Thermoresponsive star-like γ-substituted poly(caprolactone)s for micellar drug delivery

Article abstract of DOI:10.1039/c7tb01291h

Temperature responsive drug carriers are attractive due to their ability to provide controlled release of the encapsulated cargo based on the use of external stimuli. In this work, 4- and 6-arm thermoresponsive star-like block copolymers were synthesized

Full text of DOI:10.1039/c7tb01291h

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DOI: 10.1039/C7TB01291H.
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Journal of Materials Chemistry B
ARTICLE
Thermoresponsive star-like γ-substituted poly(caprolactone)s for
micellar drug delivery
Katherine E. Washington, Ruvanthi N. Kularatne, Jia Du, Yixin Ren, Matthew J. Gillings, Calvin X.
Geng, Michael C. Biewer, and Mihaela C. Stefan*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Temperature responsive drug carriers are attractive due to their ability to provide controlled release of the encapsulated
cargo based on the use of external stimuli. In this work, 4- and 6-arm thermoresponsive star-like block copolymers were
synthesized through the ring-opening polymerization of γ-substituted ε-caprolactone monomers γ-2-[2-(2-
methoxyethoxy)ethoxy]ethoxy-ε-caprolactone (MEEECL) and γ-ethoxy-ε-caprolactone (ECL) using pentaerythritol and myo-
inositol as multifunctional initiators. These amphiphilic block copolymers were shown to self-assemble into micelles and
were characterized in terms of their feasibility as drug carriers. Both polymers were shown to be thermodynamically stable
and demonstrated temperature responsivity in a desirable range for drug delivery, with lower critical solution temperatures
of 39.4 oC and 39.8 oC for the 4- and 6-arm polymers, respectively. It was shown that the 6-arm star polymer had a higher
drug loading capability and better stability in vitro, allowing it to function as a better vehicle for drug delivery in cytotoxicity
experiments. These star polymers show promise as drug carriers due to their biocompatibility, biodegradability, and
temperature controlled release of doxorubicin.
www.rsc.org/
responsiveness, and can play a role in micellar properties such
as stability, size, degradation rate, and drug loading capabilities.
The stability of these systems is of great importance. In order to
Introduction
Considerable efforts in recent years have aimed to develop
smart drug carriers for improved delivery of anticancer drugs to
tumor sites with increased efficiency and decreased side
effects.1-7 In particular, there is an interest in developing
systems that can release their cargo in a triggered response
either from the application of external stimuli or from a change
in environment.8-11 These systems allow for an increased
control over where and when the encapsulated cargo is
delivered and can be used to preferentially release drugs at
tumor sites. Types of stimuli responsive polymers can include
those that are sensitive to pH, temperature, reduction
conditions, light, or multiple stimuli.
In the case of polymeric micellar drug delivery systems,
polymers made from aliphatic polyesters such as
poly(glycolide)s, poly(lactide)s, and poly(caprolactone)s have
been studied extensively.12, 13 Polyesters are attractive due to
their biocompatibility and biodegradability through hydrolysis
of esters in the backbone. Among these, poly(caprolactone)s
are one of the most investigated materials due to the ease of
function as drug carriers, it is imperative that they can stay
intact upon dilution in the bloodstream.16 In addition, the size
can be used to passively target tumors through the enhanced
permeability and retention (EPR) effect. Ideally, the particles
should be in the size of 10-100 nm for this effect so that they
are large enough to avoid clearance and small enough to bypass
filtration in the spleen.17-20 In these ways, polymeric micelles
show promise for efficient delivery of poorly water soluble
anticancer drugs while minimizing toxicity.
Temperature responsive polymers can be advantageous as
polymeric micellar drug delivery systems, as they permit control
over the release of the drug based on the application of
localized heating or mild hyperthermia.21 Polymers that display
a lower critical solution temperature (LCST), provide a release
mechanism of drug based on a solubility transition of the
polymer in aqueous medium upon heating above the LCST.
Previously, our group reported the synthesis of poly{γ-2-[2-(2-
methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}
(PMEEECL)
which was synthesized by living ring-opening polymerization of
tri(ethylene glycol) substituted ε-caprolactone monomer.22 This
polymer has many advantages, including water solubility, and
thermoresponsivity. Unlike other thermoresponsive polymers,
PMEEECL is also biodegradable. When part of an amphiphilic
block copolymer, it was shown that the LCST can be tuned
depending on the hydrophobic block.23-26 In addition, PMEEECL
is an attractive candidate for the hydrophilic block on
amphiphilic star polymers because it can be used in living ring-
property tunability through addition of substituents to the
In this manner, substituents can affect the
hydrophilicity or hydrophobicity of the polymer, provide stimuli
15
backbone.14,
Department of Chemistry and Biochemistry, The University of Texas at Dallas,
Richardson, TX 75080, USA. *E-mail: mihaela@utdallas.edu; Fax: +1-972-883-
2925; Tel: +1-972-883-6581
Electronic Supplementary Information (ESI) available: 1H NMR spectra, SEC traces,
TMAFM Height Images, degradation and absorbance curves for DOX loaded
micelles.
See DOI: 10.1039/x0xx00000x
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opening polymerization which allows tunability of the system
through control over the molecular weight and composition.
