Article

pubs.acs.org/Macromolecules

Biodegradable Glycopolymeric Micelles Obtained by RAFT-

controlled Radical Ring-Opening Polymerization
Sylvia Ganda,† Yanyan Jiang,† Donald S. Thomas,‡ Jeaniffer Eliezar,† and Martina H. Stenzel*,†
†
Centre for Advanced Macromolecular Design, School of Chemistry, ‡NMR Facility, Mark Wainwright Analytical Centre, The
University of New South Wales, UNSW, Sydney, NSW 2052, Australia
*
S Supporting Information
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ABSTRACT: The design and synthesis of an entirely degradable glycopolymer micelle was presented. This design relies on the
utilization of RAFT-controlled radical ring-opening polymerization (rROP) technique to afford multiple insertions of cleavable
ester linkages onto the backbone of the corona. RAFT polymerization using a macroRAFT agent based on poly(ε-caprolactone)
PCL was employed to control the polymerization of well-defined statistical glycopolymers of 1-O-acryloyl-2,3:4,5-di-O-
isopropylidene-β-D-fructopyranose (1-O-AiPrFru) and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) monomer. Three block
copolymers were synthesized to generate poly(ε-caprolactone)-b-poly[(1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyr-
anose)-co-(5,6-benzo-2-methylene-1,3-dioxepane)] (PCL-b-P[(1-O-AiPrFru)-co-(BMDO)]) with varying block lengths. Self-
assembly of the deprotected block copolymers generated nonspherical egg shaped micelles, where the shorter chains PCL106-b-
P[(1-O-AFru)69-co-(BMDO)9] underwent self-assembly forming micelles with the hydrodynamic diameter (DH) of 106 nm. The
biodegradation of these micelles were investigated via enzymatic degradation by Lipase Pseudomonas sp., indicating entirely
degradable architectures, which are no longer visible via dynamic light scattering (DLS). SEC further confirmed the appearance
of fragmented glycopolymeric units. In vitro cell proliferation assay of the micelles and their degradation products revealed no
toxicity against healthy human fibroblast HS27 and breast cancer MDA-MB-231 cell lines. The polymer concentration range
tested was up to 0.20 mg·mL−1 with the cell viabilities of ≥95%.

■ INTRODUCTION
Glycopolymers as synthetic materials bearing natural carbohy-
drate-based derivatives have been long known and explored to
deliver therapeutics in a targeted fashion. The structure of
glycopolymers that mimic natural carbohydrates in biological
systems offer myriad advantages. Carbohydrates are involved in
many of the essential biological processes related to
reproduction, inflammation, signal transmission, and biomo-
lecular recognition events such as cell differentiation and
infection. Building upon this foundation, carbohydrate-directed
targeting has been used as a classic approach in promoting
active targeting by utilizing the carbohydrate ligands that are
recognized by the carbohydrate-binding proteins (receptors)
on cell surfaces known as lectins.1 This activity induces cell−
cell interactions, mediating an increase in cellular uptake via
receptor-endocytosis.2 The prominent feature of cell recog-
nition mediated by carbohydrate-based therapeutics draws
major attention as a potential agent for targeted drug delivery.
Although a single interaction is considerably weak, the
advantages of using glycopolymers as drug carriers can be
extended through a collective binding effect, also known as
cluster glycoside ef fect caused by multivalent ligands binding with
lectins.3,4 Carbohydrate-coated nanoparticles accommodate
extended advantages by not only promoting cell recognition
as naturally occurring polysaccharide derivatives, but also in the
ability to stimulate active targeting with carbohydrate binding
proteins and transporters. A plethora of carbohydrate decorated
nanoparticles have been demonstrated to promote selective and
active targeting.5−8 Despite significant advances from synthetic
and conceptual viewpoints followed by the promising outcomes
of carbohydrate-based nanocarriers, the persisting issue of
nondegradability of the glycopolymers may cause unwanted
immune responses and toxicity, eventually preventing their
translation toward in vivo application. The biomaterials
intended for biomedical applications require definite bio-
compatibility and biodegradability.9 Previous studies reported
Received: February 4, 2016
Revised: April 25, 2016
Published: June 2, 2016

© 2016 American Chemical Society 4136 DOI: 10.1021/acs.macromol.6b00266

Macromolecules 2016, 49, 4136−4146
Macromolecules
the synthesis of partially biodegradable amphiphilic glycopol-
ymer micelles comprising degradable poly(ε-caprolactone)
block and various sugar moieties focusing on the delivery and
release of doxorubicin in vitro,10 and the interaction between
Concanavalin A lectin receptor that binds with different
carbohydrate ligands.11
Ever since the pioneering work of Bailey and co-workers,12
cyclic monomers, specifically cyclic ketene acetals (CKA)s, have
attracted extensive research interests in the use of radical ring-
opening polymerization (rROP) technique to incorporate
degradable ester groups onto the polymer backbone enabling
complete degradation. This approach was suggested to be the
only pathway accessible to allow for the integration of ester
units along the aliphatic backbone of vinyl polymers in a
random order.13,14 However, alike the inherent characteristic of
free radical polymerization (FRP), rROP requires an extra layer
of control mechanism to enable the design and synthesis of
well-defined complex macromolecular architectures with
narrow molecular weight distribution. For this reason, various
methods of reversible deactivation radical polymerization
(RDRP) have been explored and implemented onto rROP to
promote controlled-living polymerization characteristics to a
library of cyclic ketene acetal monomers, such as 5,6-benzo-2-
methylene-1,3-dioxepane (BMDO). Among them include
nitroxide-mediated polymerization (NMP),15−17 atom-transfer
radical polymerization (ATRP)18−24 and reversible addition−
fragmentation chain transfer (RAFT) polymerization.20,25−30
The investigation into the homopolymerization and copoly-
merization of CDRP mediated rROP have established an
avenue in the design of new hydrolytically and enzymatically
degradable materials.
RAFT technique is widely established for its robustness and
versatility in generating a high degree of structurally functional
polymers with well-defined architecture and has been
demonstrated excessively in the literature.31 The feasibility of
RAFT to control rROP was demonstrated for the first time by
Pan et al., reporting the quantitative conversion of BMDO
resulting in aliphatic polyester with good control.26 The
copolymerization of BMDO monomer via RAFT technique
with common vinyl monomers such as methyl methacrylate,25
N,N-isopropylacrylamide,20 vinyl acetate,28 and poly(ethylene
glycol methyl ether methacrylate) (PEGMA)32 has also been
reported to successfully insert multiple hydrolytically degrad-
able ester bonds on the polymeric backbone. Xiao et al.
reported the synthesis of potentially degradable glycopolymers
of 1,2:3,4-di-O-isopropylidene-6-O-(2′-formyl-4′-vinylphenyl)-
33
D-galactopyranose and BMDO via RAFT polymerization.
