Structural Modification of Protease Inducible Preprogrammed

Jan 5, 2008 - Benedict Law* and Ching-Hsuan Tung†. Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School,...
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February 2008

Published by the American Chemical Society

Volume 9, Number 2

 Copyright 2008 by the American Chemical Society

Communications Structural Modification of Protease Inducible Preprogrammed Nanofiber Precursor Benedict Law* and Ching-Hsuan Tung† Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129 Received October 31, 2007; Revised Manuscript Received December 13, 2007

As many proteases such as urokinase plasminogen activator (uPA) are overexpressed in various tumors, a new type of peptide-based smart delivery system (hydrogel matrix) that could be degraded by uPA was previously described (Law, B.; Weissleder, R.; Tung, C. H. Peptide-based biomaterials for protease-enhanced drug delivery. Biomacromolecules 2006, 7 (4), 1261-1265). Subsequently, we designed nanometer-sized fluorescent nanofibers by introducing a hydrophilic component (methoxyl polyethylene glycol) to the core peptide [MPEG2000BK(FITC)SGRSANA-kldlkldlkldl-NH2]. Preliminary studies showed that these nanofibers could detect uPA activity by optical imaging in vitro (Law, B.; Weissleder, R.; Tung, C. H. Protease-sensitive fluorescent nanofibers. Bioconjugate Chem. 2007, 18 (6), 1701-1704). Here, we further extend our studies to the structural responses of these nanofiber precursors (NFP). In the presence of a model protease, the FITC-containing hydrophilic fragments were released from the NFPs that contributed to fluorescence amplification. Simultaneously, the remaining selfassembling residues were mechanically driven to transform into interfibril networking of micrometer size hydrogel. These unique morphological changes, together with the optical property, may have considerable biomedical applications as diagnostic sensors for specific protease or dual systemic and functional delivery nanoplatforms to target protease-associated diseases.

Introduction Peptide-based hydrogel has been extensively employed in three-dimensional cell cultures3–5 and has been recognized to deliver growth factors such as EGF-2, VEGF, bFGF, and laminin in tissue engineering.6,7 The hydrogels are normally constructed with β-sheet peptides that spontaneously selfassemble in aqueous to form nanofibers and subsequently to biocompatible scaffolds8–11 via hydrophobic and electrostatic interactions.12 The uniqueness of these bioinspired materials is their flexibility to functionalize; essential adhesive motifs can be integrated into a peptide construct that greatly enhance the capability for cell entrapment13 or protein delivery.14 For example, neural progenitor cells have been entrapped within a three-dimensional network of nanofibers to promote the formation of neurons.15 Recently, platelet-derived growth factor * To whom correspondence should be addressed. Tel: 701-231-7906. E-mail: [email protected]. † Current address: The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX 77030.

(PDGF)-BB tethered nanofibers were shown to decrease cardiomyocyte death and preserve systolic function after myocardial infarction in vivo.14,16 We have previously demonstrated a “preprogrammed” matrix composed with a KLD-12 peptide17 that could be activated by a specific protease to release an encapsulated anticancer peptide in a digitized manner.1 To further enhance the capability of hydrogel in nanoformulation for systemic administration, we reported a novel design of “preprogrammed” nanofiber precursor (NFP).18 The NFP construct was composed of four components (Figure 1A): (i) a nontoxic hydrophilic methoxypolyethylene glycol (with an average molecular mass equal to 2363 Da) that prevented interNFP fibrillation; (ii) a covalently attached fluorescein isothiocyanate (FITC) that reported protease activity by fluorescence dequenching at 520 nm; (iii) a hydrophobic β-sheet segment (kldlkldlkldl) that contributed to the self-assembling property;1,3,17 and (iv) an enzyme–substrate site (SGRSANA)19,20 that could be selectively cleaved by certain proteases such as trypsin and urokinase plasminogen activator (uPA).21 Upon addition of a

