Drug release from hydrazone-containing peptide amphiphiles

Article abstract of DOI:10.1039/c1cc12570b

Hydrolytically-labile hydrazones in peptide amphiphiles were studied as degradable tethers for release of the drug nabumetone from nanofiber gels. On-resin addition of the novel compound tri-Boc-hydrazido adipic acid to a lysine ε-amine allowed for precis

Full text of DOI:10.1039/c1cc12570b

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 7962–7964
www.rsc.org/chemcomm
COMMUNICATION
Drug release from hydrazone-containing peptide amphiphilesw
John B. Matsona and Samuel I. Stupp*abcd
Received 2nd May 2011, Accepted 27th May 2011
DOI: 10.1039/c1cc12570b
Hydrolytically-labile hydrazones in peptide amphiphiles were
studied as degradable tethers for release of the drug nabumetone
from nanofiber gels. On-resin addition of the novel compound
tri-Boc-hydrazido adipic acid to a lysine e-amine allowed for
precise placement of a hydrazide in a peptide sequence.
several microns in length and in most cases are always in an
assembled state in aqueous solution. Charge screening by
addition of salts, especially multivalent counterions, or a
change in pH induces network formation and gelation at
concentrations on the order of 1% by weight.13 Previous
studies on PA systems have shown the capabilities of PAs to
support, signal and deliver cells through the efficient display of
a bioactive peptide sequence.7,14 Here we explore a new
method for the delivery of soluble small molecules from PA
gels through the use of hydrolytically labile hydrazone tethers.
These small molecules may take the form of therapeutic drugs
or biological signals.
Delivery of drugs through implantable materials and devices
has transformed several areas of medicine, including diabetes
treatment, contraception, and cardiovascular repair.1 However,
increased control over drug release rates and greater bio-
compatibility and biodegradability of the delivery materials
are still necessary improvements. Especially promising new
varieties of implantable biomaterials are those made from
biodegradable, self-assembling small molecules.2,3 Not only
are such systems more biocompatible compared with traditional
polymeric gels, but all components of these systems can also be
completely characterized on a molecular scale. Despite their
promise as materials, sustained drug delivery from biodegradable,
small molecule gels is still an underexplored area in the fields
of drug delivery and regenerative medicine.4,5
Control of drug release rate can often be accomplished by
covalently attaching a drug to a hydrolytically labile bond,
such as a hydrazone, acetal or orthoester.15–17 Typically, the
release rate is governed by the pH of the surrounding media,
with faster release observed in acidic (pH 5–6) environments
compared with physiological media (pH 7.4). Hydrazone
bonds are convenient hydrolytically labile bonds due to the
usually facile incorporation of hydrazides into delivery materials.
However, site-specific incorporation of hydrazides into peptides
via solid phase peptide synthesis is still synthetically challenging.
We first attempted on-resin addition of protected hydrazides
by the previously reported strategy of adding Boc-NHNHC(O)-
CH2CH2COOH onto a lysine e-amine.18,19 However, we
found couplings with this reagent to be very inefficient due
to undesired intramolecular cyclization reactions, which others
have also noted (see Supporting Information for detailsw).20,21
A second attempted strategy was to use (Boc)2NN(Boc)CH2-
COOH to reveal peptide hydrazines after cleavage, as reported
by Melnyk.22 Though the synthesis was effective, the resulting
peptide hydrazines were found to undergo oxidative decom-
position under the reaction conditions required for hydrazone
formation. Inspired by Melnyk’s synthesis, we synthesized
triply-protected hydrazido acid 4 for use in SPPS. It was
expected that the carbonyl of the hydrazide would provide
more oxidative stability compared with the hydrazine.
Over the past decade, our laboratory has developed and
studied peptide amphiphiles (PAs) programmed to self-assemble
into high aspect ratio nanofibers.6 These biocompatible PAs
are a broad class of molecules that have recently received
great attention due to their promise as bioactive materials
in regenerative applications ranging from central nervous
system regeneration and angiogenesis to bone, cartilage, and
enamel regeneration.7–12 PAs are synthesized by attaching a
hydrophobic tail to a short peptide sequence that includes
charged residues to promote solubility and control gelation
through electrolyte screening. When the peptide sequence design
includes a strong b-sheet forming sequence, PAs can self-assemble
into long, filamentous aggregates in aqueous solution.6 These
nanostructures are approximately 10 nm in width and up to
a Institute for BioNanotechnology in Medicine,
Northwestern University, Chicago, IL 60611, USA.
