Elucidating the Molecular Interactions of Encapsulated Doxorubicin Within a Non-ionic, Thermoresponsive Polyester Coacervate

Abstract

Thermoresponsive polymers that display a lower critical solution temperature (LCST), are attractive drug delivery systems (DDS) due to their potential to encapsulate and release therapeutics in a sustained manner as a function of temperature input. To attain the full potential of such DDS, methods that illustrate the details of drug-polymer interactions are necessary. Here, we synthesized a non-ionic, coacervate-forming, thermoresponsive polyester to encapsulate doxorubicin (Dox) and used solution state NMR spectroscopy and fluorescence microscopy techniques to probe the interactions between the polymer and Dox at the molecular level. The incomplete dehydration provides a matrix for encapsulation of sensitive therapeutics and preserving their activity, while the low hysteresis properties of the polyester provides rapid transition from soluble to coacervate phase. Saturation Transfer Difference (STD) NMR revealed the Dox-polymer interactions within the coacervates. 1H-1H Nuclear Overhauser Effect Spectroscopy (NOESY) cross-peak differences of Dox confirmed the Dox-polymer interactions. Diffusion-Ordered Spectroscopy (DOSY) revealed the slower diffusion rate of Dox in the presence of polyester coacervates. These studies illustrate how the state of the polyester (below and above LCST) affects the polyester-Dox interactions and offers details of the specific functional groups involved in these interactions. Our results provide a framework for future investigations aimed at characterizing fundamental interactions in polymer-based DDS.

Full text

Elucidating the Molecular Interactions of Encapsulated Doxorubicin
within a Nonionic, Thermoresponsive Polyester Coacervate
Mangaldeep Kundu, Daniel L. Morris, Megan A. Cruz, Toshikazu Miyoshi, Thomas C. Leeper,
and Abraham Joy*
Cite This: https://dx.doi.org/10.1021/acsabm.0c00507
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sıSupporting Information
ABSTRACT: Thermoresponsive polymers that display a lower
critical solution temperature (LCST) are attractive drug delivery
systems (DDSs) due to their potential to encapsulate and release
therapeutics in a sustained manner as a function of temperature
input. To attain the full potential of such DDSs, methods that
illustrate the details of drug−polymer interactions are necessary.
Here, we synthesized a nonionic, coacervate-forming, thermores-
ponsive polyester to encapsulate doxorubicin (Dox) and used
solution state NMR spectroscopy and fluorescence microscopy techniques to probe the interactions between the polymer and Dox
at the molecular level. The incomplete dehydration provides a matrix for encapsulation of sensitive therapeutics and preserving their
activity, while the low hysteresis property of the polyester provides rapid transition from soluble to coacervate phase. Saturation
transfer difference (STD) NMR revealed the Dox−polymer interactions within the coacervates. 1H−1H nuclear Overhauser effect
spectroscopy (NOESY) cross-peak differences of Dox confirmed the Dox−polymer interactions. Diffusion-ordered spectroscopy
(DOSY) revealed the slower diffusion rate of Dox in the presence of polyester coacervates. These studies illustrate how the state of
the polyester (below and above LCST) affects the polyester−Dox interactions and offers details of the specific functional groups
involved in these interactions. Our results provide a framework for future investigations aimed at characterizing fundamental
interactions in polymer-based DDSs.
KEYWORDS: thermoresponsive polymers, nonionic coacervate, stimuli responsive materials, smart polymers, polyester coacervate,
controlled release polyester, drug delivery system
■INTRODUCTION
Pharmaceutical therapeutic efficacy can be limited by brief in
vivo half-lives and nonspecificoff-target interactions resulting
in unwanted side effects. Enhanced drug accumulation in the
target tissue and target specificity are high priorities for drug
delivery systems (DDSs).
1−5
In this regard, polymer-based
delivery systems provide advantages of controlled release,
improved drug stability, reduced side effects, and reduced
dosing frequency.
6−9
Stimuli-responsive polymers, or “smart”polymers, have
emerged as promising contenders for therapeutic drug
delivery.
10
Smart polymers are those that undergo physical
or chemical changes in response to an external stimulus such as
temperature, pH, reduction potential, enzyme, or light.
10−13
Several reported studies have examined the encapsulation and
release of drugs using coacervate-forming polymers.
14−19
These elaborate systems rely on a delicate balance between
polymer−drug interactions that are strong enough to stabilize
the complex but not so strong that they hinder the release of
the encapsulated drug. A detailed study into these interactive
binding systems is warranted and can be used to predict future
DDS behaviors in a systematic way.
Previously, we reported the synthesis and characterization of
a series of biodegradable, thermoresponsive polyesters (TR-
PEs) inspired by elastin-like peptides (ELPs).