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Figure 1. Design of protease-inducible preprogrammed NFP. (A) The components of NFP peptide conjugate. (B) Schematic representations of nanofibers indicating both the optical and the morphological changes in response to protease digestions.

target protease, the enzyme cleaved the NFP via the substrate sites and subsequently released FITC-conjugate hydrophilic fragments [MPEG2000-BK(FITC)SGR-COOH, where B ) β-alanine] that contributed to fluorescence amplification (Figure 1B). Here, we hypothesized that these NFPs in their native form did not have fibril-to-fibril interactions but could be induced with a target protease to form interfibril networking of micrometer size hydrogels that compensate the loss of their intrinsic hydrophilicity. The entire peptide construct was synthesized by solid phase chemistry, purified by high-performance liquid chromatography (HPLC), and were further self-assembled spontaneously via hydrophobic and electrostatic interactions in aqueous buffer. The length of the resulting fibers varied from 200 nm to >1 µm but could be homogenized to 100 nm NFPs by passing them through a polycarbonate membrane pore filter (Figure 4A). Along with the self-assembling process, a high concentration of fluorophores oriented locally in close proximity and thus experienced fluorescence quenching. Cleavage of NFPs by protease released the FITC-containing fragments that were free from proximitybased quenching and resulted in fluorescence amplification. This enzyme activation approach has been previously employed in poly-L-lysine and polyethyleneglycol cocopolymer to image uPA activities in vivo.19,20 Here, a highly efficient trypsin was employed to activate the NFPs to completion within 12 h. As expected, fluorescence intensities increased with time in an enzyme-dependent manner (Figure 2A,B). Further addition of the trypsin inhibitor truncated the enzyme activities and thus inhibited the fluorescence activations (Figure 2C). Interestingly, regardless of the amount of enzyme, fluorescence activation reached the plateau after 8 h. This could be attributed to a loss of enzyme activity with time or the structural reorientation of the activated NFPs (see below). To confirm the cleavage site of the NFPs, different amounts of trypsin were incubated with the nanofibers in PBS buffer, and the solution mixtures were further monitored by analytical reverse-phase HPLC for 30 min at FITC absorbance (460 nm). In the absence of enzyme, the NFP appeared as a single peak at 18.8 min (Figure 3A, panel a). When trypsin was added to digest the NFPs, after 24 h, an extra peak appeared at an earlier retention time (at 17.0 min), indicating the presence of digested fragments (Figure 3A, panel b). When the amount of enzyme was increased during incubation, the NFP peak intensities decreased inversely with the digested fragment peaks (Figure 3A, panels c and d). To confirm the identity of the digested

Figure 2. Optical changes of NFPs in response to enzyme activation. (A) The photo images of NFPs (30 µM) after incubation of trypsin (300 µg/mL) in PBS buffer (10 mM, pH 7.4) for 24 h. FITC dequenching (changed from orange to yellow color) could be observed by the naked eye under white light. (B) A plot from the fluorescence emissions in the solutions of NFPs (1 µM) with different time points (min), after incubation with various amounts of trypsin (µg/mL) in PBS buffer (10 mM, pH 7.4). (C) Upon addition with different amounts of trypsin inhibitor (µg), the fluorescence activation of NFPs (1 µM) with trypsin (1 µg) was inhibited. Fluorescence intensities were measured at 520 nm. The excitation wavelength was 485 nm. All experiments were performed at room temperature.

Figure 3. Enzyme activation. (A) The HPLC chromatogram of the NFP solutions (1 µM) after incubation with different amounts, (a) 0, (b) 0.1, (c) 1, and (d) 10 µg/mL, of trypsin in PBS buffer (10 mM, pH 7.4, 800 µL) for 24 h at room temperature. (e) For inhibition study, the enzyme (1 µg/mL) and trypsin inhibitor (10 µg/mL) were coincubated with NFPs. All samples solutions were freeze-dried, concentrated, and resuspended with DMSO prior to injection into the HPLC. (B) The MALDI-TOF spectra of the collected fractions from HPLC at different (f) 18.8 and (g) 17.0 min retention times that represent the intact peptide construct [MPEG2000-BK(FITC)SGRSANA-kldlkldlkldlCONH2] and its corresponding digested fragment MPEG2000conjugated digested fragment [MPEG2000-BK(FITC)SGR-COOH], respectively.

