Dual functionalized telechelic block copolymers with reproducible block sizes prepared by microwave assisted RAFT polymerization

Article abstract of DOI:10.1016/j.polymer.2015.04.013

This work demonstrates the preparation of amphiphilic block copolymers with remarkable reproducibility of each block and the dual functionalization of the chain ends with a targeting unit and a fluorescent moiety. The size of the resulting micelles can be

Full text of DOI:10.1016/j.polymer.2015.04.013

Polymer 66 (2015) 110e121
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Dual functionalized telechelic block copolymers with reproducible
block sizes prepared by microwave assisted RAFT polymerization
Kaushik Mishra, Abraham Joy*
Department of Polymer Science, The University of Akron, Akron, OH 44325, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
This work demonstrates the preparation of amphiphilic block copolymers with remarkable reproduc-
ibility of each block and the dual functionalization of the chain ends with a targeting unit and a fluo-
rescent moiety. The size of the resulting micelles can be controlled between 17 and 55 nm by tuning the
size of the hydrophobic block. The polymerization of the diblock was carried out by microwave assisted
reversible addition fragmentation chain transfer (RAFT) polymerization and results in a very high degree
of reproducibility of the number of repeat units of each block. The hydrophilic block of the polymer
comprised of poly(2-hydroxyethyl acrylate) (pHEA) while the hydrophobic block was made of poly(2-
Received 31 January 2015
Received in revised form
1 April 2015
Accepted 3 April 2015
Available online 13 April 2015
Keywords:
ethylhexyl acrylate) (pEHA). Folic acid, a folate receptor targeting moiety, was tethered to the
the polymer while anthracene was covalently linked to the end. The end covalent attachment is via a
redox cleavable disulfide bond, which is optimal for controlled drug delivery systems. High efficiencies of
- and -conjugations were obtained as determined by UV and NMR measurements.
© 2015 Elsevier Ltd. All rights reserved.
a end of
Heterotelechelic functionalization
Microwave assisted polymerization
Multifunctional micelles
u
u
a
u
1. Introduction
based micelles prepared from hydrophobic blocks of styrene,
N-vinyl formamide, butyl acrylate, N-isopropyl acrylamide, or
N,N-dimethyl acrylamide and PEG as the hydrophilic block have
been reported. Micelles based on chain growth polymers synthe-
sized by controlled radical polymerization (CRP) methods such as
RAFT and ATRP typically have very good control over the molecular
weight and polydispersity [14,15]. RAFT methods [16] enable using
the end group functionalities to tether targeting [17] units such as
peptides [18], folate [19], transferrin or galactose [20]. However,
polymeric micelles still suffer from some disadvantages that hinder
their translation to the clinic. The first is the variability in the
molecular weight from batch to batch and is often a stumbling
block with regard to FDA approval [21]. The second limitation of
current micelles is their lack of multiple functional groups that
enable targeted delivery of therapeutics and co-therapeutics to the
desired organ [22].
Here we report the synthesis of an amphiphilic diblock copol-
ymer that addresses the above drawbacks and presents a blueprint
for the synthesis of well controlled polymer micelles with sub-
100 nm size range, reproducibility of block sizes and dual func-
tionalization of polymeric chain ends. A diblock copolymer of
hydroxyethyl acrylate (HEA) and 2-ethylhexyl acrylate (EHA) was
prepared by microwave assisted RAFT polymerization which pro-
vided precise control and reproducibility of each block. The con-
version of EHA was controlled to provide micelles of sizes between
Polymeric micelles are effective therapeutic delivery systems [1]
and will see increasing use due to the large number of hydrophobic
new chemical entities (NCE) that fail to move forward due to their
extremely poor aqueous solubility [2]. Polymeric micelles have
several advantages for drug delivery such as increased circulation
time and the ability to conjugate and increase the bioavailability of
hydrophobic therapeutics. Micelles with sizes in the range of
30e100 nm are able to evade clearance mechanisms such as the
reticuloendothelial system [3e5]. Such sizes [6] also facilitate
enhanced permeation and retention (EPR) in cancerous tissues due
to their leaky vasculature [7,8]. The hydrophilic corona of micelles
also contributes to decreasing non-specific adsorption and lower
toxicity of such synthetic polymers [2].
In light of the above advantages of polymeric micelles, several
designs have been reported with variations in both the hydro-
phobic and hydrophilic block. For example, various hydrophobic
core-forming blocks such as
D,L-lactic acid [9], propylene oxide [10],
L-lysine [11], aspartic acid [12] and caprolactone [13] have been
combined with PEG as the hydrophilic block. Similarly acrylate
* Corresponding author.
E-mail address: abraham@uakron.edu (A. Joy).
http://dx.doi.org/10.1016/j.polymer.2015.04.013
0032-3861/© 2015 Elsevier Ltd. All rights reserved.

