Esterase-activated release of naproxen from supramolecular nanofibres

Article abstract of DOI:10.1039/c4cc06340f

Nanofibre forming peptide amphiphiles were conjugated to naproxen through an esterase-sensitive linker. The amount of naproxen released, in the presence of enzymes, was influenced by the linker conjugating the drug to the supramolecular assembly. In vitro studies showed the anti-inflammatory activity of the released drug was maintained. This journal is

Full text of DOI:10.1039/c4cc06340f

ChemComm
View Article Online
View Journal
COMMUNICATION
Esterase-activated release of naproxen from
supramolecular nanofibres†
Cite this:DOI: 10.1039/c4cc06340f
Martin Conda-Sheridan,‡a Sungsoo S. Lee,‡b Adam T. Preslarc and
Samuel I. Stupp*abcde
Received 12th August 2014,
Accepted 18th September 2014
DOI: 10.1039/c4cc06340f
www.rsc.org/chemcomm
Nanofibre forming peptide amphiphiles were conjugated to naproxen to a glutathione-responsive disulphylbutyrate linker and attached
through an esterase-sensitive linker. The amount of naproxen released, it to a peptide domain. This approach places the CPT segments
in the presence of enzymes, was influenced by the linker conjugating as the hydrophobic core of the nanostructure. Under tumor-
the drug to the supramolecular assembly. In vitro studies showed the relevant conditions, these nanostructures exhibited in vitro
anti-inflammatory activity of the released drug was maintained.
efficacy against cancer cell lines.16
The previous examples, hydrazine–PA and disulphylbutyrate–
Over the past few years various types of nanocarriers have been PA nanofibres, are useful platforms to deliver either ketone- or
developed to achieve controlled drug release in response to alcohol-containing therapeutic agents. However, these strategies
external stimuli such as pH, temperature, light, or enzymes.1–4 cannot be extended to the large family of drugs with an acidic
These strategies can minimize the limitations of small molecule functionality, including atorvastatin (Lipitors), pregabalin
therapies including metabolism to inactive products and side (Lyricas), and levofloxacin (Levaquins). Naproxen is an acid-
effects.5,6 Bioactive supramolecular nanostructures based on bearing nonsteroidal anti-inflammatory drug used to treat
peptide amphiphiles (PAs) are a versatile platform for the delivery osteoarthritis,17 rheumatoid arthritis,18 and cancer.19 We report
of small molecules. PAs are composed of natural amino acid here the development of a peptide-based supramolecular nano-
sequences covalently conjugated to hydrophobic segments. structure bearing sensitive naproxen–ester linkers for enzyme-
These molecules can self-assemble in aqueous solutions into triggered release. Enzymatic drug release is attractive because it
high-aspect-ratio nanostructures, such as fibres and ribbons, does not require an external stimulus and can be tailored to
with their hydrophobic segments packed away from the aqueous diseases expressing specific enzymes.20
interface.7–9 The peptide domain near the hydrophobic core can be
Esterases are ubiquitous enzymes found in the body with
varied to tune the supramolecular cohesion of the nanostructures.10 varying degrees of specificity.21 Many esterases, such as human
PAs can be functionalized to display, on the surfaces of the nano- carboxylesterase and porcine liver esterase (PLE),22 present the
structures, bioactive cues for cell signalling and targeting.11–13 conserved catalytic triad sequence SHE deep inside the protein,23
Recently, PAs have been used as drug delivery systems by around 19 Å from the surface (Fig. S1, ESI†). Thus, any nanofibre–
covalently attaching small molecules containing a ketone func- drug conjugate must have a linker long enough to access the
tionality via a pH-labile hydrazone linkage.14,15 In a similar catalytic site to be hydrolysed by the enzyme. Scheme 1 shows the
approach, Cui and co-workers conjugated camptothecin (CPT) synthesis of the PA–naproxen conjugate 3 (32.5 Å from the ester
bond to the PA core, linker in red, naproxen in blue, also Fig. S2,
a Simpson Querrey Institute for BioNanotechnology, Northwestern University,
Chicago, IL 60611, USA. E-mail: s-stupp@northwestern.edu;
Fax: +1 312 503 1222; Tel: +1 312 503 0807
ESI†). In addition, we prepared PAs 4 and 5, with linkers of with a
19.4 Å or 11.6 Å extended length, respectively, to study their
differences in enzymatic hydrolysis (Scheme 2). Such molecular
design allows the presentation of the naproxen–ester derivatives
on the surface of the nanofibres, where we expect greater confor-
mational degrees of freedom relative to the hydrophobic core for
optimal esterase accessibility.24,25
b Department of Materials Science and Engineering, Northwestern University,
Evanston, IL 60208, USA
c Department of Chemistry, Northwestern University, Evanston, IL 60208, USA
d Department of Medicine, Northwestern University, Chicago, IL 60611, USA
e Department of Biomedical Engineering, Northwestern University, Evanston,
IL 60208, USA
We also synthesized C16-V3A3E3 (E3)26 PA with the same V3A3
b-sheet-forming domain to dilute the drug-conjugated PAs 3–5
within the supramolecular nanofibres. In previous investigations,
we have found that reducing the surface density of bioactive PAs
† Electronic supplementary information (ESI) available: Full experimental details
and characterization data on new compounds, CryoTEM, CD, 1H and 13C NMR,
HPLC and LC-MS traces. See DOI: 10.1039/c4cc06340f
‡ These authors contributed equally.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun.