fragments, the two eluates at 17.0 and 18.8 min were collected from the HPLC and further analyzed by a mass spectrometer. Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) showed that the average molecular masses of the peptide conjugates and their digested fragments were 4892 and 3141 Da (Figure 3B, panels f and g), respectively. This was expected as the difference between the peptide conjugate [MPEG2000BK(FITC)SGRSANA-kldlkldlkldl-CONH2, average MW ) 4892] and the digested product [MPEG2000-BK(FITC)SGR-OH,

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Figure 4. Morphological changes of NFP in response to enzyme activation. TEM images of NFPs (1 µM) alone at (A) 0 and (B) 24 h. TEM images of nanofibers (1 µM) after incubation with (C) 1 ng, (D) 10 ng, (E) 100 ng, (F) 1 µg, and (G) 10 µg/mL of trypsin in PBS buffer (10 mM, pH 7.4) for 24 h at room temperature. Morphological changes were enzyme-dependent. With increasing the enzyme concentration, the NFPs transformed to nanometer fibers, elongates, laminates, or interfibril hydrogel networks. (H) For inhibition study, the enzyme (100 ng/mL) and trypsin inhibitor (1 µg/mL) were coincubated with the NFPs. Structural reorientation was inhibited, and no morphological change was observed. All samples were negatively stained with uranyl formate prior to TEM analysis. Images magnifications were 23k×, and the panel images were 49k×.

average MW ) 3141] was 1769 Da, which reflected the lost of a fragment NH2-SANA-kldlkldlkldl-CONH2. The multiplex of the mass was due to heterogeneity of the conjugated MPEG chain. Further addition of trypsin inhibitor inhibited the enzyme activities, thus preventing the cleavage of nanofibers (Figure 3A, panel e). Together with the data from the fluorescence activation studies, we confirmed that the NFP cleavage site was located in between the arginine and the serine residue of the peptide conjugates, which was in accordance to data previously reported.21 No significant aggregation was found after storage in PBS buffer for 24 h (Figure 4B), indicating that the hydrophilic MPEG groups attached on the NFPs were able to prevent possible interfibril interactions. However, their morphologies were changed with enzyme activation. With trypsin addition, after 24 h, NFPs elongated to submicrometer size (Figure 4D). A few of these activated fibers interacted to form laminates at higher enzyme concentrations (Figure 4E). At sufficient enzyme concentrations (10 µg/mL), the NFPs were further cross-linked and developed into larger networks of micrometer size (Figure 4G), which appeared to have similar morphologies as the digested fragment (NH2-SANA-kldlkldlkldl-CONH2) that were independently synthesized by SSPS (see Supporting Information, Figure S1). These structural reorientations could be inhibited by the addition of trypsin inhibitor (Figure 4H), thus proven to be specific with enzyme activities. To further study the lamination kinetics, 100 ng/mL of trypsin was chosen to incubate with the NFPs. This particular enzyme level was the minimum condition to initiate the lamination process (Figure 4E) with the majority of hydrophilic elements remaining intact (Figure 3A, panel c); thus, morphological changes could be observed within a 24 h time course. The NFPs appeared as elongates at 8 h (Figure 5B) and continued emerging to longer than 500 nm after 16 h (Figure 5C). At 24 h, some elongates were interacting with each other, and subsequently, laminates were formed (Figure 5D). In conclusion, our studies suggested that when NFPs were cleaved via protease substrate sites, hydrophilic fragments [MPEG2000-BK(FITC)SGR-COOH] would subsequently be released and thus contribute to a significant increase in the