E-mail: s-stupp@northwestern.edu; Fax: (+312) 503-2482;
Tel: (+312) 503-6713
Synthesis of 4 was accomplished in three steps from
previously reported adipic acid monobenzyl ester (1) according
to Scheme 1. First, tert-butylcarbazate was coupled to 1 using
EDC and DPTS, affording monoprotected hydrazide 2
(DPTS = dimethylaminopyridinium p-toluene sulfonate23).
Addition of Boc2O to 2 yielded the triprotected derivative, 3.
Hydrogenolysis of the benzyl ester with Pd/C furnished
triprotected hydrazido acid 4. All reactions showed good to
excellent yields, and no chromatography was required.
b Department of Chemistry, Northwestern University,
Evanston, IL 60208, USA
c Department of Materials Science and Engineering,
Northwestern University, Evanston, IL 60208, USA
d Feinberg School of Medicine, Northwestern University,
Chicago, IL 60611, USA
w Electronic supplementary information (ESI) available: Full experi-
mental details and characterization data on new compounds, SAXS
fittings and aggregate parameters. See DOI: 10.1039/c1cc12570b
c
7962 Chem. Commun., 2011, 47, 7962–7964
This journal is The Royal Society of Chemistry 2011

View Article Online
used to further investigate the aggregates formed from PAs 5
and 6. A slope of À1 in the low q region is indicative of
cylindrical aggregates and would be expected;24 however, the
slight deviation from the À1 slope observed in the SAXS curve
of PA 6 (Fig. 1D) is likely a result of a small contribution of
bundled aggregates. The even steeper slope in the low q region
seen in the PA 5 scattering curve (Fig. 1C) is consistent with
the higher-order aggregates (bundles) seen in the TEM. We
attribute the bundling observed in PA 5 to interfiber hydrogen
bonding between surface hydrazide groups. Addition of Nb
yields a hydrazone that is less capable of hydrogen bonding,
which may explain the reduced number of higher-order
aggregates observed in PA 6. Excluding the low q region, the
SAXS curves can be reasonably fitted to a polydisperse
core-shell cylinder form factor (Supporting Informationw).
The best fit parameters yielded an average fiber diameter of
7.1 nm for PA 5 and 7.0 nm for PA 6, supporting TEM results
that addition of Nb did not alter fiber dimensions.
Scheme 1 Synthesis of triply-protected hydrazide building block for
solid phase peptide synthesis. Reagents and conditions: (i) tert-butyl-
carbazate, EDC, DPTS, CH2Cl2, 2 h, 83%; (ii) Boc2O, DMAP, NEt3,
CH2Cl2, 2 h, 94%; (iii) H2 (1 atm), Pd/C, EtOH, 4 h, 86%.
Applying building block 4 to the synthesis of potentially
bioactive materials, hydrazide-containing PA 5 was produced
using standard Fmoc-based solid-phase peptide synthesis
conditions. A free amine was generated on-resin by addition
of a Lys(Mtt) residue (Mtt = 4-methyltrityl) with selective
removal of the Mtt protecting group using 5% TFA in
CH2Cl2. Hydrazino acid 4 was activated using HBTU and
DIEA and added on-resin to the exposed lysine e-amine.
Following cleavage using 95% TFA, which removed all
protecting groups, PA 5 was purified by HPLC. The unprotected
hydrazide was found to be oxidatively stable in air in organic
solvents at 40 1C for at least 24 h.
The ketone-containing, non-steroidal anti-inflammatory
drug nabumetone (Nb) was chosen as the model drug to serve
as a release probe. Condensation of Nb with PA 5 was carried
out in DMSO at room temperature to form hydrazone-
containing PA 6, which was purified by HPLC (Scheme 2).
Structural characterization of 5 and 6 was then carried out to
assess the self-assembly of the PAs.