In an effort to expand on previously reported linear
polymers featuring PMEEECL, 4- and 6- arm star-like block
copolymers containing PMEEECL as the hydrophilic block and
poly(γ-ethoxy-ε-caprolactone) (PECL) as the hydrophobic block
were synthesized. These systems were examined to determine
the effects of the architecture on the thermoresponsivity,
stability, and drug loading. Star-like polymers have an increased
density of functional units compared to linear polymers, and
View Article Online
DOI: 10.1039/C7TB01291H
have been shown to have many promising attributes for drug
28
delivery, including increased drug loading.27,
Herein, we
Scheme 2. Synthesis of 4- and 6-arm star-like PECL-b-PMEEECL amphiphilic diblock
copolymers
report the first thermoresponsive star polymers synthesized
using PMEEECL, and compare the resulting properties with their
previously reported linear counterpart.
Table 1. Summary of polymer compositions and molecular weight
Mn
mol %
ECLb
mol %
PDIa
(g mol-1)a
MEEECLb
Results and Discussion
Polymer Synthesis
4A
6A
20,400
28,800
1.1
1.4
49.7
47.2
50.3
52.8
In a previous report from our group, a linear block
copolymer consisting of ECL and MEEECL was examined for its
thermoresponsivity, thermodynamic stability, and drug loading
capabilities.24 The linear block copolymer was synthesized
through living ring-opening polymerization of γ-ethoxy-ε-
caprolactone (ECL) and γ-2-[2-(2-methoxyethoxy)ethoxy]
ethoxy-ε-caprolactone (MEEECL) monomers using stannous (II)
2-ethylhexanoate (Sn(Oct)2) as the catalyst and benzyl alcohol
as the initiator. This polymer showed promise in terms of
temperature responsivity and thermodynamic stability,
however it was shown to have limited drug loading capacity
(2.05 wt. %). In an attempt to increase the drug loading and the
stability, two functionalized star-like diblock polycaprolactones
were synthesized. The ECL and MEEECL monomers were
synthesized according to previous published procedures
(Scheme 1) and were used for the hydrophobic and hydrophilic
block respectively.22, 24 The star polymers were synthesized with
Sn(Oct)2 as the catalyst and two different multifunctional
alcohol initiators were employed to form the 4-arm
(pentaerythritol initiator) and 6-arm (myo-inositol initiator)
block copolymers (Scheme 2). The polymerizations were carried
aDetermined by size exclusion chromatography with THF as eluent bDetermined by
1H NMR Spectroscopy
50:50 molar composition of hydrophilic to hydrophobic block:
[Initiator]: [Sn(Oct)2]: [ECL]: [MEEECL], [1]: [4]: [50]: [50] for the
4-arm polymer (4A) and [1]: [6]: [50]: [50] for the 6-arm polymer
(
6A). This composition was targeted in an effort to generate
comparable composition and molecular weights to the
previously published linear block copolymer.24 The 1H NMR
spectra are shown in Figures S1 and S2, and a summary of the
molecular weights and composition of the polymers is shown in
Table 1. The compositions were determined by the integration
of the peaks of the substituents of the block copolymers, the
methoxy group on the oligo ethylene glycol substituent at ~3.37
ppm was integrated versus the methyl group of the ethoxy
substituent at ~1.17 ppm. The molecular weights of the two
polymers were determined by size exclusion chromatography
(SEC) equipped with a triple detection system, allowing for the
determination of the absolute molecular weight. Additional
information from SEC measurements, including the SEC traces
and hydrodynamic radius, can be found in the supporting
material (Figures S3 and S4 and Table S1). The polymers were
shown to have comparable compositions to each other as well
as the linear polymer, with 6A having a slightly higher molecular
weight and both polymers having fairly narrow PDI.
o
out at 110 C with sequential monomer addition, starting with
the polymerization of the hydrophobic ECL monomer. The
polymerizations used the following molar ratios to target a
Self-Assembly and Thermoresponsivity
The lower critical solution temperature (LCST) of the
polymers was determined by measuring the change in
transmittance with increasing temperature. Transmittance
decreases above the LCST due to the dehydration and
precipitation of the polymer from the aqueous solution. The
transmittance was measured with a UV-vis spectrophotometer
at 600 nm. The LCST was taken as the temperature where there
is a 50% drop in the transmittance during heating (Figure 1 A
Scheme
1.
Synthesis
of
γ-2-ethoxy-ε-caprolactone
and
ε-caprolactone
γ-2-[2-(2-
methoxyethoxy)ethoxy]ethoxy-ε-caprolactone,
monomers
functionalized
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Table 2. Summary of polymeric micelle properties
CMC
LCST
(oC)b
Dh
Size
(g L-1)a
(nm)c Dispersityc
4A
6A
1.68 x 10-3
1.37 x 10-3
39.8
39.4
80.85
50.98
0.085
0.131
aDetermined by fluorescence spectroscopy with pyrene as a fluorescent probe
bMeasured with UV-vis as the 50% drop in transmittance at 600 nm upon heating
c
aqueous polymer solution Hydrodynamic diameter of micelles at 25 oC
determined from dynamic light scattering
Fig. 2 Size distribution (Dh) for 4A (A) and 6A (B) polymeric micelles at 25 oC obtained
from DLS.