Figure 5. Kinetics of morphological changes of NFP in response to enzyme activation. TEM images of NFPs (1 uM) after incubation with trypsin (100 ng/mL) for (A) 0, (B) 8, (C) 16, and (D) 24 h at room temperature. The structural reorientation was sequential; with time, NPRs became elongates, then laminates, and finally transformed into interfibril hydrogel networks. All samples were negatively stained with uranyl formate prior to TEM analysis. Image magnifications were 13k×, and the panel images were 23k×.

fluorescence. In reaction to a loss of intrinsic hydrophilicity, the NFPs changed to different architects. Depending on the activities of the presented proteases, only elongates or laminates were formed if the NFPs were partially digested, since the hydrophilic elements remaining in the fibers were sufficient to prevent interfibril interaction. If the majority of the hydrophilic elements were digested by protease, the remaining hydrophobic NFPs sequentially transformed from nanometer sizes to submicrometer elongations, laminates, and finally became hydrogels of micrometer sizes with time (Figure 1B). Recently, various supramolecular hydrogel systems have been developed as hydrogelators to detect different hydrolytic enzymes such as β-lactamases, glycosidases, and phosphatases.22,23 We foresee that our ultrasmall NFPs would be useful as diagnostic sensors for protease activities2 and functional delivery nanoplatforms. Potentially, the proposed concept could be applied to prepare various enzyme-responsive biomaterials.

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For example, FITC can be substituted with near-infrared fluorophores to image uPA activities in breast cancers24 and the NFPs peptide construct can be replaced by fine-tuning the substrate sequence for imaging different protease-associated diseases. Because nanotechnology has gained increased recognition for disease diagnosis, treatments, and prevention, biomolecules such as peptides, proteins, DNA, and antibodies could be further attached to the developed NFPs, thus providing an alternative approach for nanomedicine that will be based on protease expressions.

Materials and Methods Chemicals. All solvents were purchased from Fisher Scientific (Fair Lawn, NJ). All reagents for peptide synthesis were supplied by Novabiochem (San Diego, CA). R-Methyl-substituted polyethyleneglycol N-hydroxysuccinimide ester (mPEG-NHS ester, avgerage MW ) 2477 Da) was purchased from Nektar Therapeutics (San Carlos, CA). Anisole, ethandithiol, fluorescein isothiocyanate (FITC), thioanisole, trypsin (type IX-S from porcine pancreas), trypsin inhibitor (type I-S from soybean), and all other chemicals were obtained from SigmaAldrich (St. Louis, MO). Peptide Synthesis. All peptides were synthesized on rink amide resin (0.1 mmol). Peptide synthesis was performed on an automated peptide synthesizer (Fastmoc mode, ABI-433A, Applied Biosystems, Foster City, CA) employing the traditional NR-Fmoc methodology as previously described.1 To selectively introduce FITC into the peptide [BK(Fitc)SGRSANA[kldl]3] N terminus, Fmoc-Lys(ivDde)-OH [N-Rfmoc-N--1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)-3-methylbutylL-lysine], Fmoc-D-Asp(OtBu)-OH (d), Fmoc-D-Lys(Boc)-OH (l), Fmocβ-Ala-OH (B), Fmoc-L-Ser(tBu)-OH (S), Fmoc-L-Gly OH, Fmoc-LArg(Pbf)-OH (R), Fmoc-Ala-OH (A), and Fmoc-Asn(Trt)-OH (N) were employed as amino acid building blocks. After peptide elongation, MPEG-NHS (500 mg, 0.2 mmol) dissolved in DMSO (4 mL) was conjugated to the peptide N terminus. DIEPA (1 mL) was added as the catalyst. The sample mixture was allowed to gently shake for 3 h at room temperature and was washed three times with 2% (v/v) hydrazine monohydrate in DMF to selectively deprotect ivDde. FITC (151 mg, 0.4 mmol), N,N-diisopropylyethylamine (DIEPA) (1 mL), and DMSO (4 mL) were then added to the resin and allowed to further react overnight at room temperature. Cleavage of peptides from the resin and side chain deprotection employed a mixture of TFA/ thioanisole/ethanedithiol/anisole 90/5/3/2 (5 mL) for 4 h at room temperature. Peptides were then precipitated by methyl-tert-butyl ether at room temperature, redissolved in DMSO, and further purified by HPLC. The purified fractions were freeze-dried into yellow foams with >95% homogeneity. All final products were characterized by analytic HPLC and MALDI-TOF mass spectrometry (Tufts Protein Chemistry Facility, Boston, MA). Synthesis of NFP. Nanofiber precursors were freshly prepared by self-assembling mPEG2000-BK(FITC)SGRSANA-kldlkldlkldl-CONH2 (10 mg) with 50% (v/v) acetonitrile in water (5 mL). The solution mixtures were then stirred vigorously overnight to evaporate the organic solvent. To control the lengths of the preparations, the NFP solutions were passed through a mini-extruder (Avanti polar lipids Ltd., Alabaster, AL) using polycarbonate membrane (Whatman, Florham Park, NJ) with 0.1 µm pore size. After five passages, the solutions were collected and further purified by size-exclusion chromatography (P-10 biogel, BioRad, Hercules, CA) to remove any free peptides and smaller fragments. The amounts of fibers were then determined by UV absorbance for their attached FITC concentrations. Analytical HPLC and MALDI-TOF. Trypsin (200 µg/mL) was prepared by dissolving the enzyme (1 mg) in PBS (5 mL, 10 mM, pH 7.4). The stock solutions were further diluted to different concentrations (200, 20, 2, and 0 µg/mL). Different amount of enzymes (40 µL) and PBS buffer (40 uL) were then transferred to microcentrifuge tubes containing NFPs (11.1 µM) in PBS (720 µL, 10 mM, pH 7.4). For