To investigate the release of Nb from PA 6, a mixture of
PA 6 with diluent PA C16V2A2E2 (1 : 1 w/w, see Supporting
Informationw) was prepared and gelled with CaCl2. Scanning
electron microscopy (SEM) of the gel (Fig. 2A) shows the
expected fibrous morphology. The release kinetics of Nb
were measured into a sink solution of buffer by monitoring
absorbance at 330 nm (Fig. 2B). As we are interested in using
drug-delivering PA materials as injectable gels for the long-term,
sustained delivery of drugs, signals, and cells, we chose to
investigate release kinetics at physiological pH. Although
hydrazones are often designated as exhibiting high hydrolytic
stability at physiological pH, slow hydrolysis is often still
observed.25 Release was observed over 24 d, with an initial
burst release but eventual conversion to a zero-order release
profile with t1/2 = 33.9 d, as calculated from the near-linear
region in Fig. 2B. Burst release is likely due to free Nb in the
gel that occurs as a result of sample preparation. Future work
will focus on developing methods to control drug release kinetics.
In summary, a small molecule building block (4) for
incorporation of hydrazides into peptides has been synthesized.
Under standard coupling conditions, 4 can be added on-resin
PAs 5 and 6 were characterized in aqueous solution by
conventional transmission electron microscopy (TEM) and
small angle X-ray scattering (SAXS) (Fig. 1). TEM of PA 5
showed long, cylindrical nanofibers typical of many PAs
synthesized in our laboratory.3 Some bundling of fibers can
be observed in the TEM image. TEM of PA 6 showed similar
cylindrical nanofibers, though they appeared shorter and less
bundled than those of PA 5. SAXS in aqueous solution was
Scheme 2 Synthesis of drug-tethered PA by hydrazone formation.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7962–7964 7963

View Article Online
Fig. 2 SEM (A) and release profile (B) of Nb from gel of PA 6 mixed
1 : 1 with diluent PA into buffer at physiological pH (n = 5).
Notes and references
1 D. A. LaVan, T. McGuire and R. Langer, Nat. Biotechnol., 2003,
21, 1184–1191.
2 P. Y. W. Dankers and E. W. Meijer, Bull. Chem. Soc. Jpn., 2007,
80, 2047–2073.
3 H. G. Cui, M. J. Webber and S. I. Stupp, Biopolymers, 2010, 94,
1–18.
4 M. R. Kapadia, L. W. Chow, N. D. Tsihlis, S. S. Ahanchi,
J. A. Hrabie, J. Murar, J. Martinez, D. A. Popowich, Q. Jiang,
J. E. Saavedra, L. K. Keefer, J. F. Hulvat, S. I. Stupp and
M. P. Kibbe, J. Vasc. Surg., 2008, 47, 173–182.
5 M. C. Branco, D. J. Pochan, N. J. Wagner and J. P. Schneider,
Biomaterials, 2010, 31, 9527–9534.
Fig. 1 TEM and SAXS of PA 5 (A and C) and PA 6 (B and D).
to a lysine e-amine, revealing a free hydrazide upon cleavage
from the resin. We demonstrated an application of this
chemistry by developing a hydrazide-containing PA, to which
a small molecule drug could be tethered via hydrazone formation.
Drug tethering was found not to alter the assembly of the PA
into filamentous aggregates. The small molecule drug could
then be slowly released from the PA gel into aqueous solution.
Further studies on small molecule drug release from PA gels
are currently underway in our laboratory. We expect that building
block 4 will be useful in several areas of peptide and polymer-
based medicine, including peptide-based biomaterials, peptide
labeling and polymeric drug conjugation.
6 J. D. Hartgerink, E. Beniash and S. I. Stupp, Science, 2001, 294,
1684–1688.
7 G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. A. Harrington,
J. A. Kessler and S. I. Stupp, Science, 2004, 303, 1352–1355.
8 K. Rajangam, H. A. Behanna, M. J. Hui, X. Q. Han, J. F. Hulvat,
J. W. Lomasney and S. I. Stupp, Nano Lett., 2006, 6, 2086–2090.
9 M. J. Webber, X. Q. Han, S. N. P. Murthy, K. Rajangam,
S. I. Stupp and J. W. Lomasney, J. Tissue Eng. Regen. M., 2010,
4, 600–610.
