Towards smart polymeric drug carriers: Self-assembling γ-substituted polycaprolactones with highly tunable thermoresponsive behavior

Article abstract of DOI:10.1039/c3tb21488e

Synthesis and ring opening polymerization of a new γ-substituted ε-caprolactone monomer, γ-(2-methoxyethoxy)-ε-caprolactone is reported. Amphiphilic diblock copolymers comprised of poly[γ-(2- methoxyethoxy)-ε-caprolactone] and thermosensitive poly{γ-2-[2-

Full text of DOI:10.1039/c3tb21488e

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Materials Chemistry B
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Towards smart polymeric drug carriers: self-assembling
g-substituted polycaprolactones with highly tunable
thermoresponsive behavior†
Cite this: J. Mater. Chem. B, 2013, 1,
6532
Elizabeth A. Rainbolt, Katherine E. Washington, Michael C. Biewer
*
and Mihaela C. Stefan
Synthesis and ring opening polymerization of a new g-substituted 3-caprolactone monomer,
g-(2-methoxyethoxy)-3-caprolactone is reported. Amphiphilic diblock copolymers comprised of
poly[g-(2-methoxyethoxy)-3-caprolactone] and thermosensitive poly{g-2-[2-(2-methoxyethoxy)
ethoxy]ethoxy-3-caprolactone} as the hydrophobic and hydrophilic blocks, respectively, were prepared. The
copolymers exhibited fully biodegradable backbones and highly tunable thermoresponsive behavior in the
range of 31–43 ꢀC. Additionally, the copolymers were shown to self-assemble in aqueous media
Received 22nd October 2013
Accepted 27th October 2013
above their respective critical micelle concentrations, on the order of 10ꢁ2
g . Due to their
Lꢁ1
DOI: 10.1039/c3tb21488e
thermosensitive, self-assembling, and biodegradable properties, these copolymers demonstrate potential
for the use in polymeric micellar drug delivery systems.
www.rsc.org/MaterialsB
controlled release of encapsulated drugs, diblock copolymers
with thermoresponsive behavior, i.e. those that exhibit a lower
Introduction
In recent years, extensive research has focused on the develop-
ment of drug delivery systems that can solubilize, transport, and
release drug molecules in vivo in a controlled manner.1–4 These
efforts are of utmost importance as improving the delivery of
currently available drugs can improve their efficacy, minimize
their side effects, and potentially give FDA-approved molecules
new life. Synthetic organic polymers offer a wide variety of
properties and supramolecular architectures and thusly have
attracted attention for use in biomedical applications.
Polymeric micelles can encapsulate hydrophobic drug mole-
cules and transport them stealthily through the bloodstream.5–7
These micelles tend to accumulate in tumor tissues by the
enhanced permeation and retention (EPR) effect, and can be
designed to release their drug cargoes upon thermal or other
stimulus.2,5 Diblock copolymers intended for polymeric micelles
consist of a hydrophobic block and a hydrophilic block, which
drive the self-assembly in aqueous solution above their critical
micelle concentration (CMC).6,8,9 Typically, the hydrophobic core
is comprised of a biodegradable polyester while the shell is the
highly hydrophilic poly(ethylene glycol) (PEG).2,5 While the PEG
block's good biocompatibility and hydrophilicity help prolong the
micelle circulation time in the body, its inability to biodegrade
has spurred the development of alternatives. With respect to the
critical solution temperature (LCST) above which they are insol-
uble in water, are of particular interest. One of the most widely
studied thermoresponsive polymers for biomedical applications
is poly(N-isopropylacrylamide) (PNIPAAM), which exhibits an
LCST of 32 ꢀC.10,11 Although PNIPAAM has shown promise in
hydrogel-based materials for drug delivery, its low LCST and non-
biodegradable backbone largely preclude its use as an internal
drug carrier.6,12
As evidenced by numerous reports and reviews, aliphatic
polyesters are common components in drug delivery applica-
tions largely due to their biocompatibility and biodegradability
by ester hydrolysis.1,5 More specically, 3-caprolactones have
garnered much interest due to the ease with which they can be
functionalized, polymerized, and tailored to specic applica-
tions.4,13–15 While numerous copolymers have been investigated
and have shown promising potential for drug delivery applica-
tions, few amphiphilic diblock copolymers combine a fully
biodegradable backbone with physiologically relevant and
highly tunable thermoresponsive behavior.12,16 We report here
the synthesis and characterization of new micelle-forming
amphiphilic, biodegradable, and thermosensitive diblock
copolymers from g-substituted caprolactone monomers.
