Bio-orthogonal Immobilization of Fibroblast Growth Factor 2 for Spatial

Letter pubs.acs.org/journal/abseba

Bio-orthogonal Immobilization of Fibroblast Growth Factor 2 for Spatial Controlled Cell Proliferation Tessa Lühmann,† Gabriel Jones,† Marcus Gutmann,† Jens-Christoph Rybak,† Joachim Nickel,‡,§ Marina Rubini,⊥ and Lorenz Meinel*,† †

Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany Chair of Tissue Engineering and Regenerative Medicine, University Hospital of Wuerzburg, Roentgenring 11, 97070 Wuerzburg Germany § Translational Center “Regenerative Therapies in Oncology and Musculoskeletal Diseases” Wuerzburg, Branch of the Fraunhofer Institute Interfacial Engineering and Biotechnology (IGB), Wuerzburg, Germany ⊥ Institute of Organic Chemistry, University of Konstanz, Konstanz, Germany ‡

S Supporting Information *

ABSTRACT: Presentation of therapeutic proteins on material surfaces is challenged by random immobilization chemistries through lysine or cysteine residues, typically leading to heterogeneous product outcome. Pharmaceutical quality standards warrant a controlled process ideally through site specific conjugation. Therefore, we deployed genetic codon expansion to engineer a propargyl-L-lysine (Plk)-modified FGF-2 analogue, enabling site-specific copper(I)-catalyzed azide alkyne cycloaddition (CuAAC). Site-specific decoration of Plk-FGF-2 to particles sparked cell proliferation of human osteosarcoma cells in a spatially controlled manner around the decorated carrier, rendering this approach instrumental for the future design of quality-improved bioinstructive scaffold outcome. KEYWORDS: fibroblast growth factor 2 (FGF-2), bio-orthogonal immobilization, genetic codon expansion, decoration, proliferation

F

release for clinical application of such conjugates is quite challenging because of their inherent heterogeneity. These and other concerns challenge the use of unspecific immobilization chemistries, including EDC/NHS coupling.9,10 Site-specific chemistries remove these challenges through homogeneous decoration by strictly confining the covalent modification to, for example, an unnatural functional group or groups that are introduced into the biologic’s backbone at the predefined site during recombinant protein expression.11,12 This approach takes advantage of the translation machinery of archaebacteria, which incorporate the 22nd amino acid Lpyrrolysine (Pyl) in protein biosynthesis beyond the typically used 20 natural amino acid building blocks.13,14 Pyl is encoded by an amber stop codon (UAG) within a gene, being recognized by the suppressor tRNAPyl. An adjacent PylS gene encodes for a specific pyrrolysyl-tRNA synthetase (PylRS), catalyzing the transfer of pyrrolysine to its specific tRNAPyl, whose amino acid is incorporated in frame at the amber/UAG codon of the corresponding mRNA.15 The incorporation of Pyl and Pyl derived analogues into proteins recombinantly expressed in Escherichia coli (E. coli) was previously exploited

unctional immobilization of therapeutic growth factors, cytokines, or other regulatory factors combined with optimal accessibility and high reproducibility is a critical step toward the development of bioinstructive materials to successfully (re)engineer lost tissues in future clinical approaches. Decoration of biomaterials has been traditionally performed by (i) noncovalent physiochemical adsorption1,2 (ii) covalent random immobilization by, for example, endogenous amino- or carboxyl-groups, deploying EDC (1-Ethyl-3(3dimethylaminopropyl)carbodiimide)/NHS (N-hydroxysuccinimide) chemistry; 2,3 or (iii) enzymatic coupling approaches.4−7 Although the bioactivity of growth factors is quite frequently retained by noncovalent physiochemical adsorption, desorption of the protein occurs as a function of the environment into which the carrier is placed, hence in an uncontrolled and rapid manner. This challenges the reliable, sustained, and locally controlled presentation and may impact pharmacodynamics and/or toxicology of the biologic. 8 However, biomaterials onto which regulatory factors are nonspecifically and covalently immobilized also have striking limitations. First, unspecific decoration may lead to the fixation of biologics at sites relevant for their pharmacological performance. Second, random immobilization strategies lead to processes prone to variability and heterogeneous batch-tobatch product outcome. Lastly, analytical characterization and © XXXX American Chemical Society

