Application of click chemistry in the preparation of poly(ethylene oxide)-block-poly(ε-caprolactone) with hydrolyzable cross-links in the micellar core

Article abstract of DOI:10.1021/ma102548m

The aim of this study is to develop degradable core-cross-linked polymeric micelles based on poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) structures using click chemistry. Substituted monomer, that is, α-propargyl carboxylate-ε-caprolactone, was synthesized by anionic activation of ε-caprolactone and further treatment with propargyl chloroformate. Ring-opening polymerization of α-propargyl carboxylate-ε-caprolactone with methoxy PEO (5000 g/mol) as initiator and stannous octoate as catalyst was used to prepare PEO-b-poly(α-propargyl carboxylate-ε-caprolactone) (PEO-b-PPCL) block copolymer. The block copolymers were found to spontaneously associate in aqueous solution forming well-defined micelles. Stabilization of the micelles was obtained by cross-linking the core via click reaction between the azide group of tetraethylene glycol (bis)azide reagent and the alkyne group on the PPCL block in the presence of copper catalyst at room temperature. Successful cross-linking was evidenced by ¹H NMR, IR spectroscopy, and X-ray photoelectron spectroscopy (XPS). This was followed by the characterization of micellar morphology and size by transmission electron microscopy and light-scattering. Extent of bovine serum albumin (BSA) adsorption for cross-linked and non-cross-linked micelles illustrated the better stability of cross-linked micelles against protein adsorption. Finally, paclitaxel (PTX) was physically encapsulated in the micellar cores, where a similar PTX encapsulation and in vitro release profile for PTX from non-cross-linked and cross-linked micelles was observed. The results pointed to the increased stability of prepared cross-linked micelles and their potential in drug delivery.

Full text of DOI:10.1021/ma102548m

ARTICLE
pubs.acs.org/Macromolecules
Application of Click Chemistry in the Preparation of Poly(ethylene
oxide)-block-poly(ε-caprolactone) with Hydrolyzable Cross-Links in
the Micellar Core
Shyam M Garg,† Xiao-Bing Xiong,† Changhai Lu,† and Afsaneh Lavasanifar*,†,‡
†Faculty of Pharmacy and Pharmaceutical Sciences and ‡Faculty of Chemical and Material Engineering, University of Alberta, Edmonton,
Alberta, Canada
S Supporting Information
b
ABSTRACT: The aim of this study is to develop degradable
core-cross-linked polymeric micelles based on poly(ethylene
oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) structures
using click chemistry. Substituted monomer, that is, R-propar-
gyl carboxylate-ε-caprolactone, was synthesized by anionic
activation of ε-caprolactone and further treatment with propar-
gyl chloroformate. Ring-opening polymerization of R-propargyl
carboxylate-ε-caprolactone with methoxy PEO (5000 g/mol) as
initiator and stannous octoate as catalyst was used to prepare
PEO-b-poly(R-propargyl carboxylate-ε-caprolactone) (PEO-b-
PPCL) block copolymer. The block copolymers were found to
spontaneously associate in aqueous solution forming well-
defined micelles. Stabilization of the micelles was obtained by
cross-linking the core via click reaction between the azide group
of tetraethylene glycol (bis)azide reagent and the alkyne group
on the PPCL block in the presence of copper catalyst at room temperature. Successful cross-linking was evidenced by 1H NMR, IR
spectroscopy, and X-ray photoelectron spectroscopy (XPS). This was followed by the characterization of micellar morphology and
size by transmission electron microscopy and light-scattering. Extent of bovine serum albumin (BSA) adsorption for cross-linked
and non-cross-linked micelles illustrated the better stability of cross-linked micelles against protein adsorption. Finally, paclitaxel
(PTX) was physically encapsulated in the micellar cores, where a similar PTX encapsulation and in vitro release profile for PTX from
non-cross-linked and cross-linked micelles was observed. The results pointed to the increased stability of prepared cross-linked
micelles and their potential in drug delivery.
’ INTRODUCTION
modification of the hydrophobic block,4-6 introduction of
crystallinity or stereocomplex formation,7,8 covalent attachment
of hydrophobic drug,9,10 formation of glassy core,11 and cross-
linking of the micellar core or shell. Cross-linked micelles can stay
in the micellar form at concentrations below CMC of their block
copolymer; thus avoiding rapid drug release.12-15 Cross-links are
preferred to degrade in response to internal or external stimuli,
ensuring the release of incorporated drug in the vicinity of
cellular or molecular drug targets and at the same time resulting
in the biological elimination of the colloidal carrier after drug
release.
Over the last few decades, block copolymers have emerged as
an interesting class of biomaterials because of their versatile
applications in pharmaceutical science and drug delivery. Of
particular interest are amphiphilic block copolymers, which self-
assemble into polymeric micelles with core-shell architectures
above the critical micelle concentration (CMC). Polymeric
micelles are currently under investigation as nanodelivery sys-
tems for depot drug release and targeted drug delivery.1-3 The
use of polymeric micelles for the mentioned applications, how-
ever, has been hampered by the poor in vivo stability of most
micellar structures upon administration to systemic circulation,
which leads to micellar dissociation and premature release of
encapsulated drugs.