20
They exhibit
lower critical solution temperature (LCST) behavior that is
characterized by a transformation from a random coil polymer
structure to a phase-separated globular phase when the
temperature is brought above the polymer’s cloud point
temperature (Tcp). This conformational change results in the
formation of polymer-rich, dense coacervate droplets. These
coacervates are well-suited for the encapsulation and delivery
of therapeutic molecules, due to the simplicity of this system.
Merely by mixing the polymer and encapsulant in an aqueous
solvent and increasing the solution temperature, the drug of
choice can be safely encased inside the stable polymer
coacervates. The Tcp can be modulated by the identity and
ratio of the monomers. We have reported the successful
Received: May 2, 2020
Accepted: June 22, 2020
Published: June 22, 2020
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encapsulation of bovine serum albumin (BSA), within such
thermoresponsive polymers, without sacrificing its stability.
21
The present study details our efforts in characterizing the
interactions between a thermoresponsive polyester coacervate
and Dox to establish a fundamental understanding of the
molecular level relationships that govern drug encapsulation
inside polymer coacervates. The polyester was designed with
an indole pendant group and a thermoresponsive pendant
group to provide interactions with Dox and to form
coacervates above the Tcp. Simple mixing of Dox and polyester
in an aqueous solution and raising the temperature above the
Tcp provides Dox encapsulated coacervates. Fluorescence
microscopy was employed to assess the encapsulation of Dox
inside the polyester coacervates. The polyester−Dox inter-
actions were further characterized by saturation transfer
difference (STD), nuclear Overhauser effect spectroscopy
(NOESY), and diffusion-ordered spectroscopy (DOSY) NMR
techniques. All the NMR spectroscopy techniques are
explained in depth in the Results and Discussion section.
■EXPERIMENTAL SECTION
Materials. Dichloromethane (DCM), methanol (MeOH), ethyl
acetate (EtOAc), dimethylformamide (DMF), silica gel thin-layer
chromatography (TLC) plate, dialysis tubing (regenerated cellulose,
MWCO 3.5 kDa), and syringe filter (0.45 μm) were purchased from
Thermo Fisher Scientific. Triethylamine (Et3N), ethyl succinyl
chloride (EtSuCl), diethanolamine (DEA), and sodium chloride
were obtained from Acros Organics. Anhydrous sodium sulfate
(Na2SO4), sodium bicarbonate (NaHCO3), and Whatman filter paper
were bought from VWR Chemicals. bis(2-Methoxyethyl)amine
(bMoEtA), succinic acid (SA), and thionyl chloride were purchased
from TCI America. 3-(1H-Indol-3-yl)propanoic acid and anhydrous
MeOH were purchased from Chem-Impex International and EMD
Millipore, respectively. N,N′-Diisopropylcarbodiimide (DIC) was
obtained from Oakwood Chemical. Doxorubicin hydrochloride salt
was purchased from LC Laboratories. Deuterium oxide ampules were
purchased from Cambridge Isotope Laboratories. Silica gel was
obtained from Sorbent Technologies, Inc. The microscopy glass slides
were purchased from Globe Scientific. DCM was dried by distillation
process in the presence of calcium hydride. 4-(Dimethylamino)
pyridinium 4-toluene sulfonate (DPTS) was synthesized separately.
22
Synthesis of Ethyl 4-(bis(2-Methoxyethyl)amino)-4-oxobuta-
noate (bMoEtSA) (P1). Into a 100 mL round-bottom flask equipped
with a magnetic stir bar, bMoEtA (12 mL, 81.2674 mmol, 1 equiv),
Et3N (8.388 g, 82.8927 mmol, 1.02 equiv), and DCM (50 mL) were
added. The reaction mixture was purged with nitrogen (N2) gas for 15
min with magnetic stirring and then kept in N2environment
throughout the reaction. The flask was cooled in an ice−methanol
bath for 20 min, and EtSuCl (11.6 mL, 81.2674 mmol, 1 equiv) was
added dropwise through a syringe. After EtSuCl addition, the reaction
flask was removed from the ice−methanol bath and allowed to stir at
room temperature for 1 h. The reaction mixture was extracted in
DCM using deionized water (3 ×50 mL). The organic layer was
dried over anhydrous Na2SO4,filtered, and concentrated under
reduced pressure. The light-yellow product (16.801 g, 72.0629 mmol,
88.63% yield) was dried under vacuum for 14 h and characterized
using NMR. 1H NMR (300 MHz, CDCl3)δ4.13 (q, J = 7.1 Hz, 2H),
3.53 (m, J = 7.1, 6.2, 3.7 Hz, 8H), 3.32 (d, J = 6.8 Hz, 6H), 2.66 (qd, J
= 6.1, 3.1 Hz, 4H), 1.25 (t, J = 7.1 Hz, 3H).