View Article Online
Communication
ChemComm
Scheme 1 Synthesis of PA–naproxen conjugate 3. (i) PyBOP, DIPEA, DMF, DCM; (ii) TFA, TIPS, TIPS, H2O.
Fig. 1 (a) Representative cryogenic transmission electron micrograph of
1 : 3 molar mixtures of PA 3 with E3 PA. (b) Molecular graphics representa-
tion of a nanofibre assembled from PA 3 with E3 PA in 1 : 3 molar ratio.
Scheme 2 Structures of PA–naproxen conjugates 4 and 5.
In order to evaluate enzymatic hydrolysis, PA 3 was diluted
with PA E3 (1 : 3 molar ratio) and incubated in PBS (pH 7.4) with
on the nanofibres, by mixing with diluent PAs, promoted porcine liver esterase (PLE) at 20 U mLÀ1, a physiologically
enhanced biological responses in vitro and in vivo27–29 In this relevant concentration.31 Fig. 2a shows the HPLC trace of PA 3
work we co-assembled the naproxen-conjugated PAs with E3 PA before and after the addition of PLE. The peaks were identified
at 1 : 3 ratio (a higher proportion of the drug-conjugated PAs by mass spectrometry as naproxen (11.8 min), PA E3 (14.6 min),
resulted in poor water solubility). After co-assembly, cryogenic hydrolysed PA 3 (15.6 min), and non-hydrolysed PA 3 (17.3 min).
transmission electron microscopy micrographs revealed the for- Naproxen release from PA 3 was 50 Æ 4% after 5 h and 56 Æ 2%
mation of nanofibres (Fig. 1 and Fig. S3, ESI†). As previously after 24 h (Fig. 2b) with no substantial release at later time points.
reported, one-dimensional micron-long nanofibres are typical of This result indicated that PA 3, which is stable in PBS at
the C16-V3A3E3 sequence.26,28 In addition, circular dichroism of the physiological pH, can be enzymatically cleaved to release the
same systems indicated the presence of b-sheets (Fig. S4, ESI†).
drug. However, we did not observe complete hydrolysis, as
To evaluate the stability of the ester linkage, we first inves- evidenced by the presence of non-hydrolysed PA 3 in the HPLC
tigated base-promoted hydrolysis of the PA–ester conjugates 3 trace. Interestingly, we found that introducing additional PLE
diluted with a filler PA E3 (1 : 3 molar ratio) at different pH (10 U mLÀ1) after 24 h only increased naproxen hydrolysis to
values: 7.4, 9.0 and 12.0. The PA–ester conjugate exhibited 61 Æ 3% (Fig. S6, ESI†). In addition, when PA 3–PA E3 (1 : 3
chemical stability towards hydrolysis at pH 7.4, with only molar ratio) was incubated 24 h with varied amounts of PLE, we
2.8 Æ 2.3% of naproxen released after 7 d (Fig. S5, ESI†). The did not find a substantial difference in hydrolysis between 5 and
stability of the ester bond on the PAs at physiological pH was 20 U mLÀ1 of the enzyme (Fig. S7, ESI†). Since epitope crowding
comparable to previously reported polymeric systems containing on the nanofibre surface has been shown to decrease interactions
either ibuprofen or naproxen.2,30 At higher pH, the ester linker with proteins,27–29 we postulate that the 25% dilution of the drug-
was hydrolysed to a much greater extent with increased drug conjugated PA was too high for optimal esterase accessibility.
release: 22 Æ 4% at pH 9 and 76 Æ 5% at pH 12 after 7 d.
