Amine-Functionalized Polylactide-PEG Copolymers

Article abstract of DOI:10.1021/acs.macromol.7b02751

The formation of halogenated carboxylic acid intermediate followed by a ring-closing reaction led to amino-functionalized asymmetrical lactide monomer. PEG-based functional diblock and triblock polylactides were synthesized via a controlled ring-opening p

Full text of DOI:10.1021/acs.macromol.7b02751

Article
Cite This: Macromolecules XXXX, XXX, XXX−XXX
Amine-Functionalized Polylactide−PEG Copolymers
Mehmet Onur Arıcan,† Sezgi Erdogan,† and Olcay Mert*,†,‡
̆
‡
†Department of Polymer Science and Technology and Department of Chemistry, Kocaeli University, 41380 Kocaeli, Turkey
S
* Supporting Information
ABSTRACT: The formation of halogenated carboxylic acid
intermediate followed by a ring-closing reaction led to amino-
functionalized asymmetrical lactide monomer. PEG-based
functional diblock and triblock polylactides were synthesized
via a controlled ring-opening polymerization in a solvent-free
medium with high conversions (up to 96%), low polydisper-
sities as low as 1.06, monomodal GPC traces, and short reaction
time (only 1 h). No polymerization of symmetrical monomer,
synthesized via condensation of (S)-(+)-CBZ-4-amino-2-
hydroxybutyric acid, proved that the preferred site in the
mechanism of ring-opening polymerization was found as a methyl site in asymmetrical lactide monomer. A highly efficient
deprotection of copolymers was carried out in the presence of H2 gas and Pd/C catalyst without any degradation to obtain the
corresponding free amine-functionalized aliphatic poly(α-hydroxy acid)s. These biodegradable thermosensitive polymers, suitable
for any local therapy applications, were injectable around 42 °C (sol) and a gel just after cooling to body temperature. Faster
hydrolytic degradation (up to 47% in 30 days) and more effective paclitaxel release from copolymer gels (up to 95% in 20 days)
than well-known conventional PEG−PLA gels may make functional lactides a preferred candidate for developing controlled/
sustained release of drugs from delivery vehicles.
A lack of reactive functional group along the polymer
backbone is a major shortcoming for these conventional
poly(α-hydroxy acid)s. Therefore, there remains a significant
need for a diversity of functional groups like alkene,13,14
allyl,15−18 alkyne,19−21 carboxylic acid,22−28 hydroxy,23,24,29−31
and amine23,32,35−38 groups as a side chain in poly(α-hydroxy
acid)s. This inevitable necessity greatly enhances the physical
and chemical properties of conventional polymers and provides
the opportunity to further modulate them. This is also better
alternative to commonly studied aliphatic polyesters (i.e., PLA
and PLGA) because functional side chains on the polymer
backbone help to control the hydrophilicity, to adjust
mechanical strength, to increase the degradation rate, and to
bind biologically active compounds (i.e., RGD peptide) for
active targeting.3,23,33,34
Among various presenting functionalities, especially amine-
functionalized poly(α-hydroxy acid)s have drawn increasing
interests in scientific area due to its inherent proper-
ties.23,32,35−38 For example, cationic polylactides were specifi-
cally prepared from amine functionalization due to its unique
basic character in different pathways for use in medicinal
applications in recent years.35−38 Poly[α-(4-aminobutyl)-L-
glycolic acid] (PAGA) homopolymer, prepared from poly-
condensation of Nε-cbz-L-oxylysine with the elimination of
water under reduced pressure, binds DNA due to cationic
character of amine group for use in gene delivery system.
1. INTRODUCTION
The technology of controlled drug delivery systems is one of
the most rapidly developing fields of science in which
medicinal, natural, and engineering departments make great
contributions to human health care. Such delivery systems
present many advantages including improved efficacy, reduced
toxicity, and improved patient compliance and convenience
when compared to conventional methods.1 Poly(α-hydroxy
acid)s have drawn great attention in biological applications,
especially in the field of drug delivery systems because of their
biodegradable and biocompatible properties.2
Some formulations based on polylactide (PLA), polyglyco-
lide (PGA), and poly(lactide-co-glycolide) (PLGA) approved
by the Food and Drug Administration (FDA) are the most
extensively studied class of poly(α-hydroxy acid)s (or poly-
(substituted glycolide)s) with the outstanding mechanical,
physical, and thermal properties.2,3 But there is a significant
need for the development of new biomaterials making an
alternative to well-known polylactides. Contrary to the
extensive literature for PLA, there were few reports on
nonreactive poly(substituted glycolide)s, which are structural
analogous of lactic acid.4−10 Various symmetric and/or
asymmetric substituted glycolides (i.e., diethyl,5 diisobutyl,5,6
diisopropyl,4,6,7 dicyclohexyl,7 cyclohexyl methyl,7 dibenzyl,6,8,9
dihexyl,5,9,10 isopropylmethyl,9 butylmethyl,9 hexylmethyl,9−11
benzylmethyl,9,12 and trimethyl9 glycolide) were synthesized,
and then their polymerization reactions were performed with
various catalysts (i.e., Sn(Oct)2, DMAP) in the presence of
monoalcohol (i.e., benzyl alcohol) by ring-opening polymer-
ization.
Received: December 29, 2017
Revised: March 24, 2018
© XXXX American Chemical Society
A
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methane (99%), triethylamine (99%), N,N-dimethylformamide
(DMF) (99%), sodium bicarbonate (NaHCO3) (99.7%), diethyl
ether (99.5%), toluene (99.7%), ethyl acetate (99.5%), hexane (95%),
and benzene from Sigma-Aldrich (Germany) were used as received.
(3S)-cis-3,6-Dimethyl-1,4-dioxane-2,5-dione (L-lactide (LLA)) (98%)
was recrystallized three times from anhydrous ethyl acetate and dried
under vacuum before use in polymerization. Poly(ethylene glycol)
methyl ether (MePEG-2000) (Fluka), poly(ethylene glycol) (PEG-
2000) (Sigma), and tin(II) 2-ethylhexanoate (Sn(Oct)2) (Aldrich,
95%) were employed in the syntheses of copolymers. Palladium 10%
on carbon (Pd/C (10%)) (TCI Europe) catalyst was employed for the
removal of the protecting group from copolymers. The paclitaxel (ptx)
anticancer drug (99.5%) was purchased from Alfa Aesar. The
acetonitrile (Sigma-Aldrich, 99.9%), which was used as mobile
phase, was filtrated with a filtration apparatus prior to use in high
performance liquid chromatography (HPLC) for drug release
experiments.
However, poorly controlled molecular weights, broad molecular
weight distributions, and unfavorable synthesis conditions (i.e.,
5 days of reaction under very low pressure of 10−4 mmHg)
reduce the usefulness of PAGA polymers.35 Aromatic polyester
bearing pendant amine salt, capable of both cell penetration
and gene delivery, was prepared from O-carboxyanhydrides via
thiol−yne photochemistry. Multistep synthesis to the polymer
(total of seven steps: O-carboxyanhydride monomer in five
steps and polymer in two steps), overall 24% yield to final
polymer from BOC-L-tyrosine (Boc = tert-butoxycarbonyl)
starting material, some toxicity just after 100 μg/mL polymer
concentration, and degradation problem due to consisting of
aromatic group were major shortcomings of polymer.36
Tertiary amine-functionalized cationic polylactides (CPLAs)
were also prepared from allyl-functionalized lactide, synthesized
via multistep synthesis with 30% overall monomer yield,
followed by thiol−ene click functionalization by using benzyl
alcohol (BnOH) or α-methoxy-ω-hydroxyl PEG (mPEG-OH)
as initiators for gene delivery.16,37,38 Although tertiary amine
groups are suitable for electrostatic interaction, tertiary amine
restricts the reactivity to any further functionalization with any
ligand molecule for any other applications.