Synthesis of N1,N1-bis(2-Hydroxyethyl)-N4,N4-bis(2-Methoxy-
ethyl)succinamide (bMoEtDEA) (P2). P1 (15.301 g, 65.5963 mmol,
1 equiv) and DEA (13.793 g, 131.1926 mmol, 2 equiv) were added to
a 100 mL round-bottom flask with a magnetic stir bar. The reaction
mixture was allowed to heat at 80 °C under vacuum for 14 h. The
progress of the reaction was monitored using TLC plate. The crude
mixture was purified by silica gel column chromatography using a
gradient eluting solvent system (2−10% MeOH in DCM). The
purified product was concentrated under reduced pressure and dried
under vacuum with stirring to remove excess solvent. The desired
product (14.817 g, 50.6852 mmol, 77.27% yield) was confirmed using
NMR. 1H NMR (300 MHz, CDCl3)δ3.89−3.76 (m, 4H), 3.65−3.42
(m, 12H), 3.31 (d, J= 9.9 Hz, 6H), 2.84 (dd, J= 7.0, 4.9 Hz, 2H),
2.69 (dd, J= 6.9, 4.9 Hz, 2H).
Synthesis of Methyl 3-(1H-Indol-3-yl)propanoate (P3). 3-(1H-
Indol-3-yl)propanoic acid (5.753 g, 30.4053 mmol, 1 equiv) was
taken into a 250 mL round-bottom flask equipped with an addition
funnel. The reaction was stirred constantly under N2. After 5 min,
anhydrous MeOH (55 mL) was added into the flask and placed in an
ice−MeOH bath for 30 min. Thionyl chloride (3.3 mL, 45.6080
mmol, 2 equiv) was then added to the flask dropwise from the
addition funnel. After another 30 min, the cooling bath was removed,
and the reaction mixture was stirred at room temperature for 14 h.
The completion of the reaction was monitored using TLC plate.
Then, the black solution was neutralized by adding an excess amount
of solid NaHCO3. The salt was removed from the resulting light
brown solution using vacuum filtration. The product was concen-
trated under reduced pressure, yielding brown flakes. The solid was
dissolved in EtOAc (75 mL) and washed with water (50 mL) and
saturated brine solution (3 ×30 mL). The deep yellow organic layer
was concentrated under reduced pressure and dried under vacuum
oven for 14 h. The deep brown viscous product was confirmed using
NMR. 1H NMR (300 MHz, CDCl3)δ7.61 (d, J= 7.7 Hz, 1H), 7.36
(dd, J= 8.0, 0.7 Hz, 1H), 7.16 (m, J= 14.8, 11.4, 7.0 Hz, 2H), 7.01
(d, J= 1.3 Hz, 1H), 3.68 (s, 3H), 3.12 (t, J= 7.7 Hz, 2H), 2.73 (t, J=
7.7 Hz, 2H).
Synthesis of N,N-bis(2-Hydroxyethyl)-3-(1H-indol-3-yl)-
propenamide (P4). In a round-bottom flask equipped with a
magnetic stir bar, P3 (3.6057 g, 17.7459 mmol, 1 equiv) and DEA
(3.731 g, 35.4918 mmol, 2 equiv) were heated at 80 °C for 14 h under
vacuum. The progress of the reaction was supervised using TLC plate.
The brown sticky crude mixture was purified by silica gel column
chromatography using 4−10% MeOH in DCM gradient eluting
system, which yielded a pure white powder (4.2451 g, 15.3611 mmol,
86.56% yield) as confirmed by NMR. 1H NMR (300 MHz, DMSO-
d6) δ7.51 (d, J= 7.8 Hz, 1H), 7.39−7.27 (m, 1H), 7.11 (d, J= 2.3
Hz, 1H), 7.10−7.01 (m, 1H), 6.97 (dd, J= 10.8, 4.0 Hz, 1H), 4.78 (t,
J= 5.4 Hz, 1H), 4.64 (t, J= 5.4 Hz, 1H), 3.49 (dd, J= 5.6, 2.0 Hz,
4H), 3.38 (t, J= 5.7 Hz, 4H), 2.90 (dd, J= 9.4, 6.1 Hz, 2H), 2.70 (dd,
J= 9.3, 6.1 Hz, 2H).
