Synthesis, Characterization, and Evaluation of Cytotoxicity of Poly(3-methylene-2-pyrrolidone)

Article abstract of DOI:10.1021/acs.biomac.6b00210

The homo- and copolymerization of 3-methylene-2-pyrrolidone (3M2P) is introduced. 3M2P is readily polymerized via conventional free radical polymerization, and two reversible deactivation radical polymerization methods including reversible addition-fragmentation (chain) transfer and single-electron-transfer living radical polymerization. Poly(3M2P) has a high thermal stability and a very high glass transition temperature. Poly(3M2P) does not dissolve in most common organic solvents, but it has a high aqueous solubility. Cytotoxicity tests reveal that it is nontoxic to cells, even up to concentrations of 1 mg/mL. This adds poly(3M2P) to the family of water-soluble and biocompatible pyrrolidone-based vinyl polymers.

Full text of DOI:10.1021/acs.biomac.6b00210

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Article
Poly(3-methylene-2-pyrrolidone). Synthesis,
characterization and evaluation of cytotoxicity
Ingrid Marie Heyns, Rueben Pfukwa, and Bert Klumperman
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00210 • Publication Date (Web): 18 Apr 2016
Downloaded from http://pubs.acs.org on April 21, 2016
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Biomacromolecules
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Poly(3ꢀmethyleneꢀ2ꢀpyrrolidone). Synthesis, characterization
and evaluation of cytotoxicity
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Ingrid M. Heyns,† Rueben Pfukwa,† and Bert Klumperman†,‡
†Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland 7602, South
Africa.
Keywords. Pyrrolidone, 3-methylene-2-pyrrolidone, single-electron-transfer living radical polymerization, reversible addition
fragmentation (chain) transfer, biocompatible
ABSTRACT: The homo-and copolymerization of 3-methylene-2-pyrrolidone (3M2P) is introduced. 3M2P is readily pol-
ymerized via conventional free radical polymerization, and two reversible deactivation radical polymerization methods
(RDRP) including reversible addition fragmentation (chain) transfer (RAFT) and single-electron-transfer living radical
polymerization (SET-LRP). Poly(3M2P) has a high thermal stability and a very high Tg. Poly(3M2P) does not dissolve in
most common organic solvents, but it has a high aqueous solubility. Cytotoxicity tests reveal that it is non-toxic to cells,
even up to concentrations of 1 mg/ml. This adds poly(3M2P) to the family of water-soluble and biocompatible pyrroli-
done-based vinyl polymers.
1. Introduction
Vinyl polymers containing a pyrrolidone functionality in the side chain have many important applications in the
pharmaceutical, food and cosmetic industry.1–4 These applications emanate from the properties of the pyrrolidone
functionality, which include aqueous solubility, hydrogen bonding capacity, biocompatibility and coordination capacity.5–
10 Most literature reports utilise Nꢀsubstituted vinyl pyrrolidones, the preeminent example being poly(Nꢀvinylpyrrolidone)
(PVP), the polymer of Nꢀvinylpyrrolidone (NVP) (1, Figure 1). PVP is fully water soluble in all proportions and is nonꢀ
cytotoxic.7,11–13 PVP is synthesized via radical polymerization methods, including reversible deactivation radical
polymerization techniques (RDRP), in particular the reversible addition fragmentation chain transfer (RAFT)
technique.14 Other reported pyrrolidone functional vinyl polymers include derivatives of NVP, with 3ꢀalkyl substituents
(2, Figure 1), and Nꢀsubstituted systems which have spacers between the vinyl functionality and amide nitrogen (3,
Figure 1). In the latter systems, the polymerizable moiety is (meth)acrylic in nature.15,16
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O
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2
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R
O
N
N
O
O
O
O
N
O
N
N
R
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H
R
3a: R = H; n = 3
3b: R = CH3; n = 2 or 3
4a: R = CH3
4b: R = C2H5 ꢀ C8H17
4c: Ph
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1
2: R = CH3 ꢀ C4H9
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Figure 1. Representative pyrrolidone-based monomers.
