Macromolecular

Chemistry and Physics Full Paper

Oxidant-Dependent REDOX Responsiveness

of Polysulfides

Paolo Carampin, Enrique Lallana, Jureerat Laliturai, Sabrina C. Carroccio,

Concetto Puglisi, Nicola Tirelli*

Poly(propylene sulfide) (PPS) is studied as an oxidation-responsive macromolecular building

block, in the perspective of anti-inflammatory therapies. Here, we show that the nature of
the oxidant has profound effects on the outcome of the oxidation process. PPS was exposed
to hydrogen peroxide (H2O2) and hypochlorite (OCl−), which are oxidizing species commonly
encountered during inflammatory processes. It was found
that the oxidation with H2O2 converted thioethers into sulfox-
ides, producing water-soluble macromolecular products with
extremely low toxicity (L929 fibroblasts); on the contrary, the
reaction with NaOCl produced sulfones in addition to sulfox-
ides, and this was accompanied by depolymerization, which
appears to considerably affect the toxicity of the oxidation
products.

1. Introduction radical (O2•−) from oxygen and nicotinamide adenine

An ubiquitous feature of inflamed tissues is the presence
of a number of phagocyte-generated oxidants, which are
generally produced from molecular oxygen through a cas-
cade of one-electron reduction steps.[1,2] Most commonly,
this cascade starts with the generation of superoxide anion
P. Carampin, J. Laliturai[a]
School of Pharmacy and Pharmaceutical Sciences,
University of Manchester, Oxford Road, Manchester,
M13 9PT, United Kingdom
E. Lallana, N. Tirelli
School of Materials, University of Manchester,
Oxford Road, Manchester, M13 9PT, United Kingdom
E-mail: nicola.tirelli@manchester.ac.uk
S. C. Carroccio, C. Puglisi
Istituto di Chimica e Tecnologia dei Polimeri,
UOS Catania, Consiglio Nazionale delle Ricerche,
Via P. Gaifami 18, 95126 Catania, Italy
N. Tirelli
School of Biomedicine, University of Manchester,
Oxford Road, Manchester, M13 9PT, United Kingdom
[a] The Government Pharmaceutical Organization, 75/1 Rama
6 Road, Ratchathewi, Bangkok 10400, Thailand
dinucleotide phosphate (NADPH) in a reaction catalyzed
by NADPH oxidases, which are membrane-bound enzy-
matic systems typically dormant in resting phagocytes,
but activated by a variety of external and internal stimuli,
for example, bacteria and soluble factors, such as certain
inflammatory polypeptides (e.g., C5a).[3] The released O2•−
can either react with nitric oxide (NO) to form highly reac-
tive peroxynitrite (ONOO−)[4] or alternatively can be rapidly
converted into hydrogen peroxide (H2O2) under the influ-
ence of superoxide dismutase. In the presence of Fe2+, H2O2
can also produce the more damaging hydroxyl radical (•OH)
through Fenton chemistry.[5] Moreover, REDOX cycling of
Fe2+ and Fe3+ through a combination of Haber–Weiss and
Fenton chemistry can also result in the direct formation of
•
OH from O2•−.[6] The heme peroxidases such as myeloper-
oxidase and eosinophil peroxidase, secreted by inflam-
matory cells, can also catalyze the formation of the very
potent and damaging oxidants hypochlorous acid (HOCl)
and hypobromous acid (HOBr) from H2O2 in the presence of
chloride (Cl−) and bromide (Br−), respectively. On the other
hand, oxidants generated as a result of the activation of
inflammatory cells can lead to further activation processes,
either directly or through the mediation of oxidized bio-
molecules.[7] For example, the exposure of Immunoglobin