Received: June 2, 2015 Accepted: July 30, 2015

A

DOI: 10.1021/acsbiomaterials.5b00236 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 1. (A) Structure of Plk-FGF-2. Plk introduction is at position 8 as indicated. FGF-2 pbd file: 1BLD. (B) SDS PAGE of purified Plk-FGF-2 in comparison to wild-type FGF-2. Lane 1 is protein standard, lane 2 was loaded with 10 μg Plk-FGF-2, and lane 3 with 10 μg wild-type FGF-2. (C) Fragments of trypsinized Plk-FGF-2 as analyzed by LC MS/MS. The N-terminal amino acid sequence 2−25 of Plk-FGF-2 is displayed, including Plk (red) and theoretical and expected mass of the peptide. All b- and y-ions marked in the peptide sequence were found.

similar bioactivities if compared to the nontruncated protein,26,27 thus rendering the N-terminal region of FGF-2 an attractive region for further modification. Figure 1A depicts the NMR structure of FGF-2, in which the location with Plkexchange (8SerFGF-2-8PlkFGF-2) is highlighted. On the basis of this strategy, we introduced an unnatural Plk residue into FGF-2 through amber codon (UAG) suppression using a pyrrolysyl-tRNA synthethase/tRNAPyl CUA pair originated from Methanosarcina barkeri.16,28 Plk-FGF-2 was expressed in the E. coli strain BL21(DE3) and purified by heparin affinity chromatography with an average yield of 2 mg/ L (Figure 1B). The total yield is in line with the yield of other soluble proteins expressed by the native PylRS system in E. coli.17 Exchange of 8SerFGF-2−8Plk-FGF-2 did not affect heparin interaction during purification (elution at 1.5 M NaCl) and elution profiles were indistinguishable from those of wildtype FGF-2 (data not shown), indicating that heparin binding is not affected by the introduction of Plk. MALDI-MS analysis suggested that the N-terminal Met was removed upon translation by E. coli derived methionine amino peptidase (MetAP) (obs. average mass = 17140 Da, calc. average mass = 17144 Da) (Figure S2). This was corroborated by a trypsin digest of Plk-FGF-2 followed by LC-MS/MS analysis confirming the introduction of Plk at position #8 in the amino acid sequence (Figure 1C). Moreover, identity of PlkFGF-2 was shown by RP-HPLC in comparison to wild-type FGF-2 (Figure 2A). To study the structural integrity of the modified Plk-FGF-2, we used fluorescence emission following excitation of tyrosine

to provide orthogonal functionalities to perform site-directed modifications in proteins such as ubiquitin and whale sperm myoglobin via copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) in solution.16,17 Here, we report the introduction of the unnatural amino acid propargyl-L-lysine (Plk) into murine fibroblast growth factor 2 (FGF-2, basic FGF) and present a defined strategy to covalently immobilize Plk-FGF-2 in a bio-orthogonal manner to azide presenting agarose particles deploying CuAAC. FGF-2 is a basic protein (isoelectrical point = 9.6) with several surface exposed lysine residues (Lys), which are involved in low affinity receptor interaction.18 Therefore, common unspecific conjugation chemistries decorating through Lys likely jeopardize the growth factor’s bioactivity. As potent mitogenic growth factor, FGF-2 is spatially and temporarily tightly regulated during embryonal development and tissue repair19,20 and shows proliferative effects on a large number of cell types of mesodermal and ectodermal origin, including fibroblasts, mesenchymal stem cells and endothelial cells. Consequently, we evaluated the hypothesis that Plk-FGF-2 decorated particles induce spatial mitogenic responses in tissue regeneration applications, while substantially focusing on the quality outcome of the product through site specific decoration.21−24 First, we identified a suitable position in the FGF-2 amino acid (aa) sequence for unnatural amino acid introduction. The N-terminal region of the 18 kDa form of FGF-2 (1−154 aa) is highly flexible and accessible and not involved in high and low affinity receptor binding domains.25 N-terminal truncated forms of FGF-2 with 146 aa or 131 aa identified earlier, showed B


