Obviously, miktoarm star copolymers presented different properties and in vitro drug release profiles, as well as anticancer efficiency
compared to conventional linear block copolymers, many studies were studied.
have focuse on the relation between polymer architecture and their
2. Materials and Methods
properties.
Bogdanov and coꢀworkers investigated the isothermal 2.1. Materials. Methylated poly(ethylene glycol) (mPEG2k , Mw =
crystallization of three different types of PEGꢀPCL incompatible 2000 g molꢀ1), polyethylene glycol (PEG, Mw = 2000 g molꢀ1),
block copolymers in detail using DSC. The molecular weight of the stannous octoate (Sn(Oct)2), glycerol and ethylene oxide were
PEG blocks they used was 4000 or 5000 and the PEG weight purchased from SigmaꢀAldrich Co. and used as received. εꢀ
fraction was varied from 18 to 22 wt%. Results showed that the caprolactone (CL) (SigmaꢀAldrich Co.) was dehydrated by CaH2 and
diblock copolymers exhibited the highest crystallization among purified via vacuum distillation
. Doxorubicin hydrochloride
those three copolymers17. He et al. synthesized ABA type (PLAꢀ (DOX·HCl) (Zhejiang Hisun Pharmaceutical) was dissolved into
PEGꢀPLA) and BAB type (PEGꢀPLAꢀPEG) triblock copolymers and water and the pH adjusted to 9.6 to prepare doxorubicin22. Benzyl
compared their drug release properties and stealth particle bromide (BnBr) was bought from Changzhou Xinhua Active
characteristics18. The morphology, stability, thermal characteristics Material Institute. Dulbecco’s Modified Eagle’s Medium (DMEM),
and release profile of ABA and BAB type copolymer nanoparticles fetal bovine serum (FBS), trypsin solution, 100 × penicillinꢀ
based on PCL and PEG chain segments were also investigated by streptomycin solution were obtained from HyClone Inc. and used for
Zamani and coꢀworkers6. They found that the architectures of cytotoxicity assay. N, NꢀDimethylformamide (DMF) (Kermel Co.,
copolymers had a profound effect on their micellar properties. Tianjin, China) and tetrahydrofuran (THF) (Kelong Chemical Co.)
Moreover, Yin, et al. synthesized a series of AB2 type 3ꢀmiktoarm were purified before used. All the other solvents were purchased
star copolymers using PEG as the A arm and PLLA as the two B from Kelong Chemical Co. (Chengdu, China) and used as received.
arms and found that the 3ꢀmiktoarm polymers have superior vesicleꢀ
forming abilities compared to the linear diblock counterparts16.
2.2. Characterizations. 1H NMR spectra were collected on a Bruker
400MHz spectrometer using CDCl3 as solvent and tetramethylsilane
Many star polymers with different architectures which can shape
(TMS) ( 0.5%) as internal standard. FTIR spectra were recorded on a
various morphologies were also reported19ꢀ21. From these reported
FTIR spectrometer (Thermo Fisher Scientific Nicolet 8700) within
studies, it was obviously found that the extensive researches have
the range of 4000ꢀ400 cmꢀ1 and the samples were prepared using the
been concentrated on synthesis and applications of different novel
KBr disk method under the irradiation of sodium lamp. Gel
architectures. Although most of the works focused on the creation of
permeation chromatography (GPC) measurement was carried out on
new spatial structure, change of the compositions, intelligence of the
HLCꢀ8320 chromotography with THF as eluent (0.6 mL minꢀ1) at
functional group and the chain length to investigate those factors
35 °C and respect to polystyrene standards. The thermal properties
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of copolymers were determined by differential scanning calorimetry terminator when the reaction was preceded for 48 h at 0 °C. The
(DSC, Q2000 TA Instruments). About 5 mg samples in hermetically mixture was stirred vigorously for another 5 h. After THF was
closed aluminum pans were subjected to a secondary heating removed, the residues were dissolved in CHCl3, washed with
program from ꢀ80 to 100 °C at a heating rate of 10 °C minꢀ1 and a deionized water and NaCl (aq) for three times alternately. The
cooling rate of 10 °C minꢀ1. Powder Xꢀray diffractometry (XRD) organic layer was dried by MgSO4 overnight. The filtrate was
patterns were obtained on an X’Pert pro MPD Xꢀray diffractometer concentrated and recrystallized in excess cold ethyl ether. The
at 25 °C using CuKα (λ=0.154nm) as the target material with the powder (2ꢀphenylꢀ1,3ꢀdioxanꢀ5ꢀPEG2k, Intermediate 2) was
diffraction angle of 5ꢀ50°. Dynamic light scattering (DLS) tests were vacuumꢀdried at room temperature overnight.
performed on Malvern Zatasizer Nano ZS at 25 °C. The samples
were dispersed in deionized water with the concentration of 1 mg
mLꢀ1 and each sample was filtered through a 450 nm syringe filter
before analysis .The scattering angle is 90° and the coefficient of
refraction is 1.330. Each sample tested three times. The Atomic
force microscopy (AFM) pictures were received on a MFPꢀ3DꢀBIO
instrument (Oxford Instruments company) using tapping mode. The
AFM samples were prepared by casting a dilute micellar solution
(0.1 mg/mL) on a mica substrate and dried in vacuum. The scan
angle was maintained at 0°, and the images were captured in the
trace direction with a scan rate of 1 Hz. Cellular uptake was
examined by confocal laser scanning microscopy (CLSM, Leica
TCP SP5). DOX was 10 ꢁg mLꢀ1 and excited at 480 nm with the
emission at 590 nm. The flow cytometry was tested on a BD FACS
Calibur flow cytometer (Beckton Dickinson) and analyzed
approximately for 1×104 cells from each sample.