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Oxidant-Dependent REDOX Responsiveness of Polysulfides . . .
G (IgG) to •OH or HOCl in the inflamed rheumatoid joint
has been claimed to generate an aggregated IgG form that
increases neutrophil activation,[8] while complement acti-
vation is reportedly increased by the action of oxygen radi-
cals.[9] In addition, in many forms of tissue injury, including
mechanical disruption, the extent of free-radical reactions
can be increased by inactivation of cellular antioxidants, as
well as by the release of transition metal ions from their
sites of sequestration within cells.[10]
The above summary illustrates the role of oxidant spe-
cies in inflammatory processes, which is the rationale for
the use of REDOX-responsive drug delivery systems sensi-
tive to inflammation-triggered oxidation reactions.[11] In
this regard, we have focused our attention on a class of
organic polymers, poly(1,2-alkylene sulfides), where the
conversion of sulphur (II) to higher oxidation states (sul-
foxides or sulfones) results in a large change in polarity
and, consequently, in an increase of their water solu-
bility/swellability.[12–15] The resulting morphological reor-
ganization can trigger the release of encapsulated active
principles in an oxidative (inflamed) environment. This
scheme can be applied to intra- or extracellular oxidation,
depending on a number of additional factors, for instance
the recognition of the carrier and its cellular uptake.[16]
Polysulfide preparative chemistry is overwhelm-
ingly based on the living anionic polymerization of
episulfides,[17] which allows the preparation of narrow
dispersity macromolecules with an excellent control
over terminal groups,[18] possibly featuring a blocky
structure.[19–21] Episulfide polymerization can be car-
ried out under mild conditions, and can be performed in
protic media,[22] provided that disulfide impurities are
removed.[23,24] This versatility has allowed to produce a
variety of oxidation-sensitive colloidal carriers based on
poly(propylene sulfide) (PPS): vesicles[13] and micelles,[25]
which are based on the self-assembly in water of
amphiphilic block copolymer structures, and nanoparti-
cles,[14,26] which are produced through in situ emulsion
polymerization techniques. Upon exposure to H2O2, the
gradual oxidation of PPS transforms vesicles into higher
curvature structures, such as worm-like micelles, spher-
ical micelles, unimolecular micelles, eventually yielding
soluble polymers.[27,28] Cross-linked PPS nanoparticles
respond to H2O2 through a bulk oxidation mechanism,
with the oxidant diffusion into the nanoparticles occur-
ring more rapidly than their oxidation; this determines
a gradual and homogeneous swelling of the polysulfide
network in water, allowing the corresponding release of
encapsulated drugs such as doxorubicin.[29,30] PPS nano-
particles also released hydrophobic payloads such as Nile
red and Reichardt’s dye after oxidation with NaOCl.[15]
We have further demonstrated that PEGylated
PPS nanoparticles exhibit a substantially “stealth”
behavior (very slow endocytic uptake) in the presence of
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nonactivated macrophages, and can extensively circulate
in rats,[31] although this behavior may strongly depend on
the nature of the chemical groups present on the surface
of the nanoparticle.[32]
Comparatively little has been done to understand
the chemistry of the oxidation process, and specifically
whether the action of different oxidants may result not
only in different oxidation kinetics but also in different
reaction products. Few literature reports have focused
on the oxidation behavior of organic polysulfides,[33–38]
showing that they can be oxidized in vitro to polysul-
foxides,[33,37] although an earlier report from Marvel and
Weil hinted to polysulfones too.[39] It is conceivable that
under mild oxidation conditions (e.g., H2O2 as an oxidizing
agent) the reaction could generate lesser oxidized species,
that is, sulfoxides, while using harsher conditions (e.g.,
oxidation with organic peroxyacids) polymeric sulfones
can be obtained.[34,36,38,40] However, there are conflicting
evidences: for example, Orgeretravanat and Galin[37]
reported that the oxidation of PPS with meta-chloroper-
oxybenzoic acid produced poly(propylene sulfoxide) and
not sulfones; surprisingly, this was accompanied by a
severe depolymerization of the substrate, which would be
more typical of a sulfone-containing structure.[37] Addi-
tionally, the effects of other biological oxidants, such as
ClO−, or of heterogeneous conditions still remain to be
addressed.
The knowledge of the oxidized products is also impor-
tant: sulfoxide-containing polymers were initially
studied by the group of Ringsdorf in the 1970s with
very promising results in terms of extremely low in vivo
toxicity.[41,42] On the other hand, low-molecular-weight
depolymerization products and/or sulfone-containing
substrates may be considerably more toxic.
In the present work, PPS has been synthesized from a
dithiolate initiator to form linear bifunctional polymer
chains. The polymer was then oxidized under different
physicochemical environments using H2O2 and NaOCl,
which are typical markers of inflammatory reactions
but are also characterized by different solubilities in an
organic environment.
2. Experimental Section
2.1. Materials
All chemicals were used as received unless otherwise stated. Pro-
pylene sulfide (PS), acetyl chloride, ethyl bromoacetate, 2,2′-(ethy-
lenedioxy)diethanethiol, Live/Dead Cell Double Staining Kit,
NaOCl aqueous solution (available chlorine 10%–15%), tributy-
lphosphine (TBP), fungizone, dichloromethane, triethylamine
(TEA), and Dulbecco’s Modified Eagle’s Medium (DMEM) with
high glucose (4500 mg L−1) were purchased from Sigma–Aldrich
(Gillingham, UK). Ethyl acetate (AcOEt), tetrahydrofuran (THF),
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Chemistry and Physics
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and 30% (w/w) H2O2 in water were purchased from Fisher
(Leicester, UK). 0.5 M sodium methoxide (NaOMe), methanolic
solution, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were pur-
chased from Fluka (Gillingham, UK). Phosphate-buffered saline
(PBS) Dulbecco A tablets were purchased from Oxoid (Hampshire,
UK). CellTiter 96 AQueous One Solution Cell Proliferation Assay
was purchased from Promega (Southampton, UK). Foetal bovine
serum (FBS), penicillin-streptomycin and L-glutamine 200 × 10−3M
(×100) were purchased from Invitrogen (Paisley, UK).
For all operations, THF was degassed by bubbling argon under
an inert atmosphere for 1 h before use.
2.2. Physicochemical Characterization
1H NMR spectra were recorded on a Bruker 500 MHz spectro-
meter (Bruker UK Limited, UK). NMR samples were prepared in
CDCl3 at a concentration of approximately 1 wt%. Chemical shifts
are reported in ppm (δ units) upfield using internal tetramethy-
lsilane (0.00 ppm) as a reference.
FTIR spectra were recorded in ATR mode on a Tensor 27 Bruker
spectrometer (Bruker UK Limited, UK) equipped with a 3000
Series TM High Stability Temperature Controller with RS232
Control (Specac, UK). A drop of a solution of the substrate (in THF
or water) was placed on a Golden Gate Heated Diamond ATR top
plate and allowed to completely dry at 50 °C.
Molecular weight and molecular weight distribution of
polymers were determined using a Polymer Laboratories
PL-GPC50 integrated GPC (Polymer Laboratories, UK) comprising
a PLgel 5 μm Guard and two PolyPore 5 μm columns operating at
30 °C. THF was used as an eluent at a flow rate of 1.0 mL min−1.
A series of near-mono-dispersed linear polystyrene standards
(Fluka; Gillingham, UK) was used for calibration with a refractive
index detector for the analysis of the polymers.
SLS measurements were carried out on a BI-APD Brookhaven
instrument (Brookhaven, USA) equipped with a 35 mW He–Ne
laser emitting vertically polarized light at 632.8 nm, a BI-200SM
goniometer, and a BI-9000 digital correlator scanning over the
angular range 15–155°. Completely oxidized PPS with molecular
weight around 3000 g mol−1 (2 equivalents of H2O2 for 20 h) was
dissolved in MilliQ water prefiltered through a 0.2 μm pore size
syringe filter to remove dust particles (concentration in the range
4.0–9.0 mg mL−1). Each polymer solution was filtered directly
into the light scattering cell for 30 min through an on-line
filtration system composed of a Stepdos 03S liquid pump (KNF-
Flodos, Switzerland) connected to a 0.2 μm pore size filter. SLS
measurements were performed at 25 °C mounting the sample
cell in a temperature-controlled bath filled with cis–trans
decahydronaphthalene (decalin). Under these experimental
conditions, a Zimm plot was constructed using BI-ZMP software
from which the polymer Mw and second virial coefficient (A2)
were calculated. Refractive index increments (dn/dc) were
determined at 25 °C using an ABBE refractometer (Belingham
and Stanley Limited, UK).
MALDI-TOF mass spectra were recorded in linear and
reflectron mode using a Voyager-DETM STR mass spectrometer
working in positive ion mode. The instrument was equipped
with a nitrogen laser emitting at 337 nm. The accelerating
voltage was set at 20 kV, and the grid voltage and the delay time
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P. Carampin et al.
(delayed extraction, time lag) were optimized for each sample to