As rst reported by the Stefan group, the substituted
3-caprolactone monomer g-2-[2-(2-methoxyethoxy)ethoxy]-
ethoxy-3-caprolactone (MEEECL) may be polymerized by ring
opening polymerization (ROP) to generate poly{g-2-[2-(2-
methoxyethoxy)ethoxy]ethoxy-3-caprolactone} (PMEEECL), which
Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080,
USA. E-mail: mihaela@utdallas.edu; Fax: +1-972-883-2925; Tel: +1-972-883-6581
† Electronic supplementary information (ESI) available: Experimental procedures
for monomer synthesis, elemental analysis, 1H NMR spectra, CMC, DLS, and LCST
data. See DOI: 10.1039/c3tb21488e
ꢀ
displays a LCST of about 48 C, or even lower if coupled with a
6532 | J. Mater. Chem. B, 2013, 1, 6532–6537
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hydrophobic block as in a diblock copolymer.16 Previous studies spherical micelles was further evidenced by tapping mode
with MEEECL-based polymers have demonstrated the great atomic force microscopy (TMAFM) and transmission electron
potential of MEEECL as a thermoresponsive, biodegradable microscopy (TEM) of the dried micelles (Fig. 1).
hydrophilic block in micellar assemblies, as opposed to the
Upon demonstrating that MECL forms micelles with a PEG
typical PEG block in most micelles for biomedical applications. copolymer, polymers with varying ratios of MEEECL : MECL
Alkoxy- and benzyloxy-substituted caprolactones have been were prepared using ROP with benzyl alcohol initiator and
combined with MEEECL to form thermosensitive diblock copol- stannous ethylhexanoate catalyst as shown in Scheme 2. In
ymers capable of self-assembling into micelles.16,17 While these addition to the four copolymers P2–P5, two homopolymers, P1
polymers have demonstrated acceptable preliminary results, (ESI, Scheme S3†) and P6 (ESI, Scheme S4†) were prepared as
other g-substituted caprolactones are being investigated to controls. A summary of the composition and molecular weights
further enhance biocompatibility, micelle stability, and drug of polymers P1–P6 is shown in Table 1. The molecular weights
loading capacities while maintaining desirable thermosensitive are mostly in the 4500–7500 g molꢁ1 range, with polydispersity
attributes.18
indices (PDI, Mw/Mn) from 1.6 to 1.9. The mol% MEEECL
This article introduces g-(2-methoxyethoxy)-3-caprolactone increases from 0% to 100% from P1 to P6.
(MECL) as a promising candidate for the hydrophobic coun-
For the polymers containing PMEEECL (P2–P6), the LCST
terpart to the hydrophilic MEEECL in diblock copolymers. Of was determined. The polymer solutions were subjected to
particular interest was MECL's effect on the copolymer's LCST heating and cooling and monitored for changes in trans-
given the smaller difference in hydrophobicity between mittance (ESI, Fig. S10†). As the solution was heated above the
MEEECL and MECL. Additionally, the methoxyethoxy side LCST, a drop in transmittance was observed due to the dehy-
unit may interact with encapsulated drugs differently and dration and subsequent precipitation of the polymer. Homo-
could be more biologically friendly compared to alkoxy groups polymer P1 was also subjected to this protocol, but did not
and ultimately contribute to a biologically friendlier drug indicate the presence of an LCST. The LCST was taken to be
delivery system.
the temperature at which a 50% drop in transmittance was
observed upon heating. The transition is not as sharp as that
of previously reported PMEEECL-based copolymers, most
likely due to the smaller difference in hydrophobicity between
the PMEEECL block and the PMECL block. As anticipated, as
the mol% of MEEECL in the polymer increased, so did the
LCST. The data in Fig. 2 point to a linear relationship between
the mol% of hydrophobic block (MECL) and the LCST of the
polymer.