Weck’s group prepared monomers of serine, lysine, and
glutamic acid derivatives containing different lengths of side
chains having amine, alcohol, and carboxylic acid groups.23
Then, their homopolymers and copolymers with lactide were
obtained in bulk medium at 140 °C for 24 h (or 8 h for
copolymers). However, serious deviations from the theoretical
molecular weight and some oligomers due to low polymer-
ization rate and/or possible transesterification reactions when
compared to polylactide were observed. Chen et al. have
reported the synthesis of O-carboxyanhydride monomer
prepared from lysine and polymerize it to make polyesters
bearing pendant amino groups.32 On the other hand, the side
reaction products were observed due to misinsertion of the
propagating chain end into the disfavored 2-position of the O-
carboxyanhydride ring with the formation of carboxylic acid
end group having no ability further propagation.
In this article, we describe the novel synthesis of free amine-
functionalized, ready to any further functionalization with
ligands or any biological molecules, polylactide−PEG diblock
and triblock copolymers by ring-opening polymerization
(ROP) with very convenient and simple strategies (i.e., short
step of monomer synthesis, solvent-free polymerization, high
conversions, low polydispersities, and very short reaction
times). As far as we know, our study is the first of a kind,
establishing a new hydrogel-based platform composed of
amine-functionalized polylactide for use in the local therapy.
These tunable amine functional thermosensitive hydrogels
showed liquid property (sol) around 42 °C, suitable for
injection, and turned instantly into a gel form at body
temperature to cover tumor surface. Fast hydrolytic degrada-
tion and effective paclitaxel release from copolymer gels may
make these amine-functionalized lactides a preferred candidate
for developing controlled/sustained release of drug from
delivery vehicles.
Characterization. The attenuated total reflectance−Fourier trans-
form infrared (ATR-FTIR) spectra of intermediate, monomers, and
copolymers were obtained on an ATR Bruker-Tensor 27 spectrometer
between 600 and 4000 cm−1. Nuclear magnetic resonance (NMR)
spectra were acquired using Bruker Avance III 400 MHz and
VarianUNITY INOVA 500 MHz spectrometer. Liquid chromatogra-
phy/mass spectrometry-time-of-flight (LC/MS-TOF) data were
recorded on a 1260 Infinity Agilent with TOF-MS unit (Model
6230). Gel permeation chromatography (GPC) measurements were
performed on a Viscotek RI max system consisting of autosampler,
pump, oven, Viscotek RI detector (VE3580), and two LT4000L
Viscotek T-column columns (1500 A pore size, 7 μm particle size, 8
mm i.d., 300 mm length) in series. Tetrahydrofuran (THF) was used
as an eluent at flow rate of 1.0 mL min−1 at 35 °C. Refractive index
(RI) detector was calibrated with polystyrene (PS) standards having
narrow-molecular-weight distribution between 1200 and 400 000 Da.
Data were analyzed using Viscotek OmniSEC software. HPLC
measurements were conducted on a 1260 Infinity Agilent system
equipped with an ultraviolet (UV) detector and ZORBAX SB-C18 4.6
× 150 mm, 3.5 μm HPLC column. Thermal properties of the
polymers were observed with differential scanning calorimetry (DSC)
analysis using a Mettler Toledo DSC Star 1 instrument. The samples
were heated from −60 to 180 °C with heating/cooling rate of 10 °C
under a nitrogen atmosphere. Thermogravimetric analyses (TGA)
were performed on a Mettler Toledo TGA 1 Star System under a
nitrogen atmosphere between 25 and 600 °C at a heating rate of 40 °C
min−1. Rigaku Miniflex II diffractometer with Cu Kα (λ = 1.5418 Å)
radiation at 45 kV/40 mA was used for wide-angle X-ray diffraction
(WAXD) analyses. The measurements were performed at a 2θ angle of
5°−50°, a scanning rate of 1°/min, and a scanning step of 0.02°.
Synthesis of (S)-(+)-CBZ-4-Amino-2-hydroxybutyric Acid (2).
(S)-(−)-4-Amino-2-hydroxybutyric acid (14.8 g, 0.124 mol) was
dissolved in a solution of sodium hydroxide (10.4 g, 0.26 mol) in water
(100 mL). Benzyl chloroformate (CBZ) (23.6 g, 0.136 mol) was
added dropwise into the stirred solution over 2 h in an ice/salt bath,
and the reaction was maintained for a further an hour at the same
temperature. After the reaction mixture was washed with diethyl ether
(100 mL), aqueous phase was treated with diluted HCl until pH 2 was
achieved. Then, it was extracted with diethyl ether (4 × 100 mL). The
combined organic extracts were washed with saturated NaCl and dried
with Na2SO4. Solvent was removed by rotary evaporation under
reduced pressure, and the residue was recrystallized from benzene to
obtain (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid (2)39 as a white
1
solid (78%). H NMR (d6-DMSO, 500 MHz) δ: 1.54−1.7 (m, 1H),
1.74−1.92 (m, 1H), 3.05−3.17 (distorted q, 2H), 3.92−4.03 (m, 1H),
4.95−5.05 (s, 2H), 7.16−7.27 (t, J = 5.1 Hz, 1H), 7.28−7.43 (m, 5H),
11.2−14.0 (br, 1H). 13C NMR (d6-DMSO, 125 MHz) δ: 34.1, 37.1,
2. EXPERIMENTAL SECTION
65.1, 67.6, 127.6, 127.7, 128.3, 137.2, 156.1, 175.6. ATR-FTIR (νmax
/
cm−1): 3329 (NH), 3062, 3034, 2951 (CH), 1736, 1686 (CO).