obtain the highest molar mass values. The laser irradiance was
maintained slightly above threshold. Samples for MALDI-TOF
analysis (PPS and PPS oxidized with NaOCl) were prepared by
mixing 10 μL of the matrix solution [0.1 M 2-(4-hydroxyphenylazo)
benzoic acid (HABA) in THF] and the appropriate volume of 5 mg
mL−1 polymer solution in THF to obtain a 1:1 or 1:3 (v/v) (sample/
matrix) mixture. Finally, 1 μL of each sample/matrix mixture was
spotted on the sample holder and slowly dried to allow matrix
crystallization. Note: different matrices, that is HABA, dithranol,
and trans-3-indoleacrylic acid (IAA), and solvents, that is, THF
and/or THF/CHCl3, were unsuccessfully tried for the analysis of
PPS oxidized with H2O2, but the high polarity of the resulting
polymers apparently prevented their detachment.
2.3. Preparative Operations
2.3.1. Synthesis of Protected Bifunctional Initiator:
2,2′-(ethylenedioxy)diethanethioacetate
3.8 g of 2,2′-(ethylenedioxy)diethanethiol (20.8 mmol, corre-
sponding to 41.6 mmol of thiols) was dissolved in 300 mL of THF
under an inert atmosphere and cooled to 0 °C. 17.4 mL of TEA
(124.8 mmol, 3:1 molar ratio to thiols) was added under stirring.
5.9 mL of acetyl chloride (83.2 mmol, 2:1 molar ratio to thiols) was
dissolved in 30 mL of THF and added dropwise. The reaction mix-
ture was allowed to reach room temperature and stirred for 4 h.
The insoluble TEA hydrochloride was removed via filtration, and
the supernatant was concentrated under reduced pressure. The
resulting residue was dissolved in 100 mL of dichloromethane
and extracted with brine (3 × 25 mL). The organic layer was dried
over Na2SO4 and concentrated under reduced pressure to give a
crude product that was purified by column chromatography on
silica gel 230-400 mesh (hexane/AcOEt 3:1), finally recovering a
colorless oil. Yield: 3.12 g = 56% wt.
FTIR (film on ATR crystal), δ (cm−1): 2926 (νas CH2), 1687
(ν CO), 1131, 1099 (νas C—O—C), 952 (νs C—O—C).
1
H MNR (CDCl3), δ (ppm): 2.33 (s, 6, C2—C2—C2—S—
COCH3), 3.05–3.10 (t, 4,—C2—C2—S—COCH3), 3.55–3.60
(m, 8,—SCH2CH2—OCH2CH2OCH2CH2S—).
2.3.2. Synthesis of Poly(propylene sulfide)
Polymerization reactions were carried out in a FirstMate parallel
reactor (Argonaut Technologies), which was purged with argon
for 5 min prior to the introduction of reagents. The reaction ves-
sels were maintained under a positive argon pressure during
polymerization. All stock solutions employed were prepared
from solvents degassed by argon bubbling for 1 h. Following
the polymerization/end-capping procedure, PPS with molecular
weights around 3000, 6000, and 9000 g mol−1 (PPS3000, PPS6000,
and PPS9000, respectively) were prepared. In a typical procedure,
2 mL of a 2,2′-(ethylenedioxy)diethanethioacetate stock solu-
tion (133 mg mL−1 in THF, 1.0 mmol), 1.62 mL of TBP stock solu-
tion (1.25 mg mL−1 in THF, 10-fold excess of TBP per thioacetate
group), and degassed THF (5 mL) were introduced into the reactor
vessel. 2.10 equivalents of NaOMe from an aliquot of 0.5 M
NaOMe methanolic solution (4.2 mL, 2.1 mmol) were added next,
and the final mixture was stirred for 5 min at room temperature.
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Oxidant-Dependent REDOX Responsiveness of Polysulfides . . .
In order to obtain PPS with molecular weights around 3000,
6000, and 9000 g mol−1, an appropriate amount of PS (3.2, 6.3, or
12.6 mL, corresponding to 40, 80, and 120 equivalents, compared
to thioacetate groups) was added to the solution. The polymeriza-
tion mixture was stirred for 45 min at room temperature. After-
ward, 1.10 equivalents of acetic acid and 1.15 equivalents of DBU
were sequentially added to neutralize the excess of NaOMe and
adjust the pH. Finally, 3.6 equivalents of end-capping reagent
ethyl bromoacetate (0.4 mL, 3.6 mmol) were added, and the reac-
tion mixture was stirred for 2 h at room temperature. The solvent
was then evaporated under reduced pressure, and the obtained
crude product was dissolved in 30 mL of dichloromethane and
extracted with water (3 × 6 mL). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure to give a vis-
cous oil that was washed with methanol (3 × 5 mL). The polymer
was separated by decantation after centrifugation and dried
under high vacuum for 24 h at 40 °C. Yield: 65-70% wt.
End-capping yield: ≥95% (calculated by 1H NMR from the
integration of —SCH2CH2—OCH2CH2OCH2CH2S—(3.51–3.68 ppm)
and H3CH2—O(O)C—(4.05–4.26 ppm)).
FTIR (film on ATR crystal), δ (cm−1): 2959 (νas CH3), 2917 (νas
CH2), 2865 (νs CH3 and νs CH2), 1730 (ν CO ester), 1448 (δs CH2),
1372, 1263, 1221, 1172, 1103 (νas C—O—C), 943, 852 (νs C—O—C)
cm−1.
1H NMR (CDCl ), δ (ppm): 1.26–1.30 (t, 6H, CH CH —OOC—), 1.35–
3 3 2
1.45 (d, CH3 in PPS chain), 2.55–2.75 (m, PPS chain: 1 diastereotopic
CH2), 2.71–2.78 (t, 4H,—SCH2CH2OCH2CH2OCH2CH2S—), 2.85-
3.05 (m, PPS chain: CH and 1 diastereotopic CH2), 3.16-3.25
(d, 1H,—S—CH2—COO—: 1 diastereotopic CH2), 3.25-3.37
(d, 1H,—S—CH2—COO—: 1 diastereotopic CH2), 3.51–3.68
(m, 8H,—SCH2CH2—OCH2CH2OCH2CH2S—), 4.05–4.26
(q, 4H, CH3CH2—O(O)C—).
2.3.3. Oxidation Experiments
The responsiveness of PPS was studied under different physico-
chemical scenarios: H2O2 in THF, NaOCl in THF with low or high
water content, and in dichoromethane/water 1:1 mixture. Unless
otherwise stated, PPS3000 was used as a substrate. Experiments
with PPS6000 and PPS9000 were carried out under identical experi-
mental conditions as described for PPS3000.
2.3.4. Oxidation With H2O2 in THF (7.0 wt% Water
Content)
An aliquot of 30 wt% H2O2 in water (285 μL, 2.80 mmol) was
added to 3 mL of a THF solution containing 100 mg of PPS3000
(1.40 mmol of thioether groups, corresponding to a 2:1 peroxide/
thioether ratio). The mixture was stirred for 1, 5, 10, 14, 17, or
20 h at room temperature; after addition of 2 mL of deionized
water, it was dialyzed against MilliQ water (six changes during
48 h) through 1000 Da MWCO (molecular weight cut-off) dial-
ysis membranes (SpectraPor 7 regenerated cellulose tubings,
Spectrum Laboratories, Inc., USA), and finally freeze-dried. The
obtained solid was suspended in 10 mL of water, isolating the
water-soluble fraction via centrifugation at 2000 rpm for 5 min
and then freeze-drying it. The water-insoluble fraction was dried
under vacuum for 24 h at 40 °C.
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2.3.5. Oxidation With NaOCl in THF (8.0-15.9 wt% Water
Content)
An aliquot of 1.88 M NaOCl in water (373, 560 or 746 μL, corre-
sponding to 0.70, 1.05, and 1.40 mmol and then to 0.5, 0.75, and
1.0 equivalents relative to thioether groups, respectively) was
added to 5 mL of a THF solution containing 100 mg of PPS3000
(1.40 mmol of thioether groups). In typical experiments, the mix-
ture was allowed to react for 20 h at 37 ºC, but experiments con-
ducted with 1 equivalent of NaOCl were also reacted for times
ranging between 2 min and 96 h. After the addition of 2 mL of
MilliQ water (precipitation observed), the mixture was dialyzed
against MilliQ water (six changes during 48 h) through 1000 Da
MWCO dialysis membranes, and freeze-dried; the water-soluble
and water-insoluble fractions were separated as described above.
2.3.6. Oxidation With NaOCl in Dichloromethane/Water
An aliquot of 1.88 M NaOCl in water (3.7 mL, 7.0 mmol,
corresponding to 5 equivalents relative to thioethers) was
added to a solution of PPS3000 (100 mg, 0.03 mmol) in a
dichloromethane/water mixture (5.0 and 1.3 mL, respec-
tively). The mixture was stirred for 0.5, 24, 48, 72, or 96 h
at 37 °C. Afterward, the dichloromethane was evaporated
and the resulting aqueous phase was freeze-dried. Finally,
both the water-soluble and water-insoluble fractions of PPS
oxidation products were separated as described above.
2.4. Cell Culture
The mouse fibroblast L929 cell line was obtained from the Euro-
pean Collection of Animal Cell Cultures (ECACC, Rockville, UK). Cells
were routinely maintained in culture medium composed of DMEM
supplemented with 10% (v/v) FBS, 2 × 10−3M L-glutamine, 1% (v/v)
Fungizone, and 0.5% (v/v) penicillin–streptomycin. Cells in sup-
plemented DMEM medium were grown as a confluent monolayer
culture on 75 cm2 polystyrene tissue culture flasks (Falcon, Oxford,
UK). Cell cultures were maintained at 37 °C in a humidified atmos-
phere of 5% CO2. The culture medium was changed three times a
week. Adherent cells approaching 90% confluence were harvested
with trypsin and subcultured. Passages were always below 6.
2.4.1. Cytotoxicity
The oxidation of MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt]) to the corresponding colored formazan (CellTiter 96 One
Solution Cell Proliferation Assay) and the fluorescence of cal-
cein acetoxymethyl ester (calcein-AM) and propidium iodide (as
markers of live and dead cells, respectively; Live/Dead Cell Double
Staining Kit) was used to determine the cytotoxicity of PPS oxi-
dation products on L929 mouse fibroblasts. Cells in culture media
were seeded and incubated in a 96 well plate at 37 °C in a humid-
ified atmosphere of 5% CO2 at a density of 8000 cells/well. After
24 h, the culture medium was replaced with a solution of fresh
medium containing varying concentrations of the oxidation
products of linear PPS or PPS nanoparticles, which was previously
filtered through a sterile 0.22 μm filter. After 24 h, the incubation
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medium was removed, the cells were rinsed