Intermediate 2 (4 g, 2 mmol) was dissolved in DMF (45 mL),
NaH (0.19 g，8 mmol) was added in the solution. The mixture was
stirred for 4 h and then BnBr (1.0 g, 6 mmol) dissolved in DMF was
injected in the mixture. The reaction was preceded for 48 h at room
temperature. The DMF was removed completely under reduced
pressure and CDCl3 was added. The solution was filtered and the
filtrate was washed by saturated NaCl (aq) for 3 times. The pure
product (2ꢀphenylꢀ1,3ꢀdioxanꢀ5ꢀPEG2kꢀbenzyl, intermediate 3) was
obtained after the organic phase was removed in reduced pressure..
The Intermediate 3 (3 g, 1.5 mmol) was dissolved in methanol (30
mL). After 1M HCl (aq) (30 mL) was dropped in the system, the
mixture was refluxed for
6
h
and then concentrated.
Dichloromethane was used to extract the organic phase. After
washed with deionized water for three times, the organic layer was
dried by MgSO4 and precipitated in cold ethyl ether to produce
phenylꢀPEG2kꢀ1,3ꢀpropanediol (Intermediate 4).
2.3. Synthesis of phenylꢀPEGꢀ1,3ꢀpropanediol. A mixture of
glycerol (18.4 g, 0.2 mol), benzaldehyde (21.2 g, 0.2 mol) and
concentrated sulfuric acid (0.02 mL) were stirred vigorously at room
temperature (RT) for 4 h under the protection of nitrogen. The
received water was removed under reduced pressure at 40 °C. The
residues were cooled to room temperature, added in ethyl ether (25
mL), then washed with 10% Na2CO3 and saturated NaCl aqueous
solutions (aq) for three times. The organic phase was dried with
anhydrous MgSO4 overnight. The filtrate was concentrated and
recrystallized three times at ꢀ20 °C to produce white crystal 2ꢀ
2.4. Synthesis of different topologies of poly(ethylene glycol)ꢀ
poly(εꢀcaprolactone) (PEGꢀPCL) copolymers. Different types of
PEGꢀPCL copolymers were synthesized via ringꢀopening
26
polymerization
.
PhenylꢀPEG2kꢀ1,3ꢀpropanediol, mPEG2k,
PEG2k were used as macroinitiators and Sn(Oct)2 as the catalyst.
The synthesis of linear diblock copolymer (LꢀPEG2kꢀPCL4k) was
used as an example. mPEG2k (2 g, 1 mmol) was added in a roundꢀ
bottom flask with a vacuum apparatus. The flask was heated to
110 °C for 2 h with the purpose of removing the residual water
completely. When the flask was cooled to room temperature, CL (4 g,
35 mmol) and Sn(Oct)2 (0.1 wt%) were added quickly. The flask was
sealed and immersed in an oil bath at 130 °C for 48 h. The
compound was dissolved in dichloromethane and precipitated in
excess cold ethyl ether for three times. The purified white powder
was obtained when the precipitate was vacuumꢀdried. The triꢀblock
copolymer (BꢀPEG2kꢀPCL4k), star shaped copolymer (SꢀPEG2kꢀ
PCL4k) were synthesized as the same procedure above mentioned.
phenylꢀ1,3ꢀdioxanꢀ5ꢀol (Intermediate 1) 23
.
2ꢀphenylꢀ1,3ꢀdioxanꢀ5ꢀPEG2k (Intermediate 2) was synthesized
via an anionic ringꢀopening polymerization modified from the
approach previously reported 24, 25. Under the nitrogen atomosphere,
potassium (0.19 g, 5 mmol) was added in threeꢀneck flask and then
naphthalene (0.64 g, 5 mmol) was added in dropwise. After a
vigorous stirring of 2 h, Intermediate 1 (0.9 g, 5 mmol) was injected
into the mixture and cold ethylene oxide (17 mL) was added
immediately. Hydrochloric acid (1 M, 5 mL) was used as a
2.5. Critical micelle concentration (CMC). The CMC of the
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copolymers were determined by fluorescence spectrophotometer (Fꢀ were conducted in triplicate and the results were expressed as mean
7000, Hitachi HighꢀTechnologies Co.Japan) using pyrene as a probe. value with standard deviation (SD).
A series of balnk copolymers with different concentrations (0.01ꢀ
500 ꢁg mLꢀ1) containing pyrene at the fixed concentration of 6.0 ×
10ꢀ7 mol Lꢀ1 were prepared. The excitation fluorescence spectra from
cells and HepG2 liver cancer cells. The cells mentioned above were
2.8. In vitro cytotoxicity test. The cytotoxicity of blank micelles
was tested by CCKꢀ8 assay against NIH 3T3 fibroblasts, C2C12
300 to 360 nm were tested with the emission wavelength fixed at
inoculated into 96ꢀwell plates separately (4 × 104 cells mLꢀ1) in 100
390 nm. The fluorescence absorbance values of I338 and I335 at
ꢁL DMEM medium which was supplemented with 10% FBS and 1%
excitation fluorescence spectra were recorded. The CMC value was
penicillinꢀstreptomycin. The cells were cultured at 37 °C in a humid
the turning point in the logarithmic graph of the ratio of I338 and
atomosphere of 5% CO2 for 24 h. After the medium was removed,
I335 to the samples’ concentration.
four different concentrations of blank micelles in DMEM media
2.6. Preparation of DOX loaded micelles. The PEGꢀPCL
were added. The cells were incubated for another 48 h. The culture
amphiphilic macromolecules (20 mg) and DOX (5 mg) were
medium was removed and the wells were washed with PBS ( pH 7.4)
dissolved in DMF (2 mL) and stirred for 4 h, the solution was added
for three times. Afterwards, the fresh medium containing 10 ꢁL of
dropwise into pure water (20 mL) under vigorous ultrasonic
CCKꢀ8 (5 mg mLꢀ1) solution was added to each well. After incubated
agitation for 30 min. The solution was dialyzed (Spectra/Por
for another 2 h, the absorbance was measured at the wavelength of
MWCO=2000) against deionized water at 4 °C for 36 h after stirred
450 nm.
