A Single Enzyme Mediates the "quasi-Living" Formation of Multiblock Copolymers with a Broad Biomedical Potential

Article abstract of DOI:10.1021/acs.biomac.0c00126

This study describes a unique "quasi-living"block copolymerization method based on an initiation by a single enzyme. We use this term to describe a process where a preformed polymer chain can be reactivated to continue propagating with a second or third comonomer without addition of new catalyst. The presented strategy involves a laccase (oxidoreductase) mediated initial polymerization of 4-hydroxyphenylacetic acid to a homopolymer containing phenolic terminal units, which in turn can be easily reactivated by the same enzyme in the same reaction vessel to continue propagation with a second monomer (tyramine). Increased copolymer yield (up to 26.0percent) and polymer molecular mass (up to Mw = 116 ?000 Da) are achieved through the addition of previously developed micellar and hydrogel enzyme complexing agents. The produced poly(tyramine)-b-poly(4-hydroxyphenylacetic acid)-b-poly(tyramine) is water-soluble and able to self-assemble in aqueous solution. Both tyramine blocks were successfully modified with ibuprofen moieties (up to 24.6percent w/w load) as an example for potential polymer drug conjugation. The copolymerization could be further extended with addition of a third (fluorescent) comonomer in the same reaction vessel to yield a fluorescent pentablock copolymer. The successful modifications and advantageous solution behavior of the produced copolymers demonstrate their viability as versatile drug delivery and/or bioimaging agents, as confirmed by cytotoxicity and cellular uptake studies.

Full text of DOI:10.1021/acs.biomac.0c00126

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Article
A Single Enzyme Mediates the “Quasi-Living” Formation of
Multiblock Copolymers with a Broad Biomedical Potential
Dieter Michael Scheibel, Dandan Guo, Juntao Luo, and Ivan Gitsov*
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ABSTRACT: This study describes a unique “quasi-living” block copolymerization method based on an initiation by a single
enzyme. We use this term to describe a process where a preformed polymer chain can be reactivated to continue propagating with a
second or third comonomer without addition of new catalyst. The presented strategy involves a laccase (oxidoreductase) mediated
initial polymerization of 4-hydroxyphenylacetic acid to a homopolymer containing phenolic terminal units, which in turn can be
easily reactivated by the same enzyme in the same reaction vessel to continue propagation with a second monomer (tyramine).
Increased copolymer yield (up to 26.0%) and polymer molecular mass (up to Mw = 116 000 Da) are achieved through the addition
of previously developed micellar and hydrogel enzyme complexing agents. The produced poly(tyramine)-b-poly(4-hydroxyphenyl-
acetic acid)-b-poly(tyramine) is water-soluble and able to self-assemble in aqueous solution. Both tyramine blocks were successfully
modified with ibuprofen moieties (up to 24.6% w/w load) as an example for potential polymer drug conjugation. The
copolymerization could be further extended with addition of a third (fluorescent) comonomer in the same reaction vessel to yield a
fluorescent pentablock copolymer. The successful modifications and advantageous solution behavior of the produced copolymers
demonstrate their viability as versatile drug delivery and/or bioimaging agents, as confirmed by cytotoxicity and cellular uptake
studies.
limited monomer choice, recent work has focused on the
development of pseudoliving polymerizations which aim to
suppress chain termination rather than completely eliminate it.
Such techniques include atom transfer radical polymerization
(ATRP),13,14 reversible addition−fragmentation chain transfer
polymerization (RAFT),15 and stable free radical mediated
polymerization (SFRP).16 Although these polymerization
techniques have displayed good control of polymer molecular
mass and dispersity in addition to being relatively robust, the
occurrence of traditional chain termination and chain transfer
reactions could cause some loss of functionality on the
propagating polymer chain ends17,18 and thus impede to a
certain extent their applicability for the synthesis of block
copolymers.
1. INTRODUCTION
Development of “living” polymerization systems1 has been of
continuous interest to polymer chemists for the control in
molecular mass and narrow dispersity they provide when
compared with the conventional free radical- or condensation
polymerization techniques. These advantages result from rapid
and quantitative initiation, absence of side reactions, and
extremely slow or absent chain termination processes.2,3
“Living” polymerizations are also noteworthy for their ability
to produce block copolymers which often display interesting
self-assembly behavior4 and have found numerous industrial
uses including controlled drug delivery and release,5−7
patterning in lithography,8,9 and production of porous
materials,10−12 among many other applications. In contrast
to conventional free radical polymerizations, which quickly
undergo chain termination via radical coupling or dispropor-
tionation, this type of polymer synthesis can be utilized in the
formation of block copolymers due to the significantly
enhanced lifetime of the propagating active site owing to the
absence of traditional bimolecular termination processes.1,2
Because of the inherent moisture sensitivity of true “living”
anionic and cationic polymerizations, their incompatibility with
numerous solvents (specifically protic solvents), and relatively
In addition to lack of byproducts and high stereo- and
regiospecificity,18−20 enzymes are often able to mediate
Received: January 27, 2020
Revised: April 1, 2020
Published: April 1, 2020
https://dx.doi.org/10.1021/acs.biomac.0c00126
© XXXX American Chemical Society
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a
Scheme 1. Oxidation Mechanism of Phenolic Substrates by Laccase and Phenoxy Radical Coupling Products
a
Via CC ortho coupling (A), CO ortho coupling (B), CO para coupling (C), and CC para coupling (D).
polymerizations efficiently in either bulk or aqueous media at
or near ambient temperatures,21−23 minimizing solvent and
energy expenditures. Of particular importance is their ability to
induce different chemical transformations in a single-step
procedure. Using these advantages, they have been tested as
comonomer-specific initiators in chemo-enzymatic block
copolymerizations.24 This quoted paper reported the one-pot
synthesis of poly(methyl methacrylate)-b-poly(ε-caprolactone)
in supercritical CO2 at 1500 psi using a bifunctional initiator,
CuBr/bpy, and Novozym-435 as catalysts for the simultaneous
ATRP polymerization of methyl methacrylate and lipase-
mediated ring-opening polymerization of ε-caprolactone,
respectively. Undoubtedly this strategy provides good options
for the synthesis of block copolymers with unusual
compositions; the use of specialized equipment and limited
choice of monomers soluble in supercritical CO2 being the
major drawbacks. While our study was in progress, an
enzymatic cascade reaction was reported for the construction
of multiblock copolymers.25 This interesting but rather
elaborate strategy involves two enzymes: pyranose oxidase,
which sole purpose is to produce H2O2 upon being fed with D-
glucose, and a subsequently added horseradish peroxidase,
which uses the peroxide to produce a radical from
acetylacetone. This radical in turn triggers a reversible-
deactivation radical polymerization and copolymerization of
several water-soluble vinyl monomers in the presence of a
chain transfer agent (a trithiocarbonate). Besides the addition
of a comonomer, the synthesis of the block copolymers
necessitates the addition of extra portions of the two enzymes,
D-glucose and acetylacetone.25
In this current study, we report a new method of “quasi-
living” environmentally benign one-pot block copolymerization
mediated by a single enzyme without the use of any additional
cocatalysts and reagents. Of the numerous enzymes capable of
catalyzing polymerization, laccase, a benzenediol:oxygen
oxidoreductase, has proven the ability to mediate the
polymerization of numerous substrates including vinyl
monomers and more commonly phenol and aniline deriva-
tives.26−28 The polymerization of phenolic substrates by this
enzyme is known to occur via oxidation to a phenoxy radical
with concomitant reduction of oxygen to water (Scheme
1).29,30 The phenoxy radicals formed could undergo either
CC or CO coupling at the ortho- or para- position,
generating a polymer backbone composed of phenol and
phenyl ether repeating units.
