Enzyme-instructed molecular self-assembly confers nanofibers and a supramolecular hydrogel of taxol derivative

Article abstract of DOI:10.1021/ja904411z

(Figure Presented) By covalently connecting taxol with a motif that is prone to self-assemble, we successfully generate the precursor (5a), the hydrogelator (5b), and hydrogel of a taxol derivative without compromising the cytotoxic activity of the taxol.

Full text of DOI:10.1021/ja904411z

Published on Web 09/04/2009
Enzyme-Instructed Molecular Self-assembly Confers Nanofibers and a
Supramolecular Hydrogel of Taxol Derivative
Yuan Gao,† Yi Kuang,† Zu-Feng Guo,‡ Zhihong Guo,‡ Isaac J. Krauss,† and Bing Xu*,†
Department of Chemistry, Brandeis UniVersity, 415 South Street, Waltham, Massachusetts 02454, and Department
of Chemistry, The Hong Kong UniVersity of Science and Technology, Clear Water Bay, Hong Kong, China
Received May 31, 2009; E-mail: bxu@brandeis.edu
This communication reports the use of an enzymatic reaction to
initiate the self-assembly of a derivative of taxol in water to form
nanofibers that result in a supramolecular hydrogel. Composed of
a three-dimensional, elastic network spanning the volume of
aqueous media, hydrogels are an important class of biomaterials.1
Natural hydrogels exist as components in complex organisms,2 such
as the bodies of jellyfish, connective tissues in joints, the cornea in
the eye, and nuclear pore complexes inside cells,3 and man-made
hydrogels continue to find successful applications in biomedicine
such as contact lenses,4 drug delivery,5 and tissue engineering.6
Although a majority of hydrogels are polymeric hydrogels whose
networks consist of covalently cross-linked natural or synthetic
polymers,6 supramolecular hydrogels,7,8 whose networks consist
of nanofibers formed through the self-assembly of small molecules
(i.e., hydrogelators7), have emerged as promising biomaterials in
the past decade.9 Usually, changes in temperature, pH, or ionic
strength can successfully trigger the formation of supramolecular
hydrogels. It is, however, advantageous to use inherent biological
processes to create supramolecular hydrogels in ViVo or in situ for
certain biomedical applications.10 By mimicking biomacromolecular
self-assembly (e.g., formation of collagen fibrils11), the integration
of enzymatic reactions with self-assembly of small molecules
provides an effective means to form nanofiber networks and results
in hydrogels under various conditions.12-14
Scheme 1 shows the synthetic route and the structure of 5a, which
consists of a self-assembly motif,17 an enzyme-cleavable group,14
a linker, and a taxol molecule. Based on the study of the
structure-activity of taxol derivatives,18 we connected a simple
linker (succinic acid) to the C2′ hydroxyl group of taxol (1) and
obtained the intermediate (2),19 which can be activated by N-
hydroxysuccinimide (NHS) to afford 3. The reaction of 3 with a
phosphatase substrate (NapFFKYp, 4)20 that consists of the self-
assembly motif and the enzyme-cleavable group affords 5a in an
overall yield of 37.1%. Compared to taxols and the pyridinium taxol
prodrug,21 5a exhibits better aqueous solubility (7.6 mg/mL or 4.26
mM in a 100 mM phosphate buffered saline solution) and has a
distribution coefficient (octanol/water) of 0.61. Compound 5a has
excellent stability in water and shows hardly any dephosphorylation
over months in the absence of a phosphatase.20
Scheme 1a
While it is feasible to initiate hydrogelation using an enzyme that
converts a precursor into a hydrogelator,13 most precursors explored
so far bear limited biological activities. To create hydrogels with
sophisticated biological functions by design, we chose to synthesize
and examine a hydrogel precursor (5a) based on taxol (1), a well-
established antineoplastic agent that binds specifically to the ꢀ-tubulin
subunit of microtubules (MT) to arrest mitosis and result in pro-
grammed cell death (i.e., apoptosis) and that has shown remarkable
activity in the treatment of breast, lung, ovarian, bladder and head and
neck cancers.15 To connect taxol covalently with a motif that tends to
self-assemble and a group that is cleavable by an enzyme, we designed
and synthesized a precursor for producing a taxol hydrogel without
compromising activity of the taxol. As illustrated in Scheme 1, upon
the action of an enzyme, the precursor (5a) transforms into a
hydrogelator (5b), which self-assembles into nanofibers and affords a
supramolecular hydrogel of the taxol derivative (5b). This hydrogel
can slowly release 5b into an aqueous medium. In addition to
representing the first example of enzyme-instructed self-assembly and
hydrogelation of complex, bioactive small molecules, this result
demonstrates a new, facile way to formulate highly hydrophobic drugs,
such as taxol, into an aqueous form (e.g., hydrogel) without comprising
their activity and promises a general methodology to create therapeutic
molecules that have a dual role16 as the delivery vehicle and the drug
itself.