K. Mishra, A. Joy / Polymer 66 (2015) 110e121
111
17 and 55 nm. Poly(2-ethylhexyl acrylate) provides a low Tg dense
hydrophobic core and poly(hydroxyethyl acrylate) (pHEA) provides
a hydrophilic corona and was chosen as an alternative to PEO due to
the reported drawbacks of PEO [23] such as hypersensitivity after
administration [24] and rapid clearance due to the production of
anti-PEO antibodies [25,26]. The RAFT end groups of the copolymer
(SEC) in THF was performed on a Waters 150-C Plus instrument
equipped with RI and LS detector. ESI-MS was performed on a
Bruker HTC ultra QIT.
The size and distribution of the nanoparticles were measured by
Brookhaven Instruments BI-200SM research goniometer and laser
light scattering system fixed on an optical bench at a fixed angle of
90ꢀ. Fourier transform infrared spectroscopy (FTIR) was recorded
on a Shimadzu IRAffinity-1S fixed with an ATR accessory. The
UVeVis measurements were carried out on a Shimadzu UV-1800
spectrophotometer. Fluorescence measurements were carried out
on Synergy TM MX plate reader from BioTek.
A fixed amount of pyrene was added into each micellar solution
with different concentrations to provide a hydrophobic probe. The
concentration of pyrene in the samples was 2 mM. All samples were
sonicated for 3 min and then allowed to reach steady state condi-
tions. The fluorescence emission spectra (lexitation ¼ 334.0 nm) was
collected, and the intensity ratio of the peak at 384 nm to that at
373 nm (I384/I373) was plotted against the log of the micelle con-
centration to provide the critical micelle concentrations (CMCs).
were dual end functionalized at the
the -chain end with antracene.
Microwave assisted living radical polymerization is finding
a-chain end with folate and at
u
increasing use in polymer synthesis due to the rapid formation of a
homogeneous heating profile [27]. Reports on microwave assisted
CRP are still sparse among which RAFT is the best studied so far
[28,29]. It has been found in several cases that microwave irradia-
tion aids in the rate enhancement of the polymerization (kp), thus
attaining higher conversions in a shorter time while still retaining
the fidelity of the end groups [30]. In the literature this has been
attributed to occur due to higher temperature profiles [31], con-
centration of radicals, and even “non-thermal effects” of the mi-
crowave [32].
Trimethylsilyl
(TMS)-protected
2-hydroxyethyl
acrylate
(HEA_TMS) was first polymerized using prop-2-yn-1-yl 4-cyano-4-
((phenylcarbonothioyl)thio)pentanoate as the RAFT agent and AIBN
as the initiator under microwave conditions. This macro-chain
transfer agent was used to polymerize EHA in varying ratios,
generating several pHEA_TMS-b-EHA diblock copolymers with
hydrophobic EHA content ranging from 27 to 55 mol%. The TMS
groups on the hydrophilic block were deprotected under weak
acidic conditions. Micellar dispersions of the acrylate diblock co-
polymers in water were prepared via nanoprecipitation followed by
dialysis. The self-assembled nanostructures were examined by
dynamic light scattering (DLS). Finally the attachment of folate
2.3. Synthesis of poly(2-((trimethylsilyl)oxy)ethyl acrylate)
(pHEA_TMS)
In a typical experiment (Entry 3 in Table 1), HEA_TMS (1.412 g,
7.5 mmol), RAFT-p (0.024 g, 0.075 mmol), AIBN (1.724 mg,
10.50 mmol) and anhydrous 1,4-dioxane (3.0 mL) were added to a
MW reaction vessel of volume 10 mL with an appropriate stir bar.
The reaction vessel was degassed by purging the solution with N2
gas for 30 min while the solution is immersed in an ice-water bath.
Then the vessel was transferred to the microwave reactor and
heated at 70 ꢀC for 3 h under fast stirring conditions. The resulting
polymer was precipitated from cold hexanes multiple times and
dried under vacuum to yield 0.950 g of polymer (Mn ¼ 19.7 kg/mol,
PDI ¼ 1.15).
azide to the
a end (hydrophilic end) and anthracene azide to the u
end (hydrophobic end) of the polymeric chain was carried out as
outlined in Scheme 1. The dually functionalized block copolymer
was characterized by UV and NMR. The current work demonstrates
the utility of RAFT polymerization under microwave conditions as a
versatile method for the synthesis of telechelic functionalized
polymers of reproducible block sizes and dual functionalization of
both telechelic ends of the diblock copolymer (Scheme 2).