Synthesis of Thermoresponsive Polymer TR-(bMoEt-r-mTrp)APE
(p(bMoTrp)). P2 (1.5107 g, 5.1677 mmol, 0.95 equiv), P4 (75.2 mg,
0.272 mmol, 0.05 equiv), SA (642.4 mg, 5.4399 mmol, 1 equiv), and
DPTS (636 mg, 2.1759 mmol, 0.4 equiv) were added into a round-
bottom flask with a magnetic stir bar. The mixture was purged with
N2for 15 min, and dry DCM (11 mL) was added to it. Then the flask
was placed in the ice-MeOH bath for 15 min before adding DIC (2.6
mL, 16.6115 mmol, 3 equiv) dropwise. After 30 min, the ice−
methanol bath was removed, and the reaction mixture was allowed to
stir at room temperature for 3 days. In the end, the mixture was
diluted with a small amount of DCM, passed through a cotton plug to
remove the insoluble urea byproduct, concentrated under reduced
pressure, and purified by dialysis. Briefly, the concentrated crude
product was kept inside 3.5 kDa MWCO dialysis bag which was
placed in a beaker with MeOH (500 mL) with constant stirring. The
MeOH solvent was refreshed at 3, 6, 12, and 24 h. The polymer was
recovered as white amorphous solid when concentrated under
reduced pressure and was confirmed by NMR. 1H NMR (750
MHz, D2O) δ7.62 (s, 1H), 7.42 (d, J= 31.1 Hz, 1H), 7.13 (d, J=
72.8 Hz, 1H), 4.28 (s, 5H), 3.64 (d, J= 68.5 Hz, 12H), 3.40 (d, J=
25.9 Hz, 6H), 2.76 (d, J= 15.8 Hz, 9H).
Molecular Weight Measurement. p(bMoTrp) (6 mg) was
dissolved in DMF (2 mL) mixed with 25 mM LiBr for the molecular
weight measurement in TOSOH EcoSec HLC-8320 SEC instrument.
The instrument temperature was 50 °C, and the eluent flow rate was
set to 0.8 mL/min. The polymer solution was filtered using a syringe
filter (0.45 μm) to remove any nondissolvable particles. The
molecular weight was calculated relative to PMMA standard using
the RI detector.
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Cloud Point Measurement. The cloud point temperature or Tcp
was measured on a Shimadzu UV-1800 UV−vis spectrophotometer as
per standard literature procedure.
28
The spectrophotometer was
equipped with a Shimadzu S-1700 thermoelectric single cell holder to
control the temperature. p(bMoTrp) was dissolved in ultrapure water
solution at a concentration of 10 mg/mL. The absorbance was
measured at a fixed wavelength of 600 nm with a temperature ramp of
0.5 °C min−1.Tcp is measured as the temperature corresponding to
the 50% transmittance value. To calculate Tcp, the absorbance values
(A) are processed as a percentage of transmittance (%T) and plotted
against the change in temperatures.
=−
%
T10
A(1)
Fluorescence Microscopy. p(bMoTrp) (4.07 mg, 0.05 mM) and
Dox (0.2 mg, 0.5 mM) were mixed in 700 μL of ultrapure water to
form a homogeneous solution. A 10 μL portion of the solution was
added to a glass slide, which was heated above p(bMoTrp)’sTcp to
induce coacervate formation. Then, it was placed under the
fluorescence microscope, and the images were captured using a
tetramethylrhodamine isothiocyanate (TRITC) filter.
NMR Experiments. The 300 MHz NMR instrument was used to
characterize all the monomers, and other NMR experiments were
recorded on an Agilent DD2 750 MHz instrument. VNMRJ software
(version 3.2) was used to analyze all NMR data.
Variable Temperature (VT) NMR. p(bMoTrp) (10 mg) was
dissolved in 1 mL of D2O with shaking for 14 h to achieve the best
dissolution. 1H NMR experiments were measured at 15, 19, 23, 27,
29, 31, and 37 °C. Spectra were acquired using 64 scans with an
acquisition time of 1.5 s and a relaxation delay of 3s.
Saturation Transfer Difference (STD) NMR. For the STD
experiments, p(bMoTrp) (4.07 mg, 0.05 mM) was dissolved in 700
μLofD
2O by shaking it for 14 h. After that, Dox (0.2 mg, 0.5 mM)
was mixed with the polymer solution and STD NMR measurements
obtained at 23 and 50 °C. A 50 ms Gaussian-shaped pulse was used to
selectively saturate polymer resonances at 7 ppm using a saturation
power and frequency of 12 dB and 1500 Hz, respectively. Varian’s
preinstalled BioPack dpfgse_satxfer pulse sequence was used and the
on-resonance experiment was automatically subtracted from the off-
resonance experiment as part of the phase cycle. Experiments were
conducted with 64 scans using an acquisition time of 0.682 s and a
starting relaxation delay of 0.1 s. Data sets were collected as STD
build-up experiments using saturation transfer delays of 0, 0.5, 1.0, 1.5,
and 2.0 ms. The combined relaxation and transfer delay time frame
were the same for each measurement in the build-up curve.
Nuclear Overhauser Effect Spectroscopy (NOESY) NMR.
1H−1H NOESY experiments were performed for Dox, p(bMoTrp),
and p(bMoTrp)−Dox mixture at below and above Tcp, i.e. 23 and 50
°C, respectively. The Dox solution was made just prior to the
experiment by dissolving Dox (1.5 mg, 3.75 mM) in 700 μLofD
2O.