reactivate and facilitate the continued propagation of the
polymer chain was demonstrated in one of our previous
articles where an increase in polymer molecular mass was
observed upon the addition of a second portion of tyrosine
monomer to a solution of preformed unnatural poly(tyrosine)
and laccase.31 This reactivation opens an intriguing “quasi-
living” pathway to block copolymers by the simple addition of
a second comonomer to the polymer chain upon depletion of
the first substrate. To our knowledge, there is no analogue of
this strategy in the published literature.
The proof of concept for this new method is demonstrated
here by the copolymerization of two naturally derived
comonomers: 4-hydroxyphenylacetic acid, 4HPAA, (frequently
found in olive oil32) and 4-hydroxyphenylethylamine-tyramine,
(common ingredient in plants33). The copolymerization yields
poly(tyramine)-b-poly(4-hydroxyphenylacetic acid)-b-poly-
(tyramine) being conducted in an aqueous buffer solution at
pH 7 and 45 °C. It requires minimal reaction workup following
an environmentally benign procedure. The molecular mass of
the different blocks is controlled by changing the amount of
comonomer feed.
2. EXPERIMENTAL SECTION
2.1. Materials. 4-Hydroxyphenylacetic acid, 4HPAA (99%),
tyramine (99%), -2-(4-isobutylphenyl)propanoic acid (Ibuprofen,
99%), Rhodamine B Base (99%), anhydrous ethylene diamine (99%),
N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride,
EDC (98%), fluorescein (95%), 7-hydroxycoumarin (99%), pyrene
(99%), 2,5-dihydroxybenzoic acid, DHB (99.5%) and laccase from
Trametes versicolor (≥0.5 U/mg) (all from Sigma-Aldrich, St. Louis,
MO), anhydrous methanol (99.9%, Alfa Aesar), ethyl acetate (99.5%,
Spectrum Chemical), sulfuric acid (95−98%, J.T. Baker), maleic
anhydride (99%, Sigma-Aldrich), dichloromethane, DCM (A.C.S.
Reagent, J.T. Baker), sodium bicarbonate (99%, Alfa Aesar), and
acetic anhydride (98%, J.T. Baker) were used as received. HPLC
grade tetrahydrofuran, THF (from Pharmaco-Aaper, Oakland CA),
was distilled prior to use, removing the inhibitor (butylated
hydroxytoluene), which was found to retard laccase mediated
polymerizations.
The synthesis of 2-(4-isobutylphenyl)-N-(2-aminoethyl)-
propanamide maleimide (ibuprofen-EDA-maleimide) is described as
follows (Scheme 2).
Methyl 2-(4-isobutylphenyl)-N-(2-aminoethyl)propanoate (Ibu-
profen Methyl Ester, 1). To a 100 mL round-bottom flask connected
to a water-cooled condenser was added ibuprofen (5.0 g, 0.024 mol),
50 mL of anhydrous methanol, and 1 mL of sulfuric acid. The
solution was heated at reflux for 12 h after which the solution was
dissolved in ethyl acetate and washed three times with deionized
water. The solvent was removed by rotary evaporator and the product
was obtained as a colorless oil. Yield: 4.91 g, 92%.
In contrast to previously mentioned controlled/living
polymerization methods (i.e., ATRP, RAFT, and SFRP),
laccase-mediated polymerizations of phenolic monomers do
not undergo irreversible chain termination since the polymer
chain formed could be reactivated (oxidized) even if the
terminal phenoxy radical is quenched. The ability of laccase to
1H NMR (DMSO-d6). δ 7.24 (d, 2H), 7.05 (d, 2H), 3.78 (dd, 1H),
3.68 (s, 3H), 2.43 (d, 2H), 1.82 (m, 1H), 1. 61 (d, 3H), 0.91 (d, 6H).
2-(4-Isobutylphenyl)-N-(2-aminoethyl)propanamide (Ibuprofen-
EDA, 2). To a 50 mL round-bottom flask connected to a water-cooled
B
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Scheme 2. Synthesis of Ibuprofen-EDA-Maleimide
Scheme 3. Synthesis of Fluorescent Monomer Fluor-EDA-HPAA
condenser was added 1 (4.9 g, 0.022 mol) followed by ethylene
diamine (20 mL, 0.3 mol). The solution was heated at reflux for 24 h
after which the solution was dissolved in ethyl acetate and washed
three times with dilute sodium bicarbonate solution and finally with
deionized water. The solvent was removed by rotary evaporator and
product 2 was obtained as yellow oil. Yield: 4.71 g, 94%.
Fluorescein Ethylendiamine Spirolactam (Fluor-EDA, 5). Anhy-
drous ethylene diamine (50 mL) and 4 (5.0 g, 0.014 mol) were added
to a 250 mL round-bottom flask connected to a water-cooled
condenser. The solution was heated at reflux for 8 h. During that time,
the solution color gradually changed from yellow to orange. The
reaction mixture was concentrated to approximately 10 mL in volume
and precipitated into deionized water. The precipitate was filtered,
washed thoroughly with deionized water, and dried in a vacuum oven
overnight at 50 °C producing a yellow solid. Yield: 5.17 g, 96%.
1H NMR (DMSO-d6). δ 7.78 (d, 1H), 7.51 (m, 2H), 6.99 (d, 1H),
6.60 (d, 2H), 6.45 (d, 2H), 6.39 (d, 2H), 2.97 (t, 2H), 2.17 (t, 2H).
Fluorescein Ethylendiamine 4-Hydroxyphenylacetamide Spiro-
lactam Derivative (Fluor-EDA-HPAA, 6). To a 100 mL round-bottom
flask were added 5 (1.0 g, 0.027 mol), DCM (50 mL), EDC (6.2 g,
0.040 mol), and 4HPAA (6.2 g, 0.040 mol). The solution was allowed
to stir for 24 h. The reaction solution was washed with three 20 mL
portions of 0.5 M HCl, three 20 mL portions of sodium bicarbonate
solution, and finally with deionized water until a neutral pH was
attained. The organic layer was dried over anhydrous sodium sulfate,
decanted, and solvent evaporated via rotary evaporator to yield a
yellow solid. Yield: 1.09 g, 80%.
1H NMR (DMSO-d6). δ 8.03 (t, 1H), 7.25 (d, 2H), 7.03 (d, 2H),
5.18 (s, 2H), 3.48 (dd, 1H), 3.46 (t, 2H), 2.74 (t, 2H), 2.40 (d, 2H),
1.82 (m, 1H), 1.28 (d, 3H), 0.92 (d, 6H).