One of the earliest examples of cross-linking of the micellar
shell was reported by Wooley and coworkers in 1996.16 Since
then, this strategy has been investigated by many other
groups.17-21 Core-cross-linking was first reported by Tuzar
Considerable research has focused on increasing the stability
of polymeric micelles by preventing their dissociation in the
extreme dilution conditions of the bloodstream upon intrave-
nous administration. Some of the strategies currently under study
for the stabilization of polymeric micelles include chemical
Received: November 8, 2010
Revised:
February 10, 2011
Published: March 08, 2011
r
2011 American Chemical Society
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and coworkers in 197922 and 1982,23 in which poly(styrene/
butadiene/styrene) micelles with cores consisting of polybuta-
diene blocks were stabilized by UV radiation in the presence of a
photoinitiator. Similar strategies involving cross-linking of
the hydrophobic core by either thermal,24 photoinduced
polymerization,25,26 or conventional chemical reactions in the
micellar core have been carried out.24-31 More recently, a
doxorubicin methacrylamide derivative bearing a hydrolytically
sensitive hydrazone linker was covalently incorporated into the
cross-linked core of polymeric micelles composed of mPEO-b-
poly[N-(2-hydroxypropyl)methacrylamide-lactate] diblock co-
polymers by free radical polymerization.32 However, most of
these strategies included harsh conditions during the procedures
and have other disadvantages. For example, photo-cross-linkable
materials are conventionally unstable when exposed to light.33
Besides, high temperatures during thermal polymerization can
cause decomposition of the incorporated biomolecules.
Scheme 1. Synthetic Scheme for the Preparation of Core-
Crosslinked Micelles
Click chemistry has emerged as a highly efficient technique,
providing attractive possibilities for the synthesis of polymers
with different architectures. It offers advantages including ambi-
ent reaction conditions, quantitative yields, easily obtained
starting materials, and, in particular, high specificity, which make
the reaction doable for molecules bearing extra functional
groups, avoiding the need for the protection/deprotection
reactions.34-37 In 2001, Sharpless and coworkers35 introduced
the concept of click chemistry reactions, of which the Cu(I)-
catalyzed Huisgen 1,3-dipolar cycloaddition reaction between
terminal alkynes and azides is one of the most popular and
commonly used click reactions.38,39 In previous studies, click
chemistry has been used to prepare the shell as well as core-cross-
linked micelles.34,40-42
that are attached to the poly(ester) core of polymeric micelles via
“degradable” ester bonds. This is also the first report on the
application of click chemistry to prepare core-cross-linked mi-
celles made up of highly used PEO-b-PCL diblock copolymers.
’ EXPERIMENTAL SECTION
Materials. Methoxy-poly(ethylene oxide) (PEO) (average molecu-
lar weight of 5000 g/mol), diisopropylamine (99%), propargyl chlor-
oformate, sodium (in kerosin), butyllithium (Bu-Li) in hexane (2.5 M
solution), tetraethylene glycol ditosylate, and bovine serum albumin (BSA)
powder were purchased from Sigma (St. Louis, MO). PTX (purity >99.5)
was purchased from LC Laboratories (Woburn, MA). ε-Caprolactone was
purchased from Lancaster Synthesis (Heysham, U.K.). Stannous octoate
was purchased from MP Biomedicals (Eshwege, Germany). All other
chemicals were reagent grade. Tetraethylene glycol (bis)azide was synthe-
sized from tetraethylene glycol ditosylate and sodium azide according to
literature procedures and confirmed by 1H NMR.54
In this Article, we report a unique strategy leading to the
introduction of hydrolyzable cross-links to the core of poly-
(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) mi-
celles using click chemistry. Copolymers of PEO-b-PCL have
been extensively explored in drug delivery. In our research group,
both the core and shell block of PEO-b-PCL micelles have been
engineered to achieve depot or targeted drug and siRNA
delivery.43-53 Here we report the successful synthesis and
characterization of core-cross-linked PEO-b-PCL micelles by
click chemistry. Toward this, block copolymers of PEO-b-PCL
having pendent propargyl carboxylate groups on the PCL
block, that is, PEO-b-PPCL, were synthesized via ring-opening
polymerization of R-propargyl carboxylate ε-caprolactone using
PEO as the initiator. Consequently, the block copolymer self-
associated into micelles in aqueous solution. The micelle core
was then cross-linked via reaction between the azide group of
tetraethylene glycol (bis)azide reagent and the alkyne group on
the PPCL block in the presence of copper catalysts at room
temperature. The formation of cross-linked micelles was con-
Synthesis of r-Propargyl Carboxylate-ε-Caprolactone.