Letter


Figure 2. (A) RP-HPLC analysis of wild-type FGF-2 (dark gray) and Plk-FGF-2 (light gray). (B) Fluorescence spectra of wild-type FGF-2 (0.1 mg/ mL), Plk-FGF-2 (0.1 mg/mL), and (guanidinium chloride) denatured wild-type FGF-2 (0.02 mg/mL) after excitation at 280 nm. (C) MG-63 cell proliferation assay of wild-type FGF-2, Plk-FGF-2, and sourced FGF-2 (R&D systems) as control, (mean ± standard deviation, n = 3, blank n = 9). (D) Erk phosporylation of MG-63 cells cultivated in medium containing 3.3% fetal calf serum (1) and after treatment with 2.5 μg/mL and 10 ng/mL Plk-FGF-2 (2, 3) and 2.5 μg/mL and 10 ng/mL wild-type FGF-2 (4, 5) in serum-depleted medium and of unstimulated MG-63 cells cultivated in serum-depleted medium (6) as analyzed by Western blot analysis.

and tryptophan residues at 280 nm.29 In the folded FGF-2 conformation, one single tryptophan residue is buried inside of the FGF-2 structure and, therefore, quenched within the protein core. In contrast, surface-exposed tyrosine residues may emit fluorescence. Thereby, analogous folding of a Plk-FGF-2 as compared to wild-type FGF-2 was confirmed with an emission spectrum being dominated by tyrosine fluorescence (maximum peak fluorescence emission of tyrosine residues: 305 nm, Figure 2B). To further analyze the fluorescence emission behavior of the unfolded protein, wild-type FGF-2 was denaturated with 6 M guanidinium chloride and revealed an emission spectrum with a strong fluorescence emission at 355 nm (maximum peak fluorescence of tryptophan residues: 360 nm) combined with a shoulder at 306 nm (tyrosine residue emission), confirming the conformational change of FGF-2 to a random coil state after forced unfolding through treatment with chaotropic chemicals (Figure 2B). Next we tested the biological activity of Plk-FGF-2 in a cell-based assay employing human osteosarcoma MG-63 cells as previously described.30 Plk-FGF-2 stimulated the growth of MG-63 cells as potent as its wild-type analogue and as purchased FGF-2, respectively (Figure 2C).

To study downstream signaling effects of Plk-FGF-2 stimulated MG-63 cells, ERK phosphorylation (an effector kinase of the MAPK/ERK pathway) was analyzed in comparison to total ERK expression (Figure 2D). In contrast to nonstimulated cells (lane 6) and cells cultured in the presence of 3.3% FCS (lane 1), both tested concentrations of Plk FGF-2 (2.5 μg/mL (150 nM), lane 2; 10 ng/mL (590 pM, lane 3) and of wild-type FGF-2 (2.5 μg/mL (150 nM), lane 4; 10 ng/mL (590 pM, lane 5) induced strong ERK phosphorylation. These results corroborate previous reports indicating that the N-terminal region of FGF-2 (1−154) is not involved in receptor stimulation.26,27 As next, the functionality of the alkyne group for chemical decoration was analyzed by covalently attaching an azidefluorophore to Plk-FGF-2 (10 μM), using copper(II) sulfate in the presence of sodium L-ascorbate as mild reducing agent and the water-soluble base tris(3-hydroxypropyltriazolylmethyl) amine (THPTA) as previously described.31 After click reaction, a fluorescent band with the expected electrophoretical mobility (lane #6) could be visualized by SDS-PAGE and fluorescence imaging, whereas controls (wild-type FGF-2 or coupling in absence of copper(II) sulfate) did not show detectable C