Fig. 3 TEM images of empty micelles (A) 4A and (B) 6A.
Fig. 1 Transmittance and CMC plots showing thermoresponsiveness and thermodynamic
stability of 4A (A and B) and 6A (C and D) respectively.
transmission electron microscopy (TEM). Empty micelles were
prepared by dialysis method at a concentration of 1 mg mL-1.
The micelles were examined with dynamic light scattering (DLS)
to determine the hydrodynamic diameter (Dh) and the
dispersity of the sample. The micelles exhibited sizes of 80.85
nm and 50.98 nm for 4A and 6A, respectively (Table 2, Figure 2).
The micelles formed from polymer 6A were much smaller in size
when compared to those formed from 4A. In order to observe
the morphology, TEM was performed using phosphotungstic
acid for negative staining. Amphiphilic block copolymers can
assemble into various structures such as spherical micelles,
vesicles, or cylindrical micelles depending on several factors
including the composition of the block copolymer, the
and C). The LCST of 4A and 6A were comparable, with 4A
o
o
showing LCST of 39.4 C and 6A with LCST of 39.8 C (Table 2).
These LCST values are useful for drug delivery applications since
they are higher than physiological temperature (37 oC), meaning
the micelles will be stable as they circulate in the bloodstream,
and below 40 oC allowing the application of external
temperature to release the encapsulated cargo. Importantly,
these polymers show improved LCST values over the previously
reported linear polymer, which had an LCST below physiological
temperature.
The critical micelle concentration (CMC) was determined by
fluorescence spectroscopy using pyrene as a probe.29 The
pyrene excitation spectrum shows a peak shift from 334.5 nm
to 337.5 nm as pyrene goes from a hydrophilic environment into
the hydrophobic core of the micelle. The intensity ratio
(I337.5/I334.5) was plotted against the logarithm of the polymer
concentration, where the intersection of the two slopes is
estimated as the CMC (Figure 1, B and D). The estimated CMC
value for 4A is 1.68 x 10-3 g L-1 and for 6A is 1.37 x 10-3 g L-1 (Table
2). The CMCs for these polymers are fairly similar, however in
comparison with the previously synthesized linear block
copolymers (8.95 x 10-3 g L-1), the values are almost a magnitude
lower indicating that the star polymers have better
thermodynamic stability.24 This could be due to the increased
density of the functional groups in the star polymer allowing
increased hydrophobic interactions.
30
molecular weight, the solvent system, or environment. The
star block copolymers formed spherical micelles in aqueous
solution which was visualized from the TEM images (Figure 3).
This was expected based on our previous results with star
polymers formed from substituted polycaprolactones with
PMEEECL as the hydrophilic block.31 The sizes measured from
TEM were comparable to the values obtained with DLS.
However, unlike the distribution observed with DLS, there
appeared to be more variation in size observed in the TEM
images.
Doxorubicin Encapsulation
To determine the feasibility of using the star block
copolymers as drug carriers, doxorubicin (DOX) was loaded into
the micelles through dialysis method. The absorbance of the
DOX loaded micelles measured at 485 nm was fitted against a
pre-established calibration curve in order to determine the
concentration of the loaded drug. (Figure S5) The drug loading
Size and Morphology
The size and morphology of the empty polymeric micelles
were investigated by dynamic light scattering (DLS) and
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Table 3. Summary of DOX loaded polymeric micelles
content (DLC) and encapsulation efficiency (EE) were
determined using the following equations:
View Article Online
DOX
DLC
EE
Dh DOI: 10.1S0i3z9e/C7TB01291H
(wt. %)a
(wt. %)a
(nm)b Dispersityb
푤푒ꢀ푔ℎ푡 표푓 푒푛푐푎푝푠푢푙푎푡푒푑 퐷푂푋
퐷퐿퐶 =
퐸퐸 =
푥 100
푥 100
4A
6A
2.06
2.63
20.6
26.3
102.8
64.5
0.155
0.159
푤푒ꢀ푔ℎ푡 표푓 푝표푙푦푚푒푟
푤푒ꢀ푔ℎ푡 표푓 푒푛푐푎푝푠푢푙푎푡푒푑 퐷푂푋
푤푒ꢀ푔ℎ푡 표푓 푡표푡푎푙 퐷푂푋
aDetermined with UV-Vis spectroscopy, absorbance measured at 485 nm
bDetermined through dynamic light scattering at 25 oC
We speculated that the 6-arm polymer (6A) would have a
higher drug loading than the 4-arm polymer (4A) due to
increased density of functional units in the core of the micelle.