The method used for the synthesis of R-propargyl carboxylate-ε-
caprolactone is shown in Scheme 1. In brief, Bu-Li (24 mL) in hexane
was slowly added to dry diisopropylamine (8.4 mL) in 50 mL of dry
THF in a three-necked round-bottomed flask at -30 °C under vigorous
stirring with continuous argon supply. The solution was cooled to -
78 °C. ε-Caprolactone (3.42 g) was dissolved in 10 mL of dry THF and
added to the above-mentioned mixture slowly, and the reaction was
allowed to continue for 45 min. Propargyl chloroformate (3.55 g) was
added, and the temperature was allowed to increase to 0 °C. The
reaction was allowed to continue for 2 h and was quenched with 5 mL of
saturated ammonium chloride solution. The reaction mixture was
diluted with water and extracted with ethyl acetate (110 mL). The
combined extracts were dried over Na2SO4 and evaporated. The dark
yellowish oily crude mixture was purified twice over a silica gel column,
and the purity of the compound was confirmed with thin-layer chro-
matography (TLC) using hexane/ethyl acetate (2:1) as the mobile
1
firmed and characterized by H NMR and IR spectroscopy,
transmission electron microscopy (TEM), X-ray photoelectron
spectroscopy (XPS), and dynamic light scattering (DLS). The
encapsulation and in vitro release of paclitaxel (PTX) in cross-
linked micelles was compared with these properties for micelles
without cross-links. The results from this study point to the
enhanced stability of cross-linked micelles under diluted condi-
tions without any negative impact on the encapsulation and in
vitro release of PTX in the presence of cross-links.
1
phase. The chemical structure was analyzed by H NMR, 13C NMR,
and IR.
To the best of our knowledge, this is the first Article that
reports the use of click chemistry to develop core-cross-linked
polymeric micelles that contain biocompatible PEO “crosslinks”
Synthesis of PEO-block-poly(r-propargyl carboxylate-ε-
caprolactone) (PEO-b-PPCL). Block copolymers of PEO-b-PPCL
were synthesized by ring-opening polymerization of R-propargyl
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carboxylate-ε-caprolactone using PEO as initiator and stannous octoate
as catalyst (Scheme 1). In brief, PEO (Mw: 5000 g/mol) (1.5 g), R-
propargyl carboxylate-ε-caprolactone (1.5 g), and stannous octoate
(0.002 equiv of monomer) were added to a 10 mL previously flamed
ampule, nitrogen purged, and sealed under vacuum. The polymerization
reaction was allowed to proceed for 4 h at 140 °C in oven. We
terminated the reaction by cooling the product to room temperature.
The 1H NMR spectrum of R-propargyl carboxylate-ε-caprolactone
bearing block copolymer in CDCl3 at 300 MHz was used to assess the
conversion of R-propargyl carboxylate-ε-caprolactone monomer to
PPCL comparing the intensity of the -O-CH2 (δ 4.25)-related peak
for R-propargyl carboxylate-ε-caprolactone monomer to the intensity of
the same protons for PPCL (δ 4.05). The number-average molecular
The CMC was measured from a sharp rise in intensity ratios (I338/I333)
at the onset of micellization.
XPS measurements were conducted on freeze-dried sample of cross-
linked micelles using an Axis-165 spectrometer (Kratos Analytical,
Chestnut Ridge, NY) with a monochromatic Al KR X-ray source at
1486.6 eV. The analyzer was operated in constant resolution mode at a
pass energy of 20 eV and charge referencing was accomplished by setting
the C 1s line of adventitious hydrocarbon on the specimen surface at
284.8 eV.
Evaluation of Protein Adsorption on Micelles. The measure-
ment of the amount of protein adsorbed on the surface of the micelles
was carried out according to a method previously described.56 In brief,
non-cross-linked and cross-linked micellar solutions (8 mg/mL) were
mixed with equal volume of BSA solution (45 g BSA/L in 0.01 M PBS)
and incubated for 4 h at 37 °C. Micellar solutions with equal volume of
PBS were used as control and incubated for 4 h at 37 °C. After
incubation, solution samples of 20 μL were injected into a gel permea-
tion chromatography (GPC) system with a hydrogel column (Waters,
Milford, MA) at 25 °C. The elution pattern was detected at 35 °C by
light scattering detector (model 410, Waters). We used 0.01 M PBS (pH
7.4) (1 mL/min) as eluent. Eluate containing the micellar fraction was
collected, and the concentration of protein in the eluate was measured
using the Bio-Rad protein assay (Bio-Rad, Hercules, CA).
Preparation of PTX-Loaded Micelles. Encapsulation of PTX in
non-cross-linked PEO-b-PPCL was accomplished as reported
before.53,57 In brief, the block copolymer and PTX were both dissolved
in N,N-dimethyl formamide (DMF) as the organic solvent. This
solution was added to water in a dropwise manner, followed by dialysis
of the solution against water to remove DMF. For PTX loading in cross-
linked micelles, two different methods were used. In the first method
(method I), PTX, PEO-b-PPCL, and the cross-linking agent
(tetraethylene glycol (bis)azide) were all added to DMF, and this
solution was added to water containing sodium ascorbate and CuSO4.