Letter


Figure 3. Click reactions between Plk-FGF-2, wild-type FGF-2, and azide Fluor 488 as analyzed by SDS-PAGE. Protein staining by Coomassie brilliant blue is on the left, fluorescence of the gel for detection of azide Fluor 488 is on the right. Lane 1 was loaded with protein standard. Click reactions between Plk-FGF-2 and azide Fluor 488 in the presence (lanes 2 and 6) or absence of copper(I) (lanes 3 and 7). Reactions between wildtype FGF-2 and azide Fluor 488 in the presence (lanes 4 and 8) or absence of copper(I) (lanes 5 and 9).

fluorescent labeling of the protein (Figure 3). This result indicates that under the applied CuAAC conditions endogenous groups within the wild-type form of FGF-2 do not react unspecifically with azide moieties. Moreover, functionalization of Plk-FGF-2 with azide Fluor 488 was detailed by MALDI-MS analysis and revealed an equimolar decoration of Plk-FGF-2 with the fluorescent dye after CuAAC (obs. average mass = 17716 Da, calc. average mass =17719 Da) (Figure S3). Agarose was chosen as model carrier for surface immobilization of Plk-FGF-2 because of its high biocompatibility and versatility in surface functionalization.32,33 For introduction of azide and hydroxyl functional groups onto the agarose structure, we used aminoundecanazide and ethanolamine to covalently immobilize both cues on NHS preactivated agarose beads, deploying common NHS chemistry procedures (Figure 4). Successful immobilization of aminoundecane-azide onto the agarose carrier was evaluated by Cy5-alkyne conjugation, yielding into strong labeling of the agarose material (data not shown). In order to visualize and quantify Plk-FGF-2 on the agarose surface, the fluorescence dye maleinimide Atto 594 was conjugated to the free thiols of the two cysteine residues located on the FGF-2 surface, which have been previously exchanged to serine residues or conjugated with biotins without impact on bioactivity and cellular trafficking.34,35 Atto 594labeled Plk-FGF-2 was covalently immobilized by CuAAC to azide or hydroxyl groups presenting surfaces of agarose beads in the presence or absence of copper(II) sulfate (Figure 4A, B). Specific and strong fluorescent signals of Atto-594-labeled Plk-FGF-2 on azide group presenting agarose beads were observed in the presence of copper(II) sulfate (Figure 4Aa). In contrast controls (CuAAC on hydroxyl groups presenting agarose beads (Figure 4Ab) or in absence of copper(II) sulfate on azide groups presenting agarose beads (Figure 4Ac)) did not display specific fluorescence on the bead surface, indicating that the copper catalyst was essential for the reaction between the azide and the alkyne group. Fluorescence intensity on all tested conditions was quantified in the supernatant after trypsin treatment (Figure 4Ac; Bc) and revealed a significant immobilization of Atto-594-labeled Plk-FGF-2 onto azide functionalized agarose particles in the presence of copper(II) sulfate with a 2-fold higher fluorescence signal compared to Atto-594-labeled Plk-FGF-2, which was immobilized in the absence of copper(II) sulfate (Figure 4A, c). No significant difference in fluorescence intensity of Atto-594-labeled PlkFGF-2 immobilized to agarose carriers, presenting hydroxyl

Figure 4. Site-specific immobilization of Atto 594-labeled Plk-FGF-2 onto azide and hydroxyl groups presenting agarose beads. Decoration of agarose particles (A) with aminoundecane-azide and (B) with ethanolamine. Fluorescence images of Atto 594-labeled Plk-FGF-2 after CuAAC on (Aa) aminoundecane-azide and (Ab) ethanolamine in the presence of copper(II) sulfate or absence of copper (II) sulfate (Ab, Bb). Fluoresence intensity of Atto 594-labeled Plk-FGF-2 after trypsin digestion on (Ac) agarose particles with aminoundecane-azide and (Bc) for ethanolamine after CuAAC. Atto 594 is shown in orange. The asterisk highlights significant differences among groups (p < 0.05, n = 3).