Moreover, based on previous data which showed that star
polymers have increased loading over linear polymers, we
predicted that both of the star polymers would have a higher
loading than the previously synthesized linear block copolymers
(2.05 wt.%).24, 31 The DOX loading was performed in the same
conditions as the earlier published linear block copolymers, and
at the same ratio (10:1 polymer: drug) to allow comparison of
the loading. Polymer 4A was shown to have a DLC of 2.06 wt.%,
while polymer 6A had a DLC of 2.63 wt.%. Polymer 4A had a
drug loading comparable to that of the reported value of the
linear block copolymer, while polymer 6A had the highest DLC
and encapsulation efficiency. The increased loading could be
due to the increased density of functional groups in the core
provided by the 6-arms. It is worth noting that 6A had a higher
molecular weight than 4A which could influence the loading as
well. A summary of the drug loading is shown in Table 3. The
change in size and the morphology was investigated after
loading DLS, tapping mode atomic force microscopy (TMAFM),
and TEM. The micelles retained their spherical shape after
loading and both star block copolymers showed an increase in
size after loading (Fig 4). As observed before, the sizes
measured from TEM showed good correlation with those
measured from DLS. Even after loading, the micelles retained a
size ideal for passive targeting using the EPR effect, with 4A
having the largest size, at 102.8 nm. (Fig 5)
Fig. 5 Size distribution obtained through DLS for (A) 4A and (B) 6A comparing the sizes of
empty and DOX loaded micelles
Biocompatibility and Degradation
The biodegradability of the star block copolymer 6A was
examined by dissolving the polymer in PBS (pH =7.4) and stirring
the solution at 37 oC for several days. The degradation was
measured by determining the % change in molecular weight
over time (Figure S7). The polymer showed degradation over
several days indicating the polymer was biodegradable in
physiological conditions. It can be assumed that polymer 4A
would be degradable over time as well since it has similar
structure and composition. It has been shown recently that
although star-like polyesters degrade at a slower rate than
linear polymers, the number of arms does not affect the rate of
degradation as significantly.32
To evaluate the biocompatibility of these polymers,
cytotoxicity measurements were performed using various
concentrations of the empty polymers on HeLa cells. CellTiter-
Blue® cytotoxicity kit was used to examine the cell viability after
the cells had been exposed to the polymer solutions for 24
hours. The polymers were not shown to exhibit significant
toxicity to the cells even up to 0.5 mg mL-1 (Fig 6). According to
these measurements, the polymers display excellent
biocompatibility as well as biodegradability under physiological
conditions.
In Vitro Drug Release of Doxorubicin
The in vitro release was determined for 4A and 6A at
o
o
physiological temperature (37 C) and above their LCST (40 C)
in PBS buffer (pH 7.4) and is shown in Fig. 7. Although DOX was
released at both temperatures, the initial release rate was
higher for the micelles incubated at 40 oC and the overall release
was the highest for the micelles incubated at the higher
temperature, with the cumulative release reaching around 60%
after 48 hours. This indicates that there is some thermal control
over the release of DOX, especially within the first 24 hours.
Fig. 4 TEM images of DOX loaded micelles (A) 4A and (B) 6A, scale bar = 200 nm. TMAFM
images of DOX loaded polymeric micelles (C) 4A and (D) 6A deposited on mica substrate,
scan size:1 µm
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Fig. 6 Cell viability measurements of varying concentrations of empty polymeric micelles
in PBS using HeLa cells and CellTiter-Blue ® to measure cell viability, n=4.
Fig. 9 Cell viability of HeLa cells after dosing with DOX loaded micelles above and
below the LCST
environments such as the core of a micelle, and the emission
intensity decreases as it is exposed to aqueous environment.
For the micelles incubated in PBS only, there was no significant
change in fluorescence observed for the micelle solutions over
48 hours. In the case of the micelles incubated in PBS containing
FBS, there was a visible change in NR loaded 4A after 24 hours,
indicating that there could be drug released after exposure to
serum proteins. However, there was only a slight decrease in
the fluorescence of NR loaded 6A indicating more stability over
time.
Fig. 7 Release profiles of DOX from 4A and 6A above (40 oC) and below (37 oC) their LCSTs
in PBS buffer solution (pH 7.4), n=3.
Stability of Micelles in FBS
The stability of micelles in vivo can be greatly influenced by
serum proteins. To investigate the potential use of the star
polymer micelles as drug carriers in vivo, their stability over time
in PBS and PBS supplemented with 10% FBS (which is similar to
the concentration in blood plasma) was examined (Fig 8). The
micelles were prepared by loading4A and 6A with Nile Red (NR)
and the fluorescence was monitored over time. NR is a molecule
that strongly fluoresces in hydrophobic
Cytotoxicity and Cellular Uptake of DOX Loaded Micelles
The cytotoxicity of the DOX loaded micelles was examined
using HeLa cells at various concentrations. In the interest of
determining the temperature controlled release of DOX in cells,
the cells were incubated at either physiological temperature (37
oC) or at a temperature above the LCST of the polymers (40 oC).
HeLa cells were dosed with DOX loaded micelles and allowed to
incubate at either temperature for 24 hours. The cell viability
was measured using CellTiter-Blue® assay. It was observed at all
concentrations that the release of DOX was more substantial at
temperatures above the LCST causing less cells to be viable.
Free DOX was administered to HeLa cells as well in
concentrations coinciding with the amount loaded into the star
polymer micelles based on the DLC determined. In all cases, the
free DOX showed higher cytotoxicity to the cells, however the
cells exposed to DOX loaded micelles at temperatures higher
than the LCST exhibited cytotoxicity closer to that of the free
DOX dosages (Fig 8), which correlates with what was observed
in the in vitro release (Fig 7). The DOX loaded 6A micelles
exhibited higher toxicity than the DOX loaded 4A micelles,
Fig. 8 Stability of Nile Red loaded micelles over time in PBS containing either 0%
or 10% FBS, n=3.