In this work, we present the design of glycopolymer micelles
that are fully degradable opening opportunities to the design of
new nanoparticles for drug delivery. To our knowledge, this is
the first report of a fully degradable glycopolymer micelle
prepared with radical polymerization. The approach is based on
the use of RAFT-mediated radical ring-opening polymerization
of 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) and isopro-
pylidene protected 1-O-acryloyl-β-D-fructopyranose using poly-
(ε-caprolactone) (PCL) macroRAFT agent. The incorporation
of BMDO units into the main chain forms degradable links that
can be degraded via enzymatic degradation with enzyme lipase
Pseudomonas sp. In addition, the cytotoxicity against healthy
human fibroblast HS27 and breast cancer MDA-MB-231 cell
lines was investigated.
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■ EXPERIMENTAL SECTION
Chemicals. All chemicals were reagent grade purchased from
Sigma-Aldrich and used as received unless stated otherwise.
Deuterated NMR solvents (CDCl3, DMSO-d6, D2O and toluene-d8)
were purchased from Cambridge Isotope Laboratories. 1-O-acryloyl-
2,3:4,5-di-O-isopropylidene-β-D-fructopyranose, 5,6-benzo-2-methyl-
ene-1,3-dioxepane (BMDO)34 and poly(ε-caprolactone)35 were
synthesized as described previously with slight modifications. BMDO
and ε-caprolactone monomers were distilled under reduced pressure.
Anhydrous toluene was obtained from PureSolv MD 7 solvent
purification system (Innovative Technology, Inc., Galway, Ireland)
packed with activated alumina columns to remove water and trace
impurities.
General Procedures. Size Exclusion Chromatography (SEC).
SEC was carried out using a Shimandzu modular system containing a
DGU-12A degasser, a LC-10AT pump, a SIL-10AD automatic
injector, a CTO-10A column oven, and a RID-10A differential
refractive index detector. A PL 5.0 μm bead-size guard column (50 ×
7.5 mm2) followed by four 300 × 7.8 mm linear PL (Styragel)
columns (105, 104, 103 and 500 Å pore size) were used for the
analyses. N,N-dimethylacetamide [DMAC, HPLC grade; 0.03% w/v
LiBr, 0.05% 2,6-dibutyl-4-methylphenol (BHT)] with a flow rate of 1
mL·min−1 was used as the mobile phase with an injection volume of 50
μL at 50 °C. The unit calibration was conducted over commercially
available narrow molecular weight distribution polystyrene standards
(0.5−1000 kDa, Polymer Laboratories). Chromatograms were
processed using Cirrus 2.0 software (Polymer Laboratories).
Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR was
performed using either a Bruker Avance III 300, 5 mm BBFO probe
(1H: 300.17 MHz, 13C: 75.48 MHz) or an Avance III 400, 5 mm
BBFO probe (1H: 400.13 MHz, 13C: 100.62 MHz). NMR spectra
were processed using either the Bruker TOPSPIN 3.2 software or
MesRetNova NMR software. Samples were analyzed in either CDCl3,
DMSO-d6 or D2O, except for the NMR in situ kinetics experiments
where toluene-d8 was used as the polymerization solvent and lock
material. All chemical shifts are stated in parts per million ppm (δ)
relative to tetramethylsilane (δ = 0 ppm), referenced to the chemical
shifts of residual solvent resonances (1H and 13C). Data is reported as
follows: chemical shift [multiplicity (s = singlet, d = doublet, dd =
doublet of doublets, t = triplet, m = multiplet), integration is reported
as multiples of protons, proton identity, J (denotes the coupling
constants and is measured in Hertz)].
Dynamic Light Scattering (DLS). These measurements employed a
Malvern Zetasier Nano ZS instrument equipped with a 4 mV He−Ne
laser operating at λ = 632 nm and noninvasive backscatter detection at
173°. Measurements were carried out in a disposable cuvette at 25 °C,
provided 15 equilibration period prior to each set of measurements.
For a given sample, a total of five measurements were conducted with
the number of runs, attenuator, and path length being automatically
adjusted by the instrument, depending on the sample quality.
Transmission Electron Microscopy (TEM). This was carried out on
a JEOL 1400 TEM with the beam voltage of 100 kV and a Gatan CCD
for acquisition of digital image. Samples were prepared by depositing 1
drop of the solution mixture onto a copper grid. The grids were air-
dried and negatively stained with uranyl acetate (UA) solution for 5
min.
Cell Culture. MDA-MB-231 (breast cancer) and HS27 (human
fibroblast) cell lines were grown as monolayer cultures in cell culture
flasks by using RPMI-1640 media supplemented with 10% fetal bovine
serum (FBS), 1% sodium pyruvate and 1% of L-Glutamine-Penicillin-
Streptomycin solution (with 200 mM L-glutamine, 10,000 units
penicillin and 10 mg·mL−1 streptomycin in 0.9% NaCl, sterile filtered).
Penicillin was added as an antibiotic. Cell cultures were grown in a
humidified atmosphere at 5% CO2 at 37 °C. The medium was
routinely changed every 3 days. For cell subculture, cells grown in
monolayer were released by washing with phosphate buffered saline
(PBS) and detached by trypsin/EDTA treatment after they have
reached confluence. The cells were then collected, centrifuged and
resuspended in the new culture medium.
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Cytotoxicity Test and SRB Assay. The cytotoxicity of glycopolymer
micelles and their degradation products were determined by a standard
sulforhodamine B colorimetric proliferation assay (SRB assay). After
incubation with the enzyme, the degradation products were heated to
85 °C for 15 min in order to deactivate the enzyme. MDA-MB-231
(human breast carcinoma) and HS27 (human fibroblast non-
cancerous) cell lines were seeded at the corresponding density of
4,000 cells per well in 96-well microtiter plates followed by the
addition 100 μL of RPMI-1640 culture medium per well and incubated
at 37 °C in a 5% CO2 for 24 h. The specimens were sterilized by UV
irradiation for 20 min before serially diluting (sequential 2 × dilution)
with sterile water. For the cytotoxicity assay, the medium in the cell
culture plate was discarded, and 100 μL of fresh 2 × concentrated
RPMI-1640 medium was added to each well of the plate. The micelles
and degradation products were added correspondingly into the plates
at 100 μL per well. Sterile water was added to the nontreated cells as a
control. The cells were incubated with the micelles for 48 h, and the
cell viability was determined using SRB assay. The incubation with
micelles was terminated by the addition of cold trichloroacetic acid
(TCA) (10% w/v) for 30 min at 4 °C. After a complete washing with
distilled water (5 times), the TCA-fixed cells were stained with 100 μL
of 0.4% w/v sulforhodamine B (SRB) solution in 1% acetic acid (w/v)
for 15 min. After staining, unbound dye was removed by washing with
1% acetic acid for five times, and plates were air-dried. Finally, the SRB
was solubilized with 200 μL of 10 mM unbuffered Tris base to dissolve
bounded dye, and the optical density was determined by using a
multiwell scanning spectrophotometer at the wavelength of 490 nm.