overnight. The outer phase was replaced with fresh deionized water
2.9. In vitro anticancer activity. The anticancer activity of DOX
every 4 h. The mixture was centrifuged and lyophilized (3500 rpm, 5
loaded nanoparticles was evaluated with HepG2 cells. HepG2 cells
min) to obtain the DOX loaded micelles. The concentration of DOX
were harvested and seeded in 96ꢀwell plates (4 × 104 cells mLꢀ1) in
was tested using a UVꢀVis measurement (excitation at 485 nm)
100 ꢁL medium for 24 h. DOX·HCl and DOX loaded micelles with
(Figure. S1 and Table S1ꢀS2 in the ESI †) with the calibration curve
different concentrations was added to the mediumꢀremoved 96ꢀwell
of DOX/DMF solutions. Drug loading content (DLC) and drug
plates and incubated for 48 h. The cell viability was measured by
loading efficiency (DLE) were calculated according to the following
CCKꢀ8 assay.
formulas:
DLC (wt%)=(weight of DOX in micelles/weight of DOX
2.10. Cellular uptake assay. Both flow cytometry and confocal
loaded micelles) × 100%
laser scanning microscopy (CLSM) were used to examine the
cellular uptake and the distribution of drug loaded nanoparticles in
cells. To study the cellular uptake, HepG2 cells at a logarithm phase
(1 × 104 cells mLꢀ1) were seeded in glass dishes (diameter = 35 mm).
DOX·HCl and DOX loaded micelles ([DOX] = 10 ꢁg mLꢀ1) were
added into the dishes for 1 and 4 h incubation, the medium was
removed. The cells were washed by PBS for three times, followed
by adding 200 ꢁL fresh PBS into each dish. The cells were imaged
by CLSM (TCP SP5, Leica, Germany). The fluorescence of DOX
was excited at wavelength of 480 nm with emission at 590 nm.
For the flow cytometry test, HepG2 cells (1×106 cells wellꢀ1) were
seeded in 6ꢀwell plates and incubated for 24 h. The cells were treated
with DOX·HCl and DOX loaded micelles at the same concentration
(10 ꢁg mLꢀ1) for 1 and 4 h. After that, the cells were rinsed with PBS,
trypsinized and resuspended in 1 mL PBS after concentration (1000
rpm, 5 min). The fluorescence intensity was measured (excitation
485 nm/emission 590 nm) on a BD FACS Calibur flow cytometer
(Beckton Dickinson).
DLE (wt%)=(weight of DOX in micelles/weight of DOX in
feeding) × 100%
2.7. In vitro drug release test. The release of DOX loaded micelles
was evaluated at 37 °C in buffer solution (pH 7.4 and pH 5.5) with 1
mL of dialysis solution (adjust the DOX concentration to 30 ꢁg mLꢀ1)
against 25 mL of the PBS medium under sink condition (The
saturated solubility of DOX in PBS was determined to be about 20
ꢁg mLꢀ1)27. The 1 mL of fresh dialysis solution of DOX loaded
micelles was put in dialysis membrane tubing (Spectra /Por
MWCO=1000). The tubings were immersed in vials containing 25
mL of PBS solution and put in a shaking bed at 37 °C with the
shaking rate of 150 rpm. 1 mL of the buffer solution from the release
medium was taken out at designated time intervals and the same
volume of fresh medium was added into the vials. The amount of
released DOX was detected by a fluorescence spectrometer with the
excitation wavelength at 480 nm and emission wavelength at 550 nm
((Figure. S3 and Table.S3 in the ESI †). The release experiments
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2.11. Statistical analysis. The Student's Tꢀtest was employed to different spatial structures were synthesized by ring opening
assess the statistical significance and it was performed using polymerization. The 1H NMR spectra in Fig 1 clearly confirmed the
GraphPad Prism 5 Software. The results were presented as means ± successful synthesis of diꢀ, triꢀ and star shape copolymers. The
SD. Statistical significance was set at *p < 0.05, and extreme typical peaks attributed to PCL and PEG segments were all observed
26, 31
significance was set at **p < 0.01.
in Fig 1 (a, b, c), which was accordant with previous reports
.
The characteristic chemical shifts at δ=3.6 to 3.7 ppm were assigned
to PEG segment. The signals of 3, 4, 5, 6 attributed to PCL blocks at
3. Results and Discussion
3.1. Characterization of PEGꢀPCL Copolymers with different the chemical shifts δ=2.3, 1.6, 1.4, 4.1 ppm were all observed in Fig
Topologies. As the structures of copolymers affect their micellar 1. The molecular weight of PCL blocks were calculated from the
properties28ꢀ30, copolymers with different topologies, namely block, integral area ratio of characteristic peaks between PEG (multiple, ꢀ
triblock and star shaped copolymers, were synthesized (as shown in CH2CH2Oꢀ, δ=3.6ꢀ3.7) and PCL (triplet, ꢀCH2OOCꢀ, δ=4.1) by
Scheme 2) and used to study the effect of architecture on micellar using the known molecular weight of PEG. As shown in Table 1, the
properties and antitumor efficiency. Three different topological compositions of copolymer and molecular weights determined by 1H
structures of poly(ethylene glycol)ꢀpoly(εꢀcaprolactone) with nearly NMR spectra were very close to that determined by the ratio of
identical molar ratio between PEG and PCL was synthesized. Those monomer to the initiator, suggesting that the molecular weight and
amphiphilic copolymers selfꢀassembled into micelles with composition of those copolymers can be easily controlled by feeding
hydrophobic PCL segments as the core and hydrophilic PEG blocks dose.
as the shell.