For the polymer solution, p(bMoTrp) (4.1 mg, 0.05 mM) was
dissolved in 700 μLofD
2O by shaking it for 14 h. In case of
p(bMoTrp)−Dox solution mixture, p(bMoTrp) (4.1 mg, 0.05 mM)
was dissolved first, and then Dox (1.5 mg, 3.75 mM) was added to
that homogeneous solution mixture. NOESY spectra for the three
different solutions were collected with 8 scans, a 3 s relaxation delay,
256 increments, 2048 data points, and mixing time was 150 ms.
Diffusion-Ordered Spectroscopy (DOSY) NMR. DOSY experi-
ments were performed with Dox and p(bMoTrp)−Dox mixture at 23
and 50 °C using Varian’s preinstalled Dbppste sequence. The
relaxation delay for each sample was determined from a 1HT1
relaxation experiment at each temperature. For the DOSY run, the
delay was set as three times higher than the corresponding sample T1
value. Dox (2 mg, 4.9263 mM) was dissolved in 700 μLofD
2O prior
to the experiment. For the p(bMoTrp)−Dox mixture, p(bMoTrp)
(7.2 mg, 0.0859 mM) was dissolved in 700 μLofD
2O with shaking
for 14 h, and Dox (2.1 mg, 4.9263 mmol) was added.
■RESULTS AND DISCUSSION
Dox was selected as the model drug due to its widespread use
in cancer therapy protocols. Also, it has been extensively
studied by numerous NMR techniques,
23−25
which is
beneficial for the current study as polymer coacervates are
complex systems with sparse NMR characterization data.
17,26
A
newtypeofthermoresponsiverandomcopolymer(p-
(bMoTrp)) (Figure 1A) was designed to have a moderate
binding affinity with the Dox molecule (Figure 1B).
p(bMoTrp) (Mn= 116.340 kDa, Đ= 1.6) comprises a
thermoresponsive part (bMo, 95%) and an indole functional
group part (Trp, 5%) that mimics the tryptophan amino acid
side chain and was hypothesized to afford a recruitment
platform for Dox via π−πstacking. As the Tcp of the
homopolyester containing bMo is 48 °C,
27
introduction of a
relative hydrophobic Trp would be expected to reduce the Tcp
Figure 1. Chemical structure of (A) p(bMoTrp) and (B) doxorubicin hydrochloride (Dox). The letters and numbers correspond to proton NMR
peaks of p(bMoTrp) and Dox, respectively. bMo and Trp correspond to the thermoresponsive functional group and indole functional group,
respectively.
Figure 2. Simple representation of Dox encapsulation within p(bMoTrp) coacervates. (Navy blue coiled structure represents p(bMoTrp) and aqua
dots represent water molecules.) At room temperature, Dox is mixed with an aqueous solution of p(bMoTrp). Above the Tcp, p(bMoTrp) phase
separates and results in turbid coacervate complexes. Most of the Dox molecules move inside the coacervates during this step resulting in efficient
encapsulation. When T<Tcp, p(bMoTrp) reverts to its random coil configuration and the solution returns to a clear solution.
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to be below human body temperature. Keeping in mind an
eventual biological application, this modification would ensure
that p(bMoTrp) remains in its coacervate state in vivo.
Additionally, a noncharged coacervate is advantageous over a
charged one as the latter is less stable to changes in solution
pH.
21
Thus, p(bMoTrp) was devised to have noncovalent
interactions (excluding salt bridges) with Dox through a
combination of its hydrophilic (polyester backbone and bMo)
and aromatic (Trp) region and thereby result in optimum drug
encapsulation (Figure 2).
Thethermoresponsivebehaviorofp(bMoTrp)was
examined by cloud point measurement (Figure 3A). Below
the cloud point, Tcp, the clear polymer solution does not
exhibit any change in transmittance value (T). An increase in
cloudiness and hence a rapid decrease in transmittance value of
the polymer solution occurs as the temperature approaches the
Tcp due to creation of water-insoluble coacervate droplets
which scatter the incident light. At the upper-temperature
ranges, when the solution is completely opaque, the trans-
mittance value remains steady as the coacervate conforma-
tional changes reach equilibrium.
Historically, Tcp is defined as the temperature corresponding
to 50% of the transmittance value.
28
For p(bMoTrp), Tcp (at
10 mg/mL concentration) was determined to be 29.7 °C and
the 95−5% transmittance range (i.e., the change in temper-
ature from 95% to 5% transmittance; a measure of the rate of
coacervate formation) was found to be 2.1 °C(Figure 3A).
Comparatively, the Tcp and the 95−5% transmittance range for
bMo containing homopolyester were 48 and 3.5 °C,
respectively.
27
The addition of 5% of Trp greatly decreases
the Tcp value due to increased hydrophobicity in the polymer
structure which corroborates with our prior studies.
20
The
drastic change in the 95−5% transmittance range shows that
the phase transition of p(bMoTrp) occurs rapidly, indicating
that the polymer composition is homogeneous.
29
The small
hysteresis, the temperature difference between Tcp,heat(29.7
°C) and Tcp,cool(28.9 °C), is indicative of the instantaneous
phase separation process that undergoes equilibrium-type
partitioning between droplet and soluble fraction and is not
kinetically trapped in one state or the other.