In the second method (method II), PTX-encapsulated micelles were
prepared as described above. The cross-linking agent and other reagents
were then added to micellar aqueous solution. The applied drug to
polymer weight ratio was 20 for all formulations. After dialysis, the
solution was centrifuged at 11 600g for 5 min to remove any precipitate,
and an aliquot (100 μL) of the micellar solution was diluted with
acetonitrile. The solution was analyzed for PTX content using HPLC.
Reversed phase chromatography was carried out using a Varian Prostar
210 HPLC system equipped with a Microsorb-MV 5 μm C18-100 Å
column (4.6 mm ꢀ 250 mm), and Varian 335 photodiode array HPLC
detector (Mulgrave, Australia). We injected 20 μL of sample in a
gradient elution using 0.1% trifluoroacetic acid aqueous solution and
acetonitrile at a flow rate of 1.0 mL/min at room temperature. The
proportion of acetonitrile was 40% at time 0 and increased with elution
time up to 100% within 15 min.53,58 The detection was performed at
227 nm. The level of PTX loading (w/w %) and encapsulation efficiency
was calculated using the following equations
1
weight of PEO-b-PPCL was determined from the H NMR spectrum
comparing peak intensity of PEO (-CH2CH2O-, δ 3.65) to that of
PPCL (-CtCH, δ 2.55), considering a 5000 g/mol molecular weight
for PEO. The IR spectrum was obtained by dissolving the block
copolymers in dichloromethane and preparing a thin film on NaCl disk.
The polydispersity of prepared block copolymer was assessed by gel
permeation chromatography (GPC). In brief, 20 μL of polymer solution
(20 mg/mL in THF) was manually injected into a 7.8 ꢀ 300 mm
Styragel HMW 6E column (Waters, Milford, MA) which was attached to
an HP 1100 pump. The column was eluted with 1 mL/min THF. The
elution pattern was detected by refractive index (model 410; Waters)
and DLS detectors (PD 2000 DLS; Precision Detectors, Franklin, MA)
using polystyrene standard of two molecular weights (Mw) (9580 and
13 700 g/mol).
Micelle Formation and Core-Cross-Linking. First, micelliza-
tion was achieved by solvent evaporation method. In brief, the synthe-
sized block copolymer of PEO-b-PPCL (60 mg) was dissolved in acetone
(1 mL). This solution was added to 6 mL of doubly distilled water in a
dropwise manner under moderate stirring at room temperature, followed
by the evaporation of acetone under vacuum. The prepared micellar
solution was then centrifuged to remove any aggregates.
Micellar solution of block copolymers was cross-linked using the
Huisgens 1,3-dipolar cycloaddition (azide-alkyne Click chemistry)
reaction.38,39 In brief, to the prepared PEO-b-PPCL micelle solutions,
tetraethylene glycol (bis)azide (15.86 mg; 0.065 mmol) was added
under vigorous stirring, followed by the addition of sodium ascorbate
(2.57 mg; 0.013 mmol) and copper sulfate (0.21 mg; 0.0013 mmol). The
reaction mixture was stirred for 16 h and then purified by dialysis against
water and freeze-dried. The resulted polymer was subjected to 1H NMR
and IR analysis. The schematic representation of the preparation
method for core-cross-linked micelles is shown in Scheme 1.
Characterization of Micelles. Theparticlesize anddistributionof
prepared micelles were estimated by DLS using a Malvern Zetasizer 3000
at a polymer concentration of 10 mg/mL in water at 25 °C. Morphology
of the micelles was investigated by TEM. An aqueous droplet of micellar
solution (20 μL) with a polymer concentration of 1 to 2 mg/mL was
placed on a copper-coated grid. The grid was held horizontally for 20 s to
allow the colloidal aggregates to settle. A drop of 2% solution of
phototungstic acid (PTA) in PBS (pH 7) was then added to provide
the negative stain. After 1 min, the excess fluid was removed by filter
paper. The samples were then air-dried and loaded into a Hitachi H 700
TEM. Images were obtained at a magnification of 18 000ꢀ at 75 kV.
A change in the fluorescence excitation spectra of the hydrophobic
pyrene in the presence of varied concentrations of PEO-b-PPCL was
used to measure the critical micellar concentration (CMC) of the
amount of physically loaded PTX in mg
PTX loading ð%Þ ¼
amount of copolymer in mg
ꢀ 100%
amount of physically loaded PTX in mg
encapsulation efficiencyð%Þ ¼
amount of PT added in mg
ꢀ100%
prepared block copolymer according to
a method previously
described.55 The fluorescence spectra were recorded using a Varian
Cary Eclipse fluorescence spectrophotometer (Mulgrave, Australia).
Emission wavelength and excitation/emission slit were set at 390 and
5 nm, respectively. The intensity ratio of peaks at 338 nm to those at
333 nm was plotted against the logarithm of copolymer concentration.