2-(4-Isobutylphenyl)-N-(2-aminoethyl)propanamide Maleimide
(Ibuprofen-EDA-Mal, 3). Ibuprofen-EDA (4.5 g, 0.018 mol), maleic
anhydride (5.0 g, 0.05 mol), and dichloromethane (20 mL) were
added to a 50 mL round-bottom flask, connected to a water-cooled
condenser. The solution was stirred at room temperature for 24 h.
After removing the solvent by rotary evaporation, acetic anhydride
(10 mL, 0.09 mol) was added and the solution heated at reflux for 1 h
after which an excess of water was slowly added to the solution. The
aqueous solution was neutralized with sodium bicarbonate and the
product was extracted with 50 mL portions of ethyl acetate. The
removal of solvent by rotary evaporator afforded a solid off-white
product. Yield: 5.35 g, 90%.
1H NMR (DMSO-d6). δ 8.0 (t, 1H), 7.3 (d, 2H), 7.08 (d, 2H), 6.96
(s, 2H), 3.49 (dd, 1H), 3.46 (t, 2H), 3.24 (t, 2H), 2.48 (d, 2H), 1.78
(m, 1H), 1. 31 (d, 3H), 0.83 (d, 6H). See also Figure 8.
The synthesis of fluorescein-labeled 4HPAA was accomplished
using the following protocol, Scheme 3.
1H NMR (DMSO-d6). δ 9.49 (br), 7.81 (t, 1H), 7.51 (m, 2H), 6.99
(d, 1H), 6.93 (d, 2H), 6.62 (d, 2H), 6.58 (d, 2H), 6.58 (d, 2H), 6.45
(d, 2H), 6.39 (d, 2H), 3.11 (s, 2H), 3.03 (t, 2H), 2.76 (q, 2H).
The synthesis of Rhodamine-labeled 4HPAA was accomplished as
follows (Scheme 4).
Fluorescein Methyl Ester (Fluor-OMe, 4). Fluorescein (10.0 g,
0.030 mol), 50 mL of anhydrous methanol, and 5 mL of sulfuric acid
were added to a 250 mL round-bottom flask connected to a water-
cooled condenser. The solution was heated at reflux for 12 h after
which the solution was neutralized with sodium bicarbonate and
concentrated using a rotary evaporator. The residual solid was
suspended in deionized water, mixed thoroughly, filtered, and washed
with 200 mL deionized water. The product was dried overnight in a
vacuum oven at 50 °C and obtained as a red solid. Yield: 9.82 g, 94%.
1H NMR (DMSO-d6). δ 8.15 (dd, 1H), 7.94 (td, 1H), 7.76 (td, 1H),
7.38 (dd, 1H), 7.29 (dd, 2H), 7.03 (d, 2H), 6.56 (dd, 2H), 3.56 (s,
3H).
Rhodamine B Ethylenediamine Spirolactam (Rhd-EDA, 7). To a
100 mL round-bottom flask was added Rhodamine B Base (5.0 g,
0.011 mol) and 50 mL of anhydrous ethylenediamine. The solution
was heated at reflux for 24 h. The solvent was then removed via rotary
evaporator and the residual solid suspended in deionized water. The
suspension was filtered and washed with deionized water until a
colorless filtrate was obtained. It was dried overnight in a vacuum
oven at 50°°C yielding a pink solid. Yield: 5.20 g, 95%.
1H NMR (DMSO-d6). δ 7.77 (d, 1H), 7.49 (m, 2H), 6.99 (m, 1H),
6.35 (m, 6H), 3.33 (q, 8H), 2.97 (t, 2H), 2.19 (t, 2H), 1.09 (t, 12H).
Rhodamine B Ethylenediamine 4-Hydroxphenylacetamide Spi-
rolactam Derivative (Rhd-EDA-HPAA, 8). 4HPAA (4.65 g, 0.030
C
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Scheme 4. Synthesis of Fluorescent Monomer Rhd-EDA-HPAA
mol), 50 mL DCM, EDC (4.65 g, 0.030 mol), and 7 (1.0 g, 0.021
mol) were added to a 100 mL round-bottom flask. The solution was
allowed to stir at room temperature for 24 h. The reaction mixture
was washed with three 20 mL portions of 0.05 M HCl, three 20 mL
portions of sodium bicarbonate solution, and finally with deionized
water until a neutral pH was achieved. The organic layer was dried
over anhydrous sodium sulfate, decanted, and solvent evaporated in
rotary evaporator to yield a pink solid. Yield: 0.79 g, 62%.
detector. Spectra were recorded under ambient conditions at a
resolution of 4 cm−1. A total of 64 scans were recorded for each
spectrum in addition to the background.
2.2.6. Dynamic Light Scattering (DLS). All DLS measurements
were performed on a Malvern Zetasizer ZS instrument. The
instrument used a 633 nm laser source with a fixed backscattering
detector at 173°. Size calculations were performed using a CONTIN
procedure.
2.2.7. Thermogravimetric Analysis (TGA). TGA measurements
were conducted on a TGA Q5000IR (TA Instruments). A
temperature ramp of 10 °C/min was used from room temperature
to 700 °C under a nitrogen flow (10 mL/min).
1H NMR (DMSO-d6). δ 9.21 (s, 1H), 7.80 (s, 1H), 7.52 (m, 3H),
6.97 (s, 1H), 6.93 (d, 2H), 6.60 (d, 2H), 6.35 (m, 6H), 3.30 (q, 8H),
3.10 (s, 2H), 3.05 (t, 2H), 2.79 (q, 2H), 1.06 (t, 12H).
Linear(A)−linear(B) and linear−hyperbranched complexing agents
composed of poly(ethylene glycol), PEG, and either poly(styrene),
PS, or poly(p-chloromethylstyrene), PPCMS, were synthesized by
atom transfer radical polymerization of either styrene or p-
chloromethylstyrene initiated by α-chlorophenylacetyl capped poly-
(ethylene glycol).34 Their designations are PPCMS2.3k-13k-
PPCMS2.3k and PS4.2k-PEG20k-PS4.2k, where the numbers reflect
the molecular mass of the corresponding block (PPCMS, PEG, and
PS).
2.2.8. Matrix-Assisted Desorption and Ionization Time-of-Flight
Analysis (MALDI-TOF). MALDI-TOF MS spectra were collected on a
Bruker Autoflex III having Smartbeam II laser source (Nd:YAG laser,
266 or 355 nm). Spectra were acquired in linear positive mode with
the attenuation set to the lowest value capable of obtaining high-
resolution spectra. A matrix solution was formed by dissolving DHB
in deionized water (18.1 MΩ) at a concentration of 80 mg/mL.
Sample solutions were prepared in deionized water (18.1 MΩ) at a
concentration of 1 mg/mL. Samples were spotted by mixing matrix
solution and sample solution in a 1:1 ratio and spotting 5 μL of the
resulting solution on a MTP 384 target plate (polished steel, Bruker
Daltronics).