Communications inhibition study, the enzyme (20 µg/mL, 40 µL) and trypsin inhibitor (200 µg/mL, 40 uL) were added to the NFPs. All solution mixtures were allowed to incubate for 24 h at room temperature. All samples solutions were then frozen with liquid nitrogen and further freeze-dried to white powders. The solids were then resuspended with 80% (v/v) DMSO in deionized water (100 µL) and further analyzed by analytical HPLC (Vydac C-18, No. 218TP5415, Deerfield, IL) with a linear gradient from 20 to 80% (v/v) acetonitrile in deionized water containing 0.1% (v/v) TFA (pH 2) for 30 min. Elution peaks (at 17.0 and 18.8 min) were monitored by absorbance at FITC channel (460 nm), and the collected fractions were further analyzed by MALDI-TOF (Tufts Protein Chemistry Facility, Boston, MA). Fluorescence Activation Kinetic. Trypsin (200 µg/mL) was prepared by dissolving the enzyme (1 mg) in PBS (5 mL, 10 mM, pH 7.4). The stock solutions were further diluted to different concentrations (200, 20, 2, 0.2, and 0.02 µg/mL). Different amount of enzymes (10 µL) and PBS buffers (10 µL) were then transferred to a 96 well clear bottom plate (Costar, Corning, NY) containing NFPs (11.1 µM) in PBS (180 µL, 10 mM, pH 7.4). Fluorescence intensities were recorded on a computer-controlled fluorescence plate reader (GENios Pro, TECAN, Durham, NC) at different time points (30, 60 120, 240, 480, and 720 min). All data were acquired and analyzed by the installed computer software (Magellan, TECAN). For inhibition studies, trypsin inhibitors (type I-S, 200 µg/mL, 40 uL) were prepared in PBS. The stock solutions were further diluted to different concentrations (200, 20, and 2 µg/ mL). Enzymes (20 µg/mL, 10 µL) and trypsin inhibitors (10 µL) were then transferred to a 96 well clear bottom plate containing NFPs (11.1 µM) as described above. Fluorescence intensities were recorded at 720 min. Transmission Emission Microscopy. Trypsin (200 µg/mL) was prepared by dissolving the enzyme (1 mg) in PBS (5 mL, 10 mM, pH 7.4). The stock solutions were further diluted to different concentrations (200, 20, 2, 0.2, and 0 µg/mL). The enzymes (5 µL), together with buffers (5 µL), were added to NFPs (11.1 µM) in PBS (90 µL, 10 mM, pH 7.4), followed by incubation for 24 h at room temperature. For inhibition study, the enzyme (20 µg/mL, 5 µL) and trypsin inhibitor (200 µg/mL, 5 µL) were added to the NFPs. All solution mixtures (20 µL) were then added directly onto the Formvar/carbon-coated grids. Excess droplets were blotted off on a filter paper and washed twice with deionized water. To negatively stain the samples, all grids were floated on top of uranyl formate solutions (2% w/v, 20 µL) for 1 min at room temperature. The resulting grids were allowed to dry and were examined in the TEM. All images were recorded by transmission electron microscopy (Tecnai G2 Spirit BioTWIN, Hillsboro, OR) equipped with an AMT 2k CCD camera at Harvard Medical School (Department of Cell Biology, Boston, MA). For kinetic experiments, trypsin (80 µL, 1 µg/mL) dissolved in PBS buffer (10 mM, pH 7.4) was added to NFPs (11.1 µM) in PBS (720 µL, 10 mM, pH 7.4), followed by further incubation at room temperature. Samples (20 µL) were then taken at different time points (0, 6, 12, 18, and 24 h) and transferred onto the Formvar/carbon-coated grids for TEM analysis.