Materials. (S)-(−)-4-Amino-2-hydroxybutyric acid (96%), benzyl
chloroformate (CBZ) (98%), 2-bromopropionyl bromide (97%), p-
toluenesulfonic acid monohydrate (PTSA.H2O) (98.5%), sodium
hydroxide (NaOH) (98−100.5%), hydrochloric acid (HCl) (36.5−
38%), sodium chloride (NaCl), sodium sulfate (Na2SO4), dichloro-
Synthesis of CBZ Protected 3-Aminoethyl-6-methyl-1,4-
dioxane-2,5-dione (ZNEtMG) (4). Synthesis of CBZ protected
asymmetric glycolide monomer was performed in two steps. In the
first step, 2-bromopropionyl bromide (0.84 mL, 8.06 mmol) was
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added to a solution of (S)-(+)-CBZ-4-amino-2-hydroxybutyric acid
(1.6 g, 6.32 mmol) in dichloromethane (80 mL) at 0 °C under a
nitrogen atmosphere. Triethylamine (1.45 mL) in dichloromethane (5
mL) was added dropwise over a period of 1 h to a vigorously stirred
solution in an ice/salt bath. The reaction was further stirred at 0 °C
over 30 min after the addition was completed. The progress of the
reaction was followed with thin layer chromatography (TLC, silica gel,
60 F254, CH2Cl2:CH3OH:CH3COOH (10:1:0.5)). Then, the TLC
plate was treated with a ninhydrin solution (Rf: 0.69). The reaction
mixture was diluted with more diethyl ether, washed with deionized
water (3 × 10 mL), and dried over Na2SO4. Ether was evaporated in
vacuo to give a pale yellow viscous liquid 4-(((benzyloxy)carbonyl)-
amino)-2-((2-bromopropanoyl)oxy)butanoic acid (3, intermediate)
poly(LLA-co-ZNEtMG) (10), Sn(Oct)2 (0.05 mmol, 20 mg),
MePEG-2000 (0.12 mmol, 240 mg), L-lactide (1.8 mmol, 260 mg),
and ZNEtMG (0.2 mmol, 62 mg) were added into the polymerization
tube. The polymerization reaction was kept at 120 °C for an hour
under a nitrogen atmosphere. Copolymer 10 was purified by dissolving
1
in methanol at 40 °C and then precipitated at −20 °C. H NMR
(CDCl3, 400 MHz) δ: 1.4−1.7 (d, J = 6.9 Hz, 3H; m, 3H), 2.0−2.4
(m, 2H), 3.35−3.39 (br, 1H; s, 3H), 3.42−3.49 (m, 1H), 3.51−3.56
(m, 1H), 3.57−3.75 (s, 4H), 5.0−5.5 (q, J = 6.7 Hz, 1H; m, 1H; m,
1H; m, 2H), 7.28−7.46 (m, 5H). 13C NMR (CDCl3, 100 MHz) δ:
16.6, 20.5, 30.7, 36.7, 66.7, 68.8, 69.0, 69.2, 70.5, 128.1−134.9, 156.4
169.5, 169.6, 169.7. ATR-FTIR (νmax/cm−1): 2978, 2938, 2862 (CH),
1755 (CO).
1
Similarly, the other copolymers (9, 11−18, 21, 22) were prepared
with the same protocol as above except for the synthesis of triblock
copolymers. The PEG homopolymer having two hydroxyl groups was
used in place of MePEG homopolymer having a methoxy group on
one end and a hydroxyl group on the other end for poly(LLA-co-
ZNEtMG)−PEG−poly(LLA-co-ZNEtMG) triblock copolymer syn-
theses.
Deprotection for Protected Copolymers. Deprotection of
copolymers was accomplished with a modified protocol.29 50 mg of
Pd/C (10%) was added into a solution of protected copolymers 10 or
15 (100 mg) in dichloromethane (8 mL). First, hydrogen gas
(balloon) was passed from the system to remove the air. Catalytic
hydrogenation was performed for 7 days with vigorously stirring at
room temperature under hydrogen atmosphere. The mixture was
filtrated over Celite to remove the catalyst from the reaction medium
followed by evaporation of solvent under vacuum to obtain the
deprotected copolymers 19 and 20.
Gel−Sol Phase Transition. Gel−sol transition temperatures of
copolymers were determined by following the procedures previously
reported.4,40 Gels at given concentrations were prepared from
copolymers and deionized water. The gel-to-sol transition temperature
of the copolymers was observed visually by inverting the vials at
different temperatures using a controlled water bath. Briefly, all
copolymers with deionized water were vortexed to determine whether
they form homogeneous mixture or not. Then, they were kept at 4 °C
for 30 min in fridge for the equilibrium before immersing them in a
temperature-controlled water bath. The gel-to-sol transition temper-
atures of the copolymers were examined from 4 to 80 °C with 2 °C
increments. The vials were maintained in water bath for 3−4 min at
each temperature before tilting.
Degradation Study. Hydrolytic degradation of block copolymers
was performed in phosphate buffered saline (PBS) at physiological
conditions (pH: 7.4, 37 °C, 200 rpm). 15 mg of polymer was added
into 5 mL of PBS in the test tubes followed by incubation. Samples
were taken out at predetermined time intervals, and then, the
supernatant was removed. They were washed thoroughly with
deionized water to remove salt residues and then stored at −20 °C
to lyophilize them. The resulting polymers were dissolved in THF for
GPC analyses. The percentage of degradation products was
determined by Lorentzian formulation using Origin 9 pro software
according to the literature.49
(70%). H NMR (CDCl3, 400 MHz) δ: 1.68−1.92 (m, 3H), 2.02−
2.32 (m, 2H), 3.18−3.48 (m, 2H), 4.28−4.54 (m, 1H), 4.98−5.12 (s,
2H), 5.12−5.2 (m, 1H), 5.2−5.3 (br, 1H), 7.22−7.44 (m, 5H), 8.5−
9.3 (br, 1H). 13C NMR (CDCl3, 100 MHz) δ: 21.4, 21.6, 21.7, 30.9,
37.1, 39.2, 39.6, 39.9, 67.1, 67.7, 70.9, 71.0, 128.1, 128.2, 128.3, 128.4,
128.6, 135.8, 136.2, 156.8, 158.2, 169.6, 169.8, 173.2, 174.6. ATR-
FTIR (νmax/cm−1): 3338 (NH), 3066, 3034, 2942 (CH), 1720 (CO).
In the second step, to a vigorously stirred suspension of NaHCO3 (2.1
g, 25 mmol) in dimethylformamide (100 mL), a solution of
intermediate 3 (5.82 g, 15 mmol) in dimethylformamide (40 mL)
was added at 40 °C over 4 h. The reaction was monitored with thin
layer chromatography (TLC, silica gel, 60 F254, hexane:ethyl acetate
(2:1)) to avoid oligomeric species. After the mixture was stirred for
further 3 h, the temperature was maintained, and solvent was
evaporated under reduced pressure. The residue was dissolved with
diethyl ether (50 mL). Obtained organic phase was washed with
deionized water (3 × 10 mL) and dried over Na2SO4. After removing
the ether, the resulting residue was recrystallized two times from
diethyl ether to afford pure benzyl (2-(5-methyl-3,6-dioxo-1,4-dioxan-
2-yl)ethyl)carbamate (ZNEtMG) as a white solid 4 (finally recovered
1
yield after double recrystallizations is 67%). H NMR (CDCl3, 500
MHz) δ: 1.5−1.7 (d, J = 6.5 Hz, 3H), 1.96−2.18 (m, 1H), 2.24−2.48
(m, 1H), 3.24−3.52 (distorted q, J = 6.1 Hz, 2H), 4.85−5.01 (q, J =
6.5 Hz, 1H), 5.01−5.06 (dd, J = 4.2, 7.5 Hz, 1H), 5.06−5.14 (s, 2H),
5.18−5.3 (br, 1H), 7.2−7.46 (m, 5H). 13C NMR (CDCl3, 125 MHz)
δ: 15.7, 30.6, 36.7, 66.9, 72.4, 73.8, 128.2, 128.3, 128.6, 136.3, 156.7,
167.0, 167.3. ATR-FTIR (νmax/cm−1): 3346 (NH), 3032, 2947, 2895
(CH), 1767, 1693 (CO). LC/MS-TOF (C15H17NO6Na): theoretical:
330.10 g/mol; experimental: 330.08 g/mol.