2.4. Synthesis of block copolymer, p(HEA_TMS-b-EHAx)
Three different monomer ratios were chosen so as to yield
different diblock sizes with varying hydrophobicity. In all experi-
ments, the amount of macroRAFTagent (pHEA_TMS) used was fixed
and monomer amounts were varied accordingly. Three different
monomer to macroRAFT agent ratios of 50, 100 and 150 were
employed. As a representative example when the ratio was 100
(Entry 8 in Table 1), MacroRAFT agent pHEA_TMS (0.800 g,
2. Experimental section
2.1. Materials
Folic acid was obtained from Sigma Aldrich. All other reagents
were purchased from Alfa Aesar. All reagents were used as received
without further purification unless otherwise stated. 2,20-Azobi-
s(isobutyronitrile) (AIBN) was recrystallized multiple times from
methanol before use. 4-Cyano-4-(phenylcarbonothioylthio) pen-
tanoic acid (RAFT Agent) was synthesized according to previous
literature [33]. Spectra/Por 3 dialysis membrane (MWCO 3500 Da)
was purchased from Spectrum Laboratories Inc. The modified
monomer and RAFT agent are described in the SI (S1 and S2).
0.044 mmol), AIBN (1.022 mg, 6.22 mmol), EHA (0.819 g, 4.44 mmol)
and anhydrous 1,4-dioxane (3.0 mL) were added to a microwave
reaction vessel of volume 10 mL with an appropriate stir bar. The
reaction vessel was degassed by purging the solution for 30 min
with N2 gas while the solution is immersed in an ice-water bath. The
vessel was transferred to the microwave reactor and heated at 70 ꢀC
for 1.5 h under fast stirring conditions. The resulting polymer was
precipitated from cold ether multiple times and then dried under
vacuum to yield 1.2 g of polymer (Mn ¼ 27.7 kg/mol, PDI ¼ 1.21).
2.2. Methods
2.5. Preparation of micelles by dialysis method
Polymerizations were carried out in a CEM Discover SP single
mode microwave reactor. NMR spectra were recorded on a Varian
NMRS 300 or 500 MHz instrument. 1H NMR chemical shifts are
reported in ppm relative to the solvent's residual 1H signal. 13C
NMR spectra were recorded at 126 MHz. Size exclusion chroma-
tography (SEC) analysis in DMF was performed on a TOSOH HLC-
8320 GPC equipped with RI and variable UV detector which was
calibrated with PMMA standards. The eluent used was a 10 mM
mixture of LiBr in DMF at 50 ꢀC. Size exclusion chromatography
As an example, pHEA_TMS-b-EHA_150 (Entry 9 in Table 1;
Mn ¼ 30.9 kg/mol, PDI ¼ 1.24) was deprotected first and then fully
dissolved in DMF at a concentration of 1.0 mg/mL. This polymer
solution was then nanoprecipitated by adding a few drops of
water until it turned blue. The solution was then dialyzed using
Millipore water for 36 h with water being exchanged every 3 h for
the first 12 h, and then exchanged then every 6 h for the next
24 h. The size of the nanoparticles was investigated by dynamic
light scattering (DLS).

Scheme 1. Synthesis of end-functionalized polymers via RAFT polymerization.

K. Mishra, A. Joy / Polymer 66 (2015) 110e121
113
Scheme 2. Synthesis of diblock copolymers.
Table 1
Synthesis of polymers via RAFT polymerization.
#
Polymera
[M]0/[CTA]o/[I]o [M]o (mmol) Time (h) Ðb
Mn,GPC (g/mol)b Mn,NMR (g/mol)c Mn,theory (g/mol)e Repeat unitsc Conv. (%)f
1
2
3
4
5
6
7
8
9
pHEA1_TMS
pHEA2_TMS
pHEA3_TMS
pHEA4_TMS
pHEAAm1_TMS
pHEAAm2_TMS
pHEA_TMS-b-EHA_50
pHEA_TMS-b-EHA_100
pHEA_TMS-b-EHA_150
100/1/0.14
100/1/0.14
100/1/0.14
100/1/0.14
100/1/0.14
100/1/0.14
50/1/0.14
7.5
7.5
7.5
7.5
7.5
7.5
2.2
4.4
6.6
6.6
6.6
3
3
3
3
3
3
1.5
1.5
1.5
1.5
1.5
1.14 19.6
1.14 19.7
1.15 19.7
1.12 19.7
1.08 37.0
1.11 36.5
1.23 23.5
1.21 27.8
1.24 31.0
1.25 30.9
1.27 31.5
17.6
17.6
17.8
17.8
16.6
16.6
24.1
31.3
38.3
38.1
38.5
18.4
18.4
18.5
18.4
17.6
17.7
24.9
28.7
32.0
31.9
31.7
92
92
93
93
87
96
96
96
96
92
92
74
58
51
50
50
87
35d
74d
112d
111d
113d
100/1/0.14
150/1/0.14
10 pHEA_TMS-b-EHA_150_1 150/1/0.14
11 pHEA_TMS-b-EHA_150_2 150/1/0.14
a
All the experiments (entry 1e11) used AIBN as initiator and 1,4-dioxane as solvent. The reaction was carried out in a microwave reactor at 70 ꢀC.