D


Letter


Figure 5. MG-63 cell proliferation on Plk-FGF-2 presenting agarose beads in serum-depleted medium. (A) Cell nuclei (DAPI) staining of MG 63 cells cultured on Atto 594-labeled Plk-FGF-2 immobilized to (a, b) aminoundecan-azide decorated agarose beads by CuAAC and (c, d) in the absence of Cu(I). Individual images of each channel are displayed in Figure S5. (B) Proliferation of MG-63 cells as determined by DAPI positive cells per bead diameter. At least 20 beads were analyzed per condition. The asterisk highlights significant differences (p < 0.05). DAPI is blue, Atto 594 is orange.

and physically adsorbed FGF-2 onto the agarose surface, on which covalently linked Plk-FGF-2 will likely generate different dose−response curves because of restricted diffusion and internalization as compared to soluble FGF-2. Previous work has addressed FGF-2 attachment to fibrin hydrogels,30 to heparin decorated materials36,37 and to sulfonated silk fibroin (SF) films38 by deploying electrostatic interactions. Although physiochemical mediated immobilization of FGF-2 to negatively charged materials permits a local pool of FGF-2, cellular stimulation is temporary and limited by diffusion of the biologic or by its cellular and extracellular metabolism. Conventional conjugation strategies, targeting thiol- or primary amine functional groups in FGF-2 during immobilization are likely to influence the growth factor’s activity by (i) covalent modification of Lys residues within the receptor binding epitope and (ii) unfavorable sterical position of FGF-2 for receptor activation by using surface located free thiol groups. Binding strategies deploying the available thiol groups of the FGF-2 for decoration would ultimately impact FGF-2 performances, as the receptor and low affinity receptor binding sites of FGF-2 would be shielded by the particular carrier. Therefore, alternatives are required to exploit the maximum benefit from FGF-2 upon immobilization. For that, we selected the flexible N-terminal region (position #8, Figure 1A) of FGF-2 for modification to allow adequate flexibility of FGF-2 for receptor interaction after immobilization on the particle surface. In this study, Plk-FGF-2 was immobilized by covalent CuAAC chemistry with the ultimate aim to spatially restrict the mitogenic effects of FGF-2 to the surrounding of a model polymeric carrier, sending proliferative signals in a paracrine fashion. We observed that Atto 594-labeled Plk-FGF2 remained immobilized onto the agarose particle for at least 48 h under cell culture conditions (experiment ended at 48 h; Figure 5A, Figure S5) and cell proliferation was significantly enhanced in the proximity of FGF-2-presenting agarose particles (Figure 5B). It will be of future interest to discover the stability of immobilized Plk-FGF-2 in respect to cell cultivation time, protease levels and temperature in more detail. The CuAAC strategy can be replaced by other bio-orthogonal chemistries. For that, the respective unnatural amino acid analogue can be replaced by the one used here within, such that other functional groups are integrated into the sequence during