Synthesis of CBZ Protected 3,6-Diaminoethyl-1,4-dioxane-
2,5-dione (ZDNEtG) (5). Synthesis of dibenzyl ((3,6-dioxo-1,4-
dioxane-2,5-diyl)bis(ethane-2,1-diyl))dicarbamate (ZDNEtG) was
performed by modifying the method in the literature.4,5 (S)-
(+)-CBZ-4-Amino-2-hydroxybutyric acid (1.5 g, 6 mmol) and p-
toluenesulfonic acid monohydrate (30 mg, 0.15 mmol) mixture in
toluene (90 mL) was refluxed with using Dean−Stark apparatus for 5
h in order to eliminate water. The proceeding of the reaction was
monitored with thin layer chromatography (TLC, silica gel, 60 F254
,
hexane:ethyl acetate (3:1) eluent mixture). Then, the TLC plate was
treated with a potassium permanganate (KMnO4) stain (Rf: 0.4). The
flask was allowed to cool room temperature, and then toluene was
evaporated under reduced pressure. The resulting residue was washed
with cold toluene, and the solvent was separated by decantation. The
obtained crystals were dissolved with ethyl acetate and recrystallized
from hexane at −20 °C to eliminate impurities. After the solvent was
removed, CBZ protected 3,6-diaminoethyl-1,4-dioxane-2,5-dione
In Vitro Paclitaxel Release Study. Release studies were
conducted for poly(LLA-co-ZNEtMG)-PEG, poly(LLA-co-NEtMG)-
PEG, and PLLA−PEG block copolymers according to the same
methodology that was described in a previously published work.4
Paclitaxel, which was selected as an anticancer drug, was loaded into
copolymer gels effectively with loading ratio of 1.0%. Briefly, for the
preparation of MePEG−poly(LLA-co-ZNEtMG) diblock copolymer
10 gel, 1.17 mg of paclitaxel, 117 mg of compound 10, and 233 μL of
deionized water were added to the 1.5 mL vial, and the sample was
vortexed at room temperature for 5 min to get homogeneous drug
loaded gel. Then drug loaded gels were allowed to stand at 4 °C for 30
min in fridge for equilibrium. Other drug loaded copolymer gels were
prepared in the same manner as shown in Table 1. Then, 650 μL of
2% Tween 80 in PBS buffer at pH 7.4 was added to the surface of the
drug loaded gels at room temperature for drug release studies. These
samples were kept in incubator at 37 °C with a constant speed of 200
1
(ZDNEtG) was obtained (58%). H NMR (CDCl3, 400 MHz) δ:
1.91−2.1 (m, 2H), 2.4−2.52 (m, 2H), 2.9−3.48 (br, 2H), 3.53−3.64
(m, 2H), 3.86−3.96 (m, 2H), 4.35−4.45 (dd, J = 8.2, 10.5 Hz, 2H),
5.28−5.33 (s, 4H), 7.32−7.48 (m, 10H). 13C NMR (CDCl3, 100
MHz) δ: 27.1, 42.1, 68.4, 70.4, 128.3, 128.5, 128.6, 135.0, 151.2, 174.4.
ATR-FTIR (νmax/cm−1): 3445 (NH), 3000, 2940, 2880 (CH), 1742
(CO). LC/MS-TOF (C24H26N2O8Na): theoretical: 493.16 g/mol;
experimental: 493.18 g/mol.
General Procedure for Copolymer Syntheses. Syntheses of
diblock and triblock copolymers were carried out in bulk as previously
described.4 Briefly, in order to perform the synthesis of MePEG−
C
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there were no oligomeric products in the purified monomers.
In addition, the purified monomer was analyzed with GPC to
check oligomeric species, and no oligomeric peaks were
obtained.
Table 1. Gel Preparation with Paclitaxel Drug at 1% Drug
Loading Ratio
copolymer
(mg)
drug
(mg)
deionized
water (μL)
ID
Chemical analysis of intermediate 3 was performed by ATR-
MePEG−poly(LLA-co-ZNEtMG), 10
MePEG−poly(LLA-co-NEtMG), 19
117
117
115
1.17
1.17
1.15
233
233
235
1
FTIR, H, and 13C NMR techniques. A single broad band at
1720 cm−1 as a combination of three carbonyl groups was
observed in ATR-FTIR spectrum of intermediate 3, whereas
two carbonyl stretching vibrations in the starting material 2
separately appeared at 1686 and 1736 cm−1 (Supporting
Information Figures S1 and S2). Also, two new resonances at
poly(LLA-co-ZNEtMG)−PEG−
poly(LLA-co-ZNEtMG), 15
poly(LLA-co-NEtMG)−PEG−
poly(LLA-co-NEtMG), 20
115
1.15
235
MePEG−PLLA, 21
PLLA−PEG−PLLA, 22
112
140
1.12
1.4
238
210
1
1.68−1.92 ppm (CH3) and 4.28−4.54 ppm (CH) in the H
NMR spectrum and three new resonances at 21.4−21.6−21.7
ppm (CH3), 39.2−39.6−39.9 ppm (CH), and a new carbonyl
peak at 169.6−169.8 ppm were observed in the 13C NMR
spectrum of intermediate 3 when compared to starting material
2. Moreover, the shifting of the CH proton and carbon in
starting material 2 from 3.92−4.03 and 67.6 ppm to 5.12−5.2
and 70.9−71.0 ppm in 1H and 13C NMR proved the formation
of intermediate 3, respectively (Figures S13, S14, S22, and
rpm. At different time periods, 650 μL of supernatant was taken out
from the vial, and the same amount of fresh supernatant was added.
Before the measurements, collected supernatants in Eppendorf tubes
were kept in a fridge at −20 °C. The amounts of paclitaxel in the
supernatants were analyzed via HPLC at 227 nm using a UV detector.
HPLC assays were repeated three times for each release group, and
drug-free gels were also analyzed to eliminate the influence of low
characteristic signals of copolymers.
1
S23). ZNEtMG 4 monomer was analyzed by ATR-FTIR, H,
13C, COSY 2-D, HMQC 2-D NMR, and LC/MS-TOF
techniques. Its chemical structure was confirmed by the shifting
of the CH proton and carbon in intermediate 3 from 4.28−4.54
to 4.85−5.01 ppm and from 39.2−39.6−39.9 to 72.4 ppm in
3. RESULTS AND DISCUSSION
Syntheses of Protected Functional Monomer. CBZ
protected 3-aminoethyl-6-methyl-1,4-dioxane-2,5-dione
(ZNEtMG) (4) was synthesized via two-step reaction
sequence: the formation of halogenated carboxylic acid
intermediate 3 from (S)-(+)-CBZ-4-amino-2-hydroxybutyric
acid39 with 2-bromopropionyl bromide in dichloromethane
(DCM) at 0 °C and cyclocondensation reaction of the
intermediate 3 with NaHCO3 in dimethylformamide at 40 °C
(Scheme 1). According to NMR analyses, intermediate 3
yielded a 1:1 mixture of diastereomers (R,S and S,S) while a
stereoselectivity was observed for final monomer 4 (R,S or S,S).