A much less reported system of vinyl pyrrolidones is based on monomers in which the vinyl/methylene group is directly
attached to the pyrrolidone ring in position 3 (4 and 5, Figure 1).17–20 Monomer 4a has been polymerized anionically,21
yielding well defined polymers, and via free radical polymerization (FRP).19 In the latter study, the aqueous solubility of
polymers from 4a was also demonstrated.19 In other studies, monomers 4aꢀb,17 and 4c,18 were polymerized via FRP,
and the effect of the Nꢀsubstituent on the thermal properties and aqueous solubility was assessed. Monomer 5 was
homoꢀ and copolymerized with styrene and methyl methacrylate via FRP.20
Figure 2. 3M2P (6), N-methyl methacrylamide (7) and tulipalin A (8).
To extend the versatility of pyrrolidoneꢀfunctional vinyl polymers, we focused on 3ꢀmethyleneꢀ2ꢀpyrrolidone (3M2P), (6,
Figure 2). To the best of our knowledge there are no literature reports that demonstrate the polymerization of 3M2P by
chain growth methods such as FRP and RDRP techniques. We drew inspiration from the fact that the lactone
analogue, known as tulipalin A (8, Figure 2), which can be considered to be the cyclic version of MMA, has been
polymerized via FRP,22 and RDRP techniques including ATRP,23 yielding well defined polymers with a high Tg and very
high thermal stability. We anticipated that 3M2P, which can also be considered to be the cyclic version of Nꢀmethyl
methacrylamide (NꢀMMAAm) (7, Figure 2), could also be polymerized via FRP and RDRP techniques, similarly to Nꢀ
MMAAm.24 The exoꢀcyclic methylene group is conjugated with the carbonyl group, this should stabilize the propagating
radical. Furthermore we anticipated that the subsequent polymers would also be endowed with the desirable qualities
of pyrrolidone rings, adding this monomer class to the family of pyrrolidoneꢀbased vinyl monomers which have
significant academic, industrial and pharmaceutical interest. In this work we demonstrate the radical polymerization of
3M2P, via FRP and RDRP techniques such as RAFT and single electron transfer living radical polymerization (SETꢀ
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LRP), to form homopolymers. We demonstrate its copolymerization and block copolymer formation (via RAFT) with 2ꢀ
(dimethylamino)ethyl methacrylate (DMAEMA). We characterized poly(3M2P)’s thermal and solubility properties, and
finally we explored its potential biocompatibility by conducting cytotoxicity tests.
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2. Experimental section
2.1 Materials
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Ethyl chloroformate (Aldrich, 97 %), potassium carbonate (Fluka, 99 %), 3ꢀaminoꢀ1ꢀpropanol (Aldrich, 99 %),
phosphorus tribromide (Aldrich, 99 %), triphenylphosphine (Acros, 99 %), potassium tertꢀbutoxide (Aldrich, 98 %),
paraformaldehyde (Aldrich, 95 %), 2ꢀpyrrolidone (Fluka, 99 %), diꢀtertꢀbutyl dicarbonate (Aldrich, 97 %), 4ꢀ
dimethylaminopyridine (Merck, 99 %), diethyl oxalate (Aldrich, 99 %), sodium hydride (Aldrich, 95 %), trifluoroacetic
acid (Sigma Aldrich, 99 %) were used as received. Solvents; methanol, toluene, dimethylsulfoxide, N,Nꢀ
dimethylformamide, diisopropyl ether and acetonitrile were purchased from Sigma Aldrich and used without further
purification. PPh₃ was recrystallized from anhydrous acetone. Azobisisobutyronitrile (AIBN) was purchased from Merck
and recrystallized from methanol. Me6TREN and 1,2ꢀdihydroxypropaneꢀ3ꢀoxyꢀ2ꢀbromoꢀ2ꢀmethylpropionyl were
synthesized as described in literature.25,26 The synthesis of the RAFT agent, 2ꢀhydroxyethyl 2ꢀ
(butylthiocarbonothioylthio)ꢀ2ꢀmethylpropanoate is described in the SI. TLC plates (0.20 mm Silica gel 60, with
fluorescent indicator UV254) and Silica gel 60(0.063 ‐ 0.2 mm/70 ‐ 230 mesh) were purchased from Machery‐Nagel.