Dose−response curves were plotted accordingly where the values were
expressed as percentage of control (nontreated cells were used as
controls). The optical density was used to calculate cell viability.
OD490,sample − OD490,blank
cell viability (%) = × 100
OD490,control − OD490,blank
Synthesis. Protection of D-Fructose. Synthesis of 2,3:4,5-Di-O-
isopropylidene-β-D-fructopyranose.36 Dry and finely powdered D-
fructose (5.0 g, 27.75 mmol) was added to a cooled mixture of
concentrated sulfuric acid (5.0 mL) and acetone (100.0 mL) in an
Erlenmeyer flask. The suspended mixture was left stirring on a
magnetic stirring plate at room temperature until all the sugar had
dissolved. The solution was left sitting at room temperature for an
additional 80 min before cooling with ice. Following, an ice-cold
solution of 2.75 M NaOH (15.3 g, 1.1 equiv) was added gradually
upon stirring. The crude product was a crystalline solid, isolated via
recrystallization by dissolving in boiling ether, cooling and adding
hexane to give the final product of rosette of needles. 1H NMR
(300.17 MHz, DMSO-d6) δ (ppm): 5.06 (t, 1H, J = 5.8 Hz), 4.56 (dd,
1H, H-4, 3J4,3 = 2.5 Hz), 4.27 (d, 1H, H-3, 3J3,4 = 2.5 Hz), 4.21 (dd,
1H, H-5), 3.73 (dd, 1H, H-2, 2J2,2′ = 4.9 Hz), 3.53 (dd, 1H, H-2′, 2J2′,2
= 4.5 Hz), 3.39 (m, 2H, H-6), 1.44, 1.34, 1.27 (4s, 12H, 4 × CH3).
Glycosylation of Protected D-Fructose Derivative. Synthesis of 1-
O-Acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (1-O-
AiPrFru). First, 3.0 g (11.53 mmol) of 2,3:4,5-di-O-isopropylidene-β-
D-fructopyranose and 3.23 mL of triethylamine (Et3N, 23.05 mmol)
were added to a three-neck round-bottom flask equipped with a
magnetic stirrer bar and dissolved in anhydrous dichloromethane
(270.0 mL) under argon atmosphere and cooled in an ice bath.
Acryloyl chloride (1.44 mL, 5.85 mmol) dissolved in 90.0 mL of dry
DCM was added dropwise with stirring under argon flow. The
solution was allowed to warm to room temperature overnight. The
reaction mixture was poured into ice-cold water and extracted three
times with DCM (270.0 mL) and dried over Na2SO4. The solvent was
removed by rotovapping to give a sticky orange residue, which was
purified by silica gel column chromatography by using a mixture of
ethyl acetate:n-hexane = 1:2 v/v % as the eluent to obtain maximum
separation. The product fractions were combined and solvent removed
under reduced pressure to yield pure sticky clear gel glycomonomer.
1
H NMR (300.17 MHz, CDCl3), δ (ppm): 6.51 (dd, 1H, 1, 2J1, 1′ = 1.4
Hz), 6.19 (dd, 1H, 3, 3J3, 1,1′ = 10.4 Hz), 5.88 (dd, 1H, 2, 2J1′,1 = 1.4
Hz), 4.64 (dd, 1H, 6, J = 7.9, 3J6,5 = 2.6 Hz), 4.53 (dd, 1H, 8, 2J8,8′ =
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11.8 Hz), 4.38 (dd, 1H, 5, 3J5,6 = 2.6 Hz), 4.27 (m, 1H, 7), 4.16 (dd,
1H, 8′, 2J8′,8 = 11.8 Hz), 3.87 (m, 1H, 4), 1.57, 1.51, 1.41, 1.37 (4s,
12H, 4 × 9) (Figure S1, Supporting Information).
Synthesis of 5,6-Benzo-2-(bromomethyl)-1,3-dioxepane.34 A 10.0
g sample of o-benzene dimethanol (72.4 mmol, 1.0 equiv) and 12.3 g
bromodimethyl acetaldehyde (72.4 mmol, 1.0 equiv) were mixed in a
predried nitrogen flask equipped with a Claisen bridge under argon
atmosphere. Then, 0.16 g of p-toluenesulfonic acid (0.93 mmol) was
added, and the mixture was heated to 120 °C for 18 h. Following, the
product obtained was of dry solid and was left to cool down. The
crude solid was dissolved in 60.0 mL of CHCl3 and washed with 30.0
mL of saturated NaHCO3− solution and water. The excess water was
removed by drying over MgSO4. The solution was then filtered and
solvent removed by evaporation under reduced pressure. The product
obtained was recrystallized from 0.1 L cyclohexane to yield 14.5 g
(82%). 1H NMR (300.17 MHz, CDCl3), δ (ppm): 7.16−7.24 (m,
4H), 5.12 (t, 1H, 3J = 5.12 Hz), 4.93 (d, 4H, 3J = 1.8 Hz), 3.46−3.44
(d, 2H, 3J = 5.2 Hz).
Synthesis of 5,6-Benzo-2-methylene-1,3-dioxepane (BMDO).34
First, 14.47 g of 5,6-benzo-2-(bromomethyl)-1,3-dioxepane (59.54
mmol, 1 equiv) was dissolved in 100.0 mL of tert-butanol in a predried
round-bottom Schlenk flask under nitrogen. Then, 6.83 g of potassium
tert-butylate (60.86 mmol, 1.0 equiv) was added and the solution was
heated to reflux for 18 h. The reaction mixture was left for cooling
before the addition of 100.0 mL of Et2O to establish separation and
filtered through vacuum filtration. The solvents were then evaporated
with the resulting oil distilled at reduced pressure (2.6 mbar) at 107 °C
to yield colorless liquid which solidified to form white crystal solids on
standing. 1H NMR (400.13 MHz, toluene-d8), δ (ppm): 3.92 (s, 2H,
B1), 4.76 (s, 4H, B2) 6.57−6.59 (m, 2H, Ar), 6.91−6.93 (m, 2H, Ar)
(Figure S2, Supporting Information).