twice with PBS at pH 7.4, and their viability
was analyzed. For the MTS assay, a mixture
containing MTS and culture medium without
FBS and phenol red was added into each well.
After 3 h of incubation, the absorbance was
read on a microplate reader (μQuant, Biotek,
UK) at 490 nm. The relative cell viability
(%) was calculated by the formula [A]test/
[A]control∗100, where [A]test is the absorbance Figure 1. Preparation of PPS by in situ deprotection of the dithioacetate precursor, ani-
of the test sample, and [A]control is the absorb- onic ring-opening episulfide polymerization, and end-capping of the resulting bifunc-
ance of the control cells incubated solely with tional PPS.
culture medium. For the live/dead assay, cells
were simultaneously incubated with calcein-AM and propidium this precursor in the presence of a reducing agent (TBP) allows
iodide in PBS (pH 7.4) for 10 min at 37 °C, washed three times to minimize the presence of disulfides, which behave as
with PBS (pH 7.4), and immediately analyzed for green (live cells) chain transfer agents in episulfide polymerization. The addi-
and red (dead cells) fluorescence using a fluorescence microscope tion of PS produced a bis(thiol)-terminated polysulfide that
(Leica DMI 6000 B, Leica Microsystem). The relative cell viability
was finally end-capped with ethyl bromoacetate following
(%) was calculated as the ratio between the number of live cells
a recently optimized protocol.[18,22] Using this procedure, PPS
(green) and the total number of cells (live and death: green and
with a targeted degree of polymerization of 40, 80, and 120
red, respectively) multiplied by 100.
(PPS3000, PPS6000, and PPS9000, respectively) were prepared, with
a 65%–70% overall yield and polydispersity values around 1.1
3. Results and Discussion (GPC and MALDI-TOF; Table 1), confirming the living and con-
trolled character of the episulfide polymerization.
3.1. Synthesis of PPS
3.2. Oxidation Behavior of PPS
PPS was synthesized from a bifunctional initiator [2,2′-(ethy-
lenedioxy)diethanethiol], which was generated in situ from Since PPS is hydrophobic, its oxidation may proceed in
its acetylated precursor (Figure 1); the in situ methanolysis of homogeneous or heterogeneous conditions depending on