Determination of CMC was performed using pyrene, a small
uorescent molecule whose excitation peak shis from a
maximum intensity at about 335 nm in hydrophilic environ-
ments to a maximum intensity at about 338 nm in hydrophobic
environments. The ratio of the intensities of these two peaks
was monitored as a function of polymer concentration; a sharp
increase in I338/I335 signied the CMC (ESI, Fig. S11†). Copoly-
mers P2–P5 displayed values on the order of 10ꢁ2 g Lꢁ1 for their
respective CMC, which is within an acceptable range for block
copolymer micelles. Homopolymers P1 and P6 were subjected
to the same protocol but gave no indication of self-assembly.
Polymers P2 and P3, with higher hydrophobic PMECL content
Results and discussion
The synthesis of the monomer MECL (Scheme 1) was modied
from the previously reported MEEECL procedure.16 Briey,
2-methoxyethanol was reacted with tosyl chloride to produce
2-methoxyethyl 4-methylbenzenesulfonate (1). Next, 1,4-
cyclohexanediol was deprotonated with NaH then reacted with the
product from the previous step to generate 4-(2-methoxyethoxy)
cyclohexanol (2). Treatment with chromic acid followed by column
purication yielded 4-(2-methoxyethoxy)cyclohexanone (3),
which was then subjected to Baeyer–Villager oxidation to form
g-(2-methoxyethoxy)-3-caprolactone (MECL). 1H NMR, 13C
NMR, and C/H elemental analysis conrmed the formation of
g-(2-methoxyethoxy)-3-caprolactone (ESI, Fig. S4 and Table S1†).
As
a proof-of-concept experiment to demonstrate the
hydrophobicity of MECL, and thus its ability to act as a hydro-
phobic core, a diblock copolymer of PEG (57 mol%) and PMECL
(43 mol%) was prepared (ESI, Scheme S2 and Table S2†). As
anticipated, this polymer P0 self-assembled in aqueous solution
to form micelles of about 50 nm in diameter, and displayed a
critical micelle concentration (CMC) of 9.04 ꢂ 10ꢁ4 g Lꢁ1, both
of which are reasonable values for PEG-containing amphiphilic
diblock copolymer micelles (ESI, Fig. S6†). The formation of
Fig. 1 (a) 3D TMAFM images of polymer P0 micelles deposited on mica
substrate, scan size: 500 nm2; (b) TEM image of polymer micelles, P0 deposited on
copper mesh grid stained with 1% phosphotungstic acid.
Scheme 1 Synthesis of g-2-(methoxyethoxy)-3-caprolactone (MECL).
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Scheme 2 Synthesis of diblock copolymers P2–P5 from g-substituted capro-
lactone monomers MEEECL and MECL.
show slightly lower CMC values as compared to those of P4 and
P5, which contain lower amounts of PMECL.
Dynamic light scattering (DLS) was utilized to estimate the
hydrodynamic diameter (Dh) of the self-assembled polymeric
micelles. For each of the copolymer micelle solutions at room
temperature, the Dh values were centered around 100 nm with
comparable standard deviations of 1 to 3 nm (Fig. 3). The
difference in sizes from the smallest (P2, 81 nm) to the largest
(P4, 113 nm) was attributed to the decrease in size of the
hydrophobic block. The chains of P4 with shorter PMECL
blocks likely pack less tightly than chains of P2 with larger
MECL content.
Fig. 2 Change in percent transmittance at 600 nm upon heating for aqueous
solutions of polymers P2–P6; inset, LCST as a function of mol% MECL.
The effect of temperature changes on the diameter of poly-
meric micelles was investigated using DLS (ESI, Fig. S12†). For
each copolymer, the average diameter increased as the
temperature approached and exceeded the LCST. While the
mechanics of this behavior are beyond the scope of this report,
this change in size may occur with a change in the assemblies'
morphologies as the polymer chains dehydrate. Such
a
Fig. 3 Hydrodynamic diameter (Dh) analysis of polymeric micelles at room
temperature using dynamic light scattering: (a) P2, (b) P3, (c) P4, (d) P5.
phenomenon may be further analyzed and exploited for the
temperature-triggered release of encapsulated drug molecules.