Synthesis of Poly(ε-caprolactone) Macro-RAFT Agent.35 Poly(ε-
caprolactone) macro-RAFT agent was synthesized by adopting a
procedure previously conducted by Bourissou et al. with slight
modifications.
The monomer ε-caprolactone was predistilled prior to reaction and
anhydrous toluene was obtained from a MBraun solvent purification
system packed with aluminum oxide reactor filter column. 0.933 g of ε-
caprolactone (8.18 mmol, 80.0 equiv) was added into a Schlenk flask
equipped with a magnetic stirrer bar that was previously flame-dried
under vacuum. Benzyl 2-hydroxyethyl carbonotrithioate (BHCT)37
RAFT agent (0.102 mmol, 1 equiv) as the initiator and anhydrous
toluene were then added to the flask under inert atmosphere and
mixed. The solvent was then removed under reduced pressure via
rotary evaporator and was repeated for three times until all the solvent
was removed. Subsequently, methanesulfonic acid (0.307 mmol, 3
equiv) was added to the flask under N2 flow to catalyze the reaction,
followed by the addition of anhydrous toluene (0.5 M). The reaction
flask was then immersed into a preheated oil bath at 30 °C and left to
react under N2 atmosphere for 4 h. Finally, the reaction was quenched
by the addition of N,N-diisopropylethylamine in excess.
Synthesis of Poly[(1-O-AiPrFru)-co-(BMDO)] via RAFT Polymer-
ization. Below is provided a typical procedure for the statistical radical
ring-opening RAFT polymerization of 1-O-acryloyl-2,3:4,5-di-O-
isopropylidene-β-D-fructopyranose (1-O-AiPrFru) monomer with
5,6-benzo-2-methylene-1,3-dioxepane (BMDO) monomer. This gen-
eral procedure was employed for a series of statistical glycopolymer
syntheses by varying the monomer feed ratios.
1-O-Acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (1-O-
AiPrFru) (0.307 g, 0.976 mmol), benzyl 2-hydroxyethyl carbon-
otrithioate RAFT agent (BHCT, 0.057 g, 0.00610 mmol), and 1,1′-
azobis(cyclohexanocarbonitrile) (VAZO88, 0.3 × 10−3 g, 1.22 × 10−3
mmol) were added into a Schlenk flask that was previously flame-
dried, equipped with a magnetic stirrer bar under N2 flow. The flask
was transferred into the glovebox and 5,6-benzo-2-methylene-1,3-
dioxepane (BMDO, 0.0396 g, 0.244 mmol) was added, followed by
the addition of 0.60 mL of anhydrous anisole to give [1-O-AiPrFru]0:
[BMDO]0:[BHCT]:[VAZO88] = 80:20:1.0:0.1 with the total
concentration of 2.0 M. The flask was then sealed with a glass
stopper and deoxygenated by 3 cycles of freeze−pump−thaw until all
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Macromolecules
Scheme 1. Synthetic Pathway of the Degradable Glycopolymers, i.e., Poly(ε-caprolactone)-b-poly[(1-O-acryloyl-β-D-
fructopyranose)-co-(5,6-benzo-2-methylene-1,3-dioxepane)] (PCL-b-P[1-O-AFru-co-BMDO]), Followed by Self-Assembly to
Degradable Micelles
the dissolved gas was removed. The flask was then immersed in an oil
bath preheated to 90 °C and left to react for 24 h. The polymerization
was stopped by immersing the tube into an ice bath and introduced to
air. The crude sample was diluted in CHCl3 and precipitated for 3
times into chilled methanol and isolated by centrifugation before
drying in vacuo to obtain pure statistical glycopolymer.
Real-Time 1H and 1H−13C HSQC NMR Monitoring of RAFT
Statistical Copolymerization Kinetics. All the kinetic experiments
including 1H and 1H−13C HSQC NMR spectra respectively were
recorded on a Bruker Avance III 400 MHz spectrometer equipped
with an ultrashielded magnet and BBFO probe. Tetramethylsilane
(TMS) was used as internal standard. 1H NMR data were acquired
with 8 scans with a relaxation delay of 10 s. Meanwhile, 2D NMR data
were acquired with 1 scan and 2048 points in t2, and the number of
increments for t1 was 128. Below is a typical procedure for the online
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monitoring of RAFT statistical copolymerization kinetics via NMR
spectroscopy.
The statistical copolymerization was performed under a similar
procedure as described previously, unless for the substitution of
anhydrous anisole as the polymerization solvent to toluene-d8. Upon
the addition of BMDO monomer into the reaction mixture in the
glovebox, the reaction mixture was transferred into a J-Young NMR
tube and sealed under N2 and taken out of the glovebox. The solution
was deoxygenated via 3 cycles of freeze−pump−thaw. The NMR tube
was then subjected to in situ online NMR kinetics experiment carried
out on a Bruker Avance III 400 MHz spectrometer equipped with an
ultrashielded magnet and BBFO probe. Tetramethylsilane (TMS) was
used as internal standard. The magnet was preheated to 90 °C before
the tube was injected and the reaction started. Polymerization kinetics
were monitored and quantified by comparing the integral ratio of
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Macromolecules
Figure 1. 1H NMR spectra (400.13 MHz, toluene-d8) of the online in situ kinetics experiment of RAFT statistical copolymerization of 1-O-AiPrFru
and BMDO monomers with [1-O-AiPrFru]0:[BMDO]0:[CTA]:[VAZO88] = 65:35:1:0.1 at 90 °C for 15.4 h.
polymeric to monomeric peaks accordingly with respect to time. The
polymerization was quenched by immersing the tube into an ice bath
and exposing it to air. The crude sample was purified and
glycopolymer isolated according to the similar procedure described
previously.
Synthesis of Block Copolymer Poly(ε-caprolactone)-block-poly-
[(1-O-AiPrFru)-co-(BMDO)]. 1-O-Acryloyl-2,3:4,5-di-O-isopropylidene-
β-D-fructopyranose (1-O-AiPrFru) (0.307 g, 0.976 mmol), poly(ε-
caprolactone) (PCL)106, MnTheo = 12 084 Da (0.057 g, 0.00610 mmol)
as macro-RAFT agent, and 1,1′-azobis(cyclohexanocarbonitrile)
(VAZO88, 0.3 × 10−3 g, 1.22 × 10−3 mmol) were added into a
previously flame-dried Schlenk flask equipped with a magnetic stirrer
bar. The Schlenk flask used was previously evacuated and refilled with
N2 gas for 3 cycles on the Schlenk line upon drying by flaming with a
carbon monoxide flame torch. Following, the flask was evacuated
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before transferring it into the glovebox. 5,6-Benzo-2-methylene-1,3-
dioxepane (BMDO, 0.0396 g, 0.244 mmol) was added into the flask
upon the addition of 0.600 mL of anhydrous Anisole to give a[1-O-
AiPrFru]0:[BMDO]0 [macroRAFT]:[VAZO88] = 80:20:1.0:0.1 and
[M] = 2.0 M. The tube was sealed with a glass stopper and
deoxygenated by 3 cycles of freeze−pump−thaw until all the dissolved
gas was removed. The flask was then immersed in an oil bath
preheated to 90 °C for 24 h. Following, the flask was immersed into an
ice bath and exposed to air in order to stop the polymerization. The
crude sample was precipitated for 3 times into chilled methanol and
isolated by centrifugation before drying in vacuo to obtain pure block
copolymer subjected toward characterization.