Table 1. Composition and molecular weight of PPS3000 oxidized with H2O2 in THF.

Reaction time Water-insoluble fractiona) Conversion to sulfoxide Mw d) Mn d) Mw / M

n d)
[h] [wt%] [mol%] [g mol−1] [g mol−1]
1H NMRb) FTIRc)
0 100 0 0 3420 (3228)e) 3120 (3046)e) 1.09 (1.06)e)
1 100 4.1 5.8 3190 2890 1.11
5 100 7.0 6.0 3010 2700 1.12
10 100 10.4 6.3 3280 2970 1.12
14 100 55.5 40.1 1490 430 3.43
17 45 52.9f) 68.6f) 1320f) 430f) 3.06f)
77.6g) 83.0g) = = =
20 0 93.5 93.5 3800h) = =
a)The THF solution containing the oxidized polymer was transferred into a water environment and then dialyzed against water (1000
Da MWCO) and freeze-dried. The water-soluble fraction was isolated by suspending the obtained solid in 10 mL of water followed by
filtration (water-insoluble fraction) and freeze-drying of supernatant; b)Calculated from the integration of signals corresponding to the
methyl group next to sulfoxides (1.38–1.58 ppm) and the total content of the methyl groups (1.29–1.58 ppm); c)Calculated from the
height of the band at 1030 cm−1 (sulfoxide stretching). The intensity of the signals was normalized against the νas CH3 band at 2959 cm−1.
To provide absolute values, the conversion of the sample at 20 h was assumed equal to that obtained from 1H NMR (93.5%); d)Calculated
from GPC measurements in THF, unless otherwise stated; e)Calculated from MALDI-TOF measurements;[43] f)THF-soluble fraction; g)THF-
insoluble (water-dispersible) fraction. Although insoluble in THF, this material aggregated in water and most polar solvents, hindering
SLS analysis; h)Calculated from SLS measurements in water. Mw = 3780 ± 52 and 3826 ± 46 g mol−1 were obtained from the extrapola-
tions to zero angle and zero concentration, respectively. A2 = 1.14 ± 0.24 was calculated from the Zimm plot, a positive value that sug-
gests lack of aggregation in water. dn/dc = 0.14 ± 0.005 mg mL−1.

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oxidized species) and methyl resonances

(1.29–1.38 ppm for sulfides, 1.38–1.58
ppm for oxidized species) (Figure 2A to
C). It is noteworthy that the development
of a νSO band can be related to the
increasing presence of sulfinic acids or
of sulfoxides, which respectively imply
or not depolymerization reactions. How-
ever, no significant change was recorded
below 1000 cm−1, which would exclude
the presence of sulfinic acids (νS—O). At
the same time, no signal possibly related
to sulfone (or sulfonic) groups could be
recorded in FTIR spectra.
Both NMR and FTIR revealed the
formation of oxidized sulphur species
already after 1 h (Table 1 and Figure 2A
to C), with an initially slow kinetics and
a significant acceleration after 10 h
(Figure 3), to yield an almost quantita-
tive conversion after 20 h (Figure 2C,
disappearance of the initial methyl
Figure 2. Top left (A): FTIR spectra of oxidized PPS3000 in THF treated with H2O2 (2:1 molar
ratio with sulfides). Top right (B): 900-1400 cm−1 window of the same spectra, showing
resonance).
the dramatic increase in absorbance of the SO stretching vibration. Arrows indicate After 14 h of reaction all samples were
variations in diagnostic bands. The intensity of all spectra was normalized against the opaque, with significant flocculation
νas CH3 band at 2959 cm−1. Bottom left (C):1H NMR spectra of PPS3000 in THF treated at later stages. Correspondingly, GPC in
with H2O2 (2:1 molar ratio with sulfides) as a function of the reaction time. The broader THF showed a markedly higher reten-
appearance of the resonances associated to the oxidized polymers is an indication of the
aggregation of the high dipole moment sulfoxides also in CDCl3. Bottom right (D): GPC
tion with increasing oxidation, which
traces in THF for PPS3000 treated with H2O2 (2:1 molar ratio with sulfides) as a function could be ascribed to coil contraction due
of time. The shift to longer retention times is particularly evident for the broad peaks to poorer solvation or depolymerization
recorded after 14 and 17 h of oxidation. This phenomenon is likely related to the poor (Figure 2D). MALDI-TOF analysis was not
expansion of the sulfoxide-containing polymer coils in THF. possible, due to poor desorption of the
polymers from the matrix, but the fully
the polarity of the oxidant. We have studied PPS oxidation oxidized product (20 h) presented a Mw = 3800 g mol−1 in
using two model oxidants, H2O2 and NaOCl: on one hand, water (SLS, Table 1; consider the increase in mass due to
they are among the most common reactive oxygen species
in inflammation processes[6]; on the other hand, due to
their different polarity and charge, they allow to study the
reactions, respectively, under homogeneous and heteroge-
neous conditions.

3.2.1. Oxidation With H2O2 (Homogeneous Oxidation)

THF was chosen as one of the few organic solvents where
both PPS and H2O2 are soluble and it is also easy to remove
via evaporation. The experiments were carried out at room
temperature after addition of H2O2 ([H2O2] ≈ 3 wt%, 2:1
molar ratio with sulfides; the medium contained about 7
wt% of water). The progress of the reaction was monitored
at different time intervals by FTIR (Figure 2A and B) and
1
H NMR (Figure 2C), respectively using the SO stretching
vibration band at 1030 cm−1 and a vibration at 1170 cm−1 Figure 3. Conversion of sulfide to sulfoxide (expressed as mol%
(possibly related to the sulfide group), and methylene and of the total sulphur atoms) in PPS3000 oxidized in THF with H2O2
methine (2.54–2.96 ppm for sulfides, 2.58–3.49 ppm for (2:1 molar ratio with sulfides).