FTIR was also employed to characterize the structure of
The structures of those copolymers were characterized by 1H copolymers. As shown in Fig 2, the absorption bands at 2866 cmꢀ1
NMR, and the spectra were presented in Fig 1. First, poly(ethylene belonged to the characteristic CꢀH stretching vibration of the
glycol) with expected terminal group was synthesized and the repeated ꢀOCH2CH2 units of PEG backbone. The peaks at 2943 cmꢀ1
spectra (Intermediate 1, 2, 3 and 4) were shown in Fig.S3ꢀS5 in the could be attributed to the CꢀH absorption of ester bonds in the
ESI †. As the primary and secondary hydroxyl group in glycerol repeated units of PCL. More importantly, the characteristic
possessed the identical activity, the mixture products of intermediate stretching vibration absorbance of ꢀC=O in ester bond at around
1 (I, II, III, IV) were obtained as shown in Fig S3 in the ESI † and 1725 cmꢀ1 and ꢀCH2OCH2 at 1105 to 1243 cmꢀ1 were all detected,
the corresponding proton signals of Intermediate 1 were all detected indicating the successful synthesis of the designed copolymers. It is
in Fig S3. The mass spectrum in Fig S4 further demonstrated the known that FTIR can not only be used to characterize the functional
successful synthesis of Intermediate 1 (+c ESIꢀMS, m/Z: 181.01 group of the copolymer qualitatively, it can also be applied to
[M+H], 202.98 [M+Na], calculated m/Z: 180.08). The Intermediate confirm the composition ratio of the compound. Therefore, as shown
2 was synthesized via an anionic ringꢀopening polymerization in Fig 2, the corrected areas of CꢀH stretching vibrations of ether
(AROP) of ethylene oxide using unprotected hydroxyl of bond in PEG at 2886 cmꢀ1 (S2) and in PCL at 2943 cmꢀ1 (S1) were
intermediate 1 as the initiator and the 1H NMR spectrum was shown calculated. The area ratio of S1 to S2 is 2.03 for SꢀPEG2kꢀPCL4k,
in Fig.S5 a†. After the terminal hydroxyl group of Intermediate 2 1.83 for BꢀPEG2kꢀPCL4k, 1.91 for LꢀPEG2kꢀPCL4k, which
was protected by benzyl, the double signals attributed to corresponded to the theoretical value 1.95. That further confirmed
cyclobenzene (C6H5ꢀ) were observed at 7.3~7.8 ppm. Meanwhile, a the designed copolymers were successfully obtained.
single peak ascribed to benzyloxy (C6H5CH2Oꢀ, 9) at 4.6 ppm
The molecular weight and molecular weight distribution of the
appeared (Fig.S5 b†). After the Intermediate 3 was acidized, the synthetic copolymers were monitored by Gel Permeation
signal at δ=5.2 ppm assigned to C6H5CH(O2)ꢀ (4) disappeared, Chromotography (GPC) as shown in Table 1 and Fig. S6†. A
whereas the peak ascribed to cyclobenzene was halved and the signal unimodal peak with high molecular weight of copolymers was
corresponding to proton of OH (14) appeared at δ=3.8 (Fig.S5 c†), detected in the GPC spectra, indicating no or negligible PEG or PCL
1
indicating the Intermediate 4 was successfully obtained.
oligomers existed. Nevertheless, unlike the H NMR, the molecular
After the macroinitiators (poly(ethylene glycol) with expected weights tested by GPC were all larger than the designed molecular
terminal groups) were obtained, the designed copolymers with weights, especially for starꢀcopolymers. That was because the
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molecular weight measured by GPC was a relative molecular weight, formation was monitored by using pyrene as a hydrophobic probe.
and the star shaped copolymer has a bigger steric hindrance than From the plot of fluorescence intensity ratio of I338/I335 versus log
linear or block copolymers due to GPC adopt sizeꢀexclusion method. C of the copolymer, the CMC values were obtained, which were
1
Combined with H NMR, FTIR and GPC results, the copolymers compiled in Table 4 and Fig.S7† . The low CMCs (10ꢀ3 g/L) in the
with different spatial structures were successful synthesized.
table suggested that the micelles were highly thermodynamically
stable and could be potentially used as candidates for drug delivery.
3.2. Crystallization of PEGꢀPCL Copolymers with different
Topologies. The effect of topologies of copolymers on their Among them, starꢀshaped copolymer micelles exhibited a little lower
crystallization was studied by XRD, FTIR and DSC and the results CMC, indicating starꢀshaped copolymers can easily form micelles,
were shown in Fig 3 and Table 1. In the spectra of XRD, strong which was in accordance with other reports that the star shape
peaks at 2θ = 21.7° and 24° attributed to PCL blocks and peaks at 2θ copolymers facilitated the micellization as the unimer state of a star
= 19.4° and 23.7° assigned to PEG blocks were clearly observed in shape copolymer with many arms resembled the micellar state,
Fig 3A, which was in good accordance with other studies reported32. which resulted in easier formation of micelles 37
.