2.2.9. Determination of Critical Aggregation Concentration.
Critical aggregation concentration (CAC) was determined using two
methods. CAC was first determined using pyrene as a fluorescent
probe and was conducted by making polymer solutions of various
concentrations in deionized water and adjusting their pH to 7 with
dilute HCl and NaOH. Five microliters of 0.1 mM pyrene in ethanol
was then added. After a 30 min incubation period, the fluorescence
spectra of the solutions were recorded from 360 to 500 nm using an
excitation wavelength of 334 nm and slit width of 1 nm. The ratio of
peak intensity at 384 to 373 nm was plotted against concentration and
the lower inflection point designated as the CAC.
CAC of the synthesized copolymers was verified using DLS and
measuring hydrodynamic diameter. Polymer solutions of similar
concentrations as above were made in deionized water and similarly
adjusted to pH 7 with dilute HCl and NaOH. The hydrodynamic
diameter of the polymer solutions was plotted against concentration,
and the lower inflection point in the curve was used to estimate the
CAC.
2.3. Syntheses. 2.3.1. Block Copolymerization of 4-Hydrox-
yphenylacetic Acid and Tyramine. A typical copolymerization was
conducted by first preparing a 0.2 M pH 7 potassium phosphate
buffer. To 200 mL of buffer solution, 200 mg of laccase is added. In
some cases, 200 mg of complexing agent, dissolved in 2 mL of
distilled THF, was added dropwise under vigorous stirring. The
solution was allowed to acclimate for 4 h at 45 °C. 4HPAA (600 mg,
3.95 mmol) was supplemented under stirring. The polymerization was
allowed to proceed for 72 h at 45 °C after which the polymerization
mixture was divided into 20 mL portions. An aliquot was taken to
2.2. Instrumentation and Methods. 2.2.1. Chromatography.
Size exclusion chromatography (SEC) was conducted on a system
consisting of a Waters 1515 isocratic pump, a Waters 1500 series
manual injector, two PolyPore 5 μm 300 mm × 7.5 mm mixed bed
columns (Polymer Laboratories, Ltd.), and a Waters 2414 refractive
index detector. All analyses were conducted at 60 °C with dimethyl
sulfoxide (DMSO) as eluent containing 0.1% (1 mg/mL) LiBr at a
flow rate of 0.8 mL/min. Sample solutions were filtered using a 0.45
μm cellulose acetate filters prior to injection. Calibration was
performed using 15 monodisperse poly(ethylene glycol) and
poly(ethylene oxide) standards (1.98−452 kDa). Molecular mass
calculations were accomplished with Waters Breeze 3.3 software.
1
2.2.2. NMR Spectroscopy. H NMR and 13C NMR spectra were
recorded using HCl-doped DMSO-d6 as solvent at 22 °C on a Bruker
AVANCE 600 MHz instrument with the solvent signal as the internal
standard.
2.2.3. Fluorescence Spectroscopy. All fluorescence measurements
were performed on a Horiba Fluorolog-3 spectrophotometer with a
SpectrAcq system controller. The instrument used a xenon arc lamp
source. Fluorescence measurements were conducted at 90° using an
excitation wavelength of 490 nm for fluorescein derivatives or 334 nm
for pyrene in water. Quantum yield of polymer samples were
calculated using fluorescein and 7-hydroxycoumarin (umbelliferone)
as standards.
2.2.4. UV−vis Spectroscopy. All UV−vis spectroscopic measure-
ments were conducted on an Agilent 8453 UV−vis spectropho-
tometer at room temperature in deionized water unless otherwise
specified.
2.2.5. FT-IR ATR Spectroscopy. Fourier transform infrared
spectroscopy (FT-IR ATR) spectra were obtained on a Bruker
Tensor 27 spectrophotometer with a MIR source and a DLaTGS
D
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Table 1. Effect of Comonomer Feed on Copolymer Molecular Mass and Yield
a
b
c
b
d
e
entry
4HPAA (mg)
Tyramine (mg)
Mw
Mp
Mn
Mn
Đ
yield (%)
N1
N2
N3
N4
N5
N6
N7
N8
N9
60
60
60
60
60
60
60
60
60
0
5
12700
116000
95500
51500
27800
29000
25500
26800
7000
23500
22000
19900
16500
15400
14900
10700
9400
7500
41000
35700
24200
16500
15800
15000
13200
11700
1.70
2.83
2.67
2.13
1.68
1.82
1.70
1.92
2.08
97.0
87.5
79.4
75.6
70.2
67.7
62.8
59.7
58.4
7300
8300
8700
9300
9400
10000
10500
10700
10
15
20
25
30
35
40
24500
a
b
c
Native laccase used as biocatalyst without complexing agents. Calculated via SEC using PEG standards. Molecular mass at the peak apex in the
d
1
SEC traces. Calculated via H NMR spectroscopy assuming Mn = 7500 for entry N1 and using it as the reference value to compare the integral
intensity ratio between the Hd protons in poly(4-HPAA) and Hf + He protons in the poly(tyramine) block, see Figure 4. (Co)polymer dispersity
e
(Mw/Mn)
a
Scheme 5. Synthesis and Post-Polymerization Modification of Poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine)
a
Only the CC coupling adducts are shown for simplicity.
serve as a reference for the molecular mass of the initial block
(molecular mass was determined via SEC using PEG standards). A
predetermined amount of tyramine was then added to each 20 mL
portion and the copolymerization was allowed to proceed for an
additional 72 h at 45 °C. Polymer isolation was conducted by initially
precipitating the copolymer via acidification of the solution to a pH <
2. The precipitate was collected via centrifugation and washed three
times with 0.2 M HCl and last with deionized water. The acidic
washes served to remove unreacted monomer, biocatalyst, and any
homo poly(tyramine) produced. MALDI-TOF and SEC analysis of
the supernatant revealed that residual material was primarily
unreacted tyramine monomer with little to no poly(tyramine) formed
(Figure S16). In some cases, the sediment was washed with THF to
remove complexing agent. Polymer yield was calculated from the ratio
of the isolated and purified product to the initial amounts of
comonomers added (see Table 1).
Fluor-EDA-HPAA or Rhd-EDA-HPAA comonomer was then
immediately added to the polymerization vessel and the reaction
was allowed to proceed for 72 h. Reaction workup was performed as
described in Section 2.3.1. To remove unreacted comonomer,
additional washes with THF were conducted.
2.3.3. Random Copolymerization of 4HPAA and Tyramine.
Random copolymerizations of 4HPAA and tyramine were conducted
using a similar procedure as described in Section 2.3.1; however, both
monomers were added in one portion and the reaction halted after 72
h.
2.3.4. Postpolymerization Modification of Poly(tyramine)-b-
poly(4HPAA)-b-poly(tyramine). Conjugation of ibuprofen-EDA-mal-
eimide to poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine) was
conducted immediately after the incorporation of the poly(tyramine)
blocks via Aza-Michael addition of the ibuprofen-EDA-maleimide to
the primary amines of the tyramine repeating units in the same
copolymerization flask. The reaction was conducted at 45 °C for 72 h.
Purification of the modified copolymer was accomplished as described
in Section 2.3.1. 1H NMR was used to quantify ibuprofen attachment
to the block copolymer.