SnakeSkin® dialysis tubing with a MW cutꢀoff of 3 500 was purchased from Thermoscientific.
2.2 Characterization
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Nuclear Magnetic Resonance (NMR) Spectroscopy. H and 13C NMR spectra were obtained with a Varian VXRꢀUnity
(300 MHz) spectrometer. NMR samples were prepared in deuterated solvents. Proton and carbon chemical shifts are
reported in parts per million (ppm) (δ) downfield from tetramethylsilane (TMS) using residual solvent protons as internal
standards.
Size Exclusion Chromatography (SEC). Molar mass and dispersities (Ð) of the homopolymer, poly(3M2P), were
obtained by 1,1,1,3,3,3ꢀhexafluoro isopropanol (HFIP) SEC. The HFIP SEC instrument was equipped with a Waters
1515 Isocratic HPLC pump, a Waters 2414 refractive index detector, a Waters 2707 autosampler, PSS PFG guard
column and two PFGꢀlinearꢀXL (7 ꢁm, 8 × 300 mm) columns in series. The HFIP contained lithium chloride (2 g/L), the
flow rate was 0.8 mL/min and the injection volume was 75 ꢁL. The calculated molar masses were relative to
poly(methyl methacrylate) (PMMA) standards with low dispersities, obtained from Polymer Laboratories. Statistical and
block copolymers were analysed with a N,Nꢀdimethylacetamide (DMAc) SEC. The DMAc SEC setup consisted of a
Waters 717 plus autosampler connected to a Shimadzu LCꢀ10AT pump with the following column configuration: 1×PSS
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GRAM analytical precolumn (10 ꢂm particle size, 8.0×50 mm), 1×PSS GRAM analytical column (10 ꢂm particle size,
100 Å pore size, 8.0×300 mm), 2×PSS GRAM analytical column (10 ꢂm particle size, 3000 Å pore size, 8.0×300 mm.)
with a Waters 410 differential refractometer. The system was calibrated using low dispersity PMMA standards.
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Electrospray Ionization Mass Spectrometry (ESIꢀMS). Analysis was performed on a Waters Synapt G2 with
electrospray ionization (ESI) in the positive mode. The column used was Waters UPLC C18, 2.1x100 mm. Samples
were dissolved in chloroform.
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Differential Scanning Calorimetry (DSC). Thermal analysis studies were obtained by DSC with a TA Instruments Q100
calorimeter calibrated with an indium standard under N2 atmosphere. Cooling and heating rates were fixed at 10 °C/min
and were obtained from ꢀ60 to 300 °C.
Thermal gravimetric analysis (TGA). Samples were analysed using a Perkin Elmer TGA 7, under an inert nitrogen
atmosphere to prevent oxidation. The heating rate was 20 °C per minute up to 900 °C.
Cell Imaging. Fluorescence and light transmission micrographs image acquisition was performed on an Olympus Cell
system attached to an IX81 inverted fluorescence microscope equipped with an FꢀviewꢀII cooled CCD camera (Soft
Imaging Systems) using a XenonꢀArc burner (Olympus Biosystems GmbH) as light source.
2.3 Methods
The monomer, 3M2P, was prepared as described in Scheme 1. Further experimental details and characterization are
included in the supplementary information (SI).
Scheme 1. Synthesis routes for 3M2P
Route 1:
PPh3
O
O
O
Br
c
b
a
Ph3P
O
Ph3P 3N
H
OEt
Br
3N
H
OEt
2N
OEt
99 %
60 %
N
H
H 12
9
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Route 2:
35 %
d
O
O
O
Na
O
a'
b'
c'
d'
Boc
Boc
N
NH
N
O
EtO
79 %
51 %
78 %
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N
O
N
16Boc
H 6
13
15
14
a) PPh₃, acetonitrile, 100 °C; b) potassium tert-butoxide, toluene, r.t.; c) 50 °C; d) paraformaldehyde, toluene, 50 °C to r.t.; a’) di-
tert-butyl dicarbonate, acetonitrile, 0 °C; b’) diethyl oxalate, sodium hydride, diisopropyl ether, 35 °C; c’) paraformaldehyde,
dimethylformamide, 100 °C; d’) trifluoroacetic acid, dichloromethane.