Determined by SEC using THF as the eluent and PS as the standard.
Repeating units calculated by using end-group analysis of homopolymers.
End-group analysis of diblock copolymer.
b
c
d
e
f
Calculated based on the monomer conversion.
Monomer conversion determined by 1H NMR.
2.6. Synthesis of folate azide (6)
solution of 4-bromobut-1-yne (0.704 mL, 7.52 mmol) in dry DMF
(2 mL) was added to the sodium methane thiosulfonate solution
(Scheme 3). The mixture was stirred overnight at 40 ꢀC. Precipi-
tated NaBr was removed by filtration. DMF was removed under
vacuum and the residue was extracted with diethyl ether
(3 ꢁ 100 mL). Upon removal of the solvent, S-(but-3-yn-1-yl)
methane thiosulfonate (8) (0.976 g, 5.94 mmol, 79% yield) was
Folic acid (0.883 g, 2 mmol) (5) was dissolved in a 50:50 mixture
of DMF and DMSO (30 mL) by continuous stirring under N2. 4-
methylmorpholine (0.440 mL, 4.00 mmol) was added to this solu-
tion at room temperature followed by TBTU (0.642 g, 2.000 mmol)
while cooling in an ice-water bath. This reaction was stirred for 1 h
at 0 ꢀC (Scheme 1) and the resulting mixture of activated folic acid
was slowly added to a water-ice cooled solution of 2-azidoethan-1-
amine (0.344 g, 4.00 mmol) in dry DMF (10 mL). This reaction
mixture was stirred for 2 h at 0 ꢀC (Synthesis of 2-azidoethan-1-
amine is described in the S3b). After being warmed to room tem-
perature, the reaction mixture was evaporated in vacuo to remove
most of the DMF and DMSO. This resulting mixture was precipi-
tated into cold diethyl ether (500 mL ꢁ 2) and then filtered through
a 250 mL glass frit funnel. The solids were washed with hexanes
and THF to remove excess 2-azidoethan-1-amine and then washed
obtained as a viscous liquid. 1H NMR (300 MHz; CDCl3):
d 3.37 (d,
J ¼ 0.3 Hz, 3H), 3.32 (t, J ¼ 7.0 Hz, 2H), 2.73e2.68 (m, 2H), 2.10 (t,
J ¼ 2.6 Hz, 1H) (Scheme 4).
2.8. Synthesis of anthracene-MTS (11)
S-(but-3-yn-1-yl) methane thiosulfonate (8) (0.194 g,
1.179 mmol), 9-(azidomethyl)anthracene (10) (0.250 g, 1.072 mmol)
(synthesis described in S4c), and anhydrous THF (3 mL) were added
to a 25 mL round bottom flask (Scheme 1) and subjected to three
freezeepumpethaw cycles. To the frozen mixture, copper (I) bro-
mide (2 mg, 0.014 mmol) was added and vacuum was applied. The
reaction was carried out for 24 h at room temperature under N2.
THF was removed under reduced pressure and the product was
recrystallized from ethanol to yield pure Anthracene methane
thiosulfonate (Anthracene-MTS) (11) (0.242 g, 0.609 mmol, 56.8%
with water to remove unreacted folic acid. g-functionalized folate
azide (6), was obtained as a solid yellow product and was dried in a
vacuum oven to yield 0.958 g (1.880 mmol) of the product in 94%
yield. 1H NMR (500 MHz; DMSO-d6):
d 11.40 (s, 1H), 8.64 (s, 1H),
8.03 (s, 1H), 7.66 (d, J ¼ 8.7 Hz, 2H), 6.90 (s, 1H), 6.64 (d, J ¼ 6.6 Hz,
2H), 4.48 (d, J ¼ 6.0 Hz, 2H), 4.38e4.34 (m, 1H), 3.22 (t, J ¼ 5.8 Hz,
2H), 2.31e1.84 (m, 6H). 13C NMR (126 MHz; DMSO-d6):
d 174.2,
yield). 1H NMR (500 MHz; DMSO-d6):
d 8.74 (s, 1H), 8.58
172.2, 166.4, 153.9, 150.9, 148.7, 129.20, 129.06, 128.1, 121.5, 111.32,
111.29, 52.8, 50.0, 46.1, 38.3, 32.1, 30.6. MS (ESI) m/z:
Calcd. ¼ 509.19; Found ¼ 532.2 ([M þ Na]þ).
2.7. Synthesis of but-3-yn-1-yl methane thiosulfonate (Butynyl-
MTS) (8)
Sodium methanesulfonothioate (7) (2.017 g, 15.04 mmol) (syn-
thesis described in S4a) was dissolved in dry DMF (25 mL). A
Scheme 3. Synthesis of butynyl-MTS.