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which can be attributed to the higher loading capabilities of the ZS instrument equipped with a He–Ne laser (633 nm) and 173°
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DOI: 10.1039/C7TB01291H
6A micelles.
backscatter detector. TEM imaging of the DOX-loaded micelles
was performed on a Tecnai G2 Spirit Biotwin microscope by FEI
and images were analyzed using Image J software. Samples
were prepared by treating copper mesh grid with 1 mg mL-1
aqueous polymer micelle solution for 2 minutes, followed by
staining with 2% phosphotungstic acid for 30 seconds. TMAFM
images were obtained by depositing the DOX loaded micelles on
freshly cleaved mica substrate and allowing to air dry and using
a VEECO-dimension 5000 Scanning Probe Microscope with
silicon cantilever with spring constant 42 nm-1. Images were
acquired at 1 Hz scan frequency and analysed with Nanoscope
7.30 software to generate the 3D renderings. Absorbance
spectra for DOX loading determination was recorded using an
Agilent UV/Vis spectrophotometer. Cytotoxicity and cellular
uptake measurements were performed with a Biotek Cytation 3
imaging reader.
Fig 10. Uptake of 4A and 6A DOX loaded micelles in HeLa cells after incubating for 4
hours. Cell nuclei were stained with DAPI.
The ability of the micelles to be taken into the cancer cell
was observed through cellular uptake experiments using HeLa
cells. The DOX loaded micelles were added to HeLa cells and
incubated for 4 hours. At that time, the cells were washed and
the nuclei were counterstained with DAPI to visualize the cell
nuclei through fluorescence microscopy, shown in Fig 10. The
red signal, attributed to DOX can be seen within the cell nuclei,
indicating the endocytosis of the micelles into the cell and the
internalization of DOX into the nucleus of the cell.
Synthetic Procedures
The synthesis of monomers MEEECL and ECL were performed
according to previously published procedures and are shown in
Scheme 1.24
Synthesis of 4-arm star-like PMEECL-b-PECL. ECL (0.387 g,
0.00245 mol) was added into a Schlenk flask and stirred under
vacuum for one hour. At that time, pentaerythritol (4.17 mg, 3.1
x 10-5 mol) and Sn(Oct)2 (53 mg, 1.2 x 10-5 mol) were added in
0.3 mL toluene under a nitrogen atmosphere to the reaction
flask. The reaction was introduced into a thermostatted oil bath
at 110 oC. The consumption of monomer was monitored using
GC/MS. After ECL was consumed, previously dried MEEECL (0.7
g, 0.00245 mol) was added in 0.2 mL of toluene to the reaction
flask under nitrogen. The polymerization was allowed to
continue over night and after the MEEECL was consumed, the
reaction was quenched by precipitation in hexane, yielding 0.8
g of clear gel-like polymer.
Experimental
Materials
All commercially available chemicals were purchased from
Sigma Aldrich or Fisher Scientific and used without further
purification unless otherwise noted. Benzyl alcohol and
Sn(Oct)2 were purified through vacuum distillation prior to use.
All polymerization reactions were conducted under purified
o
nitrogen in glassware that was dried at 120 C for at least 24
hours and cooled in a desiccator prior to use.
1H NMR (500 MHz, CDCl3, δ): 1.163 (t, 3H), 1.772 (m, 6H), 1.856
(m, 3H), 2.380 (t, 4H), 3.373 (s, 3H), 3.469 (m, 4H), 3.546 (m,
2H), 3.597 (m, 4H), 3.644 (m, 7H), 4.161 (m, 4H)
Analysis
1H NMR spectra of the synthesized monomers and polymers
were recorded on a Bruker AVANCE III 500 MHz NMR
instrument at 25 °C in CDCl3. 1H NMR data are reported in parts
per million as chemical shifts relative to tetramethylsilane (TMS)
as the internal standard. GC/MS was performed on an Agilent
6890-5973 GC/MS workstation. Molecular weight and
polydispersity indices of the synthesized polymers were
measured by SEC analysis on an OMNISEC multi-detector
system equipped with Viscotek columns (T6000M), connected
to a refractive index (RI), low angle light scattering (LALS), right
angle light scattering (RALS), and viscosity detectors with HPLC
grade THF as the eluent, and triple point calibration based on
polystyrene standards. Fluorescence spectra of the synthesized
polymers were collected with a Perkin-Elmer LS 50 BL
luminescence spectrometer at 25 °C with emission wavelength
set at 390 nm. LCST measurements were performed using a
temperature controlled Cary5000 UV-Vis spectrometer. DLS
measurements were performed using a Malvern Zetasizer Nano
Synthesis of 6-arm star-like PMEEECL-b-PECL. ECL (0.224 g,
0.0014 mol) was added into a Schlenk flask and stirred under
vacuum for one hour. At that time, myo-inositol (3.2 mg, 1.8 x
10-5 mol) and Sn(Oct)2 (45 mg, 1.1 x 10-5 mol) were added in 0.3
mL toluene under a nitrogen atmosphere to the reaction flask.