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General FRP of poly(3M2P): Homopolymerizations were carried out in a 5 mL pearꢀshaped flask; 3M2P (100 mg, 1.03
mmol) and AIBN, in a ratio of 100:1, were dissolved in DMSO (1 mL), and the mixture purged with argon. Then the flask
was immersed into an oil bath preheated to either 60 °C or 75 °C to commence the polymerization. The polymer was
isolated by slow precipitation from acetone, and was obtained as a white powder. For DSC, TGA and cytotoxicity
analyses, poly(3M2P) was dialyzed against water for ~5 days using SnakeSkin®, replacing the water every 12 h,
subsequently the polymer was freezeꢀdried for 24 h. Homopolymers were analysed by 1H NMR spectroscopy (Figure 3)
and HFIP SEC (entry 1 and 2, Table 1).
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Statistical copolymerization of N,Nꢀdimethylaminoethyl methacrylate and 3M2P: A reaction vessel was charged with
3M2P (50 mg, 0.51 mmol) and N,Nꢀdimethylaminoethyl methacrylate (DMAEMA) (81 mg, 0.52 mmol) as a comonomer.
AIBN (1.7 mg, 0.010 mmol) was added as an initiator in 1.0 mL DMF and the mixture was then purged with argon gas.
The vessel was then immersed in an oil bath, preheated to 75 °C, for 24 h. The reaction was stopped by opening the
flask to air, and cooling. The polymer was isolated by precipitation from cold diethyl ether. The copolymers were
analysed via 1H NMR spectroscopy and DMAc SEC (Figure 4c and d).
General homopolymerization via SETꢀLRP: To a 25 mL Schlenk flask was added 3M2P (0.10 g, 1.03 mmol), copper
wire wrapped around the stirring bar and 2,3ꢀdihydroxypropylꢀ2ꢀbromoꢀ2ꢀmethylpropanoate (2.47 mg, 0.10 mmol) as
initiator, in 1.0 mL DMSO. The reaction mixture was degassed by five freezeꢀpumpꢀthaw cycles whereafter the flask
was backꢀfilled with argon. A degassed microꢀsyringe was then used to transfer the ligand, Me6TREN (1.19 mg, 1.4 ꢁL,
0.01 mmol), to the already deoxygenized reaction mixture and the Schlenk flask placed in a heated oilꢀbath at 50 °C.
After 24 h, the reaction was stopped by opening the flask to air and cooling, and conversion was determined on the
1
crude polymer mixture using H NMR spectroscopy. The reaction mixture was filtered through a short basic aluminium
oxide column to remove residual copper, whereafter the polymer was precipitated from acetone to yield a white powder.
The homopolymer was analysed via 1H NMR spectroscopy (Figure 2S) and HFIP SEC (entry 4, Table 1 and Figure 4a).
General RAFT homopolymerization procedure of 3M2P: The RAFT agent utilized was 2ꢀhydroxyethyl 2ꢀ
(butylthiocarbonothioylthio)ꢀ2ꢀmethylpropanoate (SI). To a 25 mL pear flask was added 3M2P (0.11 g, 1.13 mmol),
RAFT agent (1.65 mg, 0.01 mmol) and AIBN (0.18 mg, 0.001 mmol), in 0.8ꢀ1.0 mL DMSO. After the reaction mixture
was dissolved and degassed with argon gas for 45 min, the polymerization vessel was placed in the heated oil bath at
75 °C for 24h. The reaction was stopped by opening the flask to air, and cooling, conversion was determined, on the
crude polymer mixture, using 1H NMR spectroscopy. The polymer was isolated by precipitation from acetone, to yield a
1
yellowish powder. The polymer was analysed via H NMR spectroscopy (SI, Figure 1S) and HFIP SEC (entries 3a and
3b, Table 1 and Figure 4a).