Dried micelle solutions of copolymers P2 and P4 were
imaged using tapping mode AFM and TEM with negative
staining. Polymers P2 and P4 were selected for these prelimi- 80–90 nm in diameter. TEM images of micelles aer negative
nary microscopy studies to compare the morphologies of the staining with phosphotungstic acid showed spherical assem-
self-assemblies with PMEEECL : PMECL block ratios of about blies, but with slightly smaller diameters of about 40 nm for P2
1 : 1 and 3 : 1, respectively. Fig. 4 shows 3D TMAFM and TEM and about 65 nm for P4. Future studies will delve deeper to
images of the P2 and P4 micelles. While for both polymers the better elucidate the morphology as a function of the copolymer
micelles are not completely uniform in size, they do appear to block ratios.
be spherical in shape and show diameters indicative of large
To demonstrate the biodegradability of PMEEECL-b-PMECL
micelles in the 50–100 nm range. Consistent with the DLS data block copolymers, P2 was subjected to heating at 37 ꢀC in
are the relative sizes; for P2 the micelles are approximately 40– phosphate buffer at pH 6 for ve days. Samples were taken
50 nm in diameter whereas the P4 micelles are larger, closer to periodically and analyzed by size exclusion chromatography to
Table 1 Summary of polymers P1–P6
Mn (g molꢁ1
)
PDIa (Mw/Mn)
MEEECLb (mol%)
MECLb (mol%)
LCSTc (ꢀC)
CMCd (g Lꢁ1
)
Dhe (nm)
a
Polymer
P1
P2
P3
P4
P5
P6
7200
6700
7600
7300
4400
4700
1.9
1.8
1.6
1.7
1.7
1.6
0
51
61
76
81
100
49
39
24
19
0
—
—
—
31.1
34.2
38.5
43.5
48.6
1.58 ꢂ 10ꢁ2
3.07 ꢂ 10ꢁ2
3.56 ꢂ 10ꢁ2
4.47 ꢂ 10ꢁ2
—
81 ꢃ 2
101 ꢃ 3
113 ꢃ 3
99 ꢃ 2
—
100
a
Determined by size exclusion chromatography using polystyrene calibration. b Block content mol% determined by 1H NMR. c Determined by 50%
d
e
drop in transmittance at 600 nm upon heating aqueous polymer solution. Determined by uorescence measurements with pyrene. Micelle
hydrodynamic diameter at 25 ꢀC determined using dynamic light scattering.
6534 | J. Mater. Chem. B, 2013, 1, 6532–6537
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were added to the ask, which was immediately lowered into
the oil bath. The reaction was sealed and allowed to stir over-
night at 110 ꢀC. Tetrahydrofuran (THF, 1 mL) was added to the
reaction vessel, and the polymer solution was poured into a
beaker of pentane, where it crashed out. The polymer was
ltered and dried under vacuum, then analyzed by size exclu-
sion chromatography (SEC) and 1H NMR. Fig. S5,† 1H NMR
(500 MHz, CDCl3): dH 1.81 (m, 4H), 2.04 (t, 2H), 3.36 (s, 3H), 3.55
(m, 10H), 4.18 (t, 2H).
Synthesis of poly[g-(2-methoxyethyoxy)-3-caprolactone] (P1)
To an oven-dried 10 mL Schlenk ask, a small egg-shaped stir
bar and MECL (0.40 g, 2.1 mmol) were introduced. The ask was
placed under vacuum for at least 1 hour before introducing
nitrogen atmosphere. A thermostat-controlled oil bath was
ꢀ
heated to 110 C. A stock solution of benzyl alcohol (BnOH) in
dry toluene (46 mg mLꢁ1) was prepared and 0.1 mL BnOH stock
solution (0.04 mmol BnOH) was loaded into a syringe. A solu-
tion of tin ethylhexanoate (Sn(Oct)2), (0.034 mg, 0.084 mmol),
was prepared. Promptly, the catalyst and initiator solutions
were added to the ask, which was immediately lowered into
Fig. 4 3D TMAFM images of polymer micelles deposited on mica substrate, scan
size: 500 nm2; (a) P2, (c) P4. TEM images of polymer micelles deposited on copper
mesh grid stained with 1% phosphotungstic acid; (b) P2, (d) P4.
ꢀ
the oil bath. The reaction was stirred overnight at 110 C, and
then was poured into a beaker containing pentane, where it
crashed out. The polymer was ltered and dried under vacuum,
then analyzed by SEC and 1H NMR. Fig. S7,† 1H NMR (500 MHz,
CDCl3): dH 1.80 (m, 4H), 2.39 (t, 2H), 3.35 (s, 3H), 3.51 (m, 5H),
4.16 (t, 2H).