Removal of Isopropylidene Protecting Groups. The removal of the
isopropylidene protecting groups from poly(ε-caprolactone)-b-poly-
[(1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose)-co-
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Figure 2. Plot of (a) monomer conversion and (b) total monomer conversion vs time for copolymerization of 1-O-AiPrFru and BMDO at different
feed ratios [1-O-AiPrFru]0:[BMDO]0 = 80:20 (◆) and 65:35 (○) monitored via in situ NMR experiments for 19 h at 90 °C. (c) SEC traces
(DMAC eluent, cal. to PMMA standard) of a series of statistical glycopolymers synthesized via RAFT polymerization with varying comonomer feed
ratios, i.e. [1-O-AiPrFru]0:[BMDO]0 = [100]:[0] (black line); [80]:[20] (blue line); [75]:[25] (green line); [65]:[35] (red line).
(5,6-benzo-2-methylene-1,3-dioxepane)] block copolymer is described
below. This deprotection approach was applied to all the other
glycopolymers bearing the isopropylidene protected fructose carbohy-
drate pendants. A typical deprotection for glycopolymers synthesized
via ring-opening RAFT copolymerization is described as following.
The 0.080 g of glycopolymer obtained from previous copolymeriza-
tion(s) was dissolved into 1.50 mL of 9:1% v/v TFA/H2O in a vial
equipped with a magnetic stirrer bar. The mixture was left stirring at
room temperature for 30 min. Dialysis was then performed on the
glycopolymer solution against deionized water for 48 h through a
cellulose membrane MWCO 12,000. The deprotected glycopolymer
was then lyophilized to yield a colorless low density solid.
Self-Assembly of Block Copolymers. First, 4.0 mg of poly(ε-
caprolactone)-b-poly[(1-O-acryloyl-β- D -fructopyranose)-co-(5,6-
benzo-2-methylene-1,3-dioxepane)] was added to a glass vial equipped
with a magnetic stirrer bar. 2.0 mL of DMSO was added into the vial
and mixed until all the glycopolymer was dissolved. The vial was
capped with a rubber septum and placed in a water bath heated to 20
°C. The temperature was maintained constant throughout the entire
period of micellization. 2.0 mL of DI water was added dropwise via a
syringe pump with the flow rate of 0.20 mL/h with the solution left
stirring for 10 h. The micellar solution was then dialyzed against DI
water for 2 days to remove the organic phase. The size of micelles
formed was measured by DLS measurement to obtain the hydro-
dynamic volume (DH), followed by TEM to investigate the shape and
size of the micelle in the dry state.
Enzymatic Degradation of Nanoparticles. In a typical degradation
experiment, 2.0 mg of lipase Pseudomonas sp. (≥22 units/mg solid)
was dissolved in 1.0 mL of 0.10 M phosphate buffer solution prepared
at pH 7.0. 0.50 mL of the enzyme solution was added into 0.50 mL of
micellar solution (0.60 mg·mL−1) and incubated at 37 °C for 23 h
(unless stated otherwise). Enzymatic degradation of the micelles was
observed by DLS of the solution. Online in situ DLS measurements
were also carried out to follow the disintegration of micelles by
continuously measuring for 90 min (1 min per measurement with no
delay between measurements) at 37 °C. Finally, the solution was
lyophilized and degradation products isolated by extraction with
DMAC and analyzed by size exclusion chromatography.
4141
Article
■ RESULTS AND DISCUSSION
Polymer Synthesis. The multiple insertion of main chain
ester backbones was introduced by RAFT-controlled radical
ring-opening polymerization of BMDO monomer to yield well-
defined macromolecular architecture (Scheme 1). The CKA
comonomer, i.e., 5,6-benzo-2-methylene-1,3-dioxepane
(BMDO) is comprised of a 7-membered ring, an acetal and
vinyl functional group, and was prepared following a prescribed
procedure previously conducted by Landfester and co-work-
ers34 with slight modifications. Dehydration reaction from
ortho-benzene dimethanol by bromodimethyl acetaldehyde
under inert atmosphere yielded the bromide derivative 5,6-
benzo-2-(bromomethyl)-1,3-dioxepane as a crystalline solid
upon recrystallization from cyclohexane at 82% yield. BMDO
monomer was subsequently synthesized via reduction reaction
of this analogue under dry and inert atmosphere. This is a
critical step due to BMDO monomer being highly susceptible
to hydrolysis by protonation in the presence of acid and water
molecules. Precipitation in diethyl ether gave the desired
product as suspended oil, thus requiring monomer purification
by distillation under reduced pressure to obtain BMDO
monomer as white crystalline solid at 85% purity. The final
product was characterized by 1H and 13C NMR spectroscopy
(Figure S2−S3, Supporting Information).
In order to examine the reactivity of both comonomers and
the versatility of RAFT polymerization in controlling rROP, a
series of statistical copolymerizations with varying comonomer
feed ratios was carried out. It is noteworthy that the acetal
framework of BMDO monomer is highly electrophilic, thus
making it extremely susceptible toward hydrolysis via
nucleophilic attack, leading to protonation of the vinyl bond
generating an undesired byproduct with a ring-opened structure
that can no longer be polymerized.13,25,38 Shown in Figure 1 is
an overlay of a series of 1H NMR spectra acquired from the
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Macromolecules 2016, 49, 4136−4146
Macromolecules Article

Table 1. Synthesis of Statistical Glycopolymers Based on 1-O-AiPrFru and BMDO Monomers via RAFT Polymerization and the
Solubility of the Resulting Copolymers at a Monomer Concentration of 1.0 mg·mL−1 a
monomer feed SECb solubility
entry [1-O-AiPrFru]c
[BMDO] d
Mn (Da) Đ water DMSO phosphate buffer
1 100 0 9700 1.21 yes yes yes
2 80 20 6800 1.14 yes yes yes
3 75 25 10 300 1.35 yes yes yes
4 65 35 8000 1.14 yes yes yes
a
Each polymerization was carried out at 90 °C for 24 h, in the presence of VAZO88 as the initiator and BHCT CTA; [M]0:[BHCT]:[VAZO88] =
[100]:[1]:[0.1]. bSEC analyses were obtained by using DMAc as the mobile phase, calibrated to PMMA standard. cConversion is >90%;
d
Conversion is ∼40%

Figure 3. TEM images of (a) the self-assembly of statistical glycopolymers P(1-O-AFru)50-co-P(BMDO)3 forming nanoparticles with the mean
diameter of 85 nm. (b) Higher concentration solution formed aggregation at 1.0 mg·mL−1.