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Block copolymer consisting of DMAEMA and 3M2P via RAFT: A reaction vessel was charged with DMAEMA (1.0 g,
6.36 mmol), AIBN (3.0 mg, 0.02 mmol), and 2ꢀhydroxyethyl 2ꢀ(butylthiocarbonothioylthio)ꢀ2ꢀmethylpropanoate (31 mg,
0.10 mmol) as the RAFT agent, in 1,4ꢀdioxane (2 mL). The mixture was purged with argon gas, whereafter the vessel
was placed in a heated oil bath at 75 °C for 24 h. The reaction was stopped by opening the flask to air, and cooling.
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The polymer was isolated by precipitation from cold petroleum ether. The homopolymer was analysed via H NMR
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spectroscopy and DMAc SEC (Figure 4b).
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The resulting PDMAEMA macroꢀRAFT agent was then chain extended with 3M2P. A reaction vessel was charged with
PDMAEMA macroꢀRAFT agent (0.1 g, 0.01 mmol), 3M2P (35 mg, 0.36 mmol), AIBN (0.2 mg, 0.001 mmol) were
dissolved in a mixture of 1,4ꢀdioxane (1.0 mL) and DMF (0.2 mL). The mixture was purged with argon gas, and the
flask was then immersed in a heated oil bath at 75 °C for 24 h. The reaction was stopped by opening the flask to air,
and cooling. The polymer was isolated by precipitation from cooled diethyl ether. The block copolymer was analysed
via 1H NMR spectroscopy (SI, Figure 3S) and DMAc SEC (Figure 4b).
Cytotoxicity assay: To determine the cytotoxity of poly(3M2P), different concentrations of dialyzed and freezeꢀdried
poly(3M2P) (1 mg/mL, 1 ꢂg/mL and 1 ng/mL) were dissolved Dulbecco’s modified eagle medium (DMEM) containing 1
% penicillin/streptomycin and 10 % fetal calf serum and added to GT1ꢀ7 cell (hypothalamic mouse cells) cultures. A
control (media and cells) and a positive control (media and cells treated with ethanol) were used as references.
Subsequently, the samples were incubated at 37 °C for 4 h, whereafter the cells were stained with fluorescent markers,
Hoechst 33342 dye and propidium iodide, respectively (SI, Figure 4S). Fluorescence and light transmission
micrographs were obtained and cell viability was determined (Figure 5).
3. Results and discussion
3.1 Monomer synthesis
The monomer 3M2P was prepared using two known procedures (Scheme 1). In the first approach, 3M2P was
accessed by condensing the corresponding phosphorous ylide (12, Scheme 1) with paraformaldehyde via a Wittig
reaction, as reported by Fotiadu et al. (Scheme 1S), in an overal yield of ~ 20%.27 In a second approach, similar to that
used to synthesize monomers 4aꢀc,18,19,21 3M2P was synthesized by first reacting diethyl oxalate with NꢀBocꢀ2ꢀ
pyrrolidone (14, Scheme 1) in the presence of sodium hydride, to yield an enolate intermediate (15, Scheme 1).
Subsequently, 15 was reacted with paraformaldehyde, followed by elimination to give the desired 3ꢀmethylene
functionality, in an overal yield of ~ 31%. Finally removal of the NꢀBoc protection, afforded 3M2P. Preference was given
to the second monomer synthesis route, since it has a fewer, and simpler synthesis steps (see SI for expanded
synthesis scheme), and it also afforded a higher overal yield of 3M2P.