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in a hydrophobic environment (THF,

water content ranging between 8.0 and
15.9 wt%), where it is likely present
in a dispersed state with a high local
concentration, (2) in a biphasic system
(dichloromethane/water 1:1), where the
oxidation should predominantly occur
interfacially.
(1) NaOCl dispersed in a hydrophobic
environment. No phase separation was
recorded during oxidation, while a sig-
nificant fraction of the polymer could
be precipitated at the end of reaction
by addition of water; after dialysis,
the amount of water-insoluble mate-
rial decreased with increasing amount
Figure 4 . Cell viability of L929 fibroblasts after exposure for 24 h to variable concentra-
tions of water-soluble oxidized PPS3000 with H2O2 (2:1 molar ratio with sulfides) for 20 h. of NaOCl (55% of the original weight
for 0.5 equivalents of NaOCl; 39 wt%
for 1 equivalent of NaOCl/20 h); on the
the introduction of oxygen atoms), while no appreciable other hand, no polymeric substance could be revealed by
mass loss was recorded upon dialysis (1000 Da MWCO) SLS analysis of the water phase, nor any material could
and the presence of possibly terminal groups in the form be recovered from it after freeze-drying, suggesting that
of sulfinic or sulfonic acids was already excluded on the the remaining mass of oxidized PPS3000 was converted
basis of the FTIR results. We are therefore inclined to to volatile products. In the water-insoluble phase, oxi-
dized groups were recorded (both FTIR and 1
exclude the occurrence of any depolymerization reaction. H NMR)
The autoacceleration can therefore be explained on the already after treatment with 0.5 equivalents of NaOCl per
basis of the phase separation of oxidized PPS segments thioether (Figure 6 ) and as soon as 2–3 min after the addi-
into polar and thus water-rich domains, where the higher tion of the oxidant (Figure 1SI, Supporting Information).
local concentration of H2O2 would increase the rate of In the FTIR spectra, it was possible to detect the devel-
oxidation. opment of a νSO band at 1030 cm−1 and of two bands
The cytotoxicity of the water-soluble/dispersible PPS (1215 and 1370 cm−1) likely associated to the symmetric
oxidation products was evaluated on L929 murine fibrob-
lasts by investigating the cell viability through a live/
dead and a metabolic (MTS) assay. The two tests pro-
vided a reasonably good agreement showing no cytotoxic
effects for fully oxidized PPS3000 up to a concentration of
at least 10 mg mL−1 (Figure 4). Further, while PPS3000 and
PPS6000 presented IC50 = 33–35 mg mL−1, the larger PPS9000
did not cause any significant decrease in cell viability up
to a concentration of 50 mg mL−1 (5 wt%) (Figure 5). Prod-
ucts of partial oxidation showed somehow higher tox-
icity, for example, 80% oxidized PPS3000 (17 h oxidation)
exhibited an IC50 = 15 mg mL−1; this effect is likely due
to amphiphilicity, which may confer some membrane-
disrupting activity. However, even for the partially
oxidized products, the IC50 values are far above (>1 wt%)
than the concentrations used for carriers in drug delivery.

3.2.2. Oxidation With NaOCl (Heterogeneous Oxidation)

NaOCl is substantially insoluble in any organic environ-
ment, and may therefore be useful to study the effects of
a heterogeneous oxidation process; we have specifically
used two different conditions of dispersion: (1) dispersed
Figure 5. Cell viability (MTS assay) of L929 murine fibroblast after
exposure for 24 h to various concentrations of water-soluble oxi-
dized PPS3000, PPS6000, and PPS9000 with H2O2 (2:1 molar ratio with
sulfides) for 20 h and oxidized PPS3000 with H2O2 (2:1 molar ratio
with sulfides) for 17 h.

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 Macromolecular
Oxidant-Dependent REDOX Responsiveness of Polysulfides . . . Chemistry and Physics
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ascribed to structures (SA to SD) com-

patible with alkylene sulfone depolym-
erization because they feature terminal
double bonds and sulfinic acids. This is
not a surprising phenomenon because
aliphatic polysulfones [poly(alkenyl sul-
fones)] are well known to depolymerize
under mild conditions: the weak C—S
bond can be easily cleaved thermally[44]
or via irradiation (e.g., X-rays[45,46] or
electron beams[47]), releasing ultimately
SO2 and olefins. It is unclear whether
Figure 6. Left: FTIR spectra of PPS3000 after 20 h of reaction in low water content THF the observed reduction in molecular
mixtures as a function of NaOCl concentration. Arrows indicate variations in dia- weight can be ascribed to depolymeri-
gnostic bands. The intensity of all spectra was normalized against the νas CH3 band at
zation occurring during oxidation, or to
2959 cm−1. A plot of the time evolution of the band at 1030 cm−1 is provided in the
Supporting Information, Figure 1SI. Right: H NMR spectra of oxidized PSS3000 in CDCl3 a fragmentation during MALDI-TOF
1

corresponding to the same reaction conditions. analysis. GPC analysis in THF of the
water-insoluble phase showed the same
aggregation-related problems as in
and asymmetric stretching of an SO2 group, respectively. Figure 2D. The absence of S—O stretching vibrations from
We are inclined to exclude the presence of sulfinic or sul- FTIR spectra of oxidized material apparently excluded the
fonic acids, either in protonated or deprotonated form, presence of sulfinic acids, suggesting the second option.
due to the absence of any new band below 1000 cm−1 (cor- On the other hand, the very significant weight loss and
responding to νS—O) following oxidation. The intensity of absence of polymers in the water phase would favor the
the carbonyl band increased in comparison to that of νas first hypothesis.
CH3 of PPS chain methyl groups at 2959 cm−1, which was It can be therefore concluded that the oxidation with
used to normalize the FTIR spectra. This higher weight of ClO− in THF introduced significant amounts of sulfones
terminal groups could indicate a depolymerization ran- in the PPS chain, and this led to an overall labilization
domly occurring in the PPS chain, coher-
ently with the recorded weight loss.
The 1H NMR spectra broadly con-
firmed the presence of two different oxi-
dized groups: a peak at 1.33–1.58 ppm
could be associated to methyl groups
close to sulfoxides, while one at
1.61–1.66 ppm, due to the larger down-
field shift, could be assigned to more
polar sulfones (Figure 6, right). On the
contrary, the resonances of methylene
and methyl groups were only marginally
affected (peak broadening).
MALDI-TOF analysis showed a sharp
decrease in molecular weight with
increasing amounts of NaOCl: already
at 0.5 equivalents of ClO− (per thioether),
a majority of the material appeared to
have a molar mass lower than 1500 g Figure 7. MALDI-TOF spectra of oxidized PPS3000 as a function of NaOCl concentration
mol−1 (Figure 7). The degradation prod- after 20 h of reaction in THF/water mixtures. The peak labels in the left inset corre-
ucts still exhibited a PS-based repeating spond to compounds SA-SD (showed in the right inset) and the further oxidized products
structure (74 Da spacing) with the with 16 Da spacing peaks derived thereof (sulfoxides and/or sulfones). It is noteworthy
appearance of oxygen-containing species that structures SA-SD are non- or poorly oxidized and they do not appear to contain the
central initiator-derived segment, while the (hydrolyzed) terminal groups appear to be
(presence of peaks at +16 and +32 Da). present; this suggests that even mildly oxidized larger fragments are adsorbed irrevers-
Two sets of peaks can be recognized ibly, as previously observed for the H2O2-oxidized polymers and that only the shortest
(left inset in Figure 7), which can be fragments, that is, those between a sulfone and chain terminus, can be detected.