statistical copolymerization with the monomer feed ratio of [1-
O-AiPrFru]0:[BMDO]0 = [65]:[35]. Each polymerization was
conducted under dry and inert environment in the presence of
1,1′-azobis(cyclohexanecarbonitrile) (VAZO88) as the initiator
and benzyl 2-hydroxyethyl carbonotrithioate (BHCT) as the
chain transfer agent (CTA). A detailed study of the
copolymerization kinetics was performed and monitored via
in situ NMR experiment where toluene-d8 was used as the
reaction solvent and to lock the NMR signal. NMR spectra
were recorded for 19 h via 1H and 2D 1H−13C HSQC NMR
spectroscopy. A gradual decrease in both the monomer
concentrations is evident from the loss of intensity of the
characteristic peaks of the monomers, i.e., the acrylate peaks
(CH2CH) of 1-O-AiPrFru monomer labeled “F1”, “F2”, and
“F3” at δ = 6.3−5.3 ppm, followed by the vinyl peak of BMDO
monomer labeled “B1” (CH2) at δ = 3.7 ppm (Figure
1(bottom)). The radical ring-opening polymerization of
BMDO monomer was clearly observed from the decrease of
the vinyl bond signal (CH2) at δ = 3.7 ppm (labeled “B1”),
followed by the appearance of the broad polymer peak in the
range of δ = 5.06−5.4 ppm labeled “4” (Figure 1 (top)) in the
spectra. The conversion of BMDO monomer throughout the
polymerization was monitored and quantified based on the
integration of the vinyl bond (“B1”).
The conversion for both monomers over 19 h was recorded
for feed ratios of f BMDO = 20 mol % and f BMDO = 35 mol %
(Figure 2a), respectively, revealing the significantly slower
consumption of BMDO. The amount of BMDO in the initial
feed did not show a major impact on the total monomer
conversion and polymerization rate (Figure 2b) with 1-O-
AiPrFru reaching almost complete conversion while the
conversion of BMDO levels off at 40%. It seems that the
increasing concentration of BMDO increases the rate of
polymerization. However, this might solely be the results of
various factors such as impurities as the BMDO monomer is
4142
susceptible to side reactions. A number of different studies in
the literature have also reported similar findings supporting this
outcome where reversible chain-transfer catalyzed polymer-
ization (RTCP) techniques19,20 and free radical polymer-
ization39,40 were employed. Four polymers with BMDO feed
ratio from f BMDO = 0 to 35 mol % were isolated after 24 h and
purified. The resulting glycopolymers exhibit narrow disper-
sities (Đ) within 1.14−1.35 (Table 1). It is important to note
that the presence of traces of water can result in the hydrolysis
of BMDO monomer, generating byproducts with the structure
depicted in Figure S7(a), Supporting Information. Therefore,
the incorporation of ester units on copolymer backbone was
further confirmed by the integration of the 2 protons peak
adjacent to the ester functional group at δ = 5.06−5.4 ppm
(“4”).
The resulting polymers were subsequently deprotected, and
the removal of the isopropylidene groups was confirmed by
NMR. The resulting copolymers were tested in regards to their
water-solubility as the hydrophobic BMDO may lower the
hydrophilicity of the glycopolymer. The solubility of these
glycopolymers was therefore tested using three different polar
solvents, such as pure water, dimethyl sulfoxide (DMSO) and
phosphate buffer solution, depicting good solubility with up to
35 mol % of BMDO in the monomer feed (Table 1, entry 4).
Despite the apparent water-solubility, the hydrophobic BMDO
clearly introduced amphiphilicity to the polymer as it is evident
from the TEM images as shown in Figure 3, parts a and b, using
poly[(1-O-AFru)50-co-(BMDO)3] (Table 1, entry 3). This
behavior suggested that the glycopolymers formed reorganized
and underwent aggregation to form dense hydrophobic
domains surrounded by a corona of swollen loops formed by
the hydrophilic parts of the copolymer.
Synthesis of Block Copolymers and Self-Assembly.
Although the statistical copolymers were already capable of
nanoparticle formation, the combination with a hydrophobic
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Macromolecules 2016, 49, 4136−4146
Macromolecules
polymer block that will enable the self-assembly into core−shell
structures will ensure potentially high drug loading capacity for
a hydrophobic drug. Following the statistical copolymerization
of 1-O-AiPrFru and BMDO monomer, which displayed good
control under RAFT conditions, a series of amphiphilic well-
defined PCLx-b-P[(1-O-AiPrFru)y-co-(BMDO)z] block copoly-
mers were synthesized. The block copolymers with varying
chain lengths and comonomers compositions were synthesized
via chain extension of PCL macro-RAFT agent with two
different number of repeating units (DPn = 72, MnObs. = 8.50
kDa, Mn,SEC = 5.0 kDa, Đ = 1.48 and DPn = 106, MnObs. = 12.3
kDa, Mn,SEC = 17.0 kDa, Đ = 1.12). PCL was synthesized via
organo-catalyzed ring-opening polymerization of ε-caprolac-
tone, initiated by alcohol functionalized BHCT RAFT agent in
the presence of methanesulfonic acid as the catalyst. The block
copolymerization was carried out under similar condition to the
previously conducted statistical copolymerization of 1-O-
AiPrFru and BMDO monomer with the substitution of
BHCT RAFT agent to PCL macro-RAFT agent. The
incorporation of BMDO comonomer in the feed ( f BMDO)
varied between 0 and 20 mol %.