2.3.2. Block Copolymerization of 4HPAA and Fluorescein or
Rhodamine Derivatives. Block copolymerizations of 4HPAA with
Fluor-EDA-HPAA and Rhd-EDA-HPAA were conducted by adding
60 mg of preformed poly(4-HPAA) to 20 mL of an equilibrated
solution of buffered enzyme or enzyme-complex solution at 45 °C
(preparation described in Section 2.3.1). Twenty milligrams of either
2.3.5. Cell Viability and Cellular Uptake Studies. See Supporting
Information for a detailed description
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3. RESULTS AND DISCUSSION
3.1. Synthesis of Poly(tyramine)-b-poly(4HPAA)-b-
poly(tyramine). In this study, block copolymers composed
Figure 3. Michaelis−Menten kinetics of native laccase (blue circles),
micelle−laccase complex with PPCMS2.3k-PEG13k-PPCMS2.3k (red
squares), and hydrogel−laccase complex with PS2.4k-PEG13k-PS2.4k
(green triangles). Deionized water, pH 7, room temperature.
Figure 1. SEC elution curves of 4HPAA−tyramine block copolymers
with 0.1% LiBr in DMSO as eluent. See Table 1 for sample
designations. See also Figure S1 showing SEC traces of all
copolymers.
Table 2. Effect of Complexing Agent on Maximum Enzyme
Activity and Substrate Binding
of tyramine and 4HPAA were enzymatically synthesized by
laccase, using a unique quasi-living polymerization procedure.
The interior 4HPAA block was first made by the enzyme-
mediated homopolymerization of 4HPAA. After 72 h, the
exterior polymer blocks were formed by addition of tyramine
to the homopolymer solution in the original reaction vessel still
containing the initial amount of catalytically active laccase,
Scheme 5.
complex
native
Km (mM)
Vmax (μkat)
0.04
0.035
0.03
14.2
32.7
22.1
a
micelle
b
hydrogel
a
PPCMS2.3k-PEG13k-PPCMS2.3k used as complexing agent.
PS4.2k-PEG20k-PS4.2k used as complexing agent.
b
The synthesis of the ABA block copolymers containing A
blocks with different molecular masses was monitored by SEC
(Figures 1, S1, S2, and S3) and H NMR spectroscopy. The
groups in the repeating units of the central B block (Figures 1
and 2, N3−9). The extensive hydrogen bonding and
electrostatic interaction would then shrink the copolymer
coils in the solutions. This assumption would be proven
correct if a molecular mass analysis could be performed with a
technique that does not depend on the hydrodynamic volume
of the copolymers. Indeed, Mn values of the same copolymers
1
copolymers appeared as monomodal peaks in the SEC traces
but interestingly had decreasing hydrodynamic volumes with
the increase of comonomer feed (Figure 1). A similar trend
was observed with the DLS analyses of the same samples
(Figure 2).
This seemingly strange behavior could be explained by the
occurrence of intramolecular interactions between the central
4HPAA B block and segments from the poly(tyramine) A
blocks. At low tyramine feed, those A blocks were short and
could not prevent the copolymer random coils of expanding in
the SEC eluent (Figures 1 and 2, N2). When the feed
increased, A blocks increased in length and the amino groups
in their repeating units were able to interact with the carboxylic
1
calculated by H NMR showed a clear growth tendency with
the increase of the tyramine comonomer concentration, Table
1. A notable exception is only N2, where part of the initial
central block remained unreacted (Figures 1 and 2, N1−N2).
In our previous study,34 we found that the addition of
micellar and hydrogel complexing agents to the reaction
system increases enzyme activity and polymer yields. Here, we
were using two typical representatives of these complexing
Figure 2. Hydrodynamic diameters of 4HPAA−tyramine copolymer solutions in DMSO below CAC.
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Table 3. Effect of Complexing Agent on Copolymer Molecular Mass and Yield
a
a
a
b
a
entry
complexing agent
native
4HPAA (mg)
tyramine (mg)
Mw
Mp
Mn
Mn
Đ
yield (%)
c
N7
60
60
60
30
30
30
25500
21700
17700
14900
12000
8400
15000
13900
10800
10000
10200
10900
1.70
1.55
1.64
62.8
67.9
88.8
d
f
M7
micelle
e
g
H7
hydrogel
a
b
c
Calculated via SEC using PEG standards. Calculated via 1H NMR spectroscopy with respect to the Mn of the 4HPAA homopolymer. Entry N7
d
e
f
g
from Table 1. Entry M7 from Table S1. Entry H7 from Table S2. PPCMS2.3k-PEG13k-PPCMS2.3k used as complexing agent. PS4.2k-
PEG20k-PS4.2k used as complexing agent.
1
Figure 4. H NMR spectra (600 MHz) of poly(4-HPAA) (red), poly(tyramine)-b-poly(4-hydroxyphenylacetic acid)-b-poly(tyramine) (green),
and poly(tyramine) (blue) recorded at 22 °C in DMSO-d6/HCl. Solvent impurities are marked with (×).
agents, PPCMS2.3k-PEG13k-PPCMS2.3k (micelle forming)
and PS4.2k-PEG20k-PS4.2k (hydrogel forming). The effect
these complexing agents had on both maximal enzyme activity
(Vmax) and the efficiency of substrate binding to the enzyme
complex (Km) was determined as previously described34 using
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid and Line-
weaver−Burk plot (see Figure S18), Figure 3 and Table 2.
The yield of the 4HPAA−tyramine block copolymers was
positively affected and increased by up to 9.4% (micelle) and
26.0% (hydrogel), Table 3. As the yield and molecular mass of
the interior poly(4-hydroxyphenylacetic acid) block was not
significantly affected by the addition of complexing agents
(Tables 1, S1, and S2), the improved copolymer yield could be
attributed to the formation of the poly(tyramine) blocks. This
buildup was enabled by the enzyme complexes’ ability to
actively sequester the tyramine comonomer, which was only
slightly soluble in the aqueous polymerization medium. This
low comonomer solubility explains the decrease in copolymer
yields observed when only native laccase was used, Table 1.
With the amphiphilic complexing agents, the tyramine
concentration around the enzyme active site was increased,
facilitating generation of the active tyramine phenoxy radicals
and enhancing copolymerization rate. As 4HPAA was
inherently highly water-soluble, enabling ample interaction
with the native laccase, it was not surprising that addition of
complexing agents had a minimal effect on polymerization
yield or poly(4HPAA) molecular mass, Tables 1, S1, and S2.
3.2. Characterization of Poly(tyramine)-b-poly-
(4HPAA)-b-poly(tyramine). Characterizations of poly-
(tyramine)-b-poly(4HPAA)-b-poly(tyramine) copolymers syn-
thesized using either native laccase or laccase complexes were
conducted using NMR and FT-IR ATR spectroscopy. 1H
NMR spectra of the produced block copolymers (Figure 4)
displayed broad peaks characteristic of the rigid phenyl
backbone. Signals from 8.5 to 7.75 ppm were assigned to
protons of the pronated amine of the tyramine repeat unit.