Overall synthetic yield of functional lactide monomer 4 and 5
starting from commercially available compound 2 was 47% and
58%, respectively. Some oligomerizations have been observed
at longer reaction times, whereas at shorter reaction times, the
starting material has not been converted to the monomers at
high rates, as reported previously.4 Therefore, the conversion of
the monomers to the oligomeric species was minimized by
monitoring the reactions by TLC, and thus higher yields of the
monomers were obtained. NMR measurements proved that
1
the ZNEtMG 4 in H and 13C NMR, respectively (Figure 1a,b
and Figures S14, S23). The formation of ZNEtMG 4 was also
observed in 13C NMR by the shifting from 173.2 to 174.6 ppm
(acid carbonyl group of intermediate 3) to 167.3 ppm (ester
carbonyl group of ZNEtMG 4) (Figure 1b and Figure S23).
The HMQC 2-D NMR spectrum was recorded to indicate the
direct proton−carbon shift correlation in monomer 4. It has
been proven from HMQC 2-D NMR spectrum that a-coded
peaks are bound to CH3 carbon and protons; b1 and b2 coded
protons are bound to b carbon; c and f-coded peaks refer to
peaks of CH2 carbon and protons; d and e coded peaks refer to
peaks of CH carbons and protons; and the h, i, j, and k coded
peaks show the peaks of the carbon and protons of the aromatic
ring. The l, m, and n coded peaks are also the peaks of carbonyl
groups (Figure 1c). In the COSY 2-D NMR spectrum of the
compound 4 shown in Figure 1d, the interactions of the
neighboring protons which indicate spin−spin coupling
Scheme 1. Synthesis of CBZ Protected Amine-Functionalized Monomer 4
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1
Figure 1. H NMR (a), 13C NMR (b), HMQC 2-D NMR (c), and COSY 2-D NMR (d) spectra of ZNEtMG 4.
interactions between the correlated nuclei in the structure 4
were observed in the cross-peaks of horizontal and vertical axes.
The presence or absence of these cross-peaks has been
employed effectively in the assignment of skeleton connectiv-
ities in the monomer structure. The diagonal peaks here serve
only as reference points. The off-diagonal peaks at points 1, 2,
3, 4, and 5 represent coupling of protons of “a” with “d”, “b1,
b2” with “c and e”, “c” with “b1, b2, and g”, “e” with “b1, b2”, and
“b1” with “b2” in ZNEtMG 4. There is no cross-peak for “k, l,
m, and n” because it does not possess any hydrogens.
The 1H NMR spectrum of MePEG−poly(LLA-co-ZNEtMG)
diblock copolymer 10 with the peak assignments is shown in
Figure 2a. Resonances appearing at 1.4−1.7 ppm, 2.0−2.4 ppm,
3.35−3.39 ppm, 3.42−3.49 and 3.51−3.56 ppm, 5.0−5.5 ppm,
and 7.28−7.46 ppm were assigned to methyl protons (a, b) of
poly(LLA-ZNEtMG) block, methylene protons (c) of
ZNEtMG, both amine protons (d) of ZNEtMG and methoxy
protons (e) at the end of the MePEG block, methylene protons
(f) of ZNEtMG, methine protons (h, i, j) of poly(LLA-
ZNEtMG) and methylene protons (k) of protecting group
(−O−CH2−C6H5), and aromatic protons (l, m, n), respec-
tively. The singlet signal appearing at 3.57−3.75 ppm belongs
to methylene protons (g) of MePEG block in the copolymer.
The multiplet peak at 4.18−4.42 ppm is attributed to
methylene protons of LLA-ZNEtMG units connected to the
MePEG block (poly(LLA-co-ZNEtMG)−COO−CH2−CH2−)
and the methine proton neighboring the hydroxyl end-group
(−COO−CH(CH2CH2NHCBZ)OH), which prove copolymer
Polymerization and Deprotection. MePEG−poly(LLA-
co-ZNEtMG) diblock and poly(LLA-co-ZNEtMG)−PEG−
poly(LLA-co-ZNEtMG) triblock copolymers were obtained
via the ring-opening polymerization using monomers of
ZNEtMG 4 and L-lactide and the terminal MePEG or PEG
as an initiator in the presence of Sn(Oct)2. All polymerization
reactions done under solvent-free medium at 120 °C are
presented in Scheme 2. The benzyl protective groups were
removed and free amine side chains were obtained via catalytic
hydrogenolysis by using H2 gas to give functional copolymers
19 and 20. Molecular weights of copolymers were specifically
kept in a specific range (generally less than 10K) in order to be
suitable for sol−gel preparation by changing the mole ratio of
ester monomers while keeping a constant mole ratio of initiator
and catalyst, as seen in Table 2. The repeating unit of the
ZNEtMG and LLA in the block copolymers were determined
1
formation (Figure 2a). H NMR spectroscopy also verifies the
polymerization of the ZNEtMG monomer by the appearance of
a methine protons peak at 5.0−5.5 ppm, too. In addition, very
1
high conversions (>90%) were obtained from the H NMR
spectra of copolymers by integration of the polymer peaks
relative to monomer peaks (Table 2). The 13C NMR spectrum
of compound 10 also showed resonances of main peaks at 16.6
(CH3), 69.0 (CH), 169.6 (CO) for lactide units, 70.5 (CH2)
for MePEG, and 20.5 (CH3), 30.7 (CH2−CH2−NH), 36.7
(CH2−CH2−NH), 66.7 (−CH2C6H5), 68.8 (CH−CH3), 69.2
(CH−CH2−), 128.1−134.9 (aromatic ring), 156.4
(−NHCOO−), 169.5 (−COCHCH3), and 169.7
(−COCHCH2−) for the ZNEtMG unit, and the end units,
the neighboring units to the end, and some low quantity of
1
from H NMR spectra by comparing the intensity of the
methine proton signal of polymers at δ = 5.0−5.5 ppm, the
phenyl proton signal of ZNEtMG unit at δ = 7.28−7.46 ppm
and methylene proton signal of MePEG or PEG at δ = 3.57−
3.75 ppm.
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Scheme 2. Syntheses of Protected and Deprotected Block Copolymers
heterotacticity (Figure S26). The reaction of compound 10
with 33% HBr/AcOH led to partial deprotection (∼60%) with
significant degradation. Complete removal of the protecting
group without backbone degradation was accomplished with
H2 gas using Pd/C catalyst. GPC analysis of the deprotected
polymer 19 confirmed that there was no chain scission under
these reaction conditions (Figure 2d). Deprotection of
compound 10 was easily confirmed by the disappearance of
phenyl protons and carbons at 7.28−7.46 and 128.1−134.9
ppm in 1H and 13C NMR, respectively (Figure 2b,c and Figure
S26).
The molecular weight distribution of copolymers was
determined with GPC. These copolymers exhibited a
monomodal peaks with polydispersity indices varying from
1.06 to 1.26, indicating the monodisperse properties of the
synthesized copolymer chains (Table 2). On the other hand,
LLA-ZNEtMG feed ratio was optimized at 90/10% because
beginning of homopolymerization occurred with increasing
feeding ratio of ZNEtMG monomer for copolymers 12 and 13
(Table 2). A lower reactivity of ZNEtMG than L-lactide in the
copolymerization was due to more bulky side groups in
ZNEtMG. GPC analysis also showed that longer reaction times
lead to decreasing the molecular weight and increasing the
polydispersity value of copolymer (data not shown). Therefore,
the polymerization time was optimized as 1 h. As can be seen
from the Figure 2d, MePEG homopolymer was not detected on
the GPC traces of copolymers, proving that syntheses of
copolymers were effectively accomplished with absence of
unreacted MePEG homopolymer. Also, MePEG−poly(LLA-co-
ZNEtMG) (10) was analyzed with ATR-FTIR spectroscopy.