a (a) Succinic anhydride, DIEA, chloroform; (b) NHS, DCC, chloroform;
(c) sodium carbonate, acetone, water.
Figure 1. Optical (A-C) and the corresponding transmission electron micro-
scopic (TEM) images (D-F) of the solution of 5a with [5a] ) 1.0 wt % (A, D);
the solution of 5a at 5 min after the addition of alkaline phosphatase (ALP) (B, E);
and the hydrogel of 5b overnight after the addition of ALP (C, F).
After dissolving 10 mg of 5a into 1 mL of water at pH ) 7.3
with the aid of sonication (Figure 1A), we added 5 µL of alkaline
phosphatase (10 U/µL) into the solution. The solution becomes
slightly turbid (Figure 1B) 5 min after the addition of the enzyme
† Brandeis University.
‡ The Hong Kong University of Science and Technology.
9
13576 J. AM. CHEM. SOC. 2009, 131, 13576–13577
10.1021/ja904411z CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
and turns into a translucent hydrogel (Gel5b, Figure 1C) overnight.
Mass spectroscopic (MS) and HPLC data (Figure S3) confirm the
complete conversion of 5a to 5b in the hydrogel.20 Moreover, MS
analysis indicates that 5b is stable in the gel state over weeks,20 an
important prerequisite for the sustained release of 5b from its own
hydrogel (Vide infra). As shown in the TEM image (Figure 1D), a
solution of 5a gives featureless aggregates after cryo-drying.
According to the TEM in Figure 1E, 5 min after the addition of
the enzyme, the mixture already contains the nanofibers with a width
of 20 nm, in addition to particle aggregates. The nanofibers appear
to stretch out of the amorphous area, suggesting that the nanofiber
grows from the enzymes, consistent with an enzyme-catalyzed self-
assembly process. While its scanning electron micrograph (SEM)
shows lamellar microstructures (Figure S4),20 the cryo-dried Gel5b
exhibits well-dispersed nanofiber networks with a uniform fiber
width of 29 nm in its TEM (Figure 1F). These results confirm the
self-assembly and formation of the nanofibers upon enzyme
catalysis. Circular dichroism (CD) spectra of the solution of 5a
and the corresponding Gel5b (Figure S5)20 further help elucidate
the molecular arrangement of 5b in gel phase. The spectrum of
Gel5b exhibits a positive band near 192 nm (ππ* transition of the
amide bonds) and a broad negative band near 216 nm (nπ*
transition of the amide bonds and ππ* of the naphthyl aromatics),
coinciding with the CD of NapFFGEY17 and indicating the
existence of ꢀ-sheet-like features. Moreover, the intensity of the
peak at 298 nm, a characteristic peak of taxol (1),22 decreases
dramatically in the CD of Gel5b, compared to that of the solution
of 5a, indicating that 5b might align in the nanofibers in such a
way to force the intrinsic dipole transition moments of the taxols
to opposite directions to reduce each other,23 which agrees with
the antiparallel arrangement in a ꢀ-sheet-like secondary structure.17
Collectively, CD, TEM, and SEM indicate that 5b self-assembles
into a ꢀ-sheet-like structure to afford nanofibers that reach high
density and result in sheet-like matrices in the hydrogel.
from the treatment of the solution of 5a (0.8 wt %) with alkaline
phosphatase, and a mixed gel made by adding alkaline phosphatase
into the solution of 5a (0.6 wt %) and 4 (0.6 wt %). Once in contact
with a fresh PBS buffer solution,24 Gel 5b and the mixed gel release
5b at rates of 0.13% and 0.046% per hour, respectively. These
preliminary results demonstrate the sustained release of 5b from
its own gel and suggest a way for release rate control via the
concentration of 5b in the mixed gel.
In conclusion, we have demonstrated that, with proper molecular
design, the integration of enzymatic reaction and self-assembly
provides a powerful method to create molecular hydrogels of
clinically used therapeutics without compromising their bioactivities.
This work also suggests that drug molecules are excellent candidates
for engineering functional hydrogels or soft materials for various
biomedical applications, including sustained or controlled drug
delivery. In addition, this work demonstrates enzyme-instructed self-
assembly as a facile strategy for generating the supramolecular
hydrogels of molecules that inherently have poor solubility in water.
Acknowledgment. This work was partially supported by NSF
(DMR 0820492), start-up grant from Brandeis University, RGC-
Hong Kong (663608), and an HFSP grant (RGP0056/2008).
Supporting Information Available: The experimental section, MS,
NMR, CD, SEM, optical images, HPLC traces, cell viability, drug
release procedure, and the structures of 4. This material is available
free of charge via the Internet at http://pubs.acs.org.
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To evaluate the activity of 5a, we used it to treat HeLa cells and
used taxol (1) as the control. As shown in Figure 2A, after 48 h of
incubation with HeLa cells, 5a exhibits an IC50 value of 25.2 (
2.2 nM, which is comparable to that of 1 (13.5 ( 2.2 nM). Further
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