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components that could be isolated are

still insoluble in water. For a qualitative
comparison, nanoparticles composed of
PPS of similar molecular weight (com-
pared to PPS3000) were prepared and
cross-linked via Michael-type addi-
tion, similarly to previous reports.[31]
The nanoparticles dispersed in water
at the concentration of 4 mg mL−1 and
oxidized with 1 equivalent of NaOCl
per thioether for 20 h at ambient tem-
perature showed a significantly higher
Figure 8. Left: FTIR spectra of PPS3000 oxidized with 5 equivalents of NaOCl in dichlo- toxicity than the oxidation products of
romethane/water 1:1 at different reaction times. No significant alteration of the spec- PPS with H2O2; their IC50 value was in
trum can be recorded at reaction times > 30 min. The presence of sulfone groups cannot the range of 1 mg mL−1 (Figure 3SI, Sup-
be unequivocally confirmed. Arrows indicate variations in diagnostic bands. The inten- porting Information). It must be noted
sity of all spectra was normalized against the νas CH3 band at 2959 cm−1. Right:1H NMR
that these values are, however, not dis-
spectra of PPS3000 before and after a 30 min oxidation with 5 equivalents of NaOCl for 20
h in dichloromethane/water 1:1. In analogy to what seen in Figure 6, we have tentatively tant from those of polymers often con-
ascribed the peak appearing at 1.66 ppm to methyl groups in beta to sulfones. sidered to be rather biocompatible, such
as chitosan.[48]

of the macromolecular structure, eventually causing

depolymerization.
Note also that (a) compounds of similar structure of
SA but featuring a sulfonic acid terminal group could
be also present within SA + 16 Da mass peaks; (b) the
sulfoxide in SB is placed on the terminal sulphur atom,
under the hypothesis the vicinal oxidation may promote
the sulfone cleavage; however, the same peaks may be
attributed to SC structures cationized with potassium.
(2) NaOCl in a biphasic system. FTIR and 1H NMR spectra
(Figure 8 left and right, respectively) showed signs of oxi-
dation only with a stoichiometric excess of NaOCl. As for
the reaction in THF, all the recovered oxidation products
were soluble in organic solvents. Using a large stoichio-
metric excess of NaOCl (5 equivalents), sulfoxides were
rapidly introduced in the polymer structure (FTIR peak at
1030 cm−1, NMR resonance centered at 1.49 ppm). Smaller
amounts of other oxidized species could be detected in
the 1H NMR spectrum by the presence of a resonance cen-
tered at 1.66 ppm; although it was impossible to unequiv-
ocally identify them in the FTIR spectra, we are inclined
to recognize these groups as sulfones, in analogy to the
reaction in THF. Finally, MALDI-TOF results were virtually
indistinguishable from those of the product obtained in
THF (Figure 3SI, Supporting Information), suggesting a
similar lability of the oxidized macromolecular structure.
In summary, despite requiring significantly higher
amounts of hypochlorite and introducing more sul-
foxide than sulfone groups, the results of the oxidation
in a biphasic system were qualitatively similar to those
recorded in THF.
The toxicity of the material oxidized by NaOCl could
not be easily evaluated because the only macromolecular
4. Conclusions
The reaction of PPS with hydrogen peroxide (H2O2) in a
homogeneous THF/water environment appeared to have
an autoaccelerating character, suggesting that the local
concentration of H2O2 is a major rate-determining factor
for this oxidation. This process caused the conversion of
sulfides into sulfoxides, without significant production of
sulfones or depolymerization of the macromolecule; the
resulting polysulfoxides were soluble in water and charac-
terized by very low cytotoxicity.
Hypochlorite (ClO−) was used under two heterogeneous
conditions, likely differing in the local concentration of
the oxidant; despite some differences, in both cases, the
reaction proceeded much more rapidly than with H2O2,
and provided macromolecules with clear tendency to
depolymerization due to the presence of sulfones in addi-
tion to sulfoxides.
The different response of polysulfides to different
oxidants may allow different biomedical applications:
for example, ClO− released during neutrophile oxida-
tive bursts may lead to an instantaneous response, for
example, the release of a payload, accompanied by the
rapid degradation of polysulfides carriers to low molec-
ular weight, water-soluble products; the latter may offer
the disadvantage of higher toxicity and the advantage
of an easier elimination through kidneys. On the other
hand, the action of H2O2 may cause a sustained release of
a payload, with a longer permanence of possibly less toxic
oxidation products.
We can conclude that the responsiveness of polysulfides
is sensitive to the details of the oxidative environment