Release of PTX from Polymeric Micelles. Release of PTX from
non-cross-linked and cross-linked micelles was determined in 0.01 M
phosphate buffer (pH 7.4) containing 2 M sodium salicylate at
37 °C.53,59,60 The experiment was initiated by the addition of free or
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reported on the preparation of substituted lactone monomers
such as R-benzyl carboxylate-ε-caprolactone,52 R-propargyl-δ-
valerolactone,63 R-allyl-δ-valerolactone,64 R-propargyl-ε-
caprolactone,65,66 and R-iodo-ε-caprolactone.67 For this pur-
pose, anionic activation of ε-caprolactone monomer was per-
formed using freshly prepared non-nucleophilic strong base LDA
to extract a methylene proton from the R-position (-CH2-
CdO). The generated lithium carbanion was then quenched
with propargyl chloroformate to obtain R-propargyl carboxylate-
ε-caprolactone (Scheme 1).68 After column chromatography, R-
propargyl carboxylate-ε-caprolactone was isolated as a slightly
yellow thick oily liquid. The product produced a single spot at Rf
value of 0.37 in TLC. The yield of reaction was 49.2%. The
structure was confirmed by combined analysis of 1H NMR and
IR. In 300 MHz 1H NMR spectroscopy in CDCL3, correspond-
ing proton peaks were observed at δ: 1.20-2.20 (m, 6H); δ: 2.50
(s, 1H); δ: 3.75 (d, 1H); δ: 4.15-4.40 (m, 2H); δ: 4.70-4.85
(q, 2H) (Figure 1A). The peak at 3.75 ppm for R-propargyl
carboxylate-ε-caprolactone, which corresponds to a single pro-
ton instead of two protons of ε-caprolactone monomer, indicates
the successful substitution of the propargyl carboxylate on ε-
caprolactone at the R-position. The presence of two negative
peaks for carbonyl at 168.10 and 171.38 ppm and the generation
of a new characteristic positive peak at 50.65 ppm in the 13C
NMR spectrum also confirm the chemical structure of the
reaction product (Supporting Information Figure S1). The
presence of strong peak in the IR spectrum (Figure 1B) at
1725 cm-1 corresponds to the carbonyl groups in lactone and
propargyl carboxylate. The presence of strong peak at 3270 cm-1
and weak peak at 2120 cm-1 corresponds to the alkyne group in
the monomer.
Figure 1. (A) 1H NMR spectrum of R-propargyl carboxylate-ε-capro-
lactone (substituted monomer) in CDCl3 and peak assignments. (B) IR
spectrum of R-propargyl carboxylate-ε-caprolactone. Arrow indicates
the presence of characteristic groups.
1
In 300 MHz H NMR spectroscopy conducted on PEO-b-
PPCL in CDCL3, corresponding proton peaks for the product
were observed at δ: 1.20-2.15 (m, 6H); δ: 2.50 (s, 1H); δ: 3.30-
3.45 (s, 3H; m, 1H); δ: 3.65 (s, 4H); δ: 4.05-4.25 (m, 2H); δ:
4.75 (s, 2H) (Figure 2A). The presence of peaks at 2.50 and 4.75
ppm, which are due to the alkyne and methylene protons of the
propargyl carboxylate group, respectively, confirms the polymer-
ization of R-propargyl carboxylate-ε-caprolactone and the pre-
sence of alkyne groups in the structure of block copolymer.
Furthermore, the characteristic downfield shift of the methylene
protons (-OCH2- of R-propargyl carboxylate-ε-caprolactone)
from 4.30 to 4.15 and OdC-CH- proton from 3.75 to 3.30 in
the 1H NMR spectra (Figures 1A and 2A) strongly indicates the
ring-opening polymerization of the monomer and formation of
block copolymers. The characteristic terminal alkyne peak at
3270 cm-1 and the -CtC- peak at 2120 cm-1 in the IR
spectrum of PEO-b-PPCL indicates the presence of terminal
alkyne group in the block copolymer (Figure 2B).
The molecular weight of prepared PEO-b-PPCL block copo-
lymer, measured by comparing the peak intensities of four
methylene protons of PEO (δ: 3.65) and one alkyne proton of
PPCL (δ: 2.50) in the 1H NMR spectrum, was calculated to be
8800 g/mol (equal to a degree of R-propargyl carboxylate-ε-
caprolactone polymerization of 19, that is, PEO114-b-PPCL19).
The resulting block copolymer showed a broad polydispersity
(Mw/Mn = 1.50) when measured by GPC analysis.
micellar PTX solution to the buffer. The PTX-loaded non-cross-linked
and cross-linked micelles were prepared at 20 μg/mL PTX concentra-
tion according to the previous mentioned method. Then, 10 mL
(containing 200 μg PTX) of the micellar solutions was transferred to
a dialysis bag (Spectraphor, Mw cutoff 3500 g/mol). The dialysis bags
were placed in 500 mL of 0.01 M phosphate buffer (pH 7.4). The release
study was performed at 37 °C in a Julabo SW 22 shaking water bath
(Seelbach, Germany). At selected time intervals, the whole release media
was replaced with fresh one, and aliquots of 200 μL were withdrawn
from the inside of the dialysis bag for HPLC analysis. The amount of
PTX released was calculated by subtracting the amount of PTX that
remained in the dialysis bag from the initially added PTX. The release
profiles were compared using similarity factor, f2, and the profiles were
considered to be significantly different if f2 < 50.61
0
1
"
#
-0:5
ꢀ ꢁ
n
1
2
@
A
f2 ¼ 50 ꢀ log 1 þ
∑ jRj - Tjj
ꢀ 100
n
j¼1
where n is the sampling number and Rj and Tj are the percent released of
the reference and test formulations at each time point j.