114
K. Mishra, A. Joy / Polymer 66 (2015) 110e121
(d, J ¼ 8.9 Hz, 2H), 8.17e8.16 (m, 2H), 7.77 (s, 1H), 7.67e7.56 (m, 4H),
6.62 (s, 2H), 3.39 (t, J ¼ 7.3 Hz, 5H), 2.98 (t, J ¼ 7.3 Hz, 2H).
2.9. Folate conjugation to alpha ( ) chain end of diblock copolymer
a
The diblock copolymer (Entry 9 in Table 1) (0.200 g, 7.20
and folate azide (5.50 mg,10.80 mol) were added to a 25 mL round
bottom flask and subjected to three freezeepumpethaw cycles. In
another smaller flask, copper (I) bromide (1.549 mg, 10.80 mol)
and PMDETA (1.654 l, 7.92 mol) were added as quickly as possible
mmol)
m
m
m
m
and deoxygenated. A 1 mL aliquot of anhydrous DMSO was added
to the second flask and the homogeneous solution was transferred
to the oxygen free flask containing the polymer. The reaction was
carried out for 24 h at room temperature under N2 conditions. After
the reaction, acetic acid (20 mL) and methanol (0.5 mL) were added
to this mixture and stirred vigorously for another 24 h. This reaction
mixture was subsequently dialyzed using Millipore water to yield
pure folate functionalized diblock copolymer.
2.10. Anthracene MTS conjugation to omega (u) chain end of pHEA
Anthracene-MTS (11) (21.83 mg, 0.055 mmol) and propan-1-
amine (11.29 l, 0.137 mmol) were consecutively added to a
42 mM solution of pHEA_TMS (Entry 4 in Table 1) (50 mg,
2.75 mol) in chloroform under vigorous stirring. The reaction
m
m
mixture was stirred overnight at room temperature, turning orange
and then yellow within a few hours. The TMS groups of the
resulting polymer were deprotected by adding acetic acid (20 mL)
and methanol (0.5 mL) and stirring vigorously for another 24 h. This
resulting mixture was dialyzed using methanol to yield pure
anthracene conjugated pHEA polymer.
2.11. Synthesis of telechelic functionalized diblock copolymer
Folate conjugated diblock copolymer (32 mg, 1.478
first dissolved in 100 L of DMSO and anthracene-MTS (11)
(11.75 mg, 0.030 mmol) and propan-1-amine (6.08 l, 0.074 mmol)
mmol) was
m
m
were consecutively added to the solution. The reaction mixture was
stirred for 15 h at room temperature, gradually turning pale orange.
The resulting polymer was precipitated multiple times from cold
acetone and centrifuged to provide the product.
3. Results and discussion
3.1. Monomer and RAFT agent modification
Protection of the hydroxyl group of HEA prior to polymerization
was necessary to mitigate loss of the diblock copolymer during the
precipitation steps. A modified RAFT agent based on 4-cyano-4-
(phenylcarbonothioylthio)pentanoic acid (3) was selected as the
chain transfer agent as it provided very good control over the
monomers. The propargyl terminated RAFT agent (4) enabled the
CuAAC based a-functionalization with a targeting moiety and was
prepared according to literature [34].
3.2. Synthesis of HEA_TMS and HEAAm_TMS homoblock by
microwave assisted RAFT polymerization
Microwave reactors largely owe their popularity to observed
enhanced reaction rates. There is a lack of understanding of
microwave effects in the scientific community, but significant ad-
vances have been made due to the recent systematic work by
Dudley et al. [35]. Enhanced reaction rates in multicomponent
systems can be often explained by increased reaction temperatures
in the molecular environments (cage effect) but with a lower

K. Mishra, A. Joy / Polymer 66 (2015) 110e121
115
macroscopic temperature. In addition, the direct microwave irra-
diation of molecules leads to very fast and homogeneous heating
that has resulted in the reduction of side reactions, cleaner prod-
ucts, and higher yields [36]. A high degree of end group fidelity has
been demonstrated by successfully employing the homopolymers
as macroCTAs for subsequent block copolymerizations [37]. The
ability for complete reaction synthesis and purification of polymers
in a shorter time is an attractive concept for fast screening of block
copolymers. Synthesis of the polymer was performed in a com-
mercial microwave reactor which incorporates a focused single/
mono-mode cavity for precise heating. The polymerizations were
carried out under microwave conditions to provide high yields,
faster reaction rates, and precise control over temperature in the
reaction vessel.
spectroscopy which was carried out on a 500 MHz instrument with
a relaxation delay of 30 s. A representative example is shown in
Fig. 1a with the proton assignments corresponding to the repeating
unit. As shown in the expanded region of the spectrum between 7.3
and 8.0 ppm, the signals of the RAFT end group were detected
around 7.34, 7.50 and 7.93 ppm, which confirmed the retention of
the RAFT end group. In order to calculate the monomer conversion,
an aliquot of the reaction mixture was withdrawn by syringe at the
end of the reaction and analyzed by 1H NMR spectroscopy. The
conversion was determined by the integration of the methylene
backbone proton resonances relative to the olefinic monomer
proton resonances (Eq. (1)). Theoretical number-average molecular
weights were calculated based on Eq. (2), where MWCTA and MWm
are the molecular weights of RAFT agent and monomer, respec-
tively. [M]0 and [CTA]0 are the initial concentrations of monomer
and RAFT agent, respectively. The theoretical molecular weight
shows good agreement with the experimental molecular weight.