The reaction was introduced into a thermostatted oil bath at
110 oC. The consumption of monomer was monitored using
GC/MS. After ECL was consumed, previously dried MEEECL (0.4
g, 0.0014 mol) was added in 0.2 mL of toluene to the reaction
flask under nitrogen. The polymerization was allowed to
continue over night and after the MEEECL was consumed, the
reaction was quenched by precipitation in hexane, yielding 0.5
g of clear gel-like polymer.
1H NMR (500 MHz, CDCl3, δ): 1.165 (t, 3H), 1.785 (m, 6H), 1.857
(m, 3H), 2.382 (t, 4H), 3.375 (s, 3H), 3.471 (m, 4H), 3.547 (m,
2H), 3.599 (m, 4H), 3.646 (m, 7H), 4.1632 (m, 4H)
6 | J. Name., 2012, 00, 1-3
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Journal of Materials Chemistry B
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DOI: 10.1039/C7TB01291H
Determination of LCST
DLS Analysis
To determine the LCST, 2 mg of polymer was dissolved in 10 mL Aqueous suspensions of micelles were prepared as stated
of water to make a 0.2 wt. % solution. The % transmittance at above at a concentration above the determined CMC, at 1 mg
600 nm was recorded at temperatures ranging from 25 oC to 55 mL-1.
The micelles were analyzed to determine their
oC. The LCST or cloud point was taken at the point of 50% drop hydrodynamic diameters (Dh) using dynamic light scattering.
in transmittance for each sample.
Prior to measurement, the polymer micelle solutions were
filtered with a 0.22 µm nylon syringe filter. Size measurements
were recorded at 25 °C in triplicate.
Preparation of Micelles
Polymeric micelles were formed through nanoprecipitation and
dialysis. The polymer (5 mg) was dissolved in THF (0.4 mL) and
Demonstration of Polymer Degradation
added dropwise to 5 mL of water under sonication. The A sample of polymer (15 mg) was dissolved in 3 mL of PBS (pH
resulting micelle suspension was transferred to SnakeSkin® 7.4, DNase-, RNase-, and Protease-Free) and was stirred in a
dialysis tubing (MWCO 3500 Da) and dialyzed against a closed system in a thermostatted oil bath at 37 °C over a period
minimum of 1500 mL deionized water over a 24-hour period. of 6 days. Samples were taken periodically and analyzed by SEC
The final contents of the dialysis tubing were filtered through a to monitor the change in Mn from t=0 to t=6 days. The resulting
Nylon syringe filter (0.22 μm) to obtain a polymeric micelle change in molecular weight is plotted as % of initial Mn vs. days
solution with a concentration of 1 mg mL-1.
spent in PBS solution at 37 °C.
Preparation of DOX Loaded Micelles
Biological Studies
DOX-loaded micelles were prepared in a manner similar to the Unless otherwise indicated, all cell culture experiments were
empty micelles. DOX·HCl was first neutralized by adding 3 performed using RPMI-1640 medium with L-glutamine and
equivalents of triethylamine in DMSO. An aliquot of the sodium bicarbonate supplemented with 10% fetal bovine serum
neutralized DOX solution containing 0.5 mg of DOX was added and 1% penicillin-streptomycin. Cells were grown in
a
o
to a polymer solution (5 mg in 0.4 mL DMSO). The DOX-polymer humidified environment at 37 C, 5% CO2. Cell viability studies
solution was then added dropwise into 5 mL of deionized water were performed using the CellTiter-Blue® assay (Promega)
under sonication. The resulting suspension was transferred to according to the manufacturer’s recommended protocol.
dialysis tubing and dialyzed against a minimum of 1500 mL of
In vitro DOX Release
deionized water over a 24 hour period. The contents of the
dialysis tube were finally filtered using a Nylon syringe filter A 1 mg mL-1 solution of DOX loaded micelles were prepared as
(0.45 μm) to obtain a 1 mg mL-1 solution of DOX loaded micelles. previously described. DOX loaded solution (2 mL) was
To determine the DLC and EE, the drug loaded micelle solutions transferred to dialysis membrane tubes with MWCO of 3500 Da.
were diluted with DMSO in a 1:1 ratio to break the micelles. The The tubes were immersed in beakers of pH 7.4 PBS solutions (10
o
absorbance of the solution at 485 nm was fitted to a pre- mL) and stirred at a constant speed at either 37 C or 40 oC. At
established standard curve of DOX in DMSO/ DI H2O.
specific time intervals, 2 mL of the release medium was
withdrawn and replaced with fresh solution. The DOX content
in the samples was analysed using UV-Vis spectroscopy.
Determination of CMC
The CMC was determined using pyrene, a hydrophobic Absorbance measurements in the release media were taken at
fluorescent molecule, as a probe. Various concentrations of 485 nm to calculate the cumulative DOX release.
polymer samples were combined with a small amount of pyrene
Stability of Micelles in FBS
(6.0 x 10-5 M in THF) in 0.2 mL THF. The polymer/pyrene
samples were added dropwise into 10 mL of deionized water. NR loaded micelles were prepared in the same method as DOX
The resulting solutions were stirred for 4h to allow micelle loaded micelles and were incubated in PBS containing either 0%
assembly and complete evaporation of THF. The resulting or 10% FBS. The fluorescence emission intensity was measured
solutions contained concentrations from 1 x 10-5 to 1 g L-1 of at 632 nm at desired time points. The NR emission intensities
polymer and a constant concentration of pyrene. Fluorescence are normalized to the initial fluorescence intensities (t=0) and
spectra of the polymer/pyrene solutions were collected at 25 °C plotted versus time.