Synthesis of diblock copolymers containing g-(2-methoxyethoxy)-
3-caprolactone (MECL) and g-2-[2-(2-methoxyethoxy)ethoxy]-
ethoxy-3-caprolactone (MEEECL) (P2–P5)
Fig.
5
Demonstration of biodegradability of PMEEECL-b-PMECL: number-
average molecular weight (from SEC) of P2 as a function of time spent in pH 6
phosphate buffer at 37 ꢀC.
Polymers P2–P5 were prepared with varying MEEECL : MECL
ratios. They were obtained using similar procedures, but used
different feed ratios of the monomers. For P3, the following
procedure was used. A molar ratio of MEEECL : MECL : initiator
¼ 50 : 50 : 1 was employed. To an oven-dried 10 mL Schlenk
ask, MEEECL (0.50 g, 1.8 mmol) was introduced. The ask was
placed under vacuum, and aer an hour of pumping down, the
vacuum in the Schlenk ask was cancelled with nitrogen. A
thermostat-controlled oil bath was heated to 110 ꢀC. A stock
solution of benzyl alcohol (BnOH) containing 39 mg mLꢁ1 in dry
toluene was prepared and 0.1 mL BnOH stock solution
(0.036 mmol BnOH) was loaded into a syringe. Tin ethyl-
hexanoate (Sn(Oct)2), (29 mg, 0.072 mmol), was measured and
dissolved in 0.3 mL of toluene, and loaded into a syringe.
Promptly, the catalyst and initiator solutions were added to the
monitor the change in polymer molecular weight. As seen in
Fig. 5, the polymer molecular weight decreases over several
days, corresponding to the polymer degradation by ester
hydrolysis in acidic conditions.
Experimental
Synthesis and characterization of the monomers, as well as
additional polymer analysis are included in the ESI.†
Synthesis of poly(ethylene glycol)-b-poly[g-(2-methoxyethoxy)-
3-caprolactone] (P0)
To an oven-dried 10 mL Schlenk ask hydroxy-terminated PEG ask, which was immediately lowered into the oil bath. In a
(0.085 g, 0.0425 mmol, Mn ꢄ 2000 g molꢁ1) was introduced and clean scintillation vial, MECL (0.34 g, 1.8 mmol) was measured
the ask was placed under vacuum. Aer an hour of pumping out, and placed under vacuum for 2 hours. Aer 3 hours of
down, the vacuum in the Schlenk ask was cancelled with reacting, a small sample of the reaction mixture was crashed
nitrogen. A thermostat-controlled oil bath was heated to 110 ꢀC. into a scintillation vial containing pentane. The sample was
Meanwhile, MECL (0.40 g, 2.1 mmol) was measured into a vial analyzed by GC/MS and 1H NMR to check for residual monomer.
and placed under vacuum for 1 hour, then cancelled with Upon consumption of the rst monomer, the MECL was dis-
nitrogen and dissolved in 0.2 mL of dry toluene. Similarly, a solved in 0.3 mL toluene, and then added to the reaction vessel.
solution of tin ethylhexanoate, (Sn(Oct)2), (0.034 g, 0.085 mmol) The reaction was stirred overnight at 110 ꢀC, and then was
was prepared. Promptly, the monomer and catalyst solutions poured into a beaker containing pentane, where it crashed out.
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The polymer was ltered and dried under vacuum, then the two trendlines before and aer the abrupt increase in I338
/
analyzed by SEC and 1H NMR. Fig. S8,† 1H NMR (500 MHz,
CDCl3): dH 1.79 (m, 4H), 2.39 (t, 2H), 3.36 (s, 3H), 3.53 (m, 8H),
4.16 (t, 2H).
I335 vs. log[c] was taken to be the critical micelle concentration.
Tapping mode atomic force microscopy (TMAFM) images of
micelles
Synthesis of poly{g-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-3-
caprolactone} (P6)
To a freshly cleaved mica substrate, a drop of polymeric micelle
solution was deposited and allowed to dry. AFM studies were
obtained using a VEECO-dimension 5000 Scanning Probe
Polymerization of MEEECL was performed using the same
1
Microscope with silicon cantilever with spring constant 42 nmꢁ1
.
procedure as P1. Fig. S9,† H NMR (500 MHz, CDCl3): dH 1.80
(m, 4H), 2.38 (t, 2H), 3.37 (s, 3H), 3.55 (m, 13H), 4.16 (t, 2H).