The peak at 1755 cm−1 corresponds to the stretching vibration
of carbonyl groups, while peaks at 2978, 2938, and 2862 cm−1
are related to C−H stretching vibrations (Figure S6).
Gel−Sol Transition Properties. The gel−sol transition
temperatures were determined for PEG based polyester diblock
and triblock copolymers 10, 15, 19, 20, 21, and 22 by the
changing of the concentration of copolymers.4,40 Our object
was specifically to prepare appropriate copolymers that display
a sol behavior at around 42 °C, which is proper for injection,
and then a gel with quick cooling to body temperature. Thus,
each component’s length in the diblock and triblock PEG based
polyesters was adjusted with a great attention during the
chemical syntheses. The gel-to-sol transition upon heating can
be seen in Figure 3a−c. Both 44 and 42 °C at 33.5% for
MePEG−poly(LLA-co-ZNEtMG) (10) and MePEG−poly-
(LLA-co-NEtMG) (19) and 46 and 42 °C at 33% for
poly(LLA-co-ZNEtMG)−PEG−poly(LLA-co-ZNEtMG) (15)
and poly(LLA-co-NEtMG)−PEG−poly(LLA-co-NEtMG)
(20) were found as an appropriate critical gel−sol transition
temperature, respectively. These results indicated that the gel−
sol transition temperatures were slightly lower for copolymers
19 and 20 due to removal of the hydrophobic CBZ protecting
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group. Triblock copolymers 15 and 20 showed higher gel−sol
temperature than diblock copolymers 10 and 19 at a constant
33% concentration due to more hydrophobic nature in both
sides of triblock copolymer. Also, amine-functionalized diblock
copolymers 10 and 19 were compared with MePEG−PLLA 21
in terms of gel−sol temperature and concentration. Copolymer
21, as expected, reached the desired gel−sol temperature (40
°C) at lower concentration and showed higher gel−sol
temperature at a constant 33% concentration due to hydro-
phobic nature of MePEG−PLLA 21 when compared to
copolymer 10 and 19. (Figure 3) On the other hand, PLLA−
PEG−PLLA triblock 22 copolymer indicated transitions from
gel to sedimentation41 except for very high concentrations. A
higher mole of hydrophobic ester relative to the PEG moiety in
the copolymers expectedly resulted in nonhomogenous
suspensions even at 10% concentration (i.e., copolymer 11).
Therefore, the molecular weight of the copolymers (2 mol of
ester content) was purposely kept within a specific range for the
gel−sol experiments. As a result, the aqueous polymer solutions
form a gel at high concentration levels and at a lower
temperature and became a sol at lower concentration and a
higher temperature (Figure 3d). In addition, deionized water
has a weakly acidic medium, possibly leading to the protonation
of amine groups in copolymer in the preparations of gels;50
therefore, the gel−sol transitions were also determined in a
buffer of pH: 7.4. It was observed that the change in pH led to a
decrease in gel−sol transitions of around 4.0 °C for protected
diblock copolymer 10, 2.0 °C for deprotected diblock
copolymer 19, 6.0 °C for protected triblock copolymer 15,
and 4.0 °C for deprotected triblock copolymer 20.
Release Studies. The release behavior of paclitaxel
anticancer drug from the MePEG−poly(LLA-co-ZNEtMG)
diblock 10, poly(LLA-co-ZNEtMG)−PEG−poly(LLA-co-
ZNEtMG) triblock 15, MePEG−poly(LLA-co-NEtMG) di-
block 19, and poly(LLA-co-NEtMG)−PEG−poly(LLA-co-
NEtMG) triblock 20 gels were examined with HPLC over 20
days at 37 °C. They were compared with MePEG−PLLA
diblock 21 and PLLA−PEG−PLLA triblock 22 copolymers.
There was an initial burst release of 15% to 23% for copolymers
10, 15, 19, and 20 after 48 h. It was about 6.5% and 13.5% for
PLLA−PEG copolymers 21 and 22, respectively. Briefly, side
chain length (1) and hydrophilicity (2) independently affect the
paclitaxel release rate. Longer length of the side chain (1)
increases free volume in the polymeric matrix which is a
prerequisite for a primarily diffusion controlled drug delivery.
An increase in free volume also leads to a decrease in the glass
transition temperature (i.e., copolymer 19: Tg: 17 °C). Lower
glass transition temperature than release temperature causes
more flexible and mobile chains, creating much more free
volume in the polymeric matrix,42−44 resulting in faster release
of the paclitaxel drug. These effects appear in drug release of
copolymers 19 and 21 as follows: A burst release of about 23%
paclitaxel from copolymer 19 was observed while only 6.5%
drug was released from copolymer 21. After 20 days, similar
behavior for copolymers 19 and 21 was observed as a drug
release of 72% and 28%, respectively. A similar behavior about a
close relation between the glass transition temperature and the
temperature at which the release takes place was reported for
flurbiprofen and 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin
(mTHPP) loaded PLA and PLGA nanoparticles in the
literature.42 Copolymers with CBZ group 10 and 15 had very
close Tg values to copolymers 19 and 20, and all Tgs were
below the release temperature (data not shown). All
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Figure 2. 1H NMR spectrum of protected diblock 10 (a), deprotected diblock 19 (b), 13C NMR spectrum of deprotected 19 (c), and GPC curves of
MePEG 6, deprotected diblock 19, and protected diblock 9−11 (d).
rate for copolymers with CBZ group 10 and 15 is that they are
not close-packed since they have longer side chains. The drug
was released very rapidly due to the wide spacing of these
copolymer chains in the irregular structure. The CBZ group-
removed copolymers 19 and 20 have shorter side chains and
hydrogen bonding via amino groups so they have fewer spaces
between the chains. Therefore, the drug release from the CBZ
group-removed copolymers is slower.
The hydrophilicity (2) of the copolymer 19 and 20 also leads
to faster drug release because the higher hydrophobic character
in MePEG−PLLA 21 and PLLA−PEG−PLLA 22 causes a
lower release due to the hydrophobic−hydrophobic inter-
actions between the polyester block and the paclitaxel. The
limiting factor that hinders release in the first stage is the
limited water absorption by hydrophobic unfunctionalized PLA
copolymers.45 Therefore, a hydrophilic side chain, ethylamine,
was added to enhance water absorption. On the other hand, the
release results showed that the factor of chain length was more
effective than hydrophilicity when copolymers 10 and 15 were
compared with copolymers 19 and 20. Functional copolymers
(Figure 4) show remarkable improvement in drug release
during the period of 3 weeks. As a result, paclitaxel release was
increased considerably; after 20 days, a cumulative 72%−95%
drug in the copolymers 10, 15, 19, and 20 was released as
opposed to only 28%−29% in pure PLA−PEG−PTX matrix.