with emission wavelength of 390 nm. The ratio of intensities of
the pyrene excitation peaks at 337.5 nm and 334.5 nm were
Empty Micelle Cytotoxicity Studies
recorded and plotted against the logarithm of the polymer HeLa cells were seeded in transparent flat-bottom 96-well
concentration (C). The x coordinate at the intersection of the plates at a cell density of 5,000 cells per well in 100 µL growth
two trend lines before and after the abrupt increase in the medium. After 24h to allow cell adhesion, the medium was
I337.5/I334.5 vs. Log (C) curve was taken to be the critical micelle removed, the cells were washed with 100 µL PBS, and 100 µL
concentration.
fresh growth medium was added to each well. Empty micelles
(1 mg mL-1 in PBS) were diluted to concentrations ranging from
0.06 mg mL-1 to 0.5 mg mL-1. Micelle dilutions (100 µL) were
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Journal of Materials Chemistry B
Page 8 of 10
ARTICLE
Journal Name
added to the cells via multichannel micropipette. The micelles Future optimization of these drug delivery systems will focus on
View Article Online
were incubated for 24 hours with the cells. At this time, the cell improving the drug loading capabilities eiDthOeI:r10th.1r0o3u9/gCh7TcBh0a1n2g91eHs
viability was evaluated by the CellTiter-Blue® assay, N=4.
in the substituents of the hydrophobic block or through
different loading methods.
DOX-loaded Micelle Cytotoxicity Studies
HeLa cells were seeded in transparent flat-bottom 96-well
plates at a cell density of 5,000 cells per well in 100 µL growth
medium. After 24h to allow cell adhesion, the medium was
removed, the cells were washed with 100 µL PBS, and 100 µL of
fresh growth medium was added to the cells. DOX-loaded
micelles (1 mg mL-1 in PBS) were diluted in PBS to
concentrations ranging from 0.06 mg mL-1 to 0.5 mg mL-1. DOX-
loaded micelle dilutions (100 µL) were added to the cells via
multichannel micropipette. Free DOX dosing was given
assuming the dose from the predetermined drug loading. The
Acknowledgements
We gratefully acknowledge the financial support from National
Science Foundation (CHE-1609880) and Welch Foundation (AT-
1740).
Notes and references
1.
2.
3.
4.
K. Kataoka, A. Harada and Y. Nagasaki, Advanced Drug
Delivery Reviews, 2001, 47, 113-131.
Z. Ahmad, A. Shah, M. Siddiq and H.-B. Kraatz, RSC
Advances, 2014, 4, 17028-17038.
S. Biswas, P. Kumari, P. M. Lakhani and B. Ghosh, European
Journal of Pharmaceutical Sciences, 2016, 83, 184-202.
S. Movassaghian, O. M. Merkel and V. P. Torchilin, Wiley
o
o
cells were then incubated at either 37 C or 40 C for 24 hours
with the DOX-loaded micelles. After this time, the cell viability
was evaluated using the CellTiter-Blue® assay, N=4.
Cellular Uptake
Interdisciplinary
Nanobiotechnology, 2015, 7, 691-707.
Reviews:
Nanomedicine
and
HeLa cells were seeded in a 35-mm glass bottom dish at a
density of 200,000 cells per well and allowed to adhere for 24 h
in 2 mL of growth media. At that time, the medium was
removed, the cells were washed with 2mL of PBS, and 2 mL of
fresh growth medium was added along with 1 mL of DOX-
loaded micelles (0.2 mg mL-1 in PBS). The cells dosed with DOX-
loaded micelles were allowed to incubate for 4 hours. After the
uptake period, the cells were washed 3 times with 2 mL of PBS,
fixed with 4% paraformaldehyde (room temperature, 10
minutes), washed 3 times with 2 mL of PBS, and the nuclei were
counterstained with DAPI. Images were obtained using a BioTek
Cytation 3 Cell Imaging Multi-Mode Reader.
5.
A. Rösler, G. W. M. Vandermeulen and H.-A. Klok,
Advanced Drug Delivery Reviews, 2012, 64, Supplement,
270-279.
T. M. Allen and P. R. Cullis, Science, 2004, 303, 1818.
A. Rahikkala, V. Aseyev, H. Tenhu, E. I. Kauppinen and J.
Raula, Biomacromolecules, 2015, 16, 2750-2756.
D. Schmaljohann, Advanced Drug Delivery Reviews, 2006,
58, 1655-1670.
S. Mura, J. Nicolas and P. Couvreur, Nat Mater, 2013, 12,
991-1003.
M. A. C. Stuart, W. T. S. Huck, J. Genzer, M. Muller, C. Ober,
M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M.
Urban, F. Winnik, S. Zauscher, I. Luzinov and S. Minko, Nat
Mater, 2010, 9, 101-113.
V. P. Torchilin, Nat Rev Drug Discov, 2014, 13, 813-827.
K. E. Washington, R. N. Kularatne, V. Karmegam, M. C.
Biewer and M. C. Stefan, Wiley Interdisciplinary Reviews:
Nanomedicine and Nanobiotechnology, 2016, DOI:
10.1002/wnan.1446, DOI.