The molecular structure and 1H NMR of a representative
block copolymer sample is shown in Figure 4 before and after
Figure 4. 1H NMR spectra (300.17 MHz) of (a) poly(ε-
caprolactone)-b-poly[(1-O-AiPrFru)-co-(BMDO)] (in CDCl3) and
(b) poly(ε-caprolactone)-b-poly[(1-O-AFru)-co-(5,6-benzo-2-methyl-
ene-1,3-dioxepane)] (in DMSO-d6) after deprotection of the sugar
moieties showing complete disappearance of the isopropylidene
protecting groups.
deprotection of the sugar moieties. The incorporation of ester
backbone obtained from a successful radical ring-opening
polymerization is marked by the appearance of the peak at δ =
5.0−5.3 ppm, a characteristic peak of the two protons adjacent
to the ester bond (“4”) on the glycopolymer backbone. A
summary of the physical characterization and size measure-
ments is presented in Table 2. SEC (Figure 5) depicts a clear
shift in molecular weight of the PCL macro-RAFT agent to the
higher molecular weight region, indicating successful chain
extension forming block copolymers. The dispersities (Đ) of
4143
Article
these block copolymers lie within 1.18−1.40, exhibiting a good
control over molecular weight distribution by RAFT polymer-
ization. The higher dispersity value block copolymers PCL106-b-
P[(1-O-AiPrFru)69-co-(BMDO)9] (Đ = 1.29) indicated the
presence of a minor branching on the backbone due to
backbiting leading to the formation of side chains.18 This is also
evident from the SEC chromatogram displaying monomodal
molecular weight distribution with a higher molecular weight
shoulder depicted as dashed line in Figure 5. The deprotection
of block copolymers was performed under acidic environment
in order to remove the isopropylidene protecting groups prior
to self-assembly forming micelles. The elimination of the
protecting groups is noticeably marked by the disappearance of
the isopropylidene peaks between δ = 1.2−1.6 ppm,
accompanied by a change in solubility. It should be noted
here that SEC analysis of the deprotected block copolymers is
not available due to the disparate solubility of both blocks.
NMR analysis however suggests that the BMDO esters were
not cleaved during hydrolysis in acidic conditions, indicated by
the presence of the peak at δ = 5.0−5.3 ppm which belongs to
the 2 protons adjacent to the ester bond corresponding to the
aliphatic polyester structure.
It is widely established that well-defined amphiphilic block
copolymers are capable of undergoing self-assembly forming
nanoparticles with various morphologies in aqueous solution as
a consequence to the unfavorable interaction between the
hydrophobic segments and the surrounding aqueous environ-
ment. According to the protocol introduced by Eisenberg and
co-workers, the micelles were prepared by initially dissolving
the block copolymer in a suitable organic solvent. Micellization
was induced with the dropwise addition of deionized water.41,42
The sizes and morphologies of micelles prepared from different
block copolymers (Table 2) were determined by transmission
electron microscopy (TEM), indicating ellipsoidal (egg) shaped
micelles for M-1 with an average size of 60 × 143 nm (W × H)
(Figure 6a). Meanwhile, the micelles formed by M-2 provided a
mixture of both ellipsoidal and spherical micelles with an
average width and height of 69 and 125 nm (W × H) (Figure
6b) consecutively. In comparison, M-3 micelles are greater in
size at 86 × 232 nm as a result of the longer hydrophilic
segment and were observed to be unstable in aqueous solution,
which is expected for micelles with such length ratio (Figure S8,
Supporting Information). The high crystallinity of PCL as the
core-forming block led to the formation of egg-shaped micelles
with a clear core−shell structure due to crystallization.43−46
Dynamic light scattering (DLS) provides a supporting evidence
of the hydrodynamic diameter (DH) of the micelles in aqueous
solution to be approximately 134 nm (by intensity) for M-1,
106 nm for M-2, and 269 nm for M-3. A first indication of the
stability of M-2 micelles was depicted by the enduring
hydrodynamic diameter as recorded by DLS after isolation
and redissolving in aqueous solution. The sizes observed by
TEM and DLS are in similar orders of magnitude, but they
cannot be directly compared due to the nonspherical shape.
Enzymatic Degradation. Randomly distributed ester
bonds were incorporated into the shell-forming block of the
micelles to induce biodegradability. Intracellular lysosomal
enzymes are known to promote hydrolysis toward hydrolyti-
cally labile polymeric backbones within the cells.47,48 Numerous
studies have been carried out to demonstrate and mimic the
biodegradation of these polymers, such as polyesters,
polyanhydrides,49 polypeptides50 etc. via in vitro and in vivo
pathways. Bearing this in mind, we designed our micelles to be
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Macromolecules 2016, 49, 4136−4146
Macromolecules
Table 2. Characterization and Analysis of a Series of Block Copolymers of Three Different Chain Lengths PCLx-b-P[(1-O-
AiPrFru)y-co-(BMDO)z] and the Micelle Sizes and Morphologies Formed by Self-Assembly in DMSO Followed by the
Dropwise Addition of Water
NMRa
sample block copolymer MnTheo. (Da)
M-1 PCL72-b-P(1-O-AiPrFru)88 31 700
M-2 PCL106-b-P[(1-O-AiPrFru)69-co-(BMDO)9] 32 400
M-3 PCL106-b-P[(1-O-AiPrFru)150-co-(BMDO)24] 60 500
a
Theoretical molecular weight based on monomer conversion (1H NMR, CDCl3). bSEC analyses were obtained by using THF as the mobile phase
(calibrated with PMMA standards). cThe micelles were formed after deprotection of the isopropylidene protecting groups forming PCLx-b-P[(1-O-
AFru)y-co-(BMDO)z]. dThe hydrodynamic volume (DH) of the nanoparticles as measured by dynamic light scattering (DLS) measurement based on
intensity mean. eTEM data obtained by measuring the sizes of negatively stained nanoparticles recorded as width × height.
Figure 5. SEC chromatograms of the chain extension of poly(ε-
caprolactone) macro-RAFT agent with 1-O-AiPrFru and BMDO
monomers via RAFT polymerization in anhydrous anisole at 90 °C for
24 h. [1-O-AiPrFru] 0 :[BMDO] 0 :[PCL]:[VAZO88] = (- - -)