groups, in the presence or absence of copper(II) sulfate was found (Figure 4B, c), indicating that Atto-594-labeled Plk-FGF2 is not specifically immobilized to the hydroxyl group presenting agarose carrier but likely physio-adsorbed to the agarose material. A direct comparison between azide and ethanolamine treated agarose beads was found to be difficult as the amount of beads in solution differ as a result of the modification. To study cellular responses of FGF-2 presenting agarose beads, we seeded MG-63 cells in serum depleted medium in the presence of Atto 594-labeled FGF-2-decorated agarose beads (azide presenting groups and after CuAAC) and in the presence of control agarose beads (azide presenting groups and in absence of copper(II) sulfate during CuAAC with Atto594-labeled Plk-FGF-2) for 48 h and DAPI-stained for visualization of cell nuclei as indicator for cell proliferation (Figure 5, Figure S5). Loading of Atto 594-labeled Plk-FGF-2 onto azide particles was determined in parallel and revealed an average concentration of 55 μg/mL FGF-2 per particle suspension after CuAAC, whereas control beads (prepared in absence of copper(II) sulfate during the reaction) displayed an average concentration of 5 μg/mL FGF-2 per particle suspension, respectively (data not shown). The immobilization efficiency of Atto-594-labeled Plk-FGF-2 was approximated by comparing the initial starting solution with the loaded amount of Plk-FGF-2 found after trypsin digest onto the agarose carrier and revealed an immobilization efficiency of approximately 15%. This result is associated with high standard error because of error propagation and has to be considered semiquantitatively. Cell proliferation of MG-63 cells was restricted to cells in close proximity to Atto-594-labeled Plk-FGF-2decorated agarose beads in contrast to controls (prepared in the absence of copper(II) sulfate during reaction; Figure 5A). Next, we quantified the number of cell nuclei around individual agarose particles and observed a significant increase in cell nuclei per bead for CuAAC treated agarose particles (0.65 ± 0.08) compared to agarose particles incubated with Atto 594labeled Plk-FGF-2 in the absence of copper(II) sulfate (control; 0.39 ± 0.06). The residual activity of the control is likely reflecting the activity of the physically absorbed FGF-2 (vide supra; Figure 5B). We speculate that the observed increase in cell number is caused by a combination of both immobilized E


Letter


(3) Pompe, T.; Salchert, K.; Alberti, K.; Zandstra, P.; Werner, C. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat. Protoc. 2010, 5, 1042−1050. (4) Lienemann, P. S.; Lutolf, M. P.; Ehrbar, M. Biomimetic hydrogels for controlled biomolecule delivery to augment bone regeneration. Adv. Drug Delivery Rev. 2012, 64, 1078−1089. (5) Ehrbar, M.; Rizzi, S. C.; Hlushchuk, R.; Djonov, V.; Zisch, A. H.; Hubbell, J. A.; Weber, F. E.; Lutolf, M. P. Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering. Biomaterials 2007, 28, 3856−3866. (6) Lühmann, T.; Hall, H. Cell Guidance by 3D-Gradients in Hydrogel Matrices: Importance for Biomedical Applications. Materials 2009, 2, 1058−1083. (7) Holland-Nell, K.; Beck-Sickinger, A. G. Specifically immobilised aldo/keto reductase AKR1A1 shows a dramatic increase in activity relative to the randomly immobilised enzyme. ChemBioChem 2007, 8, 1071−1076. (8) Carragee, E. J.; Hurwitz, E. L.; Weiner, B. K. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J. 2011, 11, 471−491. (9) Zhao, H.; Heusler, E.; Jones, G.; Li, L.; Werner, V.; Germershaus, O.; Ritzer, J.; Luehmann, T.; Meinel, L. Decoration of silk fibroin by click chemistry for biomedical application. J. Struct. Biol. 2014, 186, 420−430. (10) Steen Redeker, E.; Ta, D. T.; Cortens, D.; Billen, B.; Guedens, W.; Adriaensens, P. Protein engineering for directed immobilization. Bioconjugate Chem. 2013, 24, 1761−1777. (11) Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 1989, 244, 182−188. (12) Chin, J. W. Molecular biology. Reprogramming the genetic code. Science 2012, 336, 428−429. (13) Gaston, M. A.; Jiang, R.; Krzycki, J. A. Functional context, biosynthesis, and genetic encoding of pyrrolysine. Curr. Opin. Microbiol. 2011, 14, 342−349. (14) James, C. M.; Ferguson, T. K.; Leykam, J. F.; Krzycki, J. A. The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon. J. Biol. Chem. 2001, 276, 34252−34258. (15) Blight, S. K.; Larue, R. C.; Mahapatra, A.; Longstaff, D. G.; Chang, E.; Zhao, G.; Kang, P. T.; Green-Church, K. B.; Chan, M. K.; Krzycki, J. A. Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo. Nature 2004, 431, 333−335. (16) Eger, S.; Scheffner, M.; Marx, A.; Rubini, M. Formation of ubiquitin dimers via azide-alkyne click reaction. Methods Mol. Biol. 2012, 832, 589−596. (17) Nguyen, D. P.; Lusic, H.; Neumann, H.; Kapadnis, P. B.; Deiters, A.; Chin, J. W. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. J. Am. Chem. Soc. 2009, 131, 8720−8721. (18) Gospodarowicz, D.; Ferrara, N.; Schweigerer, L.; Neufeld, G. Structural characterization and biological functions of fibroblast growth factor. Endocr. Rev. 1987, 8, 95−114. (19) Tomanek, R. J.; Hansen, H. K.; Christensen, L. P. Temporally expressed PDGF and FGF-2 regulate embryonic coronary artery formation and growth. Arterioscler., Thromb., Vasc. Biol. 2008, 28, 1237−1243. (20) Uebersax, L.; Merkle, H. P.; Meinel, L. Biopolymer-based growth factor delivery for tissue repair: from natural concepts to engineered systems. Tissue Eng., Part B 2009, 15, 263−289. (21) Beenken, A.; Mohammadi, M. The FGF family: biology, pathophysiology and therapy. Nat. Rev. Drug Discovery 2009, 8, 235− 253. (22) Yun, Y. R.; Won, J. E.; Jeon, E.; Lee, S.; Kang, W.; Jo, H.; Jang, J. H.; Shin, U. S.; Kim, H. W. Fibroblast growth factors: biology, function, and application for tissue regeneration. J. Tissue Eng. 2010, 2010, 218142.