30
In addition, the
relatively sharp transition (<2 °C) for the polymer upon
heating and cooling suggests that coacervation is cooperative.
As anticipated, a lower concentration (5.8 mg/mL used for
STD and NOESY experiments) of p(bMoTrp) exhibited
higher Tcp (30.6 °C) than that of 10 mg/mL p(bMoTrp)
solution (29.7 °C) (Figure 3B). This can be attributed to the
fact that fewer polymer chains are available to form globule like
coacervates at a lower p(bMoTrp) concentration.
20,27,31
Interestingly, at approximately half the p(bMoTrp) concen-
Figure 3. LCST curve of p(bMoTrp) in (A) standard concentration (10 mg/mL, 0.09 mM) and (B) at a lower concentration (5.8 mg/mL, 0.05
mM) which was used for STD and NOESY NMR experiments. Tcp is determined as the temperature at which transmittance becomes 50%. The
term “heat”refers to the process when the temperature was increased from low to high, and the reverse process is termed “cool”. Both heat and cool
measurements were taken to assess hysteresis.
Figure 4. 1H VT NMR spectra of the polyester in the upfield region showing the backbone and hydrophilic part of p(bMoTrp). The peaks in the
spectra are assigned in accordance with the numbering on the p(bMoTrp) structure. The spectra were acquired at various temperatures as shown
against each spectrum.
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D

tration, the Tcp value only increased by 0.9 °C which indicates
the strong ability of p(bMoTrp) to form coacervates.
High resolution NMR is a useful technique for character-
izing the thermoresponsive polymer coacervates at the
molecular level. Variable temperature (VT) NMR (Figure 4)
was used to confirmthechangeinpolymerchain
conformation, and thus, coacervate formation above the Tcp.
The entire NMR spectrum of p(bMoTrp) is depicted in Figure
5. For VT NMR study, several proton NMR spectra of
p(bMoTrp) were taken across a wide range of temperatures
both below and above the Tcp of p(bMoTrp). As temperature
rises toward the Tcp, all the peaks of p(bMoTrp) become
broader due to the decrease in polymer chain mobility,
resulting in broadening of the 1H NMR signals due to
increasing dipolar coupling strength. In contrast to the
polyesters described here, the literature of thermoresponsive
polymers show that above Tcp the majority of the peaks in the
NMR spectra disappear, indicating complete dehydration and
aggregation of polymer chains.
32
However, in our system, the
proton peaks only broaden rather than completely disappear,
even well above Tcp which reproduces previous results reported
from our lab.
20,27
A substantial amount of water molecules
remain present inside the coacervate which preserves the
polymer chain mobility and permits their resolution even when
they are within the restricted coacervate complex environment.
To confirm the encapsulation, Dox-containing p(bMoTrp)
coacervate (henceforth it will be mentioned as p(bMoTrp)−
Dox) was visualized by fluorescence microscopy at 595 nm.
Below the Tcp, the p(bMoTrp)−Dox solution fluoresced with a
homogeneous red background (Figure 6A), indicating that
Dox was diffusing uniformly throughout the solution. When
the same solution was warmed above the Tcp, the appearance
of micron-sized bright, red globules confirmed the entrapment
of Dox inside the coacervate. The light red background in
some regions of Figure 6B shows that complete encapsulation
was not achieved.
Saturation transfer difference (STD) NMR is usually
employed to observe magnetization transfer from protein to
small molecules in drug discovery research.
33−36
This
technique is applied here to examine p(bMoTrp)−Dox
interactions due to their spatial association (within 5 Å)
(Figure 7). In a typical STD NMR experiment, a pair of proton
Figure 5. 1H NMR spectrum (in D2O, 750 MHz) of p(bMoTrp)−Dox mixture above Tcp,50°C. Dox peaks were assigned based on literature
reports.
Figure 6. Fluorescence microscopy image (TRITC filter) of (A) Dox
and p(bMoTrp) mixture below Tcp (at room temperature, ∼22 °C)
and (B) Dox encapsulated in p(bMoTrp) coacervate spheres above
Tcp (scale bar: 50 μm). To induce coacervate formation, the
p(bMoTrp)−Dox solution was loaded onto a glass slide which was
heated above Tcp. As the coacervate formed, the solution turned
turbid and images were recorded.
Figure 7. Illustration of the STD NMR process. Upon saturation of a
selected p(bMoTrp) peak, signal transfer can occur if Dox is within 5
Å distance of p(bMoTrp). Then STD signal is obtained when Dox
separates from the p(bMoTrp)−Dox complex. If Dox does not
interact with p(bMoTrp), there will be no STD signal. On the other
hand, if Dox stays in the complex form for a long time, it will generate
an STD signal of low intensity.