Aromatic protons Hb, Hb′’, Hc, Hc′ were visible within 7.5−6.5
ppm. Benzylic protons of the poly(4-hydroxyphenylacetic acid)
interior block Hd were assigned to the signals from 3.75 to 3.25
ppm, while the signals in the region of 3.25−2.5 ppm were
attributed to the ethylene protons He and Hf of the
poly(tyramine) blocks. The 13C NMR and FT-IR ATR spectra
of the synthesized block copolymers are provided in the
Supporting Information (Figures S6 and S7).
TGA of poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine),
Figure 5 green trace, displays two distinct decomposition
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Figure 5. Differential TGA thermogram of poly(4HPAA), black; poly(tyramine), red; poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine), green;
random poly(tyramine-co-4HPAA), blue; and a mixture of poly(4-HPAA) and poly(tyramine), pink. TGA thermograms of monomers and the
mixture of homopolymers are provided in the SI.
stages at 326.15 and 430.62 °C corresponding to degradation
of the carboxylic groups in the 4HPAA block and the amine
functionalities in the tyramine repeating units, respectively.
Similar decomposition events are observed for the homopol-
ymers poly(4HPAA) and poly(tyramine) at 367.94 and 414.10
°C (Figure 5, black and red traces). Differences in degradation
events between the block copolymer and the homopolymers
are attributed to the zwitterionic nature of the block copolymer
resulting in the presence of a carboxylate anion of decreased
stability compared to the free acid of the homopolymer.
Similarly, the protonated amine of the block copolymer
provides increased stability resulting in a higher degradation
temperature. The decomposition pattern is notably different
from the decomposition behavior of the random copolymer,
which showed a single decomposition event at 432.92 °C.
Increased thermal stability of the random copolymer may be
due to intermolecular hydrogen bonding of the side chains
stabilizing the carboxylic and amine functional groups. These
TGA analyses provide an additional independent proof that a
block copolymer formation is strongly favored over the
homopolymerization of the tyramine added.
Figure 6. Fluorescence intensity ratios of pyrene excitation bands
(I384/I373) depending on the concentration of poly(tyramine)-b-
poly(4HPAA)-b-poly(tyramine), Table 1, N7: orange triangles.
Hydrodynamic diameter of poly(tyramine)-b-poly(4HPAA)-b-poly-
(tyramine) depending on concentration measured by dynamic light
scattering at pH 7, Table 1, N7: blue squares.
Figure 7. Hydrodynamic diameter of 4HPAA−tyramine copolymer micelles at a concentration of 1 mg/mL (above CAC) formed in pH 7 buffer
solution (Table 1, N2−N9).
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1
Figure 8. H NMR spectra (600 MHz) of poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine), top green; ibuprofen conjugated poly(tyramine)-b-
poly(4HPAA)-b-poly(tyramine), middle red; and ibuprofen-EDA-maleimide (bottom, blue). Solvent impurities are marked with (×).
Table 4. Effect of Complexing Agent on Ibuprofen-EDA-
Maleimide Addition to Poly(tyramine)-b-poly(4HPAA)-b-
poly(tyramine)
dynamic light scattering (Figure 6, blue squares), where onset
micellization concentration of 0.21 mg/mL was determined.
The broad concentration range over which an increase in
fluorescence intensity ratio and hydrodynamic diameter is
observed may be attributed to the relatively disperse
copolymer. Notably the random copolymer did not change
its hydrodynamic diameter with an increase in concentration
(Figure S10), providing another independent proof that the
sequential addition of a second comonomer to the preformed
poly(4-HPAA) steered the process toward a block copolymer
formation.
All copolymers self-assemble at concentrations close to 1
mg/mL, Figure 7. It should be noted that in contrast to the
decreasing sizes at concentrations below CAC (Figure 2), the
hydrodynamic diameters of the copolymer aggregates above
CAC grow with increased molecular mass of the hydrophobic
poly(tyramine) blocks (Figure 7, N3−N9). The practical size
overlap observed with N6−N9 could be attributed to the
increased entanglement and collapse of the aggregate core with
the enlarged poly(tyramine) blocks.
a
complexing
agent
4HPAA
(mg)
tyramine
(mg)
ibuprofen incorporated
a
entry
(wt %)
N7-I
M7-I
H7-I
native
micelle
hydrogel
60
60
60
30
30
30
24.6
14.1
18.0
b
c
a
An equimolar amount of ibuprofen-EDA-maleimide relative to
tyramine was added in the same reaction flask immediately after the
b
copolymerization of the tyramine block. Calculated by 1H NMR
c
spectroscopy. PPCMS2.3k-PEG13k-PPCMS2.3k used as complexing
d
agent. PS4.2k-PEG20k-PS4.2k used as complexing agent.
3.3. Critical Aggregation Concentration Determina-
tion of Poly(tyramine)-b-poly(4HPAA)-b-poly-
(tyramine). The addition of two blocks of limited water
solubility provides interesting application possibilities based on
the characteristic amphiphilic character of these block
copolymers. Critical aggregation concentration was determined
by monitoring changes in the fluorescence spectra of pyrene.35
This change in fluorescence was quantified by measuring the
fluorescence intensity ratio at 384−373 nm (Figure 6, red
triangles). The onset of aggregation was determined by the
intersection of the regions of the low and sharp rise in the ratio
of the two intensities and was found to be 0.25 mg/mL.
This result was verified by measuring the hydrodynamic
diameter of the same copolymer at various concentrations by
These results also hint at the mechanism of propagation for
these comonomers. Both have three reactive sites: the two
ortho-positions in the benzene ring and the phenolic hydroxyl
group. Therefore, a potential three-dimensional growth
involving all possible sites could lead to loosely branched or
even hyperbranched macromolecules. However, such struc-
tures would not undergo such a dramatic size change above
CAC due to their increasingly rigid structures. That is why we
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a
Scheme 6. Block Copolymerization of Fluorescent Comonomers
a
Only CC linkages shown.
a
Table 5. Effect of Complexing Agent on Fluorescein Comonomer Addition to Poly(4HPAA)
b
entry
complexing agent
native
poly(4HPAA) feed (mg)
Fluor-EDA-HPAA feed (mg)
Fluor-EDA-HPAA incorporated (wt %)
quantum yield
N-FEH
M-FEH
H-FEH
60
60
60
20
20
20
5.7
11.3
9.9
0.07
0.15
0.13
c
micelle
d
hydrogel
a
b
c
Reaction conditions described in Section 2.3.1. Calculated via UV−vis spectroscopy. PPCMS2.3k-PEG13k-PPCMS2.3k used as complexing
d
agent. PS4.2k-PEG20k-PS4.2k used as complexing agent.