However, there were some differences in the intensity of those peaks. In order to achieve the EPR effect and longevity during systemic
When the PEG constituent was in the central block (BꢀPEGꢀPCL), circulation, the size of micelles should be appropriate for permeating
the crystallization of PEG in BꢀPEG2kꢀPCL4k copolymer was through the microvasculature of solid tumors and evading detection
strongly restrained, behaving as a weak diffraction peak at 19.4°, by the reticuloꢀendothelial system 38, 39. The morphology, size and
While two separate clear crystal peaks attributed to PCL and PEG size distribution of the micelles with or without DOX loading were
segments were observed in the copolymers of LꢀPEG2kꢀPCL4k and determined by AFM and DLS 40, 41. The results were shown in Fig 4
SꢀPEG2kꢀPCL4k. Moreover, the linear copolymer LꢀPEG2kꢀPCL4k and Table 2. All three types of copolymers can selfꢀassemble into
showed the highest intensity for the peak at 19.4°, possibly because monoꢀdispersed nanoparticles with sizes below or around 100 nm
of the weaker restriction in PCL segments compared to the other two and spherical shapes. The sizes obtained from AFM were larger due
copolymers. The relative crystallinity of PCL segments was to the aggregation of NPs in dry state and the AFM tip broadening
calculated by the area under the peak at 21.7°, and the results effect. The mean sizes of LꢀPEG2kꢀPCL4k, BꢀPEG2kꢀPCL4k, Sꢀ
showed that the crystallization of those copolymers decreased in the PEG2kꢀPCL4k were 43, 74 and 53 nm, respectively. The size of
sequence of LꢀPEG2kꢀPCL4k, BꢀPEG2kꢀPCL4k, SꢀPEG2kꢀPCL4k.
polymeric micelles selfꢀassembled by LꢀPEGꢀPCL copolymer was
It is well known that there are two peaks for the C=O vibration of the smallest among those three polymeric micelles possibly because
semiꢀcrystalline PCL, one at 1726 cmꢀ1 and the other at 1736 cmꢀ1, of high crystallinity and the topological structure. In the process of
34
corresponding to the crystalline and amorphous PCL phase33,
,
micellar formation, the PEG chains stayed in the outer phase to form
respectively. As shown in Fig.3 B, the absorption peak at 1726 cmꢀ1 the shell and the PCL chains stacked together to form the inner core.
attributed to the crystalline PCL phase was the strongest in linear As elaborated in Fig 5, due to linear structure of LꢀPEGꢀPCL, the
copolymer (LꢀPEG2kꢀPCL4k), yet the weakest was observed in star PCL segment will be intertwined tightly, exhibit the smallest space
copolymer (SꢀPEG2kꢀPCL4k). The results were in good agreement volume of inner core, thus resulted in the smallest micellar size.
with the XRD results. DSC results in Fig.3 C and Table 1 exhibited While in the formation of Bꢀmicelles, the hydrophilic PEG chain in
that the highest crystallinity of both PEG and PCL segments was the middle must be folded to make sure the hydrophobic PCL chain
observed in LꢀPEGꢀPCL copolymers due to the weakest spatial flock together, then forming an angle in the two PCL segments. Thus
hindrance in the linear copolymer 17. In addition, PCL segments in a bigger cavity was produced and the size of BꢀPEG2kꢀPCL4k was
linear LꢀPEGꢀPCL were located and combined in one end, leading to the largest. Also, the highest drug loading content for Bꢀmicelles
a longer PCL block than the other two copolymers. Therefore, PCL were further provided the evidence. In the SꢀPEG2kꢀPCL4k micelle,
blocks are more prone to be stacked tightly, thus resulted in higher although an angle was also existed in the two PCL segments, the
crystallinity 35, 36
.
PEG segment was unfolded, led to less repulsive force occurs thus
resulted in smaller cavity than BꢀPEG2kꢀPCL4k micelles. After drug
loaded, the size of copolymer micelles measured by DLS were
increased to 55, 115, 73 nm for Lꢀ, Bꢀ and Sꢀshape copolymer
3.3. Characterization of Polymeric Micelles. Critical micellar
concentration (CMC) is an effective parameter to show the stability
of micelles and a low critical value is desired. In this study, micelle
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micelles, respectively, which was similar to the research previously burst release and a slower release phase. The rapid initial release
reported 42, 43. Moreover, the size increase values of DOXꢀloaded Bꢀ might be attributed to the drug located in the cores closely to the
PEGꢀPCL micelles became bigger compared to other two copolymer interface between the core and shell of micelles as well as the large
micelles, possibly due to that the two PCL chains were separately surface areas because of their minimal diameter. After the initial
connected on both ends of PEG, which increased the chain burst release, DOX was slowly released in the following time.
flexibility. The increased flexibility promoted the hydrophobic core Among them, starꢀshaped copolymer micelles showed faster drug
became looser during the micellization, thus resulted in larger release than the other two copolymer micelles systems in pH 7.4
hydrophobic cores. This hypothesis was supported by crystallinity medium. The difference in release rate among those copolymer
results as shown in Table 1. However, although the Sꢀshaped micelles could be attributed to their crystallinity (Table 1 and Fig 3).
copolymer exhibited the lowest crystallinity, the micellar size was Lꢀ and BꢀPEGꢀPCL copolymer could form highly packed crystal in
smaller than Bꢀshaped copolymer micelles. This was because the micellar cores, preventing the drug from being dispersed
two hydrophobic PCL segments were both connected in one point, homogeneously inside the core, whereas SꢀPEGꢀPCL copolymer
resulting in the restricted movement of PCL segments. The results possessed the lowest crystallinity, which resulted in drug distributed
above revealed that both of the crystallinity and structure of homogenously inside the core, leading to the formation of more
copolymers had impacts on the micellar size. Generally speaking, hydrophilic channels 46. Those hydrophilic channels can prompt the
larger hydrophobic core can afford encapsulation of more drug release from the core of micelles. However, when the released
hydrophobic drugs, behaving as a higher drug loading content. This media was in acidic environment (pH 5.5), the drug release rates
rule was fitted for the DLC results as shown in Table 4. BꢀPEGꢀPCL were all improved. That was because the acidic environment could
copolymer micelles possessed the highest drug loading content and protonate the amino group of DOX and transformed the drug from
drug loading efficiency, whereas the linear copolymer micelles (Lꢀ hydrophobic to hydrophilic, thus made the trapped drug rapidly
PEG2kꢀPCL4k) exhibited the lowest DLC because of the highest diffused out from the core of micelles, which is desirable for
crystallinity of PCL segment as shown in Table 1. It was reported enhancing the cancer killing effects of DOX. Moreover, as
that the drug was likely to be encapsulated into the amorphous PCL protonated amino group of DOX override the effect of structure of
phase 44
.