Thermal Properties and X-ray Diffraction. The
characteristics of the copolymers featuring different polyester/
PEG contents were further assessed by TGA analyses, as
illustrated in Figure 5. Two significant weight loss steps
belonging to each block were observed when thermal stabilities
of MePEG−poly(LLA-co-ZNEtMG) diblock 9, 10, and 11
Figure 3. Image of the gel-to-sol transition upon heating (a−c); gel−
sol curves of the diblock and triblock copolymers 10, 15, 19, 20, and
21 (d). The upper left region of each curve represented the sol phase
and the opposite region (lower right) the gel phase.
copolymers 10, 15, 19, and 20 gave faster drug release, as
expected, than copolymers 21 and 22. When the release rate of
copolymers with CBZ group 10 and 15 was compared with the
copolymers without CBZ group 19 and 20 according to chain
lengths and the effect of groups, the reason for highest release
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Table 3. Characteristics of the Copolymers by TGA Analyses
at Different Polyester/PEG Contents
polyester/ polyester/ char
Tp1
Tp2
a
a
b
ID
Mn
PEG (%) PEG (%) (%) (°C) (°C)
9
1270−2000
2460−2000
3780−2000
990−2000
2290−2000
3310−2000
2260−2000
2090−2000
0−2000
39:61
55:45
65:35
33:67
53:47
62:38
53:47
51:49
0:100
0:100
40:60
57:43
65:35
35:65
55:45
64:36
55:45
52:48
0:100
5.2
4.2
5.3
7.2
3.8
4.1
0.5
0.9
0.7
0.6
286
295
302
283
285
283
320
335
421
424
427
415
416
415
413
420
415
421
10
11
14
15
16
19
20
MePEG
PEG
0−2000
0:100
b
a
1
Determined by the H NMR spectrum. Determined by TGA.
Figure 4. Paclitaxel release curves from the diblock and triblock gels.
Each point of the plot is the result of an average of at least three
independent reproductions.
char yield of copolymer 10 was found to be 4.2%. The molar
ratios of ester and ether blocks in the copolymers 9, 10, 11, 14,
15, and 16 have been calculated from the weight loss
percentage. The values thus acquired from TGA analyses
were in good agreement with those based on 1H NMR spectra
(Table 3). Also, the deprotection of amine groups in polyester
segments caused less distinctive transition between both
polyester and PEG segments (10 vs 19 and 15 vs 20 in Figure
5c) at ca. 55% mass loss, lower char yields because of the
absence of aromatic groups, and higher inflection points (Table
3).
(Figure 5a and Table 3), and poly(LLA-co-ZNEtMG)−PEG−
poly(LLA-co-ZNEtMG) triblock 14, 15, and 16 (Figure 5b and
Table 3) copolymers were investigated. For example, the first
one in diblock 10 was due to the decomposition of poly(LLA-
co-ZNEtMG) segment with 57% weight loss (T1p = 295 °C,
inflection point, temperature at which greatest rate of change
on the weight loss in the peak of first derivative has occurred).
The second stage, appearing at higher temperatures, was due to
MePEG decomposition with 43% weight loss with T2p = 424 °C
(Tp = 415 °C for pure MePEG).4 After heating to 600 °C, the
Figure 5. Thermal degradation profiles of (a) MePEG−poly(LLA-co-ZNEtMG) 9, 10, and 11 diblock copolymers; (b) poly(LLA-co-ZNEtMG)−
PEG−poly(LLA-co-ZNEtMG) 14, 15, and 16 triblock copolymers; and (c) comparison of protected 10, 15 vs deprotected 19, 20 amine di- and
triblock copolymers.
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Figure 6. DSC thermograms of (a) MePEG 6, (b) MePEG−PLLA 21, and (c) copolymer 19 in the first run and (d) MePEG 6, (e) MePEG−PLLA
21, and (f) copolymer 19 in the second run.
Thermal characteristics of MePEG−poly(LLA-co-NEtMG)
diblock 19 (MePEG 6 and MePEG−PLLA 21 for the
comparison) were also examined by DSC analysis (Figure 6).
The broadness between 30 and 48 °C in MePEG−PLLA 21 is
probably due to overlapping melting point (Tm) of PEG units
and glass transition temperature (Tg) of polyester units. Similar
results have been reported in the literature about the values of
Tg of PLA and Tm of PEG being close to each other.46 Tg for
copolymer 19 was lower and more distinctive at 17 °C due to
increased flexibility of polyester chains. On the other hand, the
Tm value of MePEG was found to be 40.2 °C for copolymer 19
while the melting temperature of pure MePEG 6 was around 56
°C (Figure 6a vs 6c). Therefore, the presence of the poly(LLA-
co-NEtMG) blocks attached to the PEG blocks diminished the
melting temperature of the corresponding PEG. This situation
proved that the crystallization of each block was remarkably
affected by the presence of the other block. Two different
melting endotherms due to crystallization behaviors of
poly(LLA-co-NEtMG) blocks were observed for copolymer
19 at 60 and 100−105 °C in the second run of DSC. The
former was probably for crystals of poly(LLA-co-NEtMG)
segments, and the latter was for only lactide units due to high
mole of lactide in the copolymer 19. Poly(LLA-co-NEtMG)−
PEG−poly(LLA-co-NEtMG) triblock 20 copolymer showed
similar behavior with diblock copolymer 19.
Figure 7. Wide-angle X-ray diffraction (WAXD) patterns of
compounds of 6, 21, 10, and 19.
The crystalline structures of MePEG 6, MePEG-PLLA 21,
MePEG−poly(LLA-co-ZNEtMG) 10, and MePEG−poly(LLA-
co-NEtMG) 19 were analyzed by wide-angle X-ray diffraction as
indicated in Figure 7. MePEG 6 showed two sharp character-
istic peaks at 2θ angles of 19.3° and 23.4° while MePEG−PLLA
21 gives an additional signal at 2θ angle of 16.9° for PLLA
segment in the copolymer. PLLA and MePEG crystallinities are
present in the copolymers 10 and 19. However, the peaks
belonging to PLLA crystallites appeared much less intense due
to the existence of ZNEtMG or NEtMG in copolymers 10 and
19 as compared with MePEG−PLLA 21. The intensity of
crystallites decreased in the order MePEG 6 > MePEG−PLLA
21 > MePEG−poly(LLA-co-NEtMG) 19 ≥ MePEG−poly-
(LLA-co-ZNEtMG) 10.
Degradation. Hydrolytic degradation study of copolymers
19 and 20 was performed according to molecular weight
change during one month at pH: 7.4 in PBS at 37 °C. The
formation of oligomeric species was observed for diblock 19
and triblock 20 copolymers due to random chain scissions
(Figure 8). Also, their degradation performances were
compared with well-known MePEG−PLLA 21, PLLA−PEG−
PLLA 22 block copolymers, and PAGA homopolymer.35 The
GPC curves in the degradation study in Figure 8 were analyzed
by Origin 9 pro software, and the three identifiable peaks were
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presence of p-toluenesulfonic acid monohydrate catalyst in
toluene via removing water from the reaction medium with a
Dean−Stark apparatus (Scheme 3). The synthesis of ZDNEtG
5 was completed in a short time like 5 h with the 58% yield.