Macromol. Chem. Phys. 2012, 213, 2052−2061

2060 © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.MaterialsViews.com

Oxidant-Dependent REDOX Responsiveness of Polysulfides . . .
and this can be used to fine tune the biomedical applica-
tions of this class of materials.
Supporting Information
Supporting Information is available from the Wiley Online Library
or from the author.
Acknowledgements: Financial support from EPSRC (grant
No. EP/C543564/1 and Advanced Research Fellowship for
NT) is gratefully acknowledged. J.L. thanks The Government
Pharmaceutical Organization of Thailand for a research grant
and a PhD studentship.
Received: May 17, 2012; Revised: July 10, 2012; Published online:
August 28, 2012; DOI: 10.1002/macp.201200264
Keywords: biomaterials; depolymerization; polysulfides; redox
polymers; sulfones
[1] A. Mantovani, P. Allavena, A. Sica, F. Balkwill, Nature 2008,
454, 436.
[2] C. Cordon-Cardo, C. Prives, J. Exp. Med. 1999, 190, 1367.
[3] B. M. Babior, Blood 1999, 93, 1464.
[4] G. L. Squadrito, W. A. Pryor, Free Radical Bio. Med. 1998, 25,
392.
[5] C. J. Morris, J. R. Earl, C. W. Trenam, D. R. Blake, Int. J.
Biochem. Cell B. 1995, 27, 109.
[6] I. Fridovich, Arch. Biochem. Biophys. 1986, 247, 1.
[7] B. Halliwell, J. M. C. Gutteridge, Methods Enzymol. 1990, 186,
1.
[8] J. Lunec, D. R. Blake, S. J. McCleary, S. Brailsford, P. A. Bacon,
J. Clin. Invest. 1985, 76, 2084.
[9] K. T. Oldham, K. S. Guice, G. O. Till, P. A. Ward, Surgery 1988,
104, 272.
[10] B. Halliwell, J. M. C. Gutteridge, D. Blake, Philos. Trans. R. Soc.
A 1985, 311, 659.
[11] C. Fang, B. Shi, Y. Y. Pei, M. H. Hong, J. Wu, H. Z. Chen,
Eur. J. Pharm. Sci. 2006, 27, 27.
[12] A. Mercanzini, S. T. Reddy, D. Velluto, P. Colin, A. Maillard,
J.-C. Bensadoun, J. A. Hubbell, P. Renaud, J. ControlledRelease
2010, 145, 196.
[13] A. Napoli, M. Valentini, N. Tirelli, M. Muller, J. A. Hubbell,
Nat. Mater. 2004, 3, 183.
[14] A. Rehor, J. A. Hubbell, N. Tirelli, Langmuir 2005, 21, 411.
[15] B. L. Allen, J. D. Johnson, J. P. Walker, Acs Nano 2011, 5, 5263.
[16] N. Tirelli, Curr. Opin. Colloid Interface 2006, 11, 210.
[17] C. D. Vo, G. Kilcher, N. Tirelli, Macromol. Rapid Commun.
2009, 30, 299.
Macromol. Chem. Phys. 2012, 213, 2052−2061
www.MaterialsViews.com © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Macromolecular
Chemistry and Physics
www.mcp-journal.de
[18] G. Kilcher, L. Wang, C. Duckham, N. Tirelli, Macromolecules
2007, 40, 5141.
[19] A. Napoli, N. Tirelli, G. Kilcher, J. A. Hubbell, Macromolecules
2001, 34, 8913.
[20] P. Hu, N. Tirelli, React. Funct. Polym. 2011, 71, 303.
[21] L. Wang, P. Hu, N. Tirelli, Polymer 2009, 50, 2863.
[22] A. Rehor, N. Tirelli, J. A. Hubbell, Macromolecules 2002, 35,
8688.
[23] G. Kilcher, L. Wang, N. Tirelli, J. Polym. Sci.,Polym. Chem.
2008, 46, 2233.
[24] L. Wang, G. Kilcher, N. Tirelli, Macromol. Chem. Phys. 2009,
210, 447.
[25] L. Wang, G. Kilcher, N. Tirelli, Macromol. Biosci. 2007, 7, 987.
[26] G. Kilcher, C. Duckham, N. Tirelli, Langmuir 2007, 23, 12309.
[27] A. Napoli, M. J. Boerakker, N. Tirelli, R. J. M. Nolte,
N. Sommerdijk, J. A. Hubbell, Langmuir 2004, 20, 3487.
[28] A. Napoli, H. Bermudez, J. A. Hubbell, Langmuir 2005, 21,
9149.
[29] A. Rehor, N. E. Botterhuis, J. A. Hubbell, N. Sommerdijk,
N. Tirelli, J. Mater. Chem. 2005, 15, 4006.
[30] V. V. Khutoryanskiy, N. Tirelli, Pure Appl. Chem. 2007, 80,
1703.
[31] A. Rehor, H. Schmoekel, N. Tirelli, J. A. Hubbell, Biomaterials
2008, 29, 1958.
[32] S. T. Reddy, A. J. van der Vlies, E. Simeoni, V. Angeli,
G. J. Randolph, C. P. O’Neill, L. K. Lee, M. A. Swartz,
J. A. Hubbell, Nat. Biotechnol. 2007, 25, 1159.
[33] T. Oyama, Y. Chujo, Macromolecules 1999, 32, 7732.
[34] K. Sato, M. Hyodo, M. Aoki, X. Q. Zheng, R. Noyori, Tetrahe-
dron 2001, 57, 2469.
[35] G. Barbieri, M. Cinquini, S. Colonna, F. Montanar, J. Chem.
Soc. C 1968, 659.
[36] M. Madesclaire, Tetrahedron 1986, 42, 5459.
[37] C. Orgeretravanat, J. C. Galin, Makromol. Chem. 1988, 189,
2017.
[38] J. C. Lee, M. H. Litt, C. E. Rogers, J. Polym. Sci. Polym. Chem.
1998, 36, 793.
[39] C. S. Marvel, E. D. Weil, J. Am. Chem. Soc. 1954, 76, 61.
[40] D. C. Dittmer, G. C. Levy, J. Org. Chem. 1965, 30, 636.
[41] H. G. Batz, V. Hofmann, H. Ringsdorf, Makromol. Chem. 1973,
169, 323.
[42] V. Hofmann, H. Ringsdorf, G. Muacevic, Makromol. Chem.
1975, 176, 1929.
[43] E. Lallana, T. Ferreri, S. Carroccio, A. M. Puga, N. Tirelli, Rapid
Commun. Mass Spectrom. 2012, 26, DOI: 10.1002/rcm.6337.
[44] M. H. Yang, Polym. Degrad. Stabil. 2000, 68, 451.
[45] M. J. Bowden, L. F. Thompson, J. P. Ballantyne, J. Vac. Sci.
Technol. 1975, 12, 1294.
[46] F. Iacona, G. Marletta, Nucl. Instrum. Methods Phys. Res. Sect.
B-Beam Interact. Mater. Atoms 2000, 166, 676.
[47] A. Watanabe, T. Sakakibara, S. Ito, H. Ono, Y. Yoshida,
S. Tagawa, M. Matsuda, Macromolecules 1992, 25, 692.
[48] S. Ungphaiboon, D. Attia, G. G. d’Ayala, P. Sansongsak,
F. Cellesi, N. Tirelli, Soft Matter 2010, 6, 4070.
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