copolymers on drug release, as shown in Fig 6B, the released rates of
In addition, we tested the size and zeta potential of the DOX three copolymer micelles were comparable.
loaded micelles in DMEM to evaluate the micellar behavior in the
3.5. In Vitro Cytotoxicity of copolymer Micelles. For the purpose
cell culture medium (Table 3). Compared to fresh micellar solution,
the micelles in the cell culture medium maintains their original mean
size while the zeta potential all decreased. As we know, the DMEM
High Glucose cell media contains glucose, glutamine, sodium
pyruvate and sodium bicarbonate. That is to say, the ionic
of evaluating the cytotoxicity, three blank micelles were incubated
with 3T3 fibroblasts, C2C12 mouse myoblast cells and HepG2
cancer cells for 48 h. Fig.7 showed that the cell viabilities of three
cell lines were higher than 90% and some of them over 100% even
the micelles concentration was as high as 600 ꢁg mLꢀ1. It implied
that the micelles were nonꢀcytotoxic to normal and cancer cells.
After drug was loaded, the cell viability of DOX loaded micelles
is significantly lower than that of blank micelles after treated with
HepG2 for 48 h via CCKꢀ8 assay (Fig.7 D), indicating higher
cytotoxicity was obtained by encapsulating DOX into micelles for
improvement of its solubility. Moreover, both DOX·HCl and DOX
loaded micelles exhibited a doseꢀdependent behavior. For three drug
loaded micelles, cell viability decreased dramatically with the DOX
concentration in the DMEM is higher than the reverseꢀosmosis water.
45
As the literature pointed out
that the higher of the ions in the
solution resulted in a thinner electrical double layer, exhibiting as
lower zeta potential. More importantly, although the zeta potential of
those micelles decreased, the decrease degree of zeta potential were
in same tendency. Moreover, zeta potential of those micelles were all
negative both in water or cell media. That meant there is little effect
on their further used in the cell media.
3.4. In Vitro Drug Release Profiles. The in vitro drug release concentrations increasing. As shown in Table 4, the IC50 values of
profiles of drug loaded copolymer micelles were investigated in free DOX·HCl, DOX loaded LꢀPEG2kꢀPCL4k, BꢀPEG2kꢀPCL4k,
different pH media (pH 7.4 and 5.5) and the results were shown in SꢀPEG2kꢀPCL4k micelles were 0.047, 1.46, 1.78, 2.03 ꢁg mLꢀ1,
Fig 6. The release profiles included two components: a rapid initial respectively. This phenomenon appeared to correspond well to the
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cellular uptake efficiency as follow discussed. Obviously, DOX·HCl endocytosis process. On the other hand, the free DOX·HCl would
expressed the lowest IC50 as DOX·HCl was passive diffused into penetrate into the tumor cells quickly without any selectivity and
HepG2 cells and it immediately accumulated in the nuclei 47, and sustainedꢀrelease property, thus resulting in stronger red
then exerted its function. Although the DOX·HCl exhibited better fluorescence in nuclei. The DOX loaded micelles was internalized
anticancer efficiency, the unexpected systemic toxicity and into cells mainly via endocytosis process and thus localized in
inevitable side effects were always induced due to DOX·HCl endocytic compartments, such as endosomes and lysosomes. With
diffused into cells without selectivity, which restricted their clinical increasing incubation time, the red fluorescence intensity became
48,
application
49. But, the encapsulation of DOX into polymeric stronger as shown in Fig 8. When the incubation time was short (1
micelles can avoid side effects. The formed nanoscale particles could h), the red fluorescence intensity measured by CLSM and FCM
accumulate in tumor tissues via the enhanced permeation and showed no significant difference (Fig.8 I, III, V), while lower
retention (EPR) effect owing to the appropriate size, which reduced intracellular DOX fluorescence intensity was observed in the cells
the poisonous side effect and meanwhile promoted the utilization treated with DOX loaded SꢀPEG2kꢀPCL4k micelles at 4 h (Fig.8 II,
50ꢀ52
ratio of the drugs
. As we expected, the DOX loaded LꢀPEGꢀ IV, V), the results were in good agreement with IC50 results as
PCL micelles exhibited the highest anticancer efficiency among shown in Fig 6. However, the internalization efficiency of DOX
three DOX loaded micelles. It was because the smallest particle sizes loaded LꢀPEGꢀPCL and BꢀPEGꢀPCL copolymer micelles was
which could foam cells more easily and advance the cellular uptake seemed to conflict with IC50 results, where lower IC50 of DOX
efficiency, it was also confirmed by CLSM and FCM results and loaded LꢀPEGꢀPCL micelles was observed compared to BꢀPEGꢀPCL
accordingly promote the inhibition effect of DOX on the micelles (Fig 6 and Table 3). One possible explanation was that the
proliferation of cancer cells significantly. However, the poorest incubation time for CLSM was only 4 h whereas that for IC50 was up
anticancer efficiency was emerged in DOX loaded SꢀPEGꢀPCL to 48 h, thus resulting in the internalization efficiency was not up to
micelles. Combing the analysis of size, release profile and DLC the best condition. Also, many studies reported that the fluorescence
results, it was not difficult to explain this phenomenon. The DOX intensity increased with the increase of incubation time 54, 55. If the
loaded SꢀPEGꢀPCL micelles exhibited faster release profiles at pH incubation time was up to 48 h, the internalization efficiency of Lꢀ
7.4 and the size and DLC were not much preferable, thus resulting in PEGꢀPCL micelles was progressed increased than that of BꢀPEGꢀ
a less dosage of DOX was in the target site when arriving at the PCL micelles.
tumor cells.