A series of identical batches of HEA_TMS based polyacrylates
were synthesized using RAFT polymerization (Entries 1e4 in
Table 1). The structures of the polymers were confirmed by 1H NMR
Fig. 1. a) 1H NMR spectra of a typical homopolymer. b) Stacked 1H NMR for four different batches and c) SEC overlay of the four batches.

116
K. Mishra, A. Joy / Polymer 66 (2015) 110e121
Fig. 2. a) 1H NMR spectra of a typical diblock copolymer. b) Stacked 1H NMR spectra for three different batches with [M]0/[CTA]0 ratio of 150. c) SEC overlay of the three batches of
ratio 150. d) Stacked 1H NMR spectra for three different batches of ratio 50,100 and 150. e) SEC overlay of the variable diblocks.

K. Mishra, A. Joy / Polymer 66 (2015) 110e121
117
microwave reactor. An acrylamide version of the same polymer
also showed good control and reproducibility but showed a higher
molecular weight value due to incompatibility with PS calibration
on the THF SEC (entries 5,6).
1.8
1.6
1.4
1.2
1.0
0.8
3.3. Synthesis of the diblock copolymer
EHA 50
EHA 100
EHA 150
The previous section demonstrated the successful microwave
assisted RAFT polymerization of the protected hydrophilic acrylate
monomer with high end group fidelity. The diblock copolymer was
synthesized by using the first block as a macro-CTA. To a fixed
amount of homopolymer, varied [M]0/[CTA]0 molar ratios were
employed to control the size of the second block. The molar ratios
used were 50, 100 and 150 respectively and yielded 35, 74 and 112
repeat units of the second block. Reproducibility of the diblock
polymer was tested by synthesizing three batches of polymer with
the [M]0/[CTA]0 fixed at 150, and as shown in entries 9e11 in
Table 1 there was a high degree of reproducibility. The structure of
the block copolymers p(HEA_TMS-b-EHAx) (where x ¼ DP of EHA)
was confirmed by 1H NMR spec-troscopy. Fig. 2a shows a typical 1H
NMR spectrum for the block copolymer with the peaks assigned for
the corresponding blocks.
-8
-6
-4
-2
0
log (C, mg mL-1)
Fig. 3. Plot of I384/I373 of pyrene vs. log C. Intersection of tangents (dotted lines) pro-
vide the CMC of the micelles with varying EHA block sizes.
From Table 1, it can be seen that different feed ratios resulted in
diblock copolymers with Mn that varied from 23.4 to 31.5 kg/mol.
While theoretical molecular weight calculations were in agreement
to SEC data for both homopolymers and diblock copolymers, there
was a discrepancy for calculated molecular weight from NMR end
group analysis. This difference was more pronounced in the larger
diblock copolymers, indicating the unreliability of end group
analysis for polymers with high DP. Repetitions of the polymeri-
zation with the same feed ratio gave polymers with reproducible
molecular weight with unimodal peaks (Fig. 2b,c). Polymerizations
with varying feed ratios produced polymers with varying block
sizes. There was good control of the polymerizations and each
proceeded without loss of living character as shown by SEC
(Fig. 2d,e). The polymers were efficiently deprotected in a weakly
acidic medium (acetic acid in THF/methanol).
Table 2
DLS and CMC data.
Polymer sample
CMCa
(m
g mLꢃ1
)
DLS sizeb (nm)
16.4 4.6
PDIc
pHEA-b-EHA_50
pHEA-b-EHA_100
pHEA-b-EHA_150
58.4
18.7
5.5
0.087
0.184
0.239
28.4
4
47.8 8.5
a
CMC determined from pyrene fluorescence experiments.
Size was reported as avg. deviation for multiple runs.
PDI obtained from DLS.
b
c
p
IðeCH2Þ
Conversionð%Þ ¼
ꢁ 100%
(1)
p
m
IðeCH2Þ þ Ið¼ CH2Þ
3.4. Preparation and characterization of micelles
½Mꢂ0 ꢁ MWm ꢁ Conversion
½CTAꢂ0
Mncalc
¼
þ MWCTA
(2)
The prepared diblock polyacrylates self-assemble into micelles
in an aqueous solution upon nanoprecipitation due to their
amphiphilic nature, as reported in the experimental section.