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3.2 Free radical polymerization of 3M2P
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We assessed the homopolymerizability of 3M2P via FRP. Under our optimized conditions, polymerization was carried
out in DMSO, and the polymer was isolated by precipitation from acetone, in about 82 % yield. We analyzed the
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polymer via H and 13C NMR (SI, Figure 4S) spectroscopy and HFIP SEC. Figure 3 compares the H NMR spectra of
the monomer 3M2P and its homopolymer (poly(3M2P)). It is evident that the exoꢀmethylene double bond of 3M2P
disappeared after polymerization. The appearance of broad signals in the lower chemical shift region (~1.5 ꢀ 3.5 ppm) is
an indication of successful polymer formation.
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A
*
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1.0
δ
(ppm)
Figure 3. Presaturation 1H NMR spectra of 3M2P (A) and poly(3M2P) (B) in D2O.
SEC analysis showed that high molar masses were obtained (entries 1 and 2, Table 1), confirming the success of the
polymerization. Ð values were greater than 1.5, as expected for conventional free radical polymerizations. Poly(3M2P)
was insoluble in a wide range of solvents, but it partially dissolved in DMSO and fully dissolved in HFIP and water. A
similar solvent resistance was observed for poly(tulipalin A) and it was attributed to structural rigidity of the polymer
chain caused by the conformational rigidity of the lactone monomer.22 Considering the similarity of the ring structures, it
is reasonable that poly(3M2P) is also similarly rigid. Additionally poly(3M2P)’s amide/lactam functionality likely
participates in intermolecular hydrogen bonding, contributing to its (organic) solvent resistance.
3.3 RDRP of 3M2P
After successfully polymerizing 3M2P via conventional FRP, we then extended the study to assess its polymerizability
under RDRP conditions, in view of preparing wellꢀdefined polymers with predetermined molar masses, low Ð, and
defined chain end functionalities. Two RDRP techniques, i.e. RAFT and SETꢀLRP, were investigated (Scheme 2). We
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used the RAFT polymerization, utilizing a trithiocarbonate RAFT chain transfer agent (Scheme 2). As 3M2P can be
considered to be the cyclic version of
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Scheme 2. Homopolymerization of 3M2P via RDRP techniques
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Top: RAFT mediated polymerization and bottom: SET-LRP.
Table 1. Experimental results for the homopolymerization of 3M2P via FRP and RDRP techniques
#
Technique
T
Time
Target
Conversion Mn,NMR
Mw,SEC
(g mol-1)
Mn,SEC
(g mol-1)e
Đf
(g mol-1)
(°C) (h)
Mn
(%)
a
d
1
FRP
FRP
60
75
60
75
50
24
16
-
82b
-
-
156 300
97 100
20 000
20 100
12 900
67 200
52 300
16 300
14 900
11 200
2.33
1.86
1.22
1.35
1.16
a
d
2
-
83b
3a RAFT
3b RAFT
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20 000
20 000
10 000
> 70c
> 65c
> 80c
18 800
13 900
8 000
4
SET-LRP
a) Monomer:AIBN (100:1). b) Conversion determined gravimetrically. c) Conversion was determined from 1H NMR spectroscopy.
d) Chain-ends were not visible on 1H NMR spectroscopy. e) HFIP SEC calibrated with PMMA standards. f) Dispersity.
NꢀMMAAm, a trithiocarbonate RAFT agent was selected, as such Zꢀgroups are known to have high chain transfer
constants and have been shown to control monomers with a stabilized propagating radical such as methacrylamides.28
RAFT mediated polymerizations were carried out in DMSO with AIBN as initiator. Characterization by SEC, in HFIP
1
(Figure 4a), and by H NMR spectroscopy (SI, Figure 1S) gave Mn values which correlate reasonably well (entries 3a
and 3b, Table 1). Additionally Đ values are low, typical of wellꢀbehaved RAFT mediated polymerizations. SETꢀLRP was
selected for polymerization of 3M2P via a transition metal catalyzed process (Scheme 2). SETꢀLRP is a versatile and
mild RDRP technique for controlling the polymerization of vinyl monomers such as methacrylamides.29,30 It can be
readily carried out in polar solvents such as DMSO and water, which enhances the disproportionation of CuΙ to Cu0,
essential to SETꢀLRP.29–31 We conducted our SETꢀLRP of 3M2P in DMSO based on the earlier successful FRP and
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RAFT of 3M2P in DMSO, and we obtained a wellꢀdefined polymer with low Đ of 1.16 (entry 4, Table 1). Representative
SEC traces for the polymer prepared by SETꢀLRP, are shown in Figure 4b and no bimodality was evident. Conversions
of the RDRP experiments could not be determined accurately, as polymerizations were performed on a very small
scale.