4. Conclusions
3.6. Cellular Internalization of Polymeric Micelles. As the cellular
In this study, three amphiphilic copolymers, LꢀPEGꢀPCL, BꢀPEGꢀ
internalization is the key step for drug loaded micelles to fulfill their
PCL and SꢀPEGꢀPCL with different topologies were successfully
anticancer activity, the effect of copolymer micelles selfꢀassembled
synthesized. Those copolymers could selfꢀassemble into micelles
by copolymers with different topologies on the internalization
with low critical micellar concentrations. The micelles were in
efficiency was compared. Confocal laser microscopy and flow
spherical shapes and nanoꢀscale particle sizes (less than 100 nm)
53
cytometry
were used to study the cellular uptake of those
with narrow size distributions. The topologies of the copolymer were
found to affect the crystallinity, particle size, encapsulation
efficiency, and in vitro drug release characteristics. BꢀPEGꢀPCL
copolymer micelles possessed larger particle size, higher DOX
encapsulation efficiency, and slower drug release rate. In contrast, Lꢀ
PEGꢀPCL copolymer micelles exhibited faster internalization and
better anticancer efficiency. In summary, these findings can provide
a new insight to rational design of amphiphilic copolymers for
anticancer drug delivery system.
copolymer micelles and the results were shown in Fig 8. From the
images of CLSM, we can see that the cells treated with DOX·HCl
showed the highest red fluorescence and most red florescence was
located in the nuclei even the incubation time was 1 h. The red
fluorescence became stronger and stronger as the time progressed (4
h). The law of increased fluorescence intensity was the same as FCM
results. Unlike DOX·HCl, the red fluorescence of drug loaded
micelles were detected in cytoplasm due to the different pathway of
cellular internalization. The free DOX·HCl was internalized into
cells by diffusion for its waterꢀsoluble property and the
internalization of passive diffusion was much faster than that of
Acknowledgements
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DOI: 10.1039/C6RA06040D
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(No. 20130181110038), Young teachers’ scientific research
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College of Chemical Engineering, Sichuan University, Chengdu 610065
China
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This journal is © The Royal Society of Chemistry 2012
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DOI: 10.1039/C6RA06040D
Figures and Tables
Scheme 1. Schematic illustration of the selfꢀassembly and drug release of polymeric micelles.
O
O
O
O
H2SO4
r.t.
HO
OH
O
(a)
,K
HO
+
HO
O
OH
O
THF
H
n
vacuum, 0 °C, THF
Intermediate 1
Intermediate 2
O
NaH,BnBr
DMF
O
O
O
n
1M HCl, CH3OH
O
OH
n
O
OH
reflux
Intermediate 3
Intermediate 4
O
O
O
H
k
O
(b)
H
O
O
O
n
n
O
LꢀPEGꢀPCL
O
O
O
O
H
O
O
O
HO
H
O
O
H
k
n
n
k
Sn(Oct)2, 130 °C, 48 h
BꢀPEGꢀPCL
O
O
O
OH
OH
O
O
n
O
O
H
k
n
O
O
SꢀPEGꢀPCL
O
H
k
Scheme 2. Synthetic routes of PEGꢀPCL copolymers with different structures.

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DOI: 10.1039/C6RA06040D
Figure 1. The 1H NMR spectra (400 MHz, CDCl3) of SꢀPEG2kꢀPCL4k (a), BꢀPEG2kꢀPCL4k (b)
and LꢀPEG2kꢀPCL4k (c) copolymers.
SꢀPEG2kꢀPCL4k
S2
S1
BꢀPEG2kꢀPCL4k
S2
S1
LꢀPEG2kꢀPCL4k
S1
S2
2943 2866
1105
1243
1725
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber (cmꢀ1)
Figure 2. FTIR spectra of LꢀPEG2kꢀPCL4k , BꢀPEG2kꢀPCL4k and SꢀPEG2kꢀPCL4k
copolymers.

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DOI: 10.1039/C6RA06040D
A
B
C
SꢀPEG2kꢀPCL4k
SꢀPEG2kꢀPCL4k
SꢀPEG2kꢀPCL4k
BꢀPEG2kꢀPCL4k
LꢀPEG2kꢀPCL4k
BꢀPEG2kꢀPCL4k
LꢀPEG2kꢀPCL4k
BꢀPEG2kꢀPCL4k
LꢀPEG2kꢀPCL4k
1737
1724
1850
1800
1750
Wavenumber (cmꢀ1
1700
1650
0
20
40
60
80
100
15
20
2θ
25
30
)
Tempereture (°C)
（
°）
Figure 3. The XRD spectra (A), enlarged segment of C=O stretching vibration in FTIR
spectrum (B) and second heating curve of DSC (C).
30
25
20
15
10
5
30
25
20
15
10
5
A
B
LꢀPEG2kꢀPCL4k
BꢀPEG2kꢀPCL4k
SꢀPEG2kꢀPCL4k
DOX/LꢀPEG2kꢀPCL4k
DOX/BꢀPEG2kꢀPCL4k
DOX/SꢀPEG2kꢀPCL4k
0
0
1
10
100
Size (d.nm)
1000
10000
1
10
100
Size (d.nm)
1000
10000
C1
D1
E1
C2
D2
E2
Figure 4. Size tested by DLS and the AFM images of NPs with or without DOX loading. (A)
blank NPs and (B) DOX loaded NPs measured by DLS. (CꢀE) AFM images of blank NPs (1) and
DOX loaded NPs (2) of Lꢀ (C), Bꢀ (D) and SꢀPEG2kꢀPCL4k (E) copolymers.