Acknowledgment. This research was supported in part by RO1 CA099385 and R33 CA114149. Supporting Information Available. TEM of NH2-SANAkldlkldlkldl-CONH2. This material is available free of charge via the Internet at http://pubs.acs.org.

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Biomacromolecules, Vol. 9, No. 2, 2008 425 (15) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 2004, 303 (5662), 1352–1355. (16) Hsieh, P. C.; MacGillivray, C.; Gannon, J.; Cruz, F. U.; Lee, R. T. Local controlled intramyocardial delivery of platelet-derived growth factor improves postinfarction ventricular function without pulmonary toxicity. Circulation 2006, 114 (7), 637–644. (17) Kisiday, J.; Jin, M.; Kurz, B.; Hung, H.; Semino, C.; Zhang, S.; Grodzinsky, A. J. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: Implications for cartilage tissue repair. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (15), 9996–10001. (18) Law, B.; Weissleder, R.; Tung, C. H. Protease-sensitive fluorescent nanofibers. Bioconjugate Chem. 2007, 18 (6), 1701–1704. (19) Law, B.; Curino, A.; Bugge, T. H.; Weissleder, R.; Tung, C. H. Design, synthesis, and characterization of urokinase plasminogen-activatorsensitive near-infrared reporter. Chem. Biol. 2004, 11 (1), 99–106. (20) Law, B.; Hsiao, J. K.; Bugge, T. H.; Weissleder, R.; Tung, C. H. Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples. Anal. Biochem. 2005, 338 (1), 151–158. (21) Ke, S. H.; Coombs, G. S.; Tachias, K.; Corey, D. R.; Madison, E. L. Optimal subsite occupancy and design of a selective inhibitor of urokinase. J. Biol. Chem. 1997, 272 (33), 20456–20462. (22) Yang, Z.; Ho, P. L.; Liang, G.; Chow, K. H.; Wang, Q.; Cao, Y.; Guo, Z.; Xu, B. Using beta-lactamase to trigger supramolecular hydrogelation. J. Am. Chem. Soc. 2007, 129 (2), 266–267. (23) Tamaru, S.; Kiyonaka, S.; Hamachi, I. Three distinct read-out modes for enzyme activity can operate in a semi-wet supramolecular hydrogel. Chemistry 2005, 11 (24), 7294–7304. (24) Duffy, M. J.; Maguire, T. M.; McDermott, E. W.; O’Higgins, N. Urokinase plasminogen activator: A prognostic marker in multiple types of cancer. J. Surg. Oncol. 1999, 71 (2), 130–135.

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