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To further enhance the application prospects of poly(3M2P), the copolymerization of 3M2P with other vinyl monomers
was investigated. We selected DMAEMA as comonomer as it forms a tertiary propagating radical similar to 3M2P, also
it is miscible with polar solvents that dissolve 3M2P. We copolymerized 3M2P with DMAEMA via FRP, producing a
polymer with a Mn of 15 700 g/mol and a Đ of 1.78 (Figure 4c). Despite the initial feed of 50:50, the final polymer had a
39 % incorporation of 3M2P monomer. It is likely that DMAEMA has a higher reactivity ratio than 3M2P, hence the
greater incorporation in the final polymer. The copolymerizability of 3M2P with a readily available ‘more activated’
monomer (MAM) is significant because the most frequently used pyrrolidone monomer, NVP, does not copolymerize
readily with MAMs.12
Block copolymer formation was pursued by chain extending poly(DMAEMA), synthesized with a trithiocarbonate RAFT
1
agent, with 3M2P. Block copolymer formation was confirmed by SEC and H NMR spectroscopic analysis. With the
former technique, the SEC trace shifted to a lower elution volume, indicating an increase in the molar mass of the
polymer (Figure 4d). The Đ value (≈ 1.26) was also reasonably low. We also evaluated the block copolymer formation
1
via H NMR spectroscopy (Figure 3S), and we observed the appearance of signals attributable to poly(DMAEMA) and
poly(3M2P) in the block copolymer.
3.5 Thermal properties
We further assessed poly(3M2P) by characterizing its thermal properties, using TGA and DSC. The TGA thermogram
revealed a 20 % weight loss near 100 °C (SI, Figure 5S), attributable to water loss, suggesting that poly(3M2P) is very
hygroscopic. TGA analysis showed that poly(3M2P) has a high thermal stability, with the main thermal decomposition
occurring between 400ꢀ500 °C.
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Mn = 16 335
Mn = 11 176
Đ
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Đ
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Retention time (min)
Retention time (min)
Poly(DMAEMA)ꢀbꢀpoly(3M2P)
Poly(DMAEMA)
c
d
M
n = 23 343
= 1.26
Mn = 15 753
Mn = 16 960
Đ
= 1.78
Đ
Đ
= 1.37
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Retention time (min)
Retention time (min)
Figure 4. SEC traces of: a) poly(3M2P) prepared RAFT (entry 3a, Table 1), b) poly(3M2P) prepared via SET-LRP (4, in
Table 1), c) random copolymer of DMAEMA and 3M2P prepared via FRP, and d) Poly(DMAEMA) and poly(DMAEMA)-
block-poly(3M2P) prepared via RAFT.
The DSC trace revealed a glass transition temperature (Tg) at 285 °C (SI, Figure 6S). Poly(tulipalin), which is
poly(3M2P)’s lactone analogue, has its thermal decomposition at 320 °C, and a Tg of 195 °C.22 The polymers from
monomers 4a and 4b (Figure 1) exhibit Tg values ranging from 110 °C to ꢀ5 °C, with the Tg values decreasing with
increasing length of the
N
ꢀalkyl substituent.17 The enhanced thermal stability and higher Tg of poly(3M2P) is likely due
to a combination of the rigidity imposed by its cyclic nature, and presumed intermolecular hydrogen bonding
interactions of the lactam’s amide linkage. Poly(3M2P) did not exhibit LCST behavior, in aqueous solution, within the
accessible experimental range, 0ꢀ80 °C.