The proceeding of the reaction was monitored with thin layer
chromatography (Rf: 0.4). Cyclization of α-hydroxyacid was a
time-dependent reaction described previously by us.4 It should
be carefully optimized because longer reaction times lead to
form oligomeric species while shorter ones cause unreacted
starting material in the reaction medium. For this reason,
formation of monomer 5 was also followed with GPC (data
were not shown). The full characterization of monomer 5 was
performed with 1H NMR, 13C NMR, HMQC 2-D NMR,
COSY 2-D NMR, ATR-FTIR, and LC/MS-TOF, as shown in
Figures S4, S15, S24, S32, S33, and S39 of the Supporting
Information. Then, the polymerization of monomer 5 with
MePEG and lactide was attempted under the same polymer-
ization conditions with monomer 4 (Scheme 3).
Although the polymerization of disubstituted lactides
catalyzed by Sn(Oct)2 has been previously reported by us
and others,4−10,24 no polymerization of disubstituted monomer
5 with MePEG (Supporting Information, Figures S45 and S46,
the molecular weight of MePEG was only observed) is probably
due to the functional long side chain of monomer 5 having
interactions with the catalyst preventing the polymerization of
itself23 or high steric hindrance of bulky substituents in both
sides, which is known to lessen the ΔG of the polymerization.23
These findings were consistent with the results of works
performed by Cohen-Arazi et al.6 and Hall et al.,47 who
reported that the polymerization of dilactones strongly depends
on substitution in the ring. For example, the polymerization
rate of lactide (−CH3 substituent) is slower than glycolide (−H
substituent) probably due to the steric hindrance of the −CH3
groups that hinders nucleophilic attack on the carbonyl groups
of the ring.6 More methyl substituent in the dilactone ring
makes it hard to polymerize. Trimethyl glycolide was
polymerized in very high temperature (180 °C) and long
reaction time (24 h).48 Even more ring substitution on
monomer, i.e., tetramethyl glycolide, may stop polymerization
in the presence of Sn(Oct)2 catalysis.47 Therefore, an increase
in the substituent’s bulkiness causes to decrease the polymer-
ization rate. Thus, steric bulky groups in both sides in
monomer 5 hindering nucleophilic attack at the carbonyl
carbon in the ring led to no polymerization. That is why the
Figure 8. GPC chromatograms of copolymer 19 after (a) and before
(b) degradation and copolymer 20 after (c) and before (d)
degradation.
fitted by a Lorentzian formulation.49 The fractional areas of the
three peaks at different degradation times were summarized.
Both diblock 19 (47%, Mn,P(LLA‑NEtMG): 1180 and
Mn,P(LLA‑NEtMG): 310) and triblock 20 (28%, Mn,P(LLA‑NEtMG):
1215 and Mn,P(LLA‑NEtMG): 320) copolymers (Figure 8) showed
faster degradation than conventional MePEG−PLLA diblock
21 (0.6% degradation, Mn,PLLA: 920, data shown in Figure S50)
and PLLA−PEG−PLLA triblock 22 (13.8% degradation,
Mn,PLLA: 2190, data shown in Figure S50) copolymers by the
end of a month, respectively. The poor hydrolytic degradability
of MePEG−PLLA and PLLA−PEG−PLLA copolymers is
likely due to being highly crystalline and hydrophobic nature
of the PLLA chains. Thus, the faster degradation rate of
synthesized novel functional copolymers that make them very
useful in variety of biological applications like drug delivery or
tissue scaffolding was due mainly to disruption of crystallinity
by the NEtMG residues and hydrophilic amine groups in the
copolymers.34 On the other hand, the copolymers 19 and 20
(17% and 19% of degradation in 30 min according to GPC
analyses) showed slower degradation than PAGA homopol-
ymer35 (interestingly, only 30 min for the intact polymer to
halve according to MALDI analyses). The difference in
degradation rate between PAGA and copolymers 19 and 20
was probably diversity in polymer structure (due to the
existence of highly lactide units in copolymer); water
penetration (i.e., the solution degradation) was studied in
Park’s work,35 and the gel was studied in the current work.
Mechanism. In order to elucidate the polymerization
mechanism of ZNEtMG 4, we also synthesized symmetrical
version of the monomer, CBZ protected 3,6-diaminoethyl-1,4-
dioxane-2,5-dione (ZDNEtG) 5, from compound 2 in the
Scheme 3. Synthesis of MePEG−Poly(LLA-co-ZDNEtG) Diblock Copolymers
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Scheme 4. Polymerization Mechanism of Asymmetrical Monomer 4 with MePEG and Lactide
polymerization of ZNEtMG 4 probably continues from only
methyl side as seen in Scheme 4. On the other hand, in
monomer 4, the temporary coordination of oxygenes in
carbonyl group of ZNEtMG 4 and in the end of MePEG
homopolymer into metal alkoxide catalyst (Sn(Oct)2) increases
the nucleophilicity of oxygen of the hydroxyl group which helps
the cleavage of the bond between the carbonyl carbon and the
endocyclic oxygen atom. Electrophilicity of the carbonyl group
in ZNEtMG 4 also plays an important role in the cleavage
during this stage. In following steps, ZNEtMG 4 continues to
open and place in the metal−oxygen bond for the growth of
copolymer chain.
functional group was found to be faster than those with free
amine groups, and the copolymers with only L-lactide showed
the slowest release. In this study, novel thermosensitive
copolymers containing free amine groups can be injected at
42 °C and can be used to treat tumors and cancerous tissues in
the local regions by forming gels at body temperature.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.macro-
mol.7b02751.
Additional data of ATR-FTIR spectra, NMR spectra,
LC/MS-TOF spectra, and GPC chromatograms (PDF)
4. CONCLUSION
PEG-based polylactide and poly(lactide-co-glycolide) copoly-
mers are among the most widely used thermosensitive
biodegradable polymers. Synthesis, characterization, and
various properties of these polymers have been extensively
studied in the literature. On the other hand, there are very few
studies on glycolide family polymers prepared by using glycolic
acid derivatives as monomers, and new biomaterials that can
undergo various modifications easily by means of free
functional groups are highly needed. For this purpose, a new
type of asymmetric substituted CBZ protected amine groups
(ZNEtMG) was synthesized for the first time. PEG-based
polymerization studies with L-lactide were carried out by the
ring-opening reaction method in the presence of Sn(Oct)2
catalyst. New biodegradable diblock and triblock copolymers
were obtained with different molar ratios of the monomers at
various molecular weights, with the keeping amounts of
initiator and catalyst constant. Removal of the CBZ protecting
group after polymer synthesis was successfully carried out
under H2 atmosphere in the presence of Pd/C (10%) catalyst.
Suspensions of various concentrations of copolymers 10, 15,
19, and 20 were prepared, and optimal concentrations for each
critical gel−sol temperature were determined. The copolymers
19 and 20 without protective groups were observed to have
gel−sol transitions at lower temperatures. Twenty days of drug
release studies were performed with using 1.0% of the
anticancer drug loaded the copolymer gels after gel-to-sol
transition temperatures and concentrations were determined.
The drug release from the copolymers with protective amine
AUTHOR INFORMATION
Corresponding Author
*(O.M.) E-mail olcay.mert@kocaeli.edu.tr; Tel
+902623032054; Fax +902623032003.
■
ORCID
Olcay Mert: 0000-0002-5769-8994
Author Contributions
M.O.A. and S.E. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was granted by TUBITAK, Turkey, with project
number 112T865.
■
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