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Figure 5. The molecular stack and micellar formation process selfꢀassembled by different
topologies copolymers.
**
A
B
50
40
30
20
10
0
**
80
60
40
20
0
L-PEG2k-PCL4k,pH=5.5
B-PEG2k-PCL4k,pH=5.5
S-PEG2k-PCL4k,pH=5.5
L-PEG2k-PCL4k,pH=7.4
B-PEG2k-PCL4k,pH=7.4
S-PEG2k-PCL4k,pH=7.4
0
20
40
60
80
0
20
40
60
80
Time (h)
Time (h)
Figure 6. The release profiles of drug loaded nanoparticles in PBS media of pH 7.4 (A) and pH
5.5 (B). The results were expressed as mean ±SD (n=3). Statistical significance was set at *p <
0.05, and extreme significance was set at **p < 0.01 in red (Bꢀ versus Sꢀ) and in black (Lꢀ versus
Sꢀ).

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A
B
L-PEG2k-PCL4k
B-PEG2k-PCL4k
S-PEG2k-PCL4k
L-PEG2k-PCL4k
B-PEG2k-PCL4k
S-PEG2k-PCL4k
120
100
80
60
40
20
0
120
100
80
60
40
20
0
100
200
400
600
100
200
400
600
Concentration(ꢁg/mL)
Concentration(ꢁg/mL)
C
D
120
100
80
60
40
20
0
L-PEG2k-PCL4k
B-PEG2k-PCL4k
S-PEG2k-PCL4k
DOX/LꢀPEG2kꢀPCL4k
100
80
60
40
20
0
DOX/BꢀPEG2kꢀPCL4k
DOX/SꢀPEG2kꢀPCL4k
DOX·HCl
100
200
400
600
1Eꢀ3
0.01
0.1
1
10
100
Concentration(ꢁg/mL)
Concentration(ꢁg/mL)
Figure 7. The cytotoxicity of blank micelles incubated with 3T3 cells (A), C2C12 cells (B) and
HepG2 cells (C) and (D) DOX·HCl and DOX loaded micelles against HepG2 cells for 48 h. The
results were expressed as mean ± SD (n=4).

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Figure 8. The cellular uptake results against HepG2 cells treated with DOX/LꢀPEG2kꢀPCL4k (A),
DOX/BꢀPEG2kꢀPCL4k (B), DOX/SꢀPEG2kꢀPCL4k (C), DOX·HCl (D). (I) and (II) were the
confocal microscopy images captured for 1 h and 4 h, respectively. For each panel, the images
from top to bottom (number 1, 2, 3) indicated cells in bright field, with DOX fluorescence, and
overlays of the two channels. (III) and (IV) were the flow cytometry results of 1 h (III) and 4 h (IV)
with the same concentration of DOX (10 µg mLꢀ1). (V) was the histogram of the mean
fluorescence intensity and the results were expressed as mean ± SD. Statistical significance was
set at *p < 0.05, and extreme significance was set at **p < 0.01

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Table 1. The molecular weights and thermal properties of PEGꢀPCL copolymers with different
structures.
Molecular weight
1H NMR
GPC
Mn Mw/Mn ꢁH (J/g) Tm (°C) ꢁH (J/g)
Thermal properties
sample
PEG segment PCL segment
Mn
Tm (°C)
LꢀPEG2kꢀPCL4k 5870
BꢀPEG2kꢀPCL4k 5942
SꢀPEG2kꢀPCL4k 6087
8188
1.69
17.01
46.79
37.83
45.12
42.29
41.38
37.14
51.18,
55.38
49.84,
54.06
54.20
9074
1.41
9.65
13915 1.66
14.82
Table 2. The size of blank and DOX loaded copolymer micelles
Blank NPs a DOX loaded NPs a
Number
mean
Number
Sample
PDI
PDI
mean (nm)
(nm)
LꢀPEG2kꢀPCL4k
BꢀPEG2kꢀPCL4k
SꢀPEG2kꢀPCL4k
43 ± 6
74 ± 12
53 ± 11
0.345 ± 0.049
0.331 ± 0.054
0.258 ± 0.036
55 ± 7
115 ± 20
73 ± 1
0.284 ± 0.008
0.315 ± 0.02
0.235 ± 0.024
a Measured by DLS. The results were expressed as mean ± SD (n=3).
Table 3. The size and Zeta potential of DOX loaded micelles in DMEM and their Zeta potential
in water
Zeta Potential (mv)
Number
mean (nm)
56 ± 12
Sample
PDI
In DMEM
In water
DOX/LꢀPEG2kꢀPCL4k
DOX/BꢀPEG2kꢀPCL4k
DOX/SꢀPEG2kꢀPCL4k
0.390 ± 0.010
0.200 ± 0.031
0.378 ± 0.015
ꢀ2.72 ± 0.75
ꢀ0.98 ± 0.13
ꢀ1.22 ± 0.20
ꢀ10.20±0.30
ꢀ7.44±0.08
ꢀ10.83±0.79
125 ± 7
75 ± 4
The results were expressed as mean ± SD (n=3).
Table 4. The CMC , drug loading capability and IC50 of copolymer micelles
b
Sample
CMC a
(ꢂg/mL)
0.833
DLC
(%)
5.4
DLE
(%)
IC50
(ꢂg/mL)
1.46
LꢀPEG2kꢀPCL4k
BꢀPEG2kꢀPCL4k
SꢀPEG2kꢀPCL4k
23.6
26.5
24.7
0.821
6.0
1.78
0.801
5.6
2.03
a The Critical Micelle Concentration was measured by pyrene.
b The half maximal inhibitory concentration.

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Graphic Abstract