To test our hypothesis, we incubated the PEG3–naproxen ligand 2
Chem. Commun.
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
cues were more functional when they were separated from the
bulk scaffold via molecular linkages.32 Moreover, D’Emanuele
and co-workers have reported that the enzymatic hydrolysis of
non-assembling dendrimers containing ester–naproxen conju-
gates increases with longer linkages.33
The anti-inflammatory activity of the hydrolysed naproxen
was evaluated using a cyclooxygenase (COX)-2 inhibitor assay.34
COX enzymes promote the synthesis of prostaglandins, which
are key mediators in inflammation, pain, and other diseases.35
For COX-2 inhibition, it is important that the drug is hydrolysed
to a carboxylic acid to promote electrostatic interactions with the
residues on the active site.36 PA 3, which exhibited the fastest
hydrolysis, was diluted with PA E3 (1: 3 molar ratio) and incu-
bated with or without 20 U mLÀ1 of PLE for 24 h at 37 1C. The
collected supernatant was indeed able to inhibit COX-2-activity by
70% (Fig. S10, ESI†). In addition, we evaluated biocompatibility of
this system and did not observe cytotoxicity in mouse mesenchymal
stem cells (Fig. S11, ESI†).
We have shown here that drugs containing carboxylic acid
functionalities can be conjugated to PAs though an ester linkage
for controlled drug release. The PA–naproxen conjugate nanofibre
exhibited stability at physiological pH and temperature, which
will be advantageous for long term storage. Enzymatic hydrolysis
of PAs shows improved released kinetics with increased spacing
between the drug and the PA backbone, likely due to increased
enzyme access to the ester linkage. The acid functionality of
naproxen is unaltered upon enzymatic action and its bioactivity
is preserved as shown by the COX-2 inhibitory assay. The bio-
compatible nature of the supramolecular assemblies make them
Fig. 2 (a) Overlap of PA 3–PA E3 (1:3 molar ratio, 1 mM final concentration)
before (black) and after (red), addition of PLE (20 U mLÀ1) at pH 7.4, 60 rpm
and 37 1C, 1 day. (b) Naproxen release as a function of time from PAs 3, 4 and
5 with PA E3 (1 : 3 molar ratio, 1 mM final concentration) in the presence of
PLE (20 U mLÀ1) at pH 7.4, 60 rpm and 37 1C.
in the presence of PLE (20 U mLÀ1) with and without PA E3 at ideal platforms for drug delivery applications. In clinical settings,
1 : 3 molar ratio to keep the same concentrations of the earlier we propose that the naproxen-conjugated nanofibres may be
experiments (Fig. S8, ESI†). In both cases we observed complete systemically delivered for cancer therapy, or locally injected to
hydrolysis of the ester after 1 d, which implies that the PLE form a gel network in situ to treat osteoarthritis or rheumatoid
activity was not disrupted when ligand 2 is not tethered to the arthritis. Furthermore, the chemistry reported here can be applied
nanostructures. Next, a further dilution of PA 3 from 25% to to many other drugs containing carboxylic acids. Since the lipid
10% by PA E3 exhibited a 15% increase in enzymatic hydrolysis tail and b-sheet regions of the PA are unaltered, it is expected that
(Fig. S9, ESI†), which suggests that naproxen crowding was an the self-assembled scaffold will be conserved upon release. There-
important factor for incomplete hydrolysis. Furthermore, when fore, this supramolecular design makes it possible to encapsulate
naproxen-conjugated PAs are co-assembled with PA E3, it is drugs37 or integrate different types of PA monomers into the same
possible that some naproxen is buried in the hydrophobic parts of nanostructure, allowing sequential release of drugs.
the nanofibre, which can also diminish the percentage of hydrolysis.