Table 6. Molecular Mass Effects of Complexing Agent on
Fluorescein Comonomer Addition to Poly(4HPAA)
Table 8. MM Effect of Complexing Agent on Rhodamine
Comonomer Addition to Poly(4HPAA)
a
a
complexing
agent
complexing
agent
entry
Mn
Mw
Mp
Đ
entry
Mn
Mw
Mp
Đ
poly(4-HPAA)
N-FEH
M-FEH
8200
9600
12300
9800
16800
18900
22200
19600
8500
8800
11600
2.05
1.97
1.81
2.01
poly(4-HPAA)
N-REH
M-REH
8200
9200
13600
10600
16800
16500
22800
19300
8500
9500
15800
2.05
1.79
1.66
1.81
native
micelle
hydrogel
native
micelle
hydrogel
b
d
b
d
c
c
H-FEH
10700
H-REH
11400
a
b
a
b
Reaction conditions described in Section 2.3.1. PPCMS2.3k-
Reaction conditions described in Section 2.3.1. PPCMS2.3k-
c
c
PEG13k-PPCMS2.3k used as complexing agent. PS4.2k-PEG20k-
PS4.2k used as complexing agent. Corresponding to addition of six
PEG13k-PPCMS2.3k used as complexing agent. PS4.2k-PEG20k-
PS4.2k used as complexing agent. Corresponds to addition of 12
d
d
monomer units
comonomer repeating units.
assume that the block copolymers most likely have linear
construction.
3.4. Ibuprofen Conjugated Poly(tyramine)-b-poly-
(4HPAA)-b-poly(tyramine). Modification of poly-
(tyramine)-b-poly(4HPAA)-b-poly(tyramine) with ibuprofen
was conducted using a maleimide derivative of ibuprofen
connected via an ethylenediamine linker. The conjugation was
conducted immediately after copolymerization of the poly-
(tyramine) blocks in the same reaction vessel and was
confirmed by 1H NMR spectroscopy (Figure 8). The
ibuprofen addition was quantified by comparing the signal of
the methyl groups of the isobutyl functional group on
ibuprofen (1−0.75 ppm) to the aromatic protons (7.3−6.9
ppm). Surprisingly, it was found that both micellar and
a
Table 7. Effect of Complexing Agents on Rhodamine Comonomer Addition to Poly(4HPAA)
b
entry
complexing agent
native
poly(4HPAA) feed (mg)
Rhd-EDA-HPAA feed (mg)
Rhd-EDA-HPAA incorporated (wt %)
quantum yield
N-REH
M-REH
H-REH
60
60
60
20
20
20
2.7
18.1
8.9
0.10
0.20
0.18
c
micelle
d
hydrogel
a
b
c
Reaction conditions described in Section 2.3.1. Calculated via UV−vis spectroscopy. PPCMS2.3k-PEG13k-PPCMS2.3k used as complexing
d
agent. PS4.2k-PEG20k-PS4.2k used as complexing agent.
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Scheme 7. Copolymerization of Fluorescein Comonomer with ABA Copolymer
Figure 9. SEC traces of poly(Fluor-EDA-HPAA)-b-poly(4HPAA)-b-poly(Fluor-EDA-HPAA) formed with different complexing agents in 0.1%
LiBr DMSO as eluent. See Table 5 for sample designations.
Figure 10. SEC traces of poly(4HPAA) (blue) and poly(Rhd-EDA-HPAA)-b-poly(4HPAA)-b-poly(Rhd-EDA-HPAA) in 0.1% LiBr DMSO as
eluent. See Table 7 for sample designations.
hydrogel enzyme complexes reduced the ibuprofen conjuga-
tion to the block copolymers (Table 4). This diminished
conjugation efficiency was probably caused by the preferential
ibuprofen derivative binding to the complexes and therefore
having a decreased ability to react with the water-soluble block
copolymer. This might initially appear to contradict the
rationale for the improved yield and molecular mass of the
block copolymers provided in Section 3.1.
It should be noted, however, that the copolymerization of
tyramine necessitates interaction of the comonomer with
laccase and therefore benefits from the increased local
tyramine concentration afforded by the enzyme complexes,
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Figure 11. SEC trace of poly(4HPAA) (blue), poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine) (green), and poly(Rhd-EDA-HPAA)-b-
poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine)-b-poly(Rhd-EDA-HPAA) (yellow) in 0.1% LiBr DMSO as eluent. See Table 9 for sample
designations.
Table 9. Molecular Mass Characteristics of Poly(Fluor-EDA-HPAA)-b-poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine)-b-
a
poly(Fluor-EDA-HPAA) Pentablock Copolymer
b
c
c
c
entry
Fluor-EDA-HPAA incorporated (wt %)
Mn
Mw
Mp
Đ
poly(4-HPAA)
triblock
pentablock
6300
19500
29400 2900
14100
43000
48700 2000
4500
2.24
2.19
1.65
13000
d
e
7.8 1.1
15100 200
a
b
c
Reaction conditions described in Section 2.3.1. SEC traces of polymers shown in Figure 11. Calculated via UV−vis spectroscopy. Calculated by
d
e
SEC using PEG standards. Conducted in triplicates. Corresponds to addition of 4 Fluor-ADA-HPAA comonomer repeating units.
copolymers (Tables 5 and 7) as reduced incorporation
would certainly result in a lower quantum yield. Although
the fluorescein comonomer (Fluor-EDA-HPAA) did contain
additional phenolic moieties on the fluorescein pendant group,
neither fluorescein or fluorescein−ethylenediamine spirolac-
tam polymerized in the presence of laccase. It can therefore be
surmised that polymerization of the fluorescein comonomer
occurred solely through the phenolic group of the amide linked
4-hydroxyphenylacetamide group.
3.6. Synthesis of poly(Fluor-EDA-HPAA)-b-poly-
(tyramine)-b-poly(4HPAA)-b-poly(tyramine)-b-poly-
(Fluor-EDA-HPAA) Pentablock Copolymers. To further
demonstrate the “quasi-living nature” of the developed block
copolymerization, pentablock copolymers were synthesized
from poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine) tri-
block copolymers and the aforementioned Fluor-EDA-HPAA
comonomer (Scheme 7).
SEC traces (Figures 9 and 10) of the synthesized block
copolymers showed a molecular mass increase indicating the
addition of the fluorescein comonomer as exterior blocks
(Figure 11). This was further confirmed via UV−vis
spectroscopy, which showed the characteristic fluorescein
absorption, Table 9.
The relatively large size of the Fluor-EDA-HPAA como-
nomer is the probable reason for the low copolymerization
efficiency (∼4 repeating units, Table 9). The notably different
hydrodynamic size of the pentablock micelles vs those of the
triblock further confirms the attachment of the Fluor-EDA-
HPAA comonomer (see DLS traces in Figure S5). The smaller
hydrodynamic diameter (58.77 nm, pentablock vs 105.7 nm,
triblock) can be explained by the voluminous fluorescein-based
blocks decreasing the extent of aggregation (aggregation
number). This assumption is also supported by the decrease
Table 10. Zeta Potential of Poly(4-HPAA), Poly(tyramine)-
b-poly(4HPAA)-b-poly(tyramine), and Poly(Fluor-EDA-
HPAA)-b-poly(tyramine)-b-poly(4HPAA)-b-
poly(tyramine)-b-poly(Fluor-EDA-HPAA)
zeta
sample
potential std dev
poly(4HPAA)
poly(tyramine)-b-poly(4HPAA)-b-poly(tyramine)
poly(Fluor)-b-poly(tyramine)-b-
poly(4HPAA)-b-poly(tyramine)-b-poly(Fluor)
−1.84
−20.5
−15.2
0.601
1.55
3.94
while the coupling of ibuprofen maleimide to tyramine
repeating units in the copolymer occurs spontaneously without
the need of a catalyst or a complexing agent and therefore
suffers from the unfavorable partition coefficient.