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spectra are collected with band selective magnetization
saturation pulses applied at two widely different frequencies:
one “on-resonance”spectrum where a polymer specific region
is saturated and another “off-resonance”spectrum where a
control saturation pulse is applied away from relevant
resonances. Subtraction of the on-resonance spectrum from
the off-resonance spectrum produces a final difference
spectrum of interest.
31
In our previous work, the STD NMR technique was
exploited to investigate protein encapsulation within polymer
coacervates.
21
In the present, protocol the p(bMoTrp) signals
are saturated so that saturation is transferred from the polymer
to Dox, whereas in our previous protocol saturation was
transferred from protein to polymer. The saturation pulses are
applied to the aromatic regions (at 7 ppm) of p(bMoTrp) as
both p(bMoTrp) and Dox proton signals overlap in the
aliphatic region (Figure 5). STD experiments were collected as
saturation build-up curves (Figure 8).
As the saturation delay increases, there is more time for
signal saturation to transfer from p(bMoTrp) to Dox and
enhance the corresponding difference spectrum peaks (from
bottom to top). The enhancement of Dox peaks in the
difference spectrum indicates that Dox is interacting with the
polymer. Below Tcp, the aromatic peaks of Dox are more
intense than they are at 50 °C, which can be attributed to the
fact that the signal intensity in the difference spectrum depends
on the exchange rate of Dox binding and releasing from the
polymer.
34,35
We hypothesize that inside the coacervates, the
interaction between p(bMoTrp) and Dox is strong and hence
the exchange rate is slow. Therefore, the signal transfer to the
pool of free Dox molecules (in terms of NMR experiment,
where the spectra are only obtained from Dox in the free state
after releasing from the p(bMoTrp)−Dox complex) is limited,
resulting in lower intensity signal in the difference spectrum at
high temperature. From the above observations, it can be
inferred that the Dox molecules are efficiently trapped inside
the coacervates.
1H−1H nuclear Overhauser effect spectroscopy (NOESY) is
useful for investigating spatial proton−proton interactions in a
system.
24,37−39
It transposes a proton spectrum in both the
horizontal and vertical axes. Therefore, all the similar peaks
meet at the same point which creates a diagonal trend in the
2D spectra. Two 1H spins with different chemical shifts but
spatially close to each other (typically if within 6 Å of each
other) can transfer magnetization between each other, and an
additional signal, known as a cross-peak (off-diagonal peak),
will be observed outside the diagonal trend line. The
magnitude of the nuclear Overhauser effect (NOE) is inversely
proportional to the sixth power of the distance between two
spins. In addition to spatial requirement, the intensity of a
NOESY cross-peak also depends on the correlation time of
molecular motions.
40
In case of small molecules, since the
tumbling rate is higher, NOE builds up at a slower rate. But,
this trend is inverse in case of high molecular weight molecules.
Interestingly, the NOE of a same molecule can be altered by
restricting its molecular motions.
40
In a typical NOESY, the
cross-peaks provide information about interactions (both intra-
and intermolecular) between two molecules.
Here, NOESY spectra of Dox and p(bMoTrp) independ-
ently (Figure 9A and B) were generated to compare with the
p(bMoTrp)−Dox mixture spectrum (Figure 9C). Notably,
there were always more Dox cross peaks present at 23 °C
compared to 50 °C in all the cases (Figure S7). Dox is well-
known to aggregate at low temperatures even in 1 mM
aqueous solution; ∼40 Dox molecules can self-aggregate
together.
41−43
However, dilute solutions of Dox and solutions
at higher temperature can restrict this aggregation phenomen-
on. At 23 °C, the self-aggregation of Dox contributes to an
increase in the intra cross-peaks due to the closer proximity of
Dox molecules. These peaks may cover up the inter cross-
peaks of Dox with p(bMoTrp). Interestingly, at 50 °C, the
intra cross-peaks of Dox in the aromatic region become more
intense in the presence of the polymer mixture (Figure 9C).
Figure 8. STD NMR of the p(bMoTrp)−Dox mixture at (A) 23 and
(B) 50 °C. Dox aromatic peaks are highlighted within the blue box.
The saturation delay increases from bottom to top. At 50 °C, the
strong interaction between Dox and p(bMoTrp) coacervate results in
low STD signal intensity.
Figure 9. 1H−1H NOESY spectra of the aromatic region of (A) Dox, (B) p(bMoTrp), and (C) p(bMoTrp)−Dox mixture at 50 °C. The Dox
cross-peaks are highlighted in the blue circle. In presence of p(bMoTrp), the intensities of those cross-peaks increase as the tumbling rate of Dox
decreases within p(bMoTrp) coacervates resulting in faster NOE buildup.
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F

This indicates that Dox is migrating into the coacervate, which
restricts its tumbling rate and enables the faster buildup of
NOEs.
Diffusion-ordered spectroscopy (DOSY) is a reliable and
powerful NMR technique used to separate individual molecule
peaks from a mixture based on differences in their translational
diffusion coefficients.