This work was supported by the National Institutes of Health
Nonetheless, we have observed here that modification of monomer NIDCR (grant 2R01DE015920-09) and NIBIB (5R01EB003806-09).
ratio allows the tuning of drug release from these supramolecular S.S.L. is supported by Samsung Scholarship Foundation Graduate
nanostructures.
Fellowship. We acknowledge the following Northwestern Univer-
The effect of linker length on enzymatic hydrolysis was sity facilities: Peptide Synthesis Core at the Simpson Querrey
evaluated using PAs 4–5 (Fig. 2). These PAs were diluted with Institute for BioNanotechnology; Integrated Molecular Structure
PA E3 (1 : 3 molar ratio) and treated with PLE at 20 U mLÀ1
.
Education and Research Center (IMSERC, NSF CHE-9871268 and
After 24 h, naproxen release from PA 4 was 36 Æ 4%, approxi- NSF CHE-0923236). The authors acknowledge Dr Liam Palmer,
mately 20% lower than that from PA 3. PA 5 exhibited the least Dr Mark Karver, and Dr Amanda Worthy for reviewing the
amount of drug release after 24 h (6 Æ 1%). Even after 48 h manuscript and scientific advice.
incubation, there was minimal release from PA 5 (7 Æ 2). Since
esterases require entry of the substrate into their catalytic
pockets, it is possible that the longer linker diminished the
steric hindrance of nanofibres to enzyme action, allowing easier
access of the naproxen conjugate to the PLE-catalytic site. This
Notes and references
1 Y. Bae, S. Fukushima, A. Harada and K. Kataoka, Angew. Chem., Int. Ed.,
2003, 42, 4640.
2 U. Hasegawa, A. J. van der Vlies, C. Wandrey and J. A. Hubbell,
is in agreement with previous findings that showed bioactive
Biomacromolecules, 2013, 14, 3314.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun.

View Article Online
Communication
ChemComm
3 Z. Y. Xiao, C. W. Ji, J. J. Shi, E. M. Pridgen, J. Frieder, J. Wu and 20 T. L. Andresen, D. H. Thompson and T. Kaasgaard, Mol. Membr.
O. C. Farokhzad, Angew. Chem., Int. Ed., 2012, 51, 11853. Biol., 2010, 27, 353.
4 Z. S. Al-Ahmady, W. T. Al-Jamal, J. V. Bossche, T. T. Bui, A. F. Drake, 21 J. M. Hatfield, M. Wierdl, R. M. Wadkins and P. M. Potter, Expert
A. J. Mason and K. Kostarelos, ACS Nano, 2012, 6, 9335. Opin. Drug Metab. Toxicol., 2008, 4, 1153.
5 Y. S. Wu, M. S. Coumar, J. Y. Chang, H. Y. Sun, F. M. Kuo, C. C. Kuo, 22 D. Hasenpusch, U. T. Bornscheuer and W. Langel, J. Mol. Model.,
Y. J. Chen, C. Y. Chang, C. L. Hsiao, J. P. Liou, C. P. Chen, H. T. Yao, 2011, 17, 1493.
Y. K. Chiang, U. K. Tan, C. T. Chen, C. Y. Chu, S. Y. Wu, T. K. Yeh, 23 S. Bencharit, C. C. Edwards, C. L. Morton, E. L. Howard-Williams,
C. Y. Lin and H. P. Hsieh, J. Med. Chem., 2009, 52, 4941. P. Kuhn, P. M. Potter and M. R. Redinbo, J. Mol. Biol., 2006, 363, 201.
6 C. Widakowich, G. de Castro, Jr., E. de Azambuja, P. Dinh and 24 J. B. Matson, C. J. Newcomb, R. Bitton and S. I. Stupp, Soft Matter,
A. Awada, Oncologist, 2007, 12, 1443. 2012, 8, 3586.
7 J. D. Hartgerink, E. Beniash and S. I. Stupp, Science, 2001, 294, 1684. 25 J. H. Ortony, C. J. Newcomb, J. B. Matson, L. C. Palmer, P. E. Doan,
8 J. D. Hartgerink, E. Beniash and S. I. Stupp, Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 5133.
9 H. Cui, T. Muraoka, A. G. Cheetham and S. I. Stupp, Nano Lett.,
2009, 9, 945.
B. M. Hoffman and S. I. Stupp, Nat. Mater., 2014, 13, 812.
26 E. T. Pashuck, H. G. Cui and S. I. Stupp, J. Am. Chem. Soc., 2010,
132, 6041.