O. Coulembier, P. Degée, J. L. Hedrick and P. Dubois,
Progress in Polymer Science, 2006, 31, 723-747.
E. A. Rainbolt, K. E. Washington, M. C. Biewer and M. C.
Stefan, Polymer Chemistry, 2015, 6, 2369-2381.
H. Jing, A. R. Elizabeth, W. Katherine, C. B. Michael and C.
S. Mihaela, Current Organic Chemistry, 2013, 17, 930-942.
M. L. Adams, A. Lavasanifar and G. S. Kwon, Journal of
Pharmaceutical Sciences, 2003, 92, 1343-1355.
H. Maeda, H. Nakamura and J. Fang, Advanced Drug
Delivery Reviews, 2013, 65, 71-79.
6.
7.
8.
9.
10.
Conclusions
11.
12.
Temperature responsive 4- and 6-arm amphiphilic star-like
polycaprolactones were successfully synthesized through the
living ring-opening polymerization of functionalized ε-
caprolactone monomers ECL and MEEECL. The use of the
MEEECL monomer enables the synthesis of star-like polymers
through with high control over the composition and molecular
weight, while also allowing the resulting polymers to be
thermoresponsive. These polymers were thermodynamically
stable with CMCs ~10-3 g L-1. Additionally, the star block
copolymers had LCST above the physiological temperature of
13.
14.
15.
16.
17.
18.
37C which is desirable for use in temperature controlled drug
delivery. These polymers were also shown to be biocompatible,
exhibiting relatively low toxicity to HeLa cells at doses up to 0.5
mg mL-1, and biodegradable over time under physiological
conditions. Drug release studies showed that the release of drug
increased with the application of external temperature in both
micellar system and cytotoxicity studies indicated increased
toxicity to cancer cells when incubated at temperatures higher
than LCST, indicating that these systems show thermally
controlled release. These drug delivery systems showed
improvement over the previously reported linear polymer in
terms of their thermodynamic stability as well as their LCSTs.
J. Wang, W. Mao, L. L. Lock, J. Tang, M. Sui, W. Sun, H. Cui,
D. Xu and Y. Shen, ACS Nano, 2015, 9, 7195-7206.
H. Maeda, Advanced Drug Delivery Reviews, 2015, 91, 3-6.
Z. L. Tyrrell, Y. Shen and M. Radosz, Progress in Polymer
Science, 2010, 35, 1128-1143.
D. Roy, W. L. A. Brooks and B. S. Sumerlin, Chemical Society
Reviews, 2013, 42, 7214-7243.
J. Hao, J. Servello, P. Sista, M. C. Biewer and M. C. Stefan,
Journal of Materials Chemistry, 2011, 21, 10623-10628.
19.
20.
21.
22.
8 | J. Name., 2012, 00, 1-3
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Page 9 of 10
Journal of Materials Chemistry B
Please do not adjust margins
Journal Name
ARTICLE
23.
24.
Y. Cheng, J. Hao, L. A. Lee, M. C. Biewer, Q. Wang and M. C.
View Article Online
DOI: 10.1039/C7TB01291H
Stefan, Biomacromolecules, 2012, 13, 2163-2173.
J. Hao, Y. Cheng, R. J. K. U. Ranatunga, S. Senevirathne, M.
C. Biewer, S. O. Nielsen, Q. Wang and M. C. Stefan,
Macromolecules, 2013, 46, 4829-4838.
25.
26.
27.
E. A. Rainbolt, J. B. Miller, K. E. Washington, S. A.
Senevirathne, M. C. Biewer, D. J. Siegwart and M. C. Stefan,
Journal of Materials Chemistry B, 2015, 3, 1779-1787.
E. A. Rainbolt, K. E. Washington, M. C. Biewer and M. C.
Stefan, Journal of Materials Chemistry B, 2013, 1, 6532-
6537.
J. M. Ren, T. G. McKenzie, Q. Fu, E. H. H. Wong, J. Xu, Z. An,
S. Shanmugam, T. P. Davis, C. Boyer and G. G. Qiao,
Chemical Reviews, 2016, 116, 6743-6836.
28.
29.
30.
31.
W. Wu, W. Wang and J. Li, Progress in Polymer Science,
2015, 46, 55-85.
G. Basu Ray, I. Chakraborty and S. P. Moulik, Journal of
Colloid and Interface Science, 2006, 294, 248-254.
J. K. Kim, S. Y. Yang, Y. Lee and Y. Kim, Progress in Polymer
Science, 2010, 35, 1325-1349.
K. E. Washington, R. N. Kularatne, J. Du, M. J. Gillings, J. C.
Webb, N. C. Doan, M. C. Biewer and M. C. Stefan, Journal
of Polymer Science Part A: Polymer Chemistry, 2016, 54,
3601-3608.
32.
J. Burke, R. Donno, R. d’Arcy, S. Cartmell and N. Tirelli,
Biomacromolecules, 2017, 18, 728-739.
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Journal of Materials Chemistry B
Page 10 of 10
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DOI: 10.1039/C7TB01291H
Fully biodegradable amphiphilic thermoresponsive star block copolymers featuring 4 and 6 arms are
reported for micellar delivery of doxorubicin.