’ RESULTS AND DISCUSSION
Synthesis and Characterization of Block Copolymers.
Block copolymers of PEO-b-PPCL were developed by ring-
opening polymerization of R-propargyl carboxylate-ε-caprolac-
tone monomer using PEO as initiator and stannous octoate as
catalyst.62 Our research group and others have previously
Micellization of Block Copolymers and Core-Cross-link-
ing. Synthesized block copolymers were assembled to polymeric
micelles by a cosolvent evaporation method as previously
described.52,62 To prevent the disassembly of micelles under
the high dilution conditions of the bloodstream, we must prevent
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core due to covalent bond formations. The TEM picture for both
micelles shows the formation of true spherical-shaped colloidal
particles having a clear boundary (Figure 3A,B). The average
diameter in the dry state based on TEM images was shown to
decrease from 19.5 nm for non-cross-linked micelles to 15.4 nm
for cross-linked micelles, which shows a similar trend to the DLS
data. The difference in size measured by these two methods (97.9
vs 19.5 nm for non-cross-linked micelles and 82.6 vs 15.4 nm for
cross-linked micelles) is attributed to the acquirement of the
TEM images under a dry state as opposed to DLS that measures
particles in a hydrated state in aqueous solutions.53,71,72
To further verify the success of core-cross-linking, we added
equal volume of acetone to a 500 μg/mL micellar solution. DLS
measurements were performed to compare the stability of the
cross-linked and non-cross-linked micelles (Figure 3C,D). The
cross-linked micelles did not dissolve in acetone, thus confirming
cross-linking of the core.73 The average size and polydispersity of
the cross-linked micelles were slightly larger in acetone, which
can be due to swelling of the core of the cross-linked micelles as a
result of being penetrated by solvent molecules, in this case
acetone.29,31 The non-cross-linked micelle, however, dissociated
and did not give any reading on the DLS. Samples maintained at
25 °C were analyzed by DLS after 21 days (data not shown). The
cross-linked micelles still maintained their nanostructure, as
opposed to the non-cross-linked micelles, but with slightly larger
size. This further verifies the stability of the core-cross-linked
micelles.
1H NMR spectroscopy was performed on the freeze-dried
micelles in CDCl3 (Figure 4). CDCl3 is a good solvent for the
PEO-b-PPCL block copolymer. The signal for the hydrophobic
PCL block is clearly visible in the non-cross-linked micelles
(1.20-2.00 ppm) (Figure 4A). However, the signal has wea-
kened considerably in the case of the cross-linked micelles
(Figure 4B). As the PCL segment is present in the micellar core,
the covalent bond formed because of cross-linking results in the
rigidity of the hydrophobic core and maintenance of micellar
structure in organic solvents like CDCl3. Because the solvent is
unable to penetrate the interior, the signal corresponding to the
intensity of the PCL block at the interior is weakened because of
lack of mobility of core segments.31 This confirms the stability of
cross-linked micelles over the non-cross-linked micelles.
The IR spectrum confirmed that a click chemistry reaction has
taken place (Figure 5). The absence of peaks at 3270 cm-1 and
weak peak at 2120 cm-1 corresponding to the alkyne group
in the PPCL core along with the appearance of sharp peak
at 810 cm-1 corresponding to dC-H bending (characteristic
of trisubstituted alkene) indicates the reaction between alkyne
groups in the PPCL core and diazide cross-linker producing the
triazole ring.
The elemental analysis by XPS is shown in Table S1
(Supporting Information). The mass concentration of nitrogen
in the sample calculated by XPS was found to be 3.65%. Figure S2
(Supporting Information) shows a broad N1s peak near 400 eV.
By multipeak fitting, the peak can be separated into two peaks at
399.9 and 401.4 eV, which corresponds to the triazole ring. The
absence of a peak at ∼405 eV indicates the absence of azide
group in the sample.74-76 This indicates the removal of any
excess azide from the cross-linked micelles. In addition, XPS
revealed an extremely small Cu2p peak denoting the presence of
residual Cu2þ ions, with a mass concentration of 0.20% in the
cross-linked micelles. This indicates that Cu ion could not be
completely removed from the sample, which may be due to the
Figure 2. (A) 1H NMR spectrum of PEO-b-PPCL in CDCl3 and peak
assignments. (B) IR spectrum of PEO-b-PPCL.