27 M. J. Webber, J. Tongers, M. A. Renault, J. G. Roncalli, D. W. Losordo
and S. I. Stupp, Acta Biomater., 2010, 6, 3.
10 C. J. Newcomb, S. Sur, J. H. Ortony, O. S. Lee, J. B. Matson,
J. Boekhoven, J. M. Yu, G. C. Schatz and S. I. Stupp, Nat. Commun., 28 S. S. Lee, E. L. Hsu, M. Mendoza, J. Ghodasra, M. S. Nickoli, A. Ashtekar,
2014, 5, 3321.
M. Polavarapu, J. Babu, R. M. Riaz, J. D. Nicolas, D. Nelson, S. Z. Hashmi,
S. R. Kaltz, J. S. Earhart, B. R. Merk, J. S. McKee, S. F. Bairstow, R. N. Shah,
W. K. Hsu and S. I. Stupp, Adv. Healthcare Mater., 2014, 452.
11 G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. A. Harrington, J. A.
Kessler and S. I. Stupp, Science, 2004, 303, 1352.
12 M. J. Webber, J. Tongers, C. J. Newcomb, K. T. Marquardt, J. Bauersachs, 29 R. N. Shah, N. A. Shah, M. M. Del Rosario Lim, C. Hsieh, G. Nuber
D. W. Losordo and S. I. Stupp, Proc. Natl. Acad. Sci. U. S. A., 2011,
108, 13438.
13 L. B. Mlinar, E. J. Chung, E. A. Wonder and M. Tirrell, Biomaterials,
2014, 35, 8678.
and S. I. Stupp, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 3293.
30 R. Rosario-Melendez, W. L. Yu and K. E. Uhrich, Biomacromolecules,
2013, 14, 3542.
31 T. Tsujita and H. Okuda, Eur. J. Biochem., 1983, 133, 215.
14 M. J. Webber, J. B. Matson, V. K. Tamboli and S. I. Stupp, Biomaterials, 32 F. Taraballi, A. Natalello, M. Campione, O. Villa, S. M. Doglia,
2012, 33, 6823.
A. Paleari and F. Gelain, Front. Neuroeng., 2010, 3, 1.
33 M. Najlah, S. Freeman, D. Attwood and A. D’Emanuele, Int. J. Pharm.,
2007, 336, 183.
34 W. J. Tsai, Y. J. Shiao, S. J. Lin, W. F. Chiou, L. C. Lin, T. H. Yang, C. M.
Teng, T. S. Wu and L. M. Yang, Bioorg. Med. Chem. Lett., 2006, 16, 4440.
35 B. F. McAdam, F. Catella-Lawson, I. A. Mardini, S. Kapoor, J. A. Lawson
and G. A. FitzGerald, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 272.
15 J. B. Matson and S. I. Stupp, Chem. Commun., 2011, 47, 7962.
16 A. G. Cheetham, P. Zhang, Y. A. Lin, L. L. Lock and H. Cui, J. Am.
Chem. Soc., 2013, 135, 2907.
17 O. Svensson, M. Malmenas, L. Fajutrao, E. M. Roos and L. S.
Lohmander, Ann. Rheum. Dis., 2006, 65, 781.
18 A. M. M. Eijsbouts, M. J. E. Kempers, F. H. J. van den Hoogen,
R. F. J. M. Laan, A. R. M. M. Hermus, H. A. Ross, F. C. G. J. Sweep 36 K. C. Duggan, M. J. Walters, J. Musee, J. M. Harp, J. R. Kiefer,
and L. B. A. van de Putte, Clin. Exp. Rheumatol., 2011, 29, 35. J. A. Oates and L. J. Marnett, J. Biol. Chem., 2010, 285, 34950.
19 M. Yao, W. Zhou, S. Sangha, A. Albert, A. J. Chang, T. C. Liu and 37 S. Soukasene, D. J. Toft, T. J. Moyer, H. Lu, H. K. Lee, S. M. Standley,
M. M. Wolfe, Clin. Cancer Res., 2005, 11, 1618.
V. L. Cryns and S. I. Stupp, ACS Nano, 2011, 5, 9113.
Chem. Commun.
This journal is ©The Royal Society of Chemistry 2014