genetic engineering opening the biologic for other chemical approaches. In fact, to date, more than 33 pyrrolysine analogues have been successfully incorporated by PylRS,39,40 demonstrating the broad versatility and exciting possibilities to sitespecifically immobilize FGF-2 or other biologics to all kind of functionalized materials. Although the spherical form of the agarose carriers used herein induced mitogenic responses of MG-63 cells, spatially patterned surfaces of Plk-FGF-2 might stimulate cell proliferation more effectively as the cell membrane is in entire contact to the surface linked growth factor. Future work will detail the mitogenic responses of cell grown on patterned and homogeneously labeled Plk-FGF-2 surfaces (e.g. azide-functionalized titanium oxide, biomaterials). Other goals may include the site-specific introduction of distinct labels (such as a fluorophore; Figure 3) or biotin to probe FGF-2 structure and function. Moreover, the Plk-FGF-2 analogue is accessible for homogeneous site-specific PEGylation for future therapeutic use. Finally, it will be of interest to translate the presented immobilization protocols of Plk-FGF-2 to other and more complex material surfaces to trigger spatial restricted mitogenic responses. In conclusion, site-specific integration of unnatural amino acids into FGF-2 is instrumental to yield a potent mitogenic biologic with unmatched spatial precision for biomaterial surface decoration. Homogenous product outcome after decoration is an inevitable demand for decorated biomaterials of the future, meeting highest pharmaceutical quality demands.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00236. Detailed experimental procedures and Figures S1−S5 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

All authors designed the experiments. T.L., G.J., M.G., M.R., and J.-C.R. performed the experiments and analyzed the data together with L.M and J.N. The manuscript was written by T.L. and L.M. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Stephanie Lamer for NanoLC-MS/MS analysis. Support by the BMBF (Federal Ministry of Education and Science, 13N13454) and by the FET Open FP7 European project MANAQA, (Magnetic Nano Actuators for Quantitative Analysis, 296679) is gratefully acknowledged.