80:20:1:0.1, MnTheo.= 32 400 Da and (···) 160:40:1:0.1, MnTheo. = 60
500 Da (THF eluent, calibrated to PMMA standard).
biologically reducible generating the known degradation
products of PCL and short glycopolymer strands that should
be approximately 6−8 repeating units in length. To evaluate the
dissociation of the micelles under physiological conditions, two
different micellar solutions based on PCL106-b-P[(1-O-AFru)69-
co-(BMDO)9] (M-2) and PCL72-b-P(1-O-AFru)88 (M-1) block
copolymers, respectively, were prepared and subjected to
enzymatic degradation in the presence of commercially
available enzyme Lipase from Pseudomonas sp. The average
concentration of serum lipase found in healthy adults lies in the
range 30−190 units·L−1.51
In a typical degradation experiment, 500 μL of the enzyme
solution (2.0 mg·mL−1 in 0.10 M PBS buffer, pH 7.0) was
added to 500 μL of micelle solution (0.70 mg·mL−1) and
incubated at 37 °C for 23 h. The degradation period was
extended up to almost 3 days for M-1 micelles due to the slow
degradation rate. The enzymatic degradation profile of the
micelles was monitored via online in situ DLS measurement
(Figure 7). Figure 7a reveals complete degradation of the entire
structure of the micelles comprising BMDO units (M-2). This
is noticeably evident from the disappearance of the original
micelle peak at 73 nm after 1 h, leaving only the residual
enzyme peak at 9 nm. It was observed that the micelles were
fully degraded by lipase Pseudomonas sp. enzyme within the first
10 min, indicated by the disappearance of the micelle signal at
73 nm. The chains reorganized forming fragments with
hydrodynamic diameters at 18 nm (4 min, −·) and 8 nm (6
min, −··) and 2 nm (10 min, red line) (Figure S9(a,b),
Supporting Information). As a control experiment, enzymatic
Article
SECb DLSc TEMc
Mn (Da) Đ DHd (nm) PdI sizee (nm) morph.
16 400 1.18 134 0.178 60 × 143 ellipsoidal
16 000 1.29 106 0.085 69 × 125 ellipsoidal and spherical
14 000 1.38 269 0.160 86 × 232 ellipsoidal
degradation was also performed on micelles bearing only
glycopolymers in the corona, but no BMDO (M-1). As
represented in Figure 7b, it was observed that the absence of
degradable ester linkages of BMDO units in the micelle shell
led to rate retardation and the inability to undergo complete
degradation. During the first 23.5 h, no degradation was
observed as the hydrodynamic diameter remained around 100
nm.
A previous study conducted by Wu et al. reported the
biodegradation of poly(ethylene oxide)-b-poly(ε-caprolactone)
(PEO-b-PCL) core−shell polymeric nanoparticles with an
essentially nondegradable corona forming block.52 In this study,
it was observed that the biodegradation of the micelles is mainly
determined by the enzyme concentration, commencing in a
one-by-one fashion. Other studies reporting similar behavior
can also be found in the literature.53,54 The mechanism was
suggested to involve adsorption of enzymes onto the core of
the nanoparticles in order for enzymatic hydrolysis of the PCL
chains to take place.55−57 Upon isolation of the degradation
products of M-2, SEC chromatograms reveal the disappearance
of the original glycopolymer peak at 16 000 Da (Figure S11,
Supporting Information) and the generation of lower molecular
weight products detected at 1200 Da. Further characterization
of the degradation products was not pursued in this study,
however previous studies reported the generation of alcohols
and carboxylic acids as byproducts of ester group hydrolysis.20
Therefore, the incorporation of ester backbones in the design of
glycopolymeric micelles has been shown to provide an
enhanced and complete biodegradation profile into fragments
of free chains that are no longer detected via dynamic light
scattering method and confirmed by SEC.
Cytotoxicity. Cytotoxicity studies were performed to
investigate the biocompatibility of the micelles M-1 and M-2
and their degradation products, M-1 degraded and M-2
degraded, (after incubation with enzymes for 2 d 22 h and
23 h respectively) against MDA-MB-231 (breast cancer) and
HS27 (healthy human fibroblast) cell lines (Figure 8).58 The in
vitro cell proliferation assay revealed no toxicity of the micelles
and degradation products in both media over the concentration
range up to 0.20 mg·mL−1 with the cell viabilities of ≥95%.
However, the presence of enzyme that was still active in the
solution containing degradation products used for in vitro
studies caused a significant decrease in cell viability at a high
polymer concentration (0.20 mg·mL−1) due to cytotoxic effects
(Figure S13, Supporting Information). Therefore, the enzy-
matic degradation solution was heated to 85 °C for 15 min after
the incubation period as a heat-deactivation method to ensure
no cytotoxic effects caused by the enzyme.
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Macromolecules 2016, 49, 4136−4146
Macromolecules Article

Figure 6. TEM images of (a) PCL72-b-P(1-O-AFru)88 block copolymers forming ellipsoidal shaped micelles and of (c) PCL106-b-P[(1-O-AFru)69-co-
(BMDO)9] block copolymers forming a mixture of spherical and ellipsoidal shaped micelles. DLS histogram of (b) PCL72-b-P(1-O-AFru)88 and (d)
PCL106-b-P[(1-O-AFru)69-co-(BMDO)9] block copolymers micelles indicating the hydrodynamic volume (DH) of micelles in aqueous solution based
on intensity mean.

Figure 7. Enzymatic degradation of the micelles of (a) PCL106-b-P[(1-O-AFru)69-co-(BMDO)9] and (b) PCL72-b-P(1-O-AFru)88 block copolymers
in number-based hydrodynamic diameter (DH) of the micelles recorded via in situ DLS measurement during enzymatic degradation. Blank micelle:
Micelle solution before enzyme addition; t0: after addition of enzyme.

Figure 8. Cytotoxicity studies of glycopolymeric micelles M-1 and M-2 and their degradation products after incubation with lipase Pseudomonas sp.
(M-1 degraded and M-2 degraded) using SRB assays against (a) HS27 and (b) MDA-MB-231 cell lines at different concentrations after 48 h
incubation period (values expressed as the percentage of nontreated cells as controls).

■ CONCLUSION
By utilizing RAFT to control radical ring-opening polymer-
biodegradable drug delivery platform that can be tailored to
achieve a high degree of main chain scission by enzymatic
attack in a short period of time. The feasibility of the RAFT
ization, we have demonstrated the synthesis of a completely process to control the polymerization was depicted from the
4145 DOI: 10.1021/acs.macromol.6b00266
Macromolecules 2016, 49, 4136−4146
Macromolecules
formation of well-defined and controlled statistical glycopol-
ymers and block copolymers as proven by NMR spectroscopy
and SEC with narrow molecular weight distributions. The ratio
of BMDO incorporation was able to be finely tuned to attain
different degrees of cleavable ester bonds while maintaining the
amphiphilic nature of the final block copolymers and integrity
of the micelles product of self-assembly. We have also
confirmed the biocompatibility of the materials and degradation
products to be nontoxic via in vitro cell proliferation assay
against healthy human fibroblast (HS27) and breast cancer
(MDA-MB-231) cell lines.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.6b00266.
NMR spectra, TEM image of the micelles and DLS and
SEC of the degradation profile (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*(M.H.S.) E-mail: M.Stenzel@unsw.edu.au.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors would like to acknowledge Dr. Krzysztof P.
Babiuch for the initial help regarding the monomer syntheses
and preliminary studies.
■
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DOI: 10.1021/acs.macromol.6b00266
Macromolecules 2016, 49, 4136−4146