3.5. Synthesis of Fluorescent Triblock Copolymers.
Fluorescent triblock copolymers were successfully produced
from the copolymerization of fluorescein and rhodamine
4HPAA derivatives (Scheme 6). The laccase complexes
showed seemingly better copolymerization efficiency (in-
creased wt % incorporated), but in general notably lower
copolymer molecular masses were observed with the
fluorescent comonomers compared to the tyramine triblocks
(Tables 6 and 8 vs Table 3). On one side, the lower apparent
molecular masses could be caused by distinctly different
hydrodynamic volumes of these fluorescent block copolymers
affecting their elution time through the SEC separation media.
The lower values, however, are most likely the result of both
the increased steric hindrance in the bulky comonomers
compared to tyramine and their significantly lower water
solubility hampering their polyaddition to the reactivated chain
ends of poly(4HPAA). Indirect proof of this assumption was
provided by the quantum yield of the produced block
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Figure 12. Cell viability of CHO (A) and SKOV3 (B) cell lines determined by MTS assays after 72 h incubation with different concentrations of
block copolymers.
Figure 13. Confocal fluorescence microscopy images of SKOV3 ovarian cancer cells and Raw 264.7 macrophage cells incubated with 1 mg/mL of
fluorescent fluorescein modified poly(tyramine-b-4-hydroxyphenylacetic acid-b-tyramine), FITC, for 24 h at 37 °C. The nuclei were stained with
DAPI in blue; the green FITC signal revealed the location of fluorescein modified polymers in the cells; the lysosomes were stained with the
LysoTracker Red in red color. The scale bar is applicable for all images.
of the pentablocks’ zeta potential compared to the triblock
precursor (Table 10).
synthetic polymers were observed to be nontoxic on CHO
cells at 1 mg/mL concentration except for poly(4HPAA),
which exhibited a mild cytotoxicity with 30% reduction of cell
viability. In SKOV3 ovarian cancer cell culture, all these block
copolymers reduced cell viability to nearly 60% at 1 mg/mL,
which may be due to the higher metabolite rates in cancer
cells. It was determined in all cases that the polymers were not
cytotoxic at concentrations up to 0.5 mg/mL indicating the
good biocompatibility of these polymers over a wide
concentration range for potential biomedical applications.
3.7. Cytotoxicity Studies. To evaluate the synthesized
block copolymers as drug delivery and bioimaging agents,
cellular toxicity of the synthesized polymers was determined by
MTS assays. Poly(4HPAA), poly(tyramine)-b-poly(4HPAA)-
b-poly(tyramine), and ibuprofen- and fluorescein-modified
block copolymers were incubated in the cell cultures of a
normal cell line Chinese hamster ovary (CHO) cells and an
ovarian cancer SKOV3 cell line, respectively (Figure 12)
(protocol described in SI). After 72 h incubation, all these
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The cell uptake properties of the nanoparticles would define
their in vivo fate and biodistribution. Cellular uptake of the
fluorescein modified poly(tyramine-b-4-hydroxyphenylacetic
acid-b-tyramine), FITC, in SKOV3 ovarian cancer cells and
Raw 264.7 macrophage cells was studied in vitro using confocal
laser scanning microscope. As shown in Figure 13, significant
cell uptake of the fluorescent copolymer was observed in the
cytosol of both cells, where the FITC signal was colocalized
with LysoTracker Red (a fluorescent probe freely permeating
cell membranes), indicating an endocytosis pathway for the
cell uptake of the nanoparticles. Interestingly, the copolymer
was observed to have a greater cell uptake in SKOV-3 ovarian
cancer cells than that in Raw 264.7 macrophage cells, which is
preferred for the in vivo delivery of antitumor drugs without
significant clearance by macrophages. In addition, the
conjugation of tumor targeting ligands on the functional
groups of the copolymer will further improve the tumor
targeted drug delivery, which will be tested in the future.
or bioimaging agents and are currently being explored in a
separate collaborative effort.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.0c00126.
Copolymerization data; SEC and DLS of copolymers,
copolymer fluorescence spectra; MALDI-TOF spectra,
SEC analysis of copolymerization supernatants, NMR
spectra of copolymers produced by post copolymeriza-
tion modification, enzyme Km and Vmax calculation
method, cytotoxicity protocol (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Ivan Gitsov − Department of Chemistry, State University of New
York − ESF, Syracuse, New York 13210, United States; The
Michael M. Szwarc Polymer Research Institute, Syracuse, New
York 13210, United States; orcid.org/0000-0001-7433-
8571; Email: igivanov@syr.edu
4. CONCLUSIONS AND OUTLOOK
Block copolymers composed of 4-hydroxyphenylacetic acid
and tyramine were successfully synthesized utilizing a single
enzyme catalyzed “quasi-living” copolymerization. To a certain
extent the process resembles the “living” polymerization as the
polymer formed could continue growing upon introduction of
another portion of the same or other monomer and without
adding or reactivating the catalyst. In the experiments
performed, laccase was first used to polymerize 4-hydrox-
yphenylacetic acid by a known mechanism (Scheme 1). To the
resulting polymer-enzyme solution tyramine was added as the
second comonomer and was able to continue polymer
propagation onto the poly(4-hydroxphenylacetic acid), which
retained reactivity via the freely mobile phenol end groups.
Molecular mass of the tyramine blocks was controlled by
adjusting comonomer feed. Although limited to phenolic
monomers, the reported method is advantageous in that it is
not inhibited by irreversible chain termination via radical
coupling or disproportionation, which are often encountered in
the conventional radical polymerization. Additionally, as the
initial homopolymerization and the subsequent copolymeriza-
tion are conducted enzymatically using water as solvent, the
described strategy is both environmentally benign and
inexpensive. Addition of previously developed micellar and
hydrogel complexing agents34 increased yields by up to 9.4%
and 26.0%, respectively, and increased copolymer molecular
mass, as well. The utilized enzyme complexes therefore provide
an efficient and low cost approach to improve copolymeriza-
tion efficiency. The scope of the method was further expanded
with the successful one-pot synthesis of a pentablock
copolymer without any supplementation of additional
biocatalyst or complexing agent.
Authors
Dieter Michael Scheibel − Department of Chemistry, State
University of New York − ESF, Syracuse, New York 13210,
United States
Dandan Guo − Department of Pharmacology, State University
of New York Upstate Medical University, Syracuse, New York
13210, United States
Juntao Luo − Department of Pharmacology, State University of
New York Upstate Medical University, Syracuse, New York
13210, United States; orcid.org/0000-0002-3538-9453
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.0c00126
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge Dr. Arthur Stipanovic
(State University of New York − ESF) for access to SEC
instrumentation. Partial funding for this study was provided by
State University of New York Network of Excellence on
Materials and Advanced Manufacturing (Award 1126892-1-
71036 to I.G.).
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