10 A. Mata, Y. B. Geng, K. J. Henrikson, C. Aparicio, S. R. Stock,
R. L. Satcher and S. I. Stupp, Biomaterials, 2010, 31, 6004–6012.
11 R. N. Shah, N. A. Shah, M. M. D. Lim, C. Hsieh, G. Nuber and
S. I. Stupp, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 3293–3298.
12 Z. Huang, C. J. Newcomb, P. Bringas, Jr., S. I. Stupp and
M. L. Snead, Biomaterials, 2010, 31, 9202–9211.
13 J. C. Stendahl, M. S. Rao, M. O. Guler and S. I. Stupp, Adv. Funct.
Mater., 2006, 16, 499–508.
14 M. J. Webber, J. Tongers, M. A. Renault, J. G. Roncalli,
D. W. Losordo and S. I. Stupp, Acta Biomater., 2010, 6, 3–11.
15 H. R. Ihre, O. L. P. De Jesus, F. C. Szoka and J. M. J. Frechet,
Bioconjugate Chem., 2002, 13, 443–452.
16 R. M. Sawant, J. P. Hurley, S. Salmaso, A. Kale, E. Tolcheva,
T. S. Levchenko and V. P. Torchilin, Bioconjugate Chem., 2006, 17,
943–949.
17 Y. Bae, S. Fukushima, A. Harada and K. Kataoka, Angew. Chem.,
Int. Ed., 2003, 42, 4640–4643.
18 J. Shao and J. P. Tam, J. Am. Chem. Soc., 1995, 117, 3893–3899.
19 A. Dirksen, S. Dirksen, T. M. Hackeng and P. E. Dawson, J. Am.
Chem. Soc., 2006, 128, 15602–15603.
20 L. Guy, J. Vidal, A. Collet, A. Amour and M. Reboud-Ravaux,
J. Med. Chem., 1998, 41, 4833–4843.
21 C. Grandjean, H. Gras-Masse and O. Melnyk, Chem.–Eur. J.,
2001, 7, 230–239.
This work was supported by the NIDCR, grant No.
2R01DE015920-06 and the NIBIB, grant No. 2R01EB003806-
06A2. JBM was supported by a Baxter Early Career Development
Award in Bioengineering. We are grateful to the Peptide
Synthesis Core in the Institute for BioNanotechnology in
Medicine (IBNAM), the Biological Imaging Facility (BIF),
the Integrated Molecular Structure Education and Research
Center (IMSERC) and the Electron Probe Instrumentation
Center (EPIC) of the Northwestern University Atomic- and
Nanoscale Characterization Experimental Center (NUANCE)
for instrument use. NUANCE Center is supported by NSF-NSEC,
NSF-MRSEC, Keck Foundation, the State of Illinois,
and Northwestern University. We acknowledge Dr Steven
Weigand and the DuPont-Northwestern-Dow Collaborative
Access Team (DND-CAT) Synchrotron Research Center at
the Advanced Photon Source (APS) at Argonne National Lab
for assistance with SAXS measurements. Use of the APS was
supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No.
DE-AC02-06CH11357. Matt Webber and Dr Ronit Bitton of
the authors’ laboratory are acknowledged for help with SEM
and SAXS, respectively.
22 D. Bonnet, C. Grandjean, P. Rousselot-Pailley, P. Joly, L. Bourel-
Bonnet, V. Santraine, H. Gras-Masse and O. Melnyk, J. Org.
Chem., 2003, 68, 7033–7040.
23 J. S. Moore and S. I. Stupp, Macromolecules, 1990, 23, 65–70.
24 O. Glatter and O. Kratky, Small Angle X-ray Scattering, Academic
Press Inc., New York, 1982.
25 J. Kalia and R. T. Raines, Angew. Chem., Int. Ed., 2008, 47,
7523–7526.
c
7964 Chem. Commun., 2011, 47, 7962–7964
This journal is The Royal Society of Chemistry 2011