44−47
The diffusion coefficient is
influenced by the molecular motion, size, and shape of the
species in solution. The acquired 1D spectral data is analyzed
based on the Stejskal−Tanner relation
48
as given in eq 2.
γδ δ
=− ∇−
Ä
Ç
Å
Å
Å
Å
Å
Å
Å
Å
Å
i
k
j
j
jy
{
z
z
z
É
Ö
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
Ñ
I
IGDexp 3
0
22 2
(2)
Iis the peak intensity under pulsed field gradient G,I0is the
signal intensity without gradient G,γis the gyromagnetic ratio,
δis gradient pulse duration, Δis pulse interval, and Dis
diffusion coefficient. After obtaining intensity information, the
data is fit by plotting intensity versus gradient strength to
obtain a diffusion constant value for each peak. The final plot
of Dversus peak chemical shift is generated, where Dvalue
increases from top to bottom on the y-axis.
The diffusivity differences between Dox alone and p-
(bMoTrp)−Dox were compared using DOSY NMR (Figure
10). As p(bMoTrp) is larger than Dox, the polymer
concentration was maintained at a lower value (0.08 mM)
than the Dox (4.92 mM), to enable clear observation of the
Dox peaks. The aromatic peaks from p(bMoTrp) were not
observed as it only comprises 5% of the total p(bMoTrp). At
50 °C, in case of p(bMoTrp)−Dox, the diffusion constant of
Dox was lower compared to that of Dox in the absence of
p(bMoTrp). As Dox molecules are being encapsulated inside
the coacervates, their molecular motion inside the solution
become constrained and hence they diffuse at a slower rate
compared to their motion in the absence of coacervates. This
difference in diffusion coefficients can be attributed to the
encapsulation phenomenon of Dox within the p(bMoTrp)
coacervates.
■CONCLUSION
In this study, a thermoresponsive polyester was designed to
interact with and encapsulate Dox efficiently and to study the
interactions between Dox and the polyester by various NMR
protocols. This work provides a compilation of polyester−drug
NMR data which offers insight into the interactions between
the polyester and Dox both below and above the Tcp. The
molecular actions of this polyester−drug model system were
evaluated using a variety of NMR techniques to better
understand the noncovalent interactions that govern DDS
behaviors. VT NMR experiments of polymer coacervate
formation showed that unlike other typical thermoresponsive
polymers, this polyester is not fully dehydrated after coacervate
formation. STD NMR proved to be an advantageous tool to
probe the interactions of Dox within the polyester coacervates.
The peak enhancements from the STD experiments clearly
indicated the tighter binding of Dox within the coacervates.
Along with STD NMR, both 1H−1H NOESY and DOSY
NMR techniques substantiated Dox−polyester interactions
above the Tcp. The NOESY experiments showed that cross-
peak intensities of Dox within the coacervates were enhanced
and the DOSY experiments confirmed that the diffusion rates
of Dox within the coacervates became slower. The protocols
described in this study can benefit future investigations into the
development of coacervate DDS and enable a detailed
understanding of the interactions between a targeted
encapsulated drug and the polymer, which in turn can be
used to optimize the efficiency of controlled release systems.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsabm.0c00507.
Detailed synthetic schemes and proton NMR spectra
(PDF)
■AUTHOR INFORMATION
Corresponding Author
Abraham Joy −Department of Polymer Science, The University
of Akron, Akron, Ohio 44325, United States; orcid.org/
0000-0001-7781-3817; Email: abraham@uakron.edu
Authors
Mangaldeep Kundu −Department of Polymer Science, The
University of Akron, Akron, Ohio 44325, United States;
orcid.org/0000-0003-2555-0474
Daniel L. Morris −Department of Chemistry and Biochemistry,
The University of Akron, Akron, Ohio 44325, United States
Megan A. Cruz −Department of Polymer Science, The
University of Akron, Akron, Ohio 44325, United States
Toshikazu Miyoshi −Department of Polymer Science, The
University of Akron, Akron, Ohio 44325, United States;
orcid.org/0000-0001-8344-9687
Thomas C. Leeper −College of Science and Mathematics,
Kennesaw State University, Kennesaw, Georgia 30144, United
States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsabm.0c00507
Notes
The authors declare no competing financial interest.
Figure 10. DOSY of Dox and p(bMoTrp)−Dox mixture at 50 °C.
The aromatic Dox peaks are highlighted inside the blue box. As
expected, diffusion of Dox within the p(bMoTrp) coacervates is
slower compared to Dox in the absence p(bMoTrp).
ACS Applied Bio Materials www.acsabm.org Article
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ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
G

■ACKNOWLEDGMENTS
The work described herein was funded in part by NSF grant
1352485. We thank Dr. Venkat Dudipala, Director of Magnetic
Resonance Center at The University of Akron, for insightful
input regarding setting up and running NMR protocols.
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