■

REFERENCES

(1) Lee, K.; Silva, E. A.; Mooney, D. J. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J. R. Soc., Interface 2011, 8, 153−170. (2) Masters, K. S. Covalent growth factor immobilization strategies for tissue repair and regeneration. Macromol. Biosci. 2011, 11, 1149− 1163. F


Letter

ACS Biomaterials Science & Engineering (23) Nugent, M. A.; Iozzo, R. V. Fibroblast growth factor-2. Int. J. Biochem. Cell Biol. 2000, 32, 115−120. (24) Gospodarowicz, D. Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature 1974, 249, 123−127. (25) Springer, B. A.; Pantoliano, M. W.; Barbera, F. A.; Gunyuzlu, P. L.; Thompson, L. D.; Herblin, W. F.; Rosenfeld, S. A.; Book, G. W. Identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis. J. Biol. Chem. 1994, 269, 26879−26884. (26) Klagsbrun, M.; Smith, S.; Sullivan, R.; Shing, Y.; Davidson, S.; Smith, J. A.; Sasse, J. Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 1839−1843. (27) Kastrup, J. S.; Eriksson, E. S.; Dalboge, H.; Flodgaard, H. X-ray structure of the 154-amino-acid form of recombinant human basic fibroblast growth factor. comparison with the truncated 146-aminoacid form. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 160− 168. (28) Eger, S.; Scheffner, M.; Marx, A.; Rubini, M. Synthesis of defined ubiquitin dimers. J. Am. Chem. Soc. 2010, 132, 16337−16339. (29) Estape, D.; van den Heuvel, J.; Rinas, U. Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor. Biochem. J. 1998, 335 (Pt 2), 343−349. (30) Campbell, P. G.; Miller, E. D.; Fisher, G. W.; Walker, L. M.; Weiss, L. E. Engineered spatial patterns of FGF-2 immobilized on fibrin direct cell organization. Biomaterials 2005, 26, 6762−6770. (31) Presolski, S. I.; Hong, V. P.; Finn, M. G. Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Curr. Protoc. Chem. Biol. 2011, 3, 153−162. (32) Cheung, H. Y.; Lau, K. T.; Lu, T. P.; Hui, D. A critical review on polymer-based bio-engineered materials for scaffold development. Composites, Part B 2007, 38, 291−300. (33) Duckworth, B. P.; Xu, J.; Taton, T. A.; Guo, A.; Distefano, M. D. Site-specific, covalent attachment of proteins to a solid surface. Bioconjugate Chem. 2006, 17, 967−974. (34) Lee, P. L.; Johnson, D. E.; Cousens, L. S.; Fried, V. A.; Williams, L. T. Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science 1989, 245, 57−60. (35) Fox, G. M.; Schiffer, S. G.; Rohde, M. F.; Tsai, L. B.; Banks, A. R.; Arakawa, T. Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine. J. Biol. Chem. 1988, 263, 18452−18458. (36) Volpato, F. Z.; Almodovar, J.; Erickson, K.; Popat, K. C.; Migliaresi, C.; Kipper, M. J. Preservation of FGF-2 bioactivity using heparin-based nanoparticles, and their delivery from electrospun chitosan fibers. Acta Biomater. 2012, 8, 1551−1559. (37) Princz, M. A.; Sheardown, H. Heparin-modified dendrimer cross-linked collagen matrices for the delivery of basic fibroblast growth factor (FGF-2). J. Biomater. Sci., Polym. Ed. 2008, 19, 1201− 1218. (38) Wenk, E.; Murphy, A. R.; Kaplan, D. L.; Meinel, L.; Merkle, H. P.; Uebersax, L. The use of sulfonated silk fibroin derivatives to control binding, delivery and potency of FGF-2 in tissue regeneration. Biomaterials 2010, 31, 1403−1413. (39) Wan, W.; Tharp, J. M.; Liu, W. R. Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool. Biochim. Biophys. Acta, Proteins Proteomics 2014, 1844, 1059−1070. (40) Lang, K.; Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 2014, 114, 4764−4806.

G


Bio-orthogonal Immobilization of Fibroblast Growth Factor 2 for Spatial

Recommend Documents