Nanoprecipitation was carried out by slowly adding water to a
stirred solvated mixture of polymer until the solution showed a
faint blue hue. The micellization was studied by both DLS and
fluorescence measurements using pyrene as an extrinsic probe.
Pyrene shows significant fine structure (vibrational bands) in its
fluorescence spectra in solution [39]. The intensities of the various
vibrational bands of pyrene show a strong dependence on solvent
Table 1 summarizes the results for the polymers prepared by
RAFT polymerization as obtained from the SEC and 1H NMR data. It
is shown that a [M]0/[CTA]0 molar ratio of 100:1 affords a molecular
weight between 19.7 and 19.6 kg/mol by SEC. The molecular
weight of each batch of polymer was found to be very similar as
evidenced by SEC and end group analysis (Fig. 1b). Entries 1e4
(Table 1) were polymerized with identical monomer to CTA feed
ratios. The results show that these RAFT polymerizations under
microwave conditions produce polymers with a high degree of
reproducibility at high conversions. While corresponding poly-
merizations carried out under traditional thermal conditions
showed reproducibility at low conversions, significant variability
was observed at higher conversions (Supplementary Information
S9). Thus it was found that microwave based RAFT polymeriza-
tion was an efficient method for obtaining reproducible polymer
chain sizes at high conversions. This is significant from a trans-
lational perspective, where batch to batch variation in molecular
weights of polymeric drug delivery systems is a major concern [38].
An overlay of the SEC spectra of the above polymerizations show
identical unimodal molecular weight distributions with Mw/Mn of
1.14 (Fig. 1c). We hypothesize that the high reproducibility of these
polymerizations is a result of homogeneous activation of radicals,
precise control of the reaction temperature, and stirring rate in the
environment [40]. The ratio of peaks at 384 and 373 nm (I384/I373
)
vary as a function of the micro-environment that pyrene experi-
ences. For example, the ratio is greater than 1 for concentrations
above CMC. The CMC concentration of the particular amphiphile
can be determined from plotting the ratio of intensities vs log of
concentration and analyzing the intersection of tangents at either
end of the curve (Fig. 3). It is seen that the CMC values of the mi-
celles are in the range of 5e58 mg/mL (Table 2). Larger hydrophobic
blocks thermodynamically favor micelle formation at lower CMCs
when compared to shorter blocks. As expected the decrease in CMC
for increasing hydrophobic block sizes is accompanied by an in-
crease in micelle size as determined by DLS. The diameters of the
micelles determined by DLS are in the range of 15e50 nm, and the
polydispersity indices (PDI) are between 0.1 and 0.25 (Table 2).

118
K. Mishra, A. Joy / Polymer 66 (2015) 110e121
Fig. 4. 1H NMR comparison between folic acid and folate azide.
Thus the nanocarriers exhibit well-dispersed individual particles as
evidenced by DLS.
of the nano-micelles. Folic acid contains two carboxylic groups at
a
and positions, of which the -carboxylic group has a higher af-
g
a
finity in the recognition of folic acid [42,43] by its receptor and
therefore should not be chemically modified. Conjugation of folate
3.5. Synthesis of folate azide
must therefore be limited to only the
g-carboxylic acid group.
While there are many papers in literature using different coupling
chemistries to link molecules to the carboxylic groups of folic acid
such as EDC coupling [44,45] and DCC coupling [46], we found that
Conjugation of targeting units to micelles is often proposed as a
method to improve therapeutic efficacy and specificity. Due to the
overexpression of folate receptors [41] in several types of cancers,
folate was chosen as a model targeting ligand for functionalization
several of these reported procedures gave a mixture of
a and g
products. The only method that provided pure -carboxylic acid
g
conjugation was the use of TBTU as reported by Santos et al. [47].
Presumably, the selectivity is provided by the steric effects of the
secondary COOH.
1.2
Using TBTU as a coupling agent, we synthesized folate azide by
coupling folic acid with 2-ethanamine azide (Scheme 1). As char-
acterized by 1H NMR, folic acid shows two carboxylic acid protons
at 12.27 and 11.40 ppm respectively, while the folate azide showed
only the carboxylic acid at 11.40 ppm peak (Fig. 4). The function-
alization was also proven by 13C NMR, HMQC and mass spectrom-
etry. Mass spectrometry revealed only one carboxylic acid product
was formed and did not show any di-functionalized product.
1.0
Folate block copolymer 0.5 mg/mL
Block copolymer 0.5 mg/mL
0.8
0.6
0.4
0.2
0.0
3.6. Alpha (
a) functionalization of RAFT based polymer
250
300
350
400
To prove that
a
end functionalization of the diblock copolymer
Wavelength (nm)
can be carried out efficiently, the diblock copolymer was reacted
with folate azide as outlined in the experimental section. The
resultant polymer was deprotected and dialyzed to obtain pure
Fig. 5. UVeVis spectra of the p(HEA92)-b-(EHA110) diblock copolymer before and after
folate conjugation.