the dissociation of the micellar core. This can be achieved by
cross-linking the micelles at the core, which has proven to be an
effective method in the past.24-26 Cross-linking of the PPCL
core was carried out using bifunctional tetraethylene glycol
bis(azide) via copper-catalyzed azide-alkyne cycloaddition
(CuAAC) click chemistry reaction, which has been previously
used for the preparation of block copolymers or polyesters with
various functional groups.69,70
The presence of terminal alkyne group and azide group is
necessary for the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddi-
tion reaction to take place. Cu(I) acts as a catalyst for the
reaction. Cu(I) is prepared in situ by the addition of Cu(II)
CuSO4, and ascorbic acid, which acts as a reducing agent and
reduces Cu(II) to Cu(I).38 Preparation of cross-linked micelles
by this method has several major advantages to those previously
reported: The reaction is known to be carried out under ambient
conditions and is highly specific. The Cu(I), ascorbic acid, and
unreacted azide can be removed by dialysis after completion of
the reaction. Finally, this method provides means for the
introduction of cross-links at desired density through potentially
degradable ester bonds to the poly(ester) core of micelles
ensuring ultimate removal of the micellar carrier from the
biological system upon degradation.
The size and morphology of the micelles were studied by DLS
and TEM (Figure 3). The average diameter for micelles was
shown to decrease from 97.9 ( 0.6 nm for non-cross-linked
micelles to 82.6 ( 0.3 nm for cross-linked micelles. The micellar
population showed a similar distribution in both cases (PI =
0.43). The decrease in size can be a result of the packing at the
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Figure 3. TEM picture of (A) non-cross-linked micelles and (B) cross-linked micelles (magnification 18 000ꢀ). Particle size distribution of (C) non-
cross-linked micelles and (D) cross-linked micelles by DLS in water and in acetone.
Figure 5. IR spectrum of (A) cross-linked micelles and (B) non-cross-
linked micelles.
photophysical properties. This property is used to measure the
CMC of block copolymers. A sharp increase in the intensity ratio
of peaks at 338 nm to those at 333 nm from the excitation spectra
of pyrene indicates the onset of micellization. Using this method,
the average CMC of PEO114-b-PPCL19 block copolymer was
Figure 4. 1H NMR spectrum and peak assignments of (A) non-cross-
found to be 0.305 ( 0.025 μM. This value was found to be higher
linked micelles and (B) cross-linked micelles in CDCl3.
than that obtained for PEO114-b-PBCL19 (0.182 μM; previously
carried out in our laboratory).52 The increase in the CMC of
fact that the dialysis was not carried out in the presence of 0.02 M
ethylenediaminetetraacetic acid disodium (EDTA).74
PEO114-b-PPCL19 compared with PEO114-b-PBCL19 is attribu-
ted to the lower hydrophobicity of the core-forming block in
PEO-b-PPCL. With regards to cross-linked micelles, the concept
of CMC is not applicable because they have a covalently attached
micellar structure, and hence the CMC assessment for cross-
linked micelles was not carried out.73
The CMC of PEO-b-PPCL block copolymer was determined
by fluorescence spectroscopy using pyrene as the fluorescent
probe. Pyrene is a strong hydrophobic probe with very low
water solubility. Because of its hydrophobicity, it preferentially
partitions into the hydrophobic domain of the micellar core
at concentrations above CMC, resulting in a change in its
Protein Adsorption on Micelles. Interaction with serum
proteins is one of the factors that influences the fate of drug
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Figure 7. In vitro release profile of physically encapsulated PTX from
different micellar formulations in phosphate buffer (pH 7.4) containing
2 M sodium salicylate at 37 °C. Each point represents mean ( SD
(n = 3).
non-cross-linked micelles in terms of preventing protein adsorp-
tion and further opsonization in the biological system.
Preparation and Characterization of Polymeric Micelles
Containing Physically Encapsulated PTX. A maximum PTX
solubility of 20.99 μg/mL was achieved with non-cross-linked
PEO-b-PPCL micelles (Table 1). The PTX encapsulation for the
PEO-b-PPCL micelles was lower than that obtained by PEO-b-
PCL micelles,53 which may be due to an increase in the rigidity of
the core with propargyl side chain in PEO-b-PPCL micelles, the
presence of shorter hydrophobic backbone in PEO-b-PPCL
micelles studied here, or both.
For PTX loading in cross-linked micelles, better solubility was
achieved when the cross-linking agent was added in DMF contain-
ing polymer and drug (method I, PTX solubility of 21.48 μg/mL)
as opposed to the method in which cross-linking agent was added
to micellar solution of drug in water (method II, PTX solubility of
1.66 μg/mL). This may be due to leaking out of the PTX from the
micellar core during the cross-linking step after the preparation of
non-cross-linked micelles. Drug loading was expected to decrease
in core-cross-linked micelles as a result of a reduction in the free
volume of the micellar core; however, this was not the case for the
cross-linked micelles prepared by method I.