3.6 Cytotoxicity
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The waterꢀsolubility of poly(3M2P) makes it an attractive polymer for biomedical applications, therefore we evaluated its
cytotoxicity at different concentrations. These studies were performed on GT1ꢀ7 cultured hypothalamic mouse cell lines
in Dulbecco’s modified eagle medium (DMEM). The cells were incubated for 4 h, and afterwards, were stained with
Hoechst 33342 dye (blue) and propidium iodide (red) prior to recording fluorescence and light microscopy images.
Hoechst 33342 dye is used to stain cells for fluorescence microscopy studies, the counterstain, propidium iodide only
penetrates the cell membranes of dead cells and is used to determine cell viability.32
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Figure 5. Fluorescence and light transmission micrographs of cells stained with Hoechst 33342 (column a) dye and
A
B
C
Control
1 mg/mL
poly(3M2P)
1 ꢁg/mL
poly(3M2P)
Positive
Control
propidium iodide (column b), and an overlay (column c). Scale bar: 100 µm.
Figure 5 shows fluorescence and light transmission micrographs of cells stained with Hoechst 33342 dye (column
A),
propidium iodide (column
(cells in media), a positive control (cells treated with ethanol in media) and cells exposed to 1 ꢁg/mL and 1 mg/mL poly
(3M2P). The cells in the control micrograph are stained blue, but were impermeable to the red propidium iodide (
Figure 5) indicating that they were viable, as expected. The cells in the positive control stained red with propidium
iodide, as expected indicating that they were not viable, micrograph , shows a pink colour, as a result of the overlay of
B) and an overlay of Hoechst 33342 dye and propidium iodide (column C); for the control
B
,
C
the red and blue stains. The micrographs of the polymerꢀcell solutions with concentrations of 1 mg/mL and 1 ꢂg/mL of
P(3M2P), did not stain red, indicating that they were not penetrated by propidium iodide, and that the presence of
polymer, even up to high concentrations of 1 mg/mL, was nonꢀtoxic to the cells. This suggests that poly(3M2P) is nonꢀ
cytotoxic, and therefore it is a promising candidate for use in the biomedical field.
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4. Conclusion
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In summary, we have presented the polymerization of 3M2P, via FRP and RDRP techniques, i.e. RAFT and SETꢀLRP.
Poly(3M2P) is highly water soluble, but is insoluble in most common organic solvents except in HFIP and in DMSO
(partially). Polymerization via the RAFT and SETꢀLRP techniques produced wellꢀdefined polymers with low dispersities.
We successfully prepared random and block copolymers of poly(DMAEMA) and poly(3M2P) via RAFT mediated
polymerization. Thermal analysis by TGA revealed that poly(3M2P) is very hygroscopic and has a very high thermal
stability, whilst DSC revealed a very high Tg of 285 °C, which we attributed to the combination of 3M2P’s cyclic rigidity
and interꢀchain hydrogen bonding interactions. We also showed that poly(3M2P) is nonꢀcytotoxic even up to
concentrations of 1 mg/mL suggesting its potential as a biomedical polymer. Finally, the ready polymerizability of 3M2P
via various RDRP techniques, and the apparent nonꢀcytotoxicity of poly(3M2P), makes this polymer an interesting
alternative to traditional pyrrolidone functional polymers such as PVP.
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ASSOCIATED CONTENT
Experimental details on the synthesis and characterization of the monomer, 3M2P; polymerization details and characterization;
molecular weights of all polymers analysed via 1H NMR spectroscopy and SEC; TGA and DSC thermograms; cytotoxic studies.
AUTHOR INFORMATION
Corresponding Author
‡E-mail: bklump@sun.ac.za
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work is based on the research supported by the South African Research Chairs Initiative of the Department of Science and
Technology and National Research Foundation of South Africa (Grant No 46855). Dr. Ben Loos is thanked for cytotoxic studies.
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FRP and RDRP
•Thermally stable
•Waterꢀsoluble
•Nonꢀcytotoxic
•Amenable to RDRP
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n
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FRP and RDRP
O
O
N
H
N
H
•ꢀ amenable to RDRP
•ꢀ thermally stable
•ꢀ water soluble
•ꢀ non-cytotoxic
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