K. Mishra, A. Joy / Polymer 66 (2015) 110e121
119
Fig. 6. A) 1H NMR of omega functionalized homopolymer. B) SEC (DMF) UV detector output for MTS homopolymer (deprotected).
folate conjugated polymer. The success of folate conjugation was
verified using 1H NMR and UVeVis spectroscopy. As seen from the
1H NMR spectra (Fig. 7) characteristic peaks of folate can be found
at 11.39, 8.65, 7.66, 6.90 and 6.65 ppm. However, the triazole proton
resulting from the CuAAC click reaction is masked by the RAFT end
groups in the region of 7.2e8.0 ppm. The functionalized diblock
copolymer is dialyzed extensively after the conjugation reaction to
remove non-covalently adsorbed folate and therefore the presence
of the folate peaks in the NMR spectra proves successful chemical
conjugation to the hydrophilic end of the polymer. The UVeVis
spectra of folate conjugated and non-conjugated micelles also
indicate the successful conjugation of folate (Fig. 5). The peak with a
lmax at 270 nm corresponding to the aromatic chromophore of
folate shows that the diblock polyacrylate micelles have been click-
functionalized with folate. Based on the calibration curve (see
Supporting Information for details) generated from the folate azide
standard solutions, the content of conjugated folate was calculated
to be 1.58 w/w%. Theoretically, the maximum possible content
would be 1.63 w/w%. Therefore, more than 96% of the chain ends
are conjugated with folate.
with detection at 325 nm. For the purposes of testing the feasibility
of the reaction, the homoblock containing 92 units (by end group
analysis) was used for verifying the conjugation. This strategy was
chosen since the deprotected diblock copolymer does not elute out
of the DMF GPC, and end group analysis by NMR is more accurate
for the homopolymer rather than for the diblock. End group anal-
ysis from the NMR spectra (Fig. 6) shows that the derivatized
anthracene substituted the RAFT agent and produced an anthra-
ceneepolymer conjugate with 94 repeating units of the homopol-
ymer, affirming the value calculated using the dithiobenzoate
group. This proved that the attachment of anthracene via methane
thiosulfonate chemistry was highly efficient. Additionally, SEC
analysis on a DMF system using a UV detector at 325 nm gave a
unimodal distribution (Fig. 6B) with similar PDI as the starting
material. On the basis of both these results, it is inferred that the
efficiency of the MTS reaction was greater than 99%.
3.8. Synthesis of telechelic functionalized polymer
As reported in the experimental section, dual functionalization
of the diblock copolymer was prepared by reacting the
a func-
3.7. Omega (
u
) functionalization of RAFT based polymer
tionalized folate polymer with 20 equivalents of anthracene MTS
and 50 equivalents of n-propyl amine. The resulting polymer was
precipitated from cold acetone multiple times to remove any excess
anthracene. Fig. 7 shows the 1H NMR spectra of the folate conju-
gated polymer and that of the dual functionalized (folate and
anthracene) polymers. A change in the downfield region of the
Anthracene was conjugated to the
u
end of the polymer using a
one-pot reaction with a methane thiosulfonate derivatized [48]
anthracene in the presence of a base such as propyl amine. Func-
tionalization with anthracene was verified using 1H NMR and SEC

120
K. Mishra, A. Joy / Polymer 66 (2015) 110e121
Fig. 7. A) 1H NMR of Folate conjugated polymer. B) 1H NMR of telechelic polymer.
spectrum was observed. Due to the complexity of end groups, it is
not possible to accurately integrate the peaks but based on the
initial results (Fig. 6) obtained from u-functionalized homopolymer
with controllable sizes. This end-group functionalizable micellar
platform with controlled reproducibility of polymer block sizes
holds promise as a synthetic tool in drug delivery systems and our
lab is currently exploring these micelles for such applications.
it is expected that the efficiency of conjugation is high. The DLS data
for the dual functionalized polymers is reported in the SI (S7c).
Acknowledgments
4. Conclusions
The authors express their gratitude to The University of Akron
for startup funding which enabled the work described here.
In the above described work, we prepared poly(hydroxyethyl
acrylate)-b-poly(2-ethylhexyl acrylate) diblock copolymers by mi-
crowave assisted RAFT polymerization and functionalized the chain
Appendix A. Supplementary data
ends with folate and anthracene at the
a and u ends respectively.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.polymer.2015.04.013.
We demonstrate that microwave assisted RAFT polymerization is
an effective method for synthesizing block copolymers with
reproducible block sizes. From a translational perspective, repro-
ducibility of polymer block sizes is a critical parameter for approval
of polymer therapeutics. The second important aspect of the cur-
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