The results of assessments on the in vitro release of PTX from
non-cross-linked and cross-linked micelles and free PTX in
phosphate buffer (pH 7.4, 0.01 M) containing 2 M sodium
salicylate at 37 °C is illustrated in Figure 7. The maximum
concentration of PTX in the medium was 0.4 μg/mL, whereas
the solubility of PTX in 2 M sodium salicylate medium was 333.1
μg/mL,60 and thus sink conditions were respected in the release
study condition. Free PTX was released from the dialysis bag at a
rapid rate, which means that the transfer of PTX through dialysis
membrane to buffer solution is not a restricting factor and the
release of PTX from the micellar formulation is the rate-limiting
step in the process. Both non-cross-linked and cross-linked
micelles showed much slower release profiles when compared
with free PTX. Owing to a decrease in free volume in the micellar
core as a result of cross-linking, we expected to see a slower
release of PTX from the core-cross-linked micelles. In reality;
however, the release profile of PTX at concentrations above the
CMC of polymers was similar for both structures. Similar release
Figure 6. (A) Gel permeation chromotagram of (a) BSA solution, (b)
cross-linked micelles, and (c) mixture of cross-linked micelles and BSA
after incubation at 37 °C for 4 h. (B) Protein adsorption of cross-linked
and non-cross-linked micelles (μg BSA/mg of micelles) (n = 3).
delivery vehicles like liposomes,77 nanoparticles,78 and micelles56
in the body. The amount of protein adsorbed on the surface of the
non-cross-linked and cross-linked micelles after incubation in a
BSA solution prepared at physiological concentration was assessed
according to a previously published method using gel permeation
chromatography for the separation of BSA adsorbed micelles from
free BSA, followed by Bio-Rad protein assay on the eluted sample
containing BSA adsorbed micelles.56,77 As shown in Figure 6A,
cross-linked micelleswerefound toelute fromthecolumn at7-10
min. Non-cross-linked micelles were found to have a similar
elution profile as cross-linked micelles (data not shown). The
elution time of BSA was found to be 10-12 min. After incubation
for 4 h with BSA, the elution peak for cross-linked and non-cross-
linked micelles remained the same. The protein binding values for
cross-linked and non-cross-linked micelles were 12.6 ( 0.7 and
19.1 ( 1.4 μg BSA/mg of micelles (n = 3), respectively. Both
cross-linked and non-cross-linked micelles showed insignificant
adsorption of BSA, suggesting that the hydrophilic PEO block
provides sufficient coverage of the hydrophobic core of the
micelles.56 Cross-linking of the micelles, however, was shown to
have caused a significant decrease (p < 0.05) in the adsorption of
proteins on the micellar surface. This can be due to the factthatthe
core of cross-linked micelles is in a fixed and more compact state
when compared with non-cross-linked micelles. This may result in
a higher density and extension of the PEO chains in the micellar
shell, leading to a better steric effect by the hydrophilic shell in the
case of cross-linked micelles. Also, the cross-linking block
(tetrathylene glycol) is hydrophilic in nature, which might be
decreasing the hydrophobicity of the core/shell interface region,
leading to less protein adsorption. These results imply
better in vivo stability of cross-linked micelles as compared with
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Table 1. Characteristics of PTX-Loaded Copolymer Micelles When DMF Was Used As the Solvent for Micellization (n = 3)
PTX loading content (%) ( SD
encapsulation
average diameter (nm)
(empty)
average diameter (nm)
(PTX loaded)
PTX released after 72
h (%)b
PTX/polymer PTX/polymer
micelles
efficiency (%)
PDIa
(mol %)
(wt%)
non-cross-linked 9.37 ( 0.13
cross-linked 9.60 ( 0.12
0.92 ( 0.01
0.94 ( 0.01
18.34 ( 0.25
18.80 ( 0.23
57.1 ( 0.6
57.5 ( 0.5
56.9 ( 0.3
56.4 ( 0.4
0.41
0.38
75.17 ( 4.43
72.20 ( 2.01
a Polydispersity index of micellar size distribution. b Release study was performed in phosphate buffer (pH 7.4) containing 2 M sodium salicylate.
profiles of PTX between non-cross-linked and cross-linked
micelles were also seen in studies carried out previously by Kissel
and group.29 This could be due to the fact that although the core
is stabilized by core-cross-linking, the drug is easily diffusible
from the micellar structure. The observation may also imply
the localization of PTX in core/shell interface rather than the
micellar core in micellar structure. Further investigations are
needed to define the possible reason behind this observation.
Even without a difference in release, the stabilization of micelles
by cross-linking is expected to lead to lower rate and extent of
drug release in vivo because dissociation of micellar structure is
now prevented by cross-linking. This hypothesis is under further
investigation in our research group through in vivo studies
comparing the pharmacokinetics of PTX formulated using
cross-linked and non-cross-linked micelles.
Studentship and the J.N. Tata Trust Scholarship. The authors
thank Alberta Centre for Surface Engineering and Science
(ACSES), University of Alberta for assistance with XPS analysis,
and Advanced Microscopy Facility (AMF), University of Alberta
for assistance with TEM analysis.
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Diblock copolymers of PEO and terminal alkyne bearing R-
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’ ASSOCIATED CONTENT
Supporting Information. 13C NMR and XPS data. This
S
b
material is available free of charge via the Internet at http://pubs.
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