Images were acquired at 1 Hz scan frequency and analyzed with
Nanoscope 7.30 soware to generate the 3D renderings.
Preparation of micelles
Transmission electron microscopy (TEM) images of micelles
Polymeric micelles of the block copolymers P0, P2, P3, P4, and
P5 were formed by nanoprecipitation. In general, the copolymer
(20 mg) was dissolved in THF (0.5 mL) and added dropwise into
10 mL of deionized water under rapid stirring. The solution was
stirred vigorously for a minimum of 3 hours to allow the poly-
mer chains to self-assemble as the THF evaporated.
A drop of polymeric micelle solution was deposited on TEM grid
(200 mesh CF200-Cu from Electron Microscopy Sciences) then
stained with phosphotungstic acid (1%). The TEM images were
obtained using a JEOL JEM-1400 transmission electron microscope.
Demonstration of PMEEECL-b-PMECL biodegradation
Analysis of micelles by dynamic light scattering (DLS)
To a Schlenk ask with 4 mL of phosphate buffer (pH 6.0), 20
mg of P2 was added. This vessel was sealed and stirred at 37 ꢀC
for 5 days. Periodically, 0.1 mL samples were removed and
analyzed by SEC to monitor the change in molecular weight.
Aqueous suspensions of polymeric micelles were prepared as
described above. To obtain the most uniform particles, micelles
were prepared and analyzed on the same day. Prior to
measuring, the micelle suspensions were passed through a
0.2 mm Nylon syringe lter. The micelles were analyzed to deter-
mine their hydrodynamic diameters using dynamic light scat-
tering with a Malvern Zetasizer Nano ZS instrument equipped
with a He–Ne laser (633 nm) and 173ꢀ backscatter detector.
Conclusions
A new g-substituted caprolactone monomer was synthesized and
used as the hydrophobic block in amphiphilic diblock copolymers.
The diblock copolymers synthesized, poly{g-2-[2-(2-methoxyethoxy)
ethoxy]ethoxy-3-caprolactone}-b-poly{g-(2-methoxyethoxy)-3-
caprolactone}, demonstrated variable properties as a func-
tion of their relative hydrophilic-hydrophobic block ratios.
These copolymers exhibited fully biodegradable backbones
and demonstrated not only self-assembly into micelles, but
also highly tunable LCSTs in the range of 31–43 ꢀC.
Considering these promising preliminary results, PMEEECL-
b-PMECL copolymers will be further optimized and studied
for use as micellar drug carriers.
Determination of lower critical solution temperature (LCST)
A solution of 0.3 wt% polymer in water was prepared and
ltered through a 0.45 mm Nylon syringe lter. The solution
was stirred and slowly heated in a thermostat-controlled
water bath. The change in % transmittance at 600 nm versus
the temperature of the solution was recorded on an Agilent
UV/Vis spectrophotometer and plotted. The temperature at
which the % transmittance sharply drops to below 50% was
taken as the LCST.
Acknowledgements
Determination of critical micelle concentration (CMC)
The critical micelle concentration was determined using the
uorescent molecule pyrene as a probe. Samples of polymer of
varying concentrations were combined with a small amount of
pyrene in less than 0.1 mL THF. These solutions were added
dropwise into 10 mL of deionized water in a scintillation vial
with a small stir bar. The solutions were stirred for a minimum
of 3 hours to allow the micelles to assemble as the THF evap-
orated. The resulting aqueous solutions contained 10ꢁ5 to 100 g
Lꢁ1 of polymer, and a constant pyrene concentration of 6.67 ꢂ
10ꢁ5 g Lꢁ1. Fluorescence spectra of the polymer–pyrene solu-
tions were collected with a Perkin-Elmer LS 50 BL luminescence
spectrometer at 25 ꢀC with emission wavelength set at 390 nm.
The ratio of the intensities of the pyrene excitation peaks at 338
nm and 335 nm were recorded and plotted against the log of
polymer concentration. The x coordinate at the intersection of
The authors gratefully acknowledge nancial support from the
Welch Foundation (AT1740), NSF-Career grant (DMR-0956116),
as well as the NSF-MRI grant (CHE-1126177), which was used to
purchase the Bruker AVANCE III 500 NMR instrument. Special
thanks to Siegwart Lab and Gao Lab at UT Southwestern for
assistance with TEM image acquisition.
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