Drug encapsulation within self-assembled microglobules formed by thermoresponsive supramolecules

Article abstract of DOI:10.1039/c0cc04063k

An 8-(phenyl)-2′-deoxyguanosine derivative self-assembles in aqueous media into discrete hexadecamers that further self-assemble above 32 °C into microglobules that encapsulate the drug doxorubicin.

Full text of DOI:10.1039/c0cc04063k

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Drug encapsulation within self-assembled microglobules formed
by thermoresponsive supramoleculesw
a
b
c
´
Jose E. Betancourt, Chandramouleeswaran Subramani, Jose L. Serrano-Velez,
Eduardo Rosa-Molinar, Vincent M. Rotello and Jose M. Rivera*
´
c
b
a
´
Received 24th September 2010, Accepted 9th October 2010
DOI: 10.1039/c0cc04063k
An 8-(phenyl)-20-deoxyguanosine derivative self-assembles in
aqueous media into discrete hexadecamers that further self-
assemble above 32 1C into microglobules that encapsulate the
drug doxorubicin.
Advances in supramolecular chemistry have led to the design
of a variety of biomimetic materials that are suitable for the
development of stimuli responsive nanocarrier systems.1–3
Light, pH, magnetic fields and temperature are among the
most frequently used stimuli.4 Nano- and microscopic globular
assemblies made from thermoresponsive polymers are likewise
promising systems for responsive drug carriers.5,6 Besides the
elastin-like polypeptides,7 most of the work in this area has
relied on the poly(N-isopropylacrylamide)8 scaffold, and
its copolymers, as environmentally responsive materials due
to their sharp coil-to-globule transition at biocompatible
temperatures of around 32 1C.9
Supramolecular self-assembly offers
a complementary
strategy to the use of polymers for the development of func-
tional nanostructures.10 To circumvent some of the limitations
shown by polymers (e.g., polydispersity, lack of self-correcting
synthesis) and to obtain new thermoresponsive scaffolds,
recently, our lab developed an 8-(m-acetylphenyl)-20-deoxy-
guanosine (mAG) derivative that self-assembles in aqueous
media into discrete supramolecular hexadecamers that exhibit
the Lower Critical Solution Temperature (LCST) phenomenon.11
Such LCST phenomenon occurs with a transition temperature
(Tt) of 58 1C, above which the supramolecular hexadecamers
engage in a temperature induced assembly to form solid
nanoscopic globules of low polydispersity.11 We hypothesized
that these globules could provide a versatile scaffold for
host–guest recognition in aqueous media. Furthermore, if
the Tt were reduced to a value closer to and below body
temperature, these systems may become suitable to prepare
thermoresponsive nano- or microcarriers for bioactive materials
such as drugs. Herein, we report our initial attempts towards
achieving these goals.
Scheme 1 (a) Representation for the (a) formation of a hexadecamer
by mECGD2OH (1) in aqueous media; and (b) stimuli-responsive
behavior of 116 that enables the encapsulation of doxorubicinꢀHCl
(DOX) within the resulting microglobule.
and composition. In recent years we have developed a family
of 8-aryl-20-deoxyguanosine (8ArG) derivatives as versatile
recognition motifs for the construction of supramolecular
nanostructures in both organic12,13 and aqueous media14
(Scheme 1). Our studies suggest that properly placed functional
groups can increase the stability and specificity of the resulting
G-quadruplex supramolecules by enhancing non-covalent
interactions such as hydrogen bonds and p-p.15 To this end,
we synthesized the 8-(m-ethoxycarbonylphenyl)-20-deoxy-
Controlling the Tt via intrinsic parameters (i.e., structural
information in the building blocks of supramolecules) enables
the reliable construction of nanostructures of well-defined size
guanosine (mECGD2OH, 1) derivative, which shows
a
a
´
Department of Chemistry, University of Puerto Rico Rıo Piedras
lower Tt than our previous mAG-based system. This new
8-(m-carbonylphenyl)-20-deoxyguanosine derivative (1) was
synthesized using a methodology similar to that previously
reported by us.11 The pure derivative 1 was obtained after
column chromatography as confirmed by NMR, IR, and mass
spectrometry (for details see ESIw, Fig. S3–S4). Monomer 1 is
Campus, San Juan, PR 00931, USA. E-mail: jmrivortz@mac.com
b Department of Chemistry, University of Massachusetts Amherst,
710 North Pleasant Street, Amherst, MA 01003, USA
´
Department of Biology, University of Puerto Rico Rıo Piedras
Campus, San Juan, PR 00931, USA
c
w Electronic supplementary information (ESI) available: Experimental
details, NMR and DLS. See DOI: 10.1039/c0cc04063k
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8537–8539 8537

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average hydrodynamic diameter (DH) for 116 is 5.0 nm
(Fig. 2c). Upon reaching Tt, the DH of the newly formed
species in solution increased by four orders of magnitude
Fig. 1 Partial 1H NMR spectrum showing the region of the N1–H
and aromatic signals of 116 (500 MHz, 10% D2O in phosphate-
buffered solution, pH 7.4, 2 M KI). The red cartoon represents 116
.
poorly soluble at room temperature (ca., 23 1C) in an aqueous
phosphate-buffered solution (pH 7.4), but goes in solution
upon addition of KI (2 M). 1H NMR experiments of
homogeneous solutions of 1 (10 mM, pH 7.4, 2 M KI) in
H2O–D2O (9 : 1, potassium buffer) reveal the signatures for its
self-assembly. The 1H NMR spectrum shows the characteristic
double set of signals corresponding to two pairs of tetrads in
different chemical environments (Fig. 1). The 2D NOESY
spectrum further supports the formation of 116, by showing
the signature cross peaks characteristic of a hexadecamer in
water (ESIw, Fig. S6).14
The thermoresponsiveness of 116 was determined by trans-
mittance experiments. Aqueous phosphate-buffered solutions
(pH 7.4, 2 M KI) of 116 were completely homogeneous and
highly soluble at room temperature. The turbidity for solutions
of 116 increased upon heating, becoming cloudy at B32 1C.
Transmittance measurements at 500 nm confirm these obser-
vations (Fig. 2a). Upon cooling it back to 25 1C, the solution
again becomes transparent. This process is reversible and
can be repeated at least ten times with no signs of fatigue
(e.g., decomposition, irreversible aggregation) (Fig. 2b).
Dynamic light scattering (DLS) studies provide essential
information regarding the sizes of the various supramolecules
as a function of temperature (25–76 1C). Below the Tt, the
Fig. 3 (a) Effect of increasing the amount of DOX in the Tt of 116
.
(b) Absorption spectra for DOX alone (3.13 mM) and 116 (10 mM)
with 5 equiv. of DOX below (25 1C) and above (40 1C) Tt. The
solutions of 116 with DOX were incubated (45 min) below or above
the Tt and then centrifuged. The supernatant was diluted (800ꢁ) with
the same buffer (phosphate buffer, pH 7.4, 2 M KI) and the absorption
spectra of the diluted solutions were measured. (c) Fluorescence
microscopy images of DOX encapsulated in the globules at 37 1C.
The white scale bar represents 20 mm.
Fig. 2 (a) Turbidity curve (measured at 500 nm) for 116 (red circles).
(b) Change in transmittance at 500 nm when alternating the tempera-
ture between 25 1C and 35 1C. (c) Average hydrodynamic diameters
(DH) of 116 as a function of temperature measured by DLS. (d) OM
micrograph of 116 at 37 1C. All measurements were performed using
an aqueous phosphate-buffered solution (pH 7.4, 2 M KI) of 1
(10 mM). The white scale bar represents 20 mm.
c
8538 Chem. Commun., 2010, 46, 8537–8539
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to >10 mm. Continued heating of the solution from 40 to
60 1C leads to a gradual decrease of the DH until the values
stabilized at o9 mm. This behavior is consistent with a steady
dehydration of the globules. Conversely, the sudden increase
in size above 70 1C and the concomitant increase in poly-
dispersity are likely due to the formation of amorphous
aggregates upon the melting of 116 (Fig. S9, ESIw). Optical
microscopy (OM), with temperature control (37 1C), showed
that these aggregates are discrete microglobules with a relatively
uniform distribution of sizes (Fig. 2d).
of DOX was encapsulated within the globules as inferred by
the diminished absorbance at 491 nm. Fluorescence micro-
scopy studies are also consistent with the encapsulation of
DOX within the microglobules (Fig. 3c).
In summary, we have demonstrated that discrete thermo-
sensitive supramolecules assembled from 8ArG derivatives are
an attractive and complementary strategy to polymer based
systems for drug encapsulation at biocompatible tempera-
tures. The scope and limitations of related systems for the
encapsulation of other bioactive molecules are currently
underway.
We next assessed the influence of binding a guest molecule
on the thermoresponsive properties from 116. Doxorubicin
hydrochloride (DOX) was chosen as the guest molecule
because of its current use as anticancer agent and its inherent
fluorescence properties.16 Previously, DOX has been conjugated
to different carrier molecules such as peptides17 and other
macromolecules.18 Its fluorescence properties has enabled
monitoring its distribution, or that of its polymer conjugates,
in micelles and even in cells.19 As shown in Fig. 3a, in the
absence of DOX, 116 exhibited a Tt of around 32 1C (pH 7.4).
However, in the presence of the drug (2 equiv. of DOX per
hexadecamer) the Tt increases modestly by up to 2 1C. DOX,
which possesses an anthraquinone moiety, could potentially
interact with the core of 116 in a manner reminiscent to that of
daunomycin, a similar anthraquinone drug that interacts with
G-quadruplex DNA structures.20 This behavior agrees with
our previous report using a hexadecamer related to 116,
in which we showed the tuning of the Tt to higher values
by its co-assembly with a more hydrophilic derivative.11
Additional DOX, however, does not seem to further increase
the Tt, underscoring the potential versatility of this system
to encapsulate other drugs with little effect in the thermo-
responsive properties.
This research was financially supported by the Center
for Hierarchical Manufacturing (DMI-0531171) JUNTO
program, NIH-SCoRE (SC1GM093994) and NCRR-NIH
(P20 RR016470). J.E.B. thanks the Alfred P. Sloan Foundation
and NSF-IFN-EPSCoR (01A-0701525) for graduate fellowships.
Notes and references
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Sedimentation experiments provide further evidence for
the encapsulation of DOX within the globules. Host 116
(0.625 mM) was mixed with DOX (5 equiv.) in a phosphate
solution (pH 7.4, 2 M KI) and incubated (45 min) either below
or above the Tt. Afterwards, the solution was centrifuged and
the absorbance of the supernatant was measured. The spectra
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below the Tt followed by centrifugation, were practically
identical (Fig. 3b). In contrast, after incubation above the Tt
(40 1C) followed by centrifugation, the supernatant registered
a decrease in the absorption from DOX. This indicates that the
sedimented globules retain the molecules of DOX. About